Patent Grant
9034642
The invention relates to the polynucleotide sequence of a nontypeable strain of Haemophilus influenzae (NTHi) genome, NTHi genes contained within the genome and polypeptides encoded by the polynucleotides. The invention also relates to uses of these NTHi polynucleotides and NTHi polypeptides including vaccines and methods of treating and preventing NTHi related disorders. The invention also relates to NTHi genes which are upregulated during or in response to NTHi infection of the middle ear or nasopharynx.
Otitis media (OM) is a highly prevalent pediatric disease worldwide and is the primary cause for emergency room visits by children (Infante-Rivand and Fernandez, Epidemiol. Rev., 15: 444-465, 1993). Recent statistics indicate that 24.5 million physician office visits were made for OM in 1990, representing a greater than 200% increase over those reported in the 1980's. While rarely associated with mortality any longer, the morbidity associated with OM is significant. Hearing loss is a common problem associated with this disease, often times affecting a child's behavior, education and development of language skills (Baldwin, Am. J. Otol., 14: 601-604, 1993; Hunter et al., Ann. Otol. Rhinol. Laryngol. Suppl., 163: 59-61, 1994; Teele et al., J. Infect. Dis., 162: 685-694, 1990). The socioeconomic impact of OM is also great, with direct and indirect costs of diagnosing and managing OM exceeding $5 billion annually in the U.S. alone (Kaplan et al., Pediatr. Infect. Dis. 16: S9-11, 1997).
Whereas antibiotic therapy is common and the surgical placement of tympanostomy tubes has been successful in terms of draining effusions, clearing infection and relieving pain associated with the accumulation of fluids in the middle ear, the emergence of multiple antibiotic-resistant bacteria and the invasive nature associated with tube placement, has illuminated the need for more effective and accepted approaches to the management and preferably, the prevention of OM. Surgical management of chronic OM involves the insertion of tympanostomy tubes through the tympanic membrane while a child is under general anesthesia. While this procedure is commonplace (prevalence rates are ˜13%; Bright et al., Am. J. Public Health, 83(7): 1026-8, 1993) and is highly effective in terms of relieving painful symptoms by draining the middle ear of accumulated fluids, it too has met with criticism due to the invasive nature of the procedure and its incumbent risks (Berman et al., Pediatrics, 93(3):353-63, 1994; Bright et al., supra.; Cimons, ASM News, 60: 527-528; Paap, Ann. Pharmacother., 30(11): 1291-7, 1996).
Progress in vaccine development is most advanced for Streptococcus pneumoniae, the primary causative agent of acute OM (AOM), as evidenced by the recent approval and release of a seven-valent capsular-conjugate vaccine, PREVNAR® (Eskola and Kilpi, Pedriatr. Infect. Dis. J. 16: S72-78, 2000). While PREVNAR® has been highly efficacious for invasive pneumococcal disease, coverage for OM has been disappointing (6-8%) with reports of an increased number of OM cases due to serotypes not included in the vaccine (Black et al., Pedriatr. Infect. Dis J., 19: 187-195; Eskola et al., Pedriatr. Infect. Dis 1, 19: S72-78, 2000; Eskola et al., N. Engl. J. Med. 344: 403-409, 2001; Snow et al., Otol. Neurotol., 23: 1-2, 2002). Less progress has been made for non-typeable Haemophilus influenzae (NTHi), the gram-negative pathogen that predominates in chronic OM with effusion (Klein, Pedriatr. Infect. Dis J., 16: S5-8, 1997; Spinola et al., J. Infect. Dis., 154: 100-109, 1986). Hampering development of effective vaccines against NTHi, is the currently incomplete understanding of the pathogenesis of NTHi-induced middle ear disease. Contributing to this delay is a lack of understanding of the dynamic interplay between microbe-expressed virulence factors and the host's immune response as the disease progresses from one of host immunological tolerance of a benign nasopharyngeal commensal, to that of an active defensive reaction to an opportunistic invader of the normally sterile middle ear space.
Although strain Rd is the exemplar organism for the current small genome sequencing rationale, and an important model organism for studying H. influenzae biology, strain Rd is a poor model for the study of pathogenicity caused by members of the genus Haemophilus. Serotype b strains of H. influenzae cause invasive diseases, for example meningitis, and nontypeable H. influenzae (NTHi) strains principally have a role in localized respiratory disease, particularly in otitis media (OM), acute sinusitis, community acquired pneumonia and have important consequences in patients with chronic obstructive pulmonary disease or cystic fibrosis (Kilpi et al., Pediatr Infect Dis J 20:654-62, 2001; Murphy, Curr Opin Infect Dis 16:129-34, 2003; Roman et al., J Clin Microbial 42:1450-9, 2004; Sethi, Clin Microbiol Rev 14:336-63, 2001; St Geme, Vaccine 19 Suppl 1:S41-50, 2000). Strain Rd, however, is a derivative of a serotype d strain. Serotype d strains are rarely associated with disease (Daines et at, J Med Microbiol 52:277-82 2003; Heath et al., Pediatr Infect Dis J 20:300-5, 2001; Rodriguez et al., Infect Immun 71:1635-42, 2003, Skoczynska et al., J Clin Microbiol 43:938-41, 2005). Because one of the most useful sets of data in the study of an organism's biology is its genomic sequence, a number of investigations have identified and characterized genes found in H. influenzae type b strains, H. influenzae Biogroup Aegyptius strains or in nontypeable strains that are not present in strain Rd (Bergman et al., Infect Immun 71:1098-108, 2003; Chang et al., Infect Immun 68:2630-7, 2000; Erdos et al., Int J Pediatr Otorhinolaryngol 67:749-55. 2003; Li et al., Mol Microbiol 47:1101-11, 2003; McMichael & Green, Curr Opin Investig Drugs 4:953-8, 2003; Pomposiello & Demple, 2001; Smoot et al., Infect Immun 70:2694-9, 2002).
Currently there is a poor understanding of how NTHi causes OM in children. The identification of putative virulence factors necessary for induction of OM will contribute significantly to the understanding of the host-pathogen interaction and ultimately, the identification of potential vaccine candidates and targets of chemotherapy. There is a tremendous need to develop more effective and accepted approaches to the management and preferably, the prevention of otitis media. Vaccine development is a very promising and cost effective method to accomplish this goal (Giebank, Pedriatr. Infect. Dis J., 13(11): 1064-8, 1994: Karma et al., Int. J. Pedritr. Otorhinolalyngol., 32(Suppl.): S127-34, 1995).
The present invention provides for the identification and characterization of the genomic sequence of NTHi H. influenzae strain 86-028NP and the polypeptide sequences encoded thereby. The 3-fold analysis of the NTHi genomic sequence is set out in a series of contig sequences denoted as SEQ ID NO: 1-576, and the subsequent 8-fold analysis of the genomic sequence is set out in a series of 11 contig sequences denoted as SEQ ID NOS: 675-685. These contigs are raw data and one of skill in the art may assemble these contigs by comparing overlapping sequences to construct the complete genome of the NTHi stain 86-028NP using routine methods.
The complete and annotated genome of NTHi strain 86-028NP is set out as SEQ ID NO: 772. The open reading frames are set in Table 6 as SEQ ID NOS: 773-2593. The amino acid sequence of the resulting gene products are set out as SEQ ID NOS: 2594-4414. The genome is approximately 1.91 kb in size, slightly larger than the strain Rd genome. A number of regions of gross genome rearrangement relative to the strain Rd genome have been identified as well as a number of genes unique to strain 86-028NP.
The present invention also provides for antibodies specific for the NTHi polypeptides of the invention. Methods of detecting NTHi bacteria in a human or in sample, such as serum, sputum, ear fluid, blood, urine, lymphatic fluid and cerebrospinal fluid are contemplated. These methods include detecting NTHi polynucleotides with specific polynucleotide probes or detecting NTHi polypeptides with specific antibodies. The invention also contemplates diagnostic kits which utilize these methods of detecting NTHi bacteria.
The present invention also contemplates methods of eliciting an immune response by administering a NTHi polypeptide of the invention or a NTHi peptide thereof. These methods include administering the NTHi polypeptide or NTHi peptide as a vaccine for treatment and/or prevention of diseases caused by NTHi infection, such as OM. The following NTHi genes are upregulated during or in response to middle ear and/or nasopharynx infections; and the polypeptides encoded by these genes and peptides thereof are contemplated as possible OM vaccine candidates and/or target of chemotherapy: hisB, lppB, sapA, lolA, rbsC, purE, ribB, arcB, uxuA, dsbB, ureH, licC, HI1647, ispZ, radC, mukF, glpR, ihfB, argR, cspD, HI0094, HI1163, HI1063, HI0665, HI1292, HI1064. NTHi hisB gene is set out as nucleotide sequence SEQ ID NO: 615 and encodes the amino acid sequence set out as SEQ ID NO: 616. NTHi sapA gene is set out as nucleotide sequence SEQ ID NO: 617 and encodes the amino acid sequence set out as SEQ ID NO: 618. NTHi rbsC gene is set out as nucleotide sequence SEQ ID NO: 619 and encodes the amino acid sequence set out as SEQ ID NO: 620. NTHi purE gene is set out as nucleotide sequence SEQ ID NO: 621 and encodes the amino acid sequence set out as SEQ ID NO: 622. NTHi ribB gene is set out as nucleotide sequence SEQ ID NO: 623 and encodes the amino acid sequence set out as SEQ ID NO: 624. NTHi arcB gene is set out as nucleotide sequence SEQ ID NO: 625 and encodes the amino acid sequence set out as SEQ ID NO: 626. NTHi uxuA gene is set out as nucleotide sequence SEQ ID NO: 627 and encodes the amino acid sequence set out as SEQ ID NO: 628. NTHi dsbB gene is set out as nucleotide sequence SEQ ID NO: 629 and encodes the amino acid sequence set out as SEQ ID NO: 630. NTHi ureH gene is set out as nucleotide sequence SEQ ID NO: 631 and encodes the amino acid sequence set out as SEQ ID NO: 632. NTHi licC gene is set out as nucleotide sequence SEQ ID NO: 633 and encodes the amino acid sequence set out as SEQ ID NO: 634. NTHi HI1647 gene is set out as nucleotide sequence SEQ ID NO: 635 and encodes the amino acid sequence set out as SEQ ID NO: 636. NTHi ispZ gene is set out as nucleotide sequence SEQ ID NO: 637 and encodes the amino acid sequence set out as SEQ ID NO: 638. NTHi radC gene is set out as nucleotide sequence SEQ ID NO: 639 and encodes the amino acid sequence set out as SEQ ID NO: 640. NTHi mukF gene is set out as nucleotide sequence SEQ ID NO: 641 and encodes the amino acid sequence set out as SEQ ID NO: 642. NTHi glpR gene is set out as nucleotide sequence SEQ ID NO: 643 and encodes the amino acid sequence set out as SEQ ID NO: 644. NTHi ihfB gene is set out as nucleotide sequence SEQ ID NO: 645 and encodes the amino acid sequence set out as SEQ ID NO: 646. NTHi argR gene is set out as nucleotide sequence SEQ ID NO: 647 and encodes the amino acid sequence set out as SEQ ID NO: 648. NTHi cspD gene is set out as nucleotide sequence SEQ ID NO: 649 and encodes the amino acid sequence set out as SEQ ID NO: 650. NTHi HI1163 gene is set out as nucleotide sequence SEQ ID NO: 651 and encodes the amino acid sequence set out as SEQ ID NO: 652. NTHi HI1063 gene is set out as nucleotide sequence SEQ ID NO: 653 and encodes the amino acid sequence set out as SEQ ID NO: 654. NTHi HI0665 gene is set out as nucleotide sequence SEQ ID NO: 655 and encodes the amino acid sequence set out as SEQ ID NO: 656. NTHi HI1292 gene is set out as nucleotide sequence SEQ ID NO: 657 and encodes the amino acid sequence set out as SEQ ID NO: 658.
The novel NTHi genes included in the polynucleotide sequences presented as SEQ ID NOS: 1-576, SEQ ID NOS: 675-685 and the nucleotide sequences set out in Tables 4 and 4B are also up-regulated during infection of the middle ear and/or the nasopharynx, and therefore are contemplated to encode OM vaccine candidates and/or targets of chemotherapy. In addition, the following NTHi genes are contemplated to be virulence-associated genes and therefore are contemplated to encode possible OM vaccine candidates and/or targets of chemotherapy: HI1386, HI1462, HI1369, lav, HI1598. NTHi HI1386 gene sequence is set out as SEQ ID NO: 659 and encodes the amino acid sequence set out as SEQ ID NO: 660. NTHi HI1462 gene sequence is set out as SEQ ID NO: 661 and encodes the amino acid sequence set out as SEQ ID NO: 662. NTHi HI1369 gene sequence is set out as SEQ ID NO: 665 and encodes the amino acid sequence set out as SEQ ID NO: 666. NTHi lav gene sequence is set out as SEQ ID NO: 663 and encodes the amino acid sequence set out as SEQ ID NO: 664. NTHi HI1598 gene sequence is set out as SEQ ID NO: 669 and SEQ ID NO: 671 and encodes the amino acid sequence set out as SEQ ID NO: 670 and SEQ ID NO: 672. Additional NTHi genes associated with virulence include the polynucleotide sequences presented as SEQ ID NO: 667 and SEQ ID NO: 673.
As a method of treating or preventing NTHi infection, the present invention contemplates administering a molecule that inhibits expression or the activity of the NTHi polypeptides, which are upregulated or active during infection. In particular, the invention contemplates methods of treating or preventing NTHi infection comprising modulating NTHi protein expression by administering an antisense oligonucleotide that specifically binds to NTHi genes that are upregulated during NTHi infections, such genes include hisB, lppB, sapA, lolA, rbsC, purE, ribB, arcB, uxuA, dsbB, ureH, licC, HI1647, ispZ, radC, mukF, glpR, ihfB, argR, cspD, HI0094, HI1163, HI1063, HI0665, HI1292, HI1064. The invention also contemplates methods of treating or preventing NTHi infection comprising administering antibodies or small molecules that modulate the activity of the proteins encoded by theses genes. The novel NTHi genes included in the polynucleotide sequences presented as SEQ ID NOS: 1-576, SEQ ID NOS: 675-685 and the nucleotide sequences set out in Tables 4 and 4B are also up-regulated during infection of the middle ear and/or the nasopharynx and therefore antisense oligonucleotides that specifically bind these polynucleotide sequences are also contemplated.
Polynucleotides and Polypeptides of the Invention
The present invention provides for the sequences of the NTHi strain 86-028NP genome. The 3-fold analysis of the genomic sequence is presented as a series of contig sequences denoted herein as “contigs 1-576”. Each contig is assigned a sequence identification number that correlates with its “contig number”. Therefore, the contigs of the present invention as set out as SEQ ID NOS: 1-576. These contig polynucleotide sequences may be assembled into the complete genome sequence of the NTHi strain 86-028NP using routine methods. Upon completion of 8-fold sequence analysis of the NTHi strain 82-028NP genome, the genomic sequence was assembled into 11 contigs which are denoted herein as SEQ ID NOS: 675-685. Finally, the complete genome is presented as one nucleic acid sequence denoted herein as SEQ ID NO: 772.
The present invention provides for the NTHi polynucleotide sequences and open reading frames contained within the contigs of SEQ ID NOS: 1-576, SEQ ID NOS: 675-685, SEQ ID NOS: 773-2593, and the nucleotide sequences set out in Table 3B, Table 4B and Table 5. The present invention also provides for the polypeptide sequences encoded by the NTHi polynucleotides of the present invention such as the amino acid sequences set out in SEQ ID NOS: 2594-4414, Table 3B, Table 4B and Table 5. The invention provides for polynucleotides that hybridize under stringent conditions to (a) the complement of the nucleotides sequence of SEQ ID NOS: 1-576; SEQ ID NOS: 675-685, SEQ ID NOS: 773-2593 and the nucleotide sequences set out in Table 3B, Table 4B and Table 5 herein (b) a polynucleotide which is an allelic variant of any polynucleotides recited above; (c) a polynucleotide which encodes a species homolog of any of the proteins recited above; or (d) a polynucleotide that encodes a polypeptide comprising a specific domain or truncation of the NTHi polypeptides of the present invention.
The NTHi polynucleotides of the invention also include nucleotide sequences that are substantially equivalent to the polynucleotides recited above. Polynucleotides according to the invention can have, e.g., at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more typically at least 90%, 91%, 92%, 93%, or 94% and even more typically at least 95%, 96%, 97%, 98% or 99% sequence identity to the NTHi polynucleotides recited above.
Included within the scope of the nucleic acid sequences of the invention are nucleic acid sequence fragments that hybridize under stringent conditions to the NTHi nucleotide sequences of SEQ ID NOS: 1-576, SEQ ID NOS: 675-685, SEQ ID NOS: 773-2593, and the nucleotide sequences set out in Table 3B, Table 4B and Table 5 herein, or compliments thereof, which fragment is greater than about 5 nucleotides, preferably 7 nucleotides, more preferably greater than 9 nucleotides and most preferably greater than 17 nucleotides. Fragments of, e.g., 15, 17, or 20 nucleotides or more that are selective for (i.e., specifically hybridize to any one of the polynucleotides of the invention) are contemplated. Probes capable of specifically hybridizing to a polynucleotide can differentiate NTHi polynucleotide sequences of the invention from other polynucleotide sequences in the same family of genes or can differentiate NTHi genes from other bacterial genes, and are preferably based on unique nucleotide sequences.
The term “stringent” is used to refer to conditions that are commonly understood in the art as stringent. Hybridization stringency is principally determined by temperature, ionic strength, and the concentration of denaturing agents such as formamide. Examples of stringent conditions for hybridization and washing are 0.015 M sodium chloride, 0.0015 M sodium citrate at 65-68° C. or 0.015 M sodium chloride, 0.0015M sodium citrate, and 50% formamide at 42° C. See Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, (Cold Spring Harbor, N.Y. 1989). More stringent conditions (such as higher temperature, lower ionic strength, higher formamide, or other denaturing agent) may also be used, however, the rate of hybridization will be affected. In instances wherein hybridization of deoxyoligonucleotides is concerned, additional exemplary stringent hybridization conditions include washing in 6×SSC 0.05% sodium pyrophosphate at 37° C. (for 14-base oligos), 48° C. (for 17-base oligos), 55° C. (for 20-base oligos), and 60° C. (for 23-base oligos).
Other agents may be included in the hybridization and washing buffers for the purpose of reducing non-specific and/or background hybridization. Examples are 0.1% bovine serum albumin, 0.1% polyvinyl-pyrrolidone, 0.1% sodium pyrophosphate, 0.1% sodium dodecylsulfate, NaDodSO4, (SDS), ficoll, Denhardt's solution, sonicated salmon sperm DNA (or other non-complementary DNA), and dextran sulfate, although other suitable agents can also be used. The concentration and types of these additives can be changed without substantially affecting the stringency of the hybridization conditions. Hybridization experiments are usually carried out at pH 6.8-7.4, however, at typical ionic strength conditions, the rate of hybridization is nearly independent of pH. See Anderson et al., Nucleic Acid Hybridisation: A Practical Approach, Ch. 4, IRL Press Limited (Oxford, England). Hybridization conditions can be adjusted by one skilled in the art in order to accommodate these variables and allow DNAs of different sequence relatedness to form hybrids.
The sequences falling within the scope of the present invention are not limited to these specific sequences, but also include allelic and species variations thereof. Allelic and species variations can be routinely determined by comparing the sequence provided in SEQ ID NOS: 1-576, SEQ ID NOS: 675-685, SEQ ID NOS: 773-2593, and nucleotide sequences out in Table 3B, Table 4B and Table 5 herein, preferably the open reading frames therein, a representative fragment thereof, or a nucleotide sequence at least 90% identical, preferably 95% identical, to the open reading frames within SEQ ID NOS: 1-576, SEQ ID NOS: 675-685, SEQ ID NOS: 773-2593, and the nucleotide sequences set out in Table 3B, Table 4B and Table 5 with a sequence from another isolate of the same species. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package, including GAP (Devereux et al., Nucl. Acid. Res., 12: 387, 1984; Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol. Biol., 215: 403-410, 1990). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894; Altschul et al., supra). The well known Smith Waterman algorithm may also be used to determine identity.
Furthermore, to accommodate codon variability, the invention includes nucleic acid molecules coding for the same amino acid sequences as do the specific open reading frames (ORF) disclosed herein. In other words, in the coding region of an ORF, substitution of one codon for another codon that encodes the same amino acid is expressly contemplated.
The isolated polypeptides of the invention include, but are not limited to, a polypeptide comprising: the amino acid sequences encoded by the nucleotide sequences included within the polynucleotide sequences set out as SEQ ID NOS: 1-576, SEQ ID NOS: 675-685, SEQ ID NOS: 773-2593, and the nucleotide sequences set out in Table 3B, Table 4B and Table 5, or the corresponding full length or mature protein. The polypeptides of the invention include the amino acid sequences of SEQ ID NO: 616, SEQ ID NO: 618, SEQ ID NO: 620, SEQ ID NO: 622, SEQ ID NO: 624, SEQ ID NO: 626, SEQ ID NO: 628, SEQ ID NO: 628, SEQ ID NO: 630, SEQ ID NO: 632, SEQ ID NO: 634, SEQ ID NO: 636, SEQ ID NO: 638, SEQ ID NO: 640, SEQ ID NO: 642, SEQ ID NO: 644, SEQ ID NO: 646, SEQ ID NO: 648, SEQ ID NO: 650, SEQ ID NO: 652, SEQ ID NO: 654, SEQ ID NO: 656, SEQ ID NO: 658, SEQ ID NO: 660, SEQ ID NO: 662, SEQ ID NO: 664, SEQ ID NO: 666, SEQ ID NO: 668, SEQ ID NO: 670, SEQ ID NO: 672, SEQ ID NO: 674, SEQ ID NO: 687, SEQ ID NO: 689, SEQ ID NO: 691, SEQ ID NO: 693, SEQ ID NO: 695, SEQ ID NO: 697, SEQ ID NO: 699, SEQ ID NO: 701, SEQ ID NO: 703, SEQ ID NO: 705, SEQ ID NO: 707, SEQ ID NO: 709, SEQ ID NO: 711, SEQ ID NO: 713, SEQ ID NO:715, SEQ ID NO: 717, SEQ ID NO: 719, SEQ ID NO: 721, SEQ ID NO:723, SEQ ID NO:725, SEQ ID NO:727, SEQ ID NO:729, SEQ ID NO: 731, SEQ ID NO: 733, SEQ ID NO: 735, SEQ ID NO: 737, SEQ ID NO: 739, SEQ ID NO: 741, SEQ ID NO: 743, SEQ ID NO: 745, SEQ ID NO: 747, SEQ ID NO: 749, SEQ ID NO: 751, SEQ ID NO: 753, SEQ ID NO: 755, SEQ ID NO: 757, SEQ ID NO: 759, SEQ ID NO: 761, 763, SEQ ID NO: 765, SEQ ID NO: 767, SEQ ID NO: 769 or SEQ ID NO: 771, SEQ ID NOS: 2594-4414 which are set out in Table 3B, Table 4B and Table 5 herein.
Polypeptides of the invention also include polypeptides preferably with biological or immunogenic activity that are encoded by: (a) an open reading frame contained within the nucleotide sequences set forth as SEQ ID NOS: 1-576, SEQ ID NOS: 675-685, SEQ ID NOS: 773-2593, and the nucleotide sequences set out in Table 3B, Table 4B and Table 5, or (b) polynucleotides that hybridize to the complement of the polynucleotides of (a) under stringent hybridization conditions.
The invention also provides biologically active or immunologically active variants of the amino acid sequences of the present invention; and “substantial equivalents” thereof (e.g., with at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, 86%, 87%, 88%, 89%, at least about 90%, 91%, 92%, 93%, 94%, typically at least about 95%, 96%, 97%, more typically at least about 98%, or most typically at least about 99% amino acid identity) that retain biological and/or immunogenic activity. Polypeptides encoded by allelic variants may have a similar, increased, or decreased activity compared to polypeptides encoded by the polynucleotides included within the nucleotide sequences presented in SEQ ID NOS: 1-576, SEQ ID NOS: 675-685, SEQ ID NOS: 773-2593, and the nucleotide sequences encoding the amino acid sequences set out as SEQ ID NOS: 2594-4414 and in Table 3B, Table 4B and Table 5 herein, and the polypeptides having an amino acid sequence set out in SEQ ID NOS: 2594-4414, Table 3B, Table 4B and Table 5 herein
NTHi peptides refer to fragments of the NTHi polypeptides encoded by the nucleotide sequences presented in SEQ ID NOS: 1-576, SEQ ID NOS: 675-685, SEQ ID NOS: 773-2593, or the nucleotide sequences set out in Table 3B, Table 4B and Table 5 herein, and the polypeptides having the amino acid sequences set out in SEQ ID NOS: 2594-4414 Table 3B, Table 4B and Table 5 herein. The preferred NTHi peptides are biologically and/or immunologically active.
The present invention further provides isolated NTHi polypeptides or NTHi peptides encoded by the NTHi nucleic acid fragments of the present invention or by degenerate variants of the nucleic acid fragments of the present invention. The term “degenerate variant” refers to nucleotide fragments which differ from a nucleic acid fragment of the present invention (e.g., an ORF) by nucleotide sequence but, due to the degeneracy of the genetic code, encode an identical NTHi polypeptide sequence. Preferred nucleic acid fragments of the present invention are the ORFs that encode proteins.
The invention also provides for NTHi polypeptides with one or more conservative amino acid substitutions that do not affect the biological and/or immunogenic activity of the polypeptide. Alternatively, the NTHi polypeptides of the invention are contemplated to have conservative amino acids substitutions which may or may not alter biological activity. The term “conservative amino acid substitution” refers to a substitution of a native amino acid residue with a nonnative residue, including naturally occurring and nonnaturally occurring amino acids, such that there is little or no effect on the polarity or charge of the amino acid residue at that position. For example, a conservative substitution results from the replacement of a non-polar residue in a polypeptide with any other non-polar residue. Further, any native residue in the polypeptide may also be substituted with alanine, according to the methods of “alanine scanning mutagenesis”. Naturally occurring amino acids are characterized based on their side chains as follows: basic: arginine, lysine, histidine; acidic: glutamic acid, aspartic acid; uncharged polar: glutamine, asparagine, serine, threonine, tyrosine; and non-polar: phenylalanine, tryptophan, cysteine, glycine, alanine, valine, proline, methionine, leucine, norleucine, isoleucine General rules for amino acid substitutions are set forth in Table 1 below.
Antisense polynucleotides complementary to the polynucleotides encoding the NTHi polypeptides are also provided.
The invention contemplates that polynucleotides of the invention may be inserted in a vector for amplification or expression. For expression, the polynucleotides are operatively linked to appropriate expression control sequence such as a promoter and polyadenylation signal sequences. Further provided are cells comprising polynucleotides of the invention. Exemplary prokaryotic hosts include bacteria such as E. coli, Bacillus, Streptomyces, Pseudomonas, Salmonella and Serratia.
The term “isolated” refers to a substance removed from, and essentially free of, the other components of the environment in which it naturally exists. For example, a polypeptide is separated from other cellular proteins or a DNA is separated from other DNA flanking it in a genome in which it naturally occurs.
Antibodies and Methods for Eliciting an Immune Response
The invention provides antibodies which bind to antigenic epitopes unique to (Le., are specific for) NTHi polypeptides. Also provided are antibodies which bind to antigenic epitopes common among multiple H. influenzae subtypes but unique with respect to any other antigenic epitopes. The antibodies may be polyclonal antibodies, monoclonal antibodies, antibody fragments which retain their ability to bind their unique epitope (e.g., Fv, Fab and F(ab)2 fragments), single chain antibodies and human or humanized antibodies. Antibodies may be generated by techniques standard in the art.
It is known in the art that antigens that mimic the capsular polysaccharide of H. influenzae will generate antibodies that exhibit the ability to kill bacteria in in vitro assays. These antibodies are also known to protect against challenge with H. influenzae in animal model systems. These studies indicate antibody to the capsular polysaccharides are likely to elicit a protective immune response in humans. The present invention provides for antibodies specific for the NTHi polypeptides of the present invention and fragments thereof, which exhibit the ability to kill both H. influenzae bacteria and to protect humans from NTHi infection. The present invention also provides for antibodies specific for the NTHi polypeptides of the invention which reduce the virulence, inhibit adherence, inhibit cell division, and/or inhibit penetration into the epithelium of H. influenzae bacteria or enhance phagocytosis of the H. influenzae bacteria.
In vitro complement mediated bactericidal assay systems (Musher et al., Infect. Immun. 39: 297-304, 1983; Anderson et al., J. Clin. Invest. 51: 31-38, 1972) may be used to measure the bactericidal activity of anti-NTHi antibodies. Further data on the ability of NTHi polypeptides and NTHi peptides to elicit a protective antibody response may be generated by using animal models of infection such as the chinchilla model system described herein.
It is also possible to confer short-term protection to a host by passive immunotherapy via the administration of pre-formed antibody against an epitope of NTHi, such as antibodies against NTHi OMP, LOS or noncapsular proteins. Thus, the contemplated vaccine formulations can be used to produce antibodies for use in passive immunotherapy. Human immunoglobulin is preferred in human medicine because a heterologous immunoglobulin may provoke an immune response to its foreign immunogenic components. Such passive immunization could be used on an emergency basis for immediate protection of unimmunized individuals exposed to special risks. Alternatively, these antibodies can be used in the production of anti-idiotypic antibody, which in turn can be used as an antigen to stimulate an immune response against NTHi epitopes.
The invention contemplates methods of eliciting an immune response to NTHi in an individual. These methods elicit immune responses which include one or more of killing NTHi bacteria, blocking NTHi attachment to cells and/or slowing NTHi replication. An “immunogenic dose” of a composition of the invention is one that generates, after administration, a detectable humoral and/or cellular immune response in comparison to the immune response detectable before administration or in comparison to a standard immune response before administration. The invention contemplates that the immune response resulting from the methods may be protective and/or therapeutic. In one embodiment, the methods comprise a step of administering an immunogenic dose of a composition comprising a NTHi protein or NTHi peptide of the invention. A NTHi protein or an antigenic peptide thereof may be fused with co-protein which may not by itself produce antibodies, but is capable of stabilizing the first protein and producing a fused protein which will have immunogenic and protective properties. Thus fused recombinant protein, preferably further comprises an antigenic co-protein, such as Glutathione-S-transferase (GST) or beta-galactosidase, relatively large co-proteins which solubilize the protein and facilitate production and purification thereof. Moreover, the co-protein may act as an adjuvant in the sense of providing a generalized stimulation of the immune system. The co-protein may be attached to either the amino or carboxy terminus of the first protein. Provided by this invention are compositions, particularly vaccine compositions, and methods comprising the NTHi polypeptides encoded by the polynucleotide of the invention or antigenic peptides thereof.
The invention also contemplates methods of eliciting an immune response to multiple H. influenzae subtypes in an individual. These methods elicit immune responses which include one or more of killing the H. influenzae bacteria, blocking H. influenzae attachment to cells and/or slowing H. influenzae replication. These methods comprise a step of administering an immunogenic dose of a composition comprising a NTHi protein or NTHi peptide of the invention which comprise an antigenic epitope that is common among multiple H. influenzae subtypes but unique with respect to any other antigenic epitopes.
In another embodiment, the methods comprise administering an immunogenic dose of a composition comprising a cell expressing a NTHi protein or NTHi peptide of the invention. In yet another embodiment, the Methods comprise administering an immunogenic dose of a composition comprising a polynucleotide encoding a NTHi protein or NTHi peptide of the invention. The polynucleotide may be a naked polynucleotide not associated with any other nucleic acid or may be in a vector such as a plasmid or viral vector (e.g., adeno-associated virus vector or adenovirus vector). The compositions of the invention may comprise one or more NTHi proteins or NTHi peptides alone or in combination with other epitopes that elicit an immune response to NTHi bacteria. The compositions of the invention may also comprise one or more NTHi proteins or peptides in combination with epitopes that elicit an immune response to one or more H. influenzae subtypes. Administration of the compositions may be by routes standard in the art, for example, parenteral, intravenous, oral, buccal, nasal, pulmonary, rectal, or vaginal. The methods may be used in combination in a single individual. The methods may be used prior or subsequent to NTHi infection of an individual.
Genes that are up-regulated in NTHi infection of the middle ear and/or the nasopharynx and genes that are associated with NTHi virulence are described herein. The polypeptides and peptides thereof which are encoded by these NTHi genes are contemplated to be useful for eliciting an immune response for treating or preventing disorders associated with NTHi infection, such as OM. Some of the polypeptides encoded by these genes include: histidine biosynthesis protein, lipoprotein B, peptide ABC transporter, periplasmic SapA precursor, outer membrane lipoproteins carrier protein precursor, ribose transport system permease protein, phosphoribosylaminoimidazole carboxylase catalytic subunit, PurE, Phosphoribosylaminoimidazole carboxylase catalytic subunit, ornithine carbamolytransferase, mannonate dehydratase, disulfide oxidoreductase, urease accessory protein, phospshocholine cytidylytransferase, putative pyridoxine biosynthesis protein, singlet oxygen resistance protein, intracellular septation protein, DNA repair protein, MukF protein, glycerol-3-phosphate regulon repressor, integration host factor beta subunit, arginine repressor, cold shock like protein, stress response protein, LicA, MukF, RadA and those hypothetical proteins encoded by HI0094, HI1163, HI0665, HI1292, HI1064 HI186, HI0352 genes. NTHi OMPs, LOS and noncapsular proteins are also contemplated to elicit an immune response for prevention and treatment of disorders associated with NTHi infection.
The invention includes methods of blocking binding of NTHi bacteria to host cells in an individual. The methods comprise administering antibodies or polypeptides of the invention that block binding of NTHi cellular attachment. Alternatively, administration of one or more small molecules that block binding of NTHi cell attachment is contemplated. In vitro assays may be used to demonstrate the ability of an antibody, polypeptide or small molecule of the invention to block NTHi cell attachment.
Pharmaceutical compositions comprising antibodies of the invention, polypeptides of the invention and/or small molecules of the invention that block NTHi cellular attachment are provided. The pharmaceutical compositions may consist of one of the foregoing active ingredients alone, may comprise combinations of the foregoing active ingredients or may comprise additional active ingredients used to treat bacterial infections. The pharmaceutical compositions may comprise one or more additional ingredients such as pharmaceutically effective carriers. Dosage and frequency of the administration of the pharmaceutical compositions are determined by standard techniques and depend, for example, on the weight and age of the individual, the route of administration, and the severity of symptoms. Administration of the pharmaceutical compositions may be by routes standard in the art, for example, parenteral, intravenous, oral, buccal, nasal, pulmonary, rectal, or vaginal.
Also provided by the invention are methods for detecting NTHi infection in an individual. In one embodiment, the methods comprise detecting NTHi polynucleotides of the invention in a sample using primers or probes that specifically bind to the polynucleotides. Detection of the polynucleotide may be accomplished by numerous techniques routine in the art involving, for example, hybridization and PCR.
The antibodies of the present invention may also be used to provide reagents for use in diagnostic assays for the detection of NTHi antigens (NTHi polypeptides and peptides thereof) in various body fluids of individuals suspected of H. influenzae infection. In another embodiment, the NTHi proteins and peptides of the present invention may be used as antigens in immunoassays for the detection of NTHi in various patient tissues and body fluids including, but not limited to: blood, serum, ear fluid, spinal fluid, sputum, urine, lymphatic fluid and cerebrospinal fluid. The antigens of the present invention may be used in any immunoassay system known in the art including, but not limited to: radioimmunoassays, ELISA assays, sandwich assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, fluorescent immunoassays, protein A immunoassays and immunoelectrophoresis assays.
Vaccines and Chemotherapeutic Targets
An aspect of the invention relates to a method for inducing an immunological response in an individual, particularly a mammal which comprises inoculating the individual with a NTHi antigen protein or an antigenic peptide thereof.
The present invention also provides for vaccine formulations which comprise an immunogenic recombinant NTHi protein or NTHi peptide of the invention together with a suitable carrier. The NTHi polypeptides and peptides thereof contemplated as vaccine candidates and/or targets of chemotherapy include, but are not limited to, histidine biosynthesis protein, lipoprotein B, peptide ABC transporter, periplasmic SapA precursor, outer membrane lipoproteins carrier protein precursor, ribose transport system permease protein, phosphoribosylaminoimidazole carboxylase catalytic subunit, PurE, 3,4-dihydroxt-2-butone 4-phosphate synthase, ornithine carbamolytransferase, marmonate dehydratase, disulfide oxidoreductase, urease accessory protein, phospshocholine cytidylytransferase, putative pyridoxine biosynthesis protein, singlet oxygen resistance protein, intracellular septation protein, DNA repair protein, MUKF protein, glycerol-3-phosphate regulon repressor, integration host factor beta subunit, arginine repressor, cold shock like protein, stress response protein, LicA, RadA and those hypothetical proteins encoded by HI0094, HI1163, HI0665, HI1292, HI1064 HI1386, HI0352 genes, NTHi OMPs, NTHi LOS and NTHi noncapsular proteins and polypeptides encoded by the novel NTHi polynucleotide sequences present in the nucleotide sequences set out as SEQ ID NOS: 1-576, SEQ ID NOS: 675-685, SEQ ID NOS: 773-2593, and the nucleotide sequences set out in Table 3B, Table 4B and Table 5 herein, and the polypeptides having the amino acid sequences set out in SEQ ID NOS: 2594-4414, Table 3B, Table 4B and Table 5 herein.
Since the protein may be broken down in the stomach, it is preferably administered parenterally, including, for example, administration that is subcutaneous, intramuscular, intravenous, or intradermal. Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the bodily fluid, preferably the blood, of the individual; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use. The vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-in water systems and other systems known in the art. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.
A. Peptide Vaccines
Peptide therapeutic agents, such as peptide vaccines, are well known in the art and are of increasing use in the pharmaceutical arts. Consistent drawbacks to the parenteral administration of such peptide compounds have been the rapidity of breakdown or denaturation. Infusion pumps, as well as wax or oil implants, have been employed for chronic administration of therapeutic agents in an effort to both prolong the presence of peptide-like therapeutic agents and preserve the integrity of such agents. Furthermore, the peptide-like agent should (with particular reference to each epitope of the peptide-like agent) ideally maintain native state configuration for an extended period of time and additionally be presented in a fashion suitable for triggering an immunogenic response in the challenged animal or immunized human.
The NTHi antigenic peptides of the invention can be prepared in a number of conventional ways. The short peptides sequences can be prepared by chemical synthesis using standard means. Particularly convenient are solid phase techniques (see, e.g., Erikson et al., The Proteins (1976) v. 2, Academic Press, New York, p. 255). Automated solid phase synthesizers are commercially available. In addition, modifications in the sequence are easily made by substitution, addition or omission of appropriate residues. For example, a cysteine residue may be added at the carboxy terminus to provide a sulfhydryl group for convenient linkage to a carrier protein, or spacer elements, such as an additional glycine residue, may be incorporated into the sequence between the linking amino acid at the C-terminus and the remainder of the peptide. The short NTHi peptides can also be produced by recombinant techniques. The coding sequence for peptides of this length can easily be synthesized by chemical techniques, e.g., the phosphotriester method described in Matteucci et al., J Am Chem Soc., 103: 3185 (1981).
Some of the NTHi peptide sequences contemplated herein may be considered too small to be immunogenic, they may be linked to carrier substances in order to, confer this property upon them. Any method of creating such linkages known in the art may be used. Linkages can be formed with heterobifunctional agents that generate a disulfide link at one functional group end and a peptide link at the other, such as a disulfide amide forming agent, e.g., N-succidimidyl-3-(2-pyridyldithio) proprionate (SPDP) (See, e.g., Jansen et al., Immun. Rev. 62:185, 1982) and bifunctional coupling agents that form a thioether rather than a disulfide linkage such as reactive esters of 6-maleimidocaproic acid, 2-bromoacetic acid, 2-iodoacetic acid, 4-(N-maleimido-methyl) cyclohexane-1-carboxylic acid and the like, and coupling agent which activate carboxyl groups by combining them with succinimide or 1-hydroxy-2-nitro-4-sulfonic acid, for sodium salt such as succinimidyl 4-(N-maleimido-methyl) cyclohexane-1-carboxylate (SMCC).
B. Vaccine Compositions and Administration
A priming dose of the immunogen that is followed by one or more booster exposures to the immunogen may be necessary to be an effective vaccine (Kramp et al., Infect. Immun., 25: 771-773, 1979; Davis et al., Immunology Letters, 14: 341-8 1986 1987). Examples of proteins or polypeptides that could beneficially enhance the immune response if co-administered include cytokines (e.g., IL-2, IL-12, GM-CSF), cytokine-inducing molecules (e.g. Leaf) or costimulatory molecules. Helper (HTL) epitopes could be joined to intracellular targeting signals and expressed separately from the CTL epitopes. This would allow direction of the HTL epitopes to a cell compartment different than the CTL epitopes. If required, this could facilitate more efficient entry of HTL epitopes into the MHC class II pathway, thereby improving CTL induction. In contrast to CTL induction, specifically decreasing the immune response by co-expression of immunosuppressive molecules (e.g. TGF-β) may be beneficial in certain diseases.
Ideally, an immunogen will exhibit two properties; the capacity to stimulate the formation of the corresponding antibodies and the propensity to react specifically with these antibodies. Immunogens bear one or more epitopes which are the smallest part of an immunogen recognizable by the combing site of an antibody. In particular instances, immunogen, fractions of immunogens or conditions under which the immunogen is presented are inadequate to precipitate the desired immunological response resulting in insufficient immunity. This is often the case with peptides or other small molecules used as immunogens. Other substances such as immunomodulators (e.g., cytokines such as the interleukins) may be combined in vaccines as well.
The vaccine art recognizes the use of certain substances called adjuvants to potentate an immune response when used in conjunction with an immunogen. Adjuvants are further used to elicit an immune response that is faster or greater than would be elicited without the use of the adjuvant. In addition, adjuvants may be used to create an immunological response using less immunogen than would be needed without the inclusion of adjuvant, to increase production of certain antibody subclasses that afford immunological protection or to enhance components of the immune response (e.g., humoral, cellular). Known adjuvants include emulsions such as Freund's Adjuvants and other oil emulsions, Bordetella pertussis, MF59, purified saponin from Quillaja saponaria (QS21), aluminum salts such as hydroxide, phosphate and alum, calcium phosphate, (and other metal salts), gels such as aluminum hydroxide salts, mycobacterial products including muramyl dipeptides, solid materials, particles such as liposomes and virosomes. Examples of natural and bacterial products known to be used as adjuvants include monophosphoryl lipid A (MPL), RC-529 (synthetic MPL-like acylated monosaccharide), OM-174 which is a lipid A derivative from E. coli, holotoxins such as cholera toxin (CT) or one of its derivatives, pertussis toxin (PT) and heat-labile toxin (LT) of E. coli or one of its derivatives, and CpG oligonucleotides. Adjuvant activity can be affected by a number of factors, such as carrier effect, depot formation, altered lymphocyte recirculation, stimulation of T-lymphocytes, direct stimulation of B-lymphocytes and stimulation of macrophages.
Vaccines are typically prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. The preparation may also be emulsified. The active immunogenic ingredient is often mixed with excipients, which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, e.g., water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants, which enhance the effectiveness of the vaccine. The vaccines are conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly. Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations. For suppositories, traditional binders and carriers may include, for example, polyalkalene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1-2%. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10%-95% of active ingredient, preferably 25-70%.
Vaccines may also be administered through transdermal routes utilizing jet injectors, microneedles, electroporation, sonoporation, microencapsulation, polymers or liposomes, transmucosal routes and intranasal routes using nebulizers, aerosols and nasal sprays. Microencapsulation using natural or synthetic polymers such as starch, alginate and chitosan, D-poly L-lactate (PLA), D-poly DL-lactic-coglycolic microspheres, polycaprolactones, polyorthoesters, polyanhydrides and polyphosphazenes polyphosphatazanes are useful for both transdermal and transmucosal administration. Polymeric complexes comprising synthetic poly-omithate, poly-lysine and poly-arginine or amphipathic peptides are useful for transdermal delivery systems. In addition, due to their amphipathic nature, liposomes are contemplated for transdermal, transmucosal and intranasal vaccine delivery systems. Common lipids used for vaccine delivery include N-(1)2,3-(dioleyl-dihydroxypropyl)-N,N,N-trimethylammonium-methyl sulfate (DOTAP), dioleyloxy-propyl-trimethylammonium chloride DOTMA, dimystyloxypropyl-3-dimethyl-hydroxyethyl ammonium (DMRIE), dimethyldioctadecyl ammonium bromide (DDAB) and 9N(N′,N-dimethylaminoethane) carbamoyl) cholesterol (DC-Chol). The combination of helper lipids and liposomes will enhance up-take of the liposomes through the skin. These helper lipids include, dioleoyl phosphatidylethanolamine (DOPE), dilauroylphosphatidylethanolamine (DLPE), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE). In addition, triterpenoid glycosides or saponins derived from the Chilean soap tree bark (Quillaja saponaria) and chitosan (deacetylated chitan) have been contemplated as useful adjuvants for intranasal and transmucosal vaccine delivery.
The proteins may be formulated into the vaccine as neutral or salt forms. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the peptide) and which are formed with inorganic acids such as, e.g., hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, e.g., sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, and procaine.
The vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic. The quantity to be administered depends on the subject to be treated, capacity of the subject's immune system to synthesize antibodies, and the degree of protection desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual. However, suitable dosage ranges are of the order of several hundred micrograms active ingredient per individual. Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed in one or three month intervals by a subsequent injection or other administration.
Upon immunization with a vaccine composition as described herein, the immune system of the host responds to the vaccine by producing large amounts of CTLs specific for the desired antigen, and the host becomes at least partially immune to later infection, or resistant to developing chronic infection. Vaccine compositions containing the NTHi polypeptide or NTHi peptides of the invention are administered to a patient susceptible to or otherwise at risk of bacterial infection to elicit an immune response against the antigen and thus enhance the patient's own immune response capabilities. In this use, the precise amounts again depend on the patient's state of health and weight, the mode of administration, the nature of the formulation, etc., but generally range from about 1.0 μg to about 5000 per 70 kilogram patient, more commonly from about 10 to about 500 mg per 70 kg of body weight. For therapeutic or immunization purposes, the NTHi polypeptide or NTHi peptides of the invention can also be expressed by attenuated viral hosts, such as vaccinia or fowlpox. This approach involves the use of vaccinia virus as a vector to express nucleotide sequences that encode the peptides of the invention. Upon introduction into an acutely or chronically infected host or into a noninfected host, the recombinant vaccinia virus expresses the immunogenic peptide, and thereby elicits a host CTL response.
Humoral immune response may be measured by many well known methods, such as Single Radial Immunodiffussion Assay (SRID), Enzyme Immunoassay (EIA) and Hemagglutination Inhibition Assay (HAI). In particular, SRID utilizes a layer of a gel, such as agarose, containing the immunogen being tested. A well is cut in the gel and the serum being tested is placed in the well. Diffusion of the antibody out into the gel leads to the formation of a precipitation ring whose area is proportional to the concentration of the antibody in the serum being tested. EIA, also known as ELISA (Enzyme Linked Immunoassay), is used to determine total antibodies in the sample. The immunogen is adsorbed to the surface of a microtiter plate. The test serum is exposed to the plate followed by an enzyme linked immunoglobulin, such as IgG. The enzyme activity adherent to the plate is quantified by any convenient means such as spectrophotometry and is proportional to the concentration of antibody directed against the immunogen present in the test sample. HAI utilizes the capability of an immunogen such as viral proteins to agglutinate chicken red blood cells (or the like). The assay detects neutralizing antibodies, i.e., those antibodies able to inhibit hemagglutination. Dilution of the test serum are incubated with a standard concentration of immunogen, followed by the addition of the red blood cells. The presence of neutralizing antibodies will inhibit the agglutination of the red blood cells by the immunogen. Tests to measure cellular immune response include determination of delayed-type hypersensitivity or measuring the proliferative response of lymphocytes to target immunogen.
Nontypeable Haemophilus influenzae (NTHi)
H. influenzae is a small, nonmotile gram negative bacterium. Unlike other H. influenzae strains, the nontypeable H. influenzae (NTHi) strains lack a polysaccharide capsule and are sometimes denoted as “nonencapsulated.” NTHi strains are genetically distinct from encapsulated strains and are more heterogenous than the type b H. influenzae isolates. NTHi presents a complex array of antigens to the human host. Possible antigens that may elicit protection include OMPs, lipopolysaccharides, lipoproteins, adhesion proteins and noncapsular proteins.
Humans are the only host for H. influenze. NTHi strains commonly reside in the upper respiratory tract including the nasopharynx and the posterior oropharynx, the lower respiratory tract and the female genital tract. NTHi causes a broad spectrum of diseases in humans, including but not limited to, otitis media, pneumonia, sinusitis, septicemia, endocarditis, epiglottitis, septic arthritis, meningitis, postpartum and neonatal infections, postpartum and neonatal sepsis, acute and chromic salpingitis, epiglottis, pericarditis, cellulitis, osteomyelitis, endocarditis, cholecystitis, intraabdominal infections, urinary tract infection, mastoiditis, aortic graft infection, conjunctivitis, Brazilian purpuric fever, occult bacteremia and exacerbation of underlying lung diseases such as chronic bronchitis, bronchietasis and cystic fibrosis.
Epidemiologic studies of NTHi have indicated that the strains are heterogeneous with respect to outer membrane protein profiles (Barenkamp et aL, Infect. Immun., 36: 535-40, 1982), enzyme allotypes (Musser et al., Infect. Inzmun., 52: 183-191, 1986), and other commonly used epidemiologic tools. There have been several attempts to subtype NTHi, but none of the methodologies have been totally satisfactory. The outer-membrane protein composition of NTHi consists of approximately 20 proteins. All NTHi strains contains two common OMP's with molecular weights of 30,000 and 16,600 daltons. NTHi strains may be subtyped based on two OMP's within the 32,000-42,000 dalton range. The NTHi liposaccharide profile is fundamentally different than the enteric gram negative bacteria and separates into 1-4 distinct bands ranging from less than 20,000 daltons.
A prototype NTHi isolate is the low passage isolate 86-028NP which was recovered from a child with chronic otitis media. This strain has been well characterized in vitro (Bakaletz et al., Infect. Immun., 53: 331-5, 1988; Holmes et al., Microb. Pathog., 23: 157-66, 1997) as well as in the chinchilla OM model (described herein) (Bakaletz et al., Vaccine, 15: 955-61, 1997; Suzuki et al., Infect. Imnzun., 62: 1710-8, 1994; DeMaria et al., Infect. Inznzun., 64: 5187-92, 1996). The 86-028NP strain was used, as described herein, to identify genes that are up-regulated in expression in the chinchilla model of otitis media and genes that are necessary for NTHi survival in the chinchilla middle ear.
DFI Strategy
A differential fluorescence induction (DFI) strategy was used herein to identify NTHi genes induced during OM in a chinchilla animal model. Several methods have been developed to identify bacterial genes that contribute to the virulence of an organism during infection. Such methods include in vivo expression technology (IVET) in which bacterial promoters regulate the expression of gene(s) required for synthesis of essential nutrients required for survival in the host; signature-tagged mutagenesis (STM) enabling tag-specific identification of genes that alter the virulence properties of a microorganism when mutated; DNA microarray technology to globally screen for transcriptionally active genes, and DFI which uses FACS analysis to select for transcriptionally active promoters (Chiang et al., Annu. Rev. Microbiol., 53: 129-154, 1999). DFI is a high-throughput method that allows for the identification of differentially regulated genes regardless of the basal level of expression and does not exclude those that are essential for growth in vitro.
DFI has been successfully utilized in many microorganisms. For example, a GFP reporter system and flow cytometry was used to study mycobacterial gene expression upon interaction with macrophages (Dhandayuthapani et al., Mol. Microbiol., 17: 901-912, 1995). A promoter trap system was used to identify genes whose transcription was increased when Salmonellae were subjected to environments simulating in vivo growth and when internalized by cultured macrophage-like cells (Valdivia and Falkow, Mol. Microbiol., 22: 367-378, 1996; Valdivia and Falkow, Science, 277: 2007-2011, 1997; Valdivia and Falkow, Curr. Opin. Microbiol., 1: 359-363, 1998). In addition, DFI has been used to identify promoters expressed in S. pneumoniae and S. aureus when grown under varied in vitro conditions simulating infection (Marra et al., Infect. Immun., 148: 1483-1491, 2002; Schneider et al., Proc. Natl. Acad. Sci. U.S.A., 97: 1671-1676, 2000). In addition, DFI has been utilized to study gene regulation in Bacillus cereus in response to environmental stimuli (Dunn and Handelsman, Gene, 226: 297-305, 1999), in S. pneumoniae in response to a competence stimulatory peptide (Bartilson et al., Mol. Microbiol., 39: 126-135, 2001), and upon interaction with and invasion of host cells in Bartonella henselae Lee and Falkow, Infect. Immun., 66: 3964-3967, 1998), Listeria monocytogenes Wilson et al., Infect. Immun., 69: 5016-5024, 2001), Brucella abortus (Eskra et al., Infect. Immun., 69: 7736-7742, 2001), and Escherichia coli (Badger et al., Mol. Microbiol., 36: 174-182, 2000).
Whereas DFI has been successfully used to identify promoters active in cell culture models of infection or in vitro conditions designed to simulate an in vivo environment, few have applied DFI to identify promoters regulated in a specific biological niche within the whole animal. This is likely due to the numerous challenges associated with sorting from an in vivo environment. The host inflammatory response, dissemination and/or clearance of bacterial cells from the site of infection, as well as adherence of bacteria to epithelial cells, possibly via biofilm formation, can make bacteria inaccessible for retrieval from the living animal. These factors, among others, contribute to the complexity of the microenvironment and the heterogeneity of gene expression as the bacteria sense and respond to these changes. Recently, DFI has been used to identify promoters expressed in S. pneumoniae when the bacteria were screened in a mouse model of respiratory tract infection and a gerbil infection model of OM (Marra et al., Infect. Inzmun. 70: 1422-33, 2002; Marra et al., Microbiol., 148: 1483-91, 2002).
Animal Model
The chinchilla model is a widely accepted experimental model for OM. In particular, a chinchilla model of NTHi-induced OM has been well characterized (Bakaletz et al., J. Infect. Dis., 168: 865-872, 1993; Bakaletz and Holmes, Clin. Diagn. Lab. Immunol., 4: 223-225, 1997; Suzuki and Bakaletz, Infect. Immun., 62: 1710-1718, 1994), and has been used to determine the protective efficacy of several NTHi outer membrane proteins, combinations of outer membrane proteins, chimeric synthetic peptide vaccine components, and adjuvant formulations as vaccinogens against OM (Bakaletz et al., Vaccine, 15: 955-961, 1997; Bakaletz et al., Infect. Immun., 67: 2746-2762, 1999; Kennedy et al., Infect. Immun., 68: 2756-2765, 2000).
In particular, there is an unique in vivo model wherein adenovirus predisposes chinchillas to H. influenzae-induced otitis media, which allowed for the establishment of relevant cell, tissue and organ culture systems for the biological assessment of NTHi (Bakaletz et al., J. Infect. Dis., 168: 865-72, 1993; Suzuki et al., Infect. Immunity 62: 1710-8, 1994). Adenovirus infection alone has been used to assess for the transudation of induced serum antibodies into the tympanum (Bakaletz et al., Clin. Diagnostic Lab Immunol., 4(2): 223-5, 1997) and has been used as a co-pathogen with NTHi, to determine the protective efficacy of several active and passive immunization regimens targeting various NTHi outer membrane proteins, combinations of OMPs, chimeric synthetic peptide vaccine components, and adjuvant formulations as vaccinogens against otitis media (Bakaletz et al., Infect Immunity, 67(6): 2746-62, 1999; Kennedy et al., Infect Immun., 68(5): 2756-65, 2000; Novotny et al., Infect Immunity 68(4): 2119-28, 2000; Poolman et al., Vaccine 19 (Suppl. 1): S108-15, 2000).
Genes Unregulated In Vivo in Response to NTHi Infection of the Middle Ear
In order to identify differentially regulated promoters in response to NTHi infection of the middle ear, a promoter trap library was constructed and sorting parameters defined. A portion of the promoter trap library was inoculated directly into the chinchilla middle ear and OM development was monitored by video otoscopy and tympanometry at 24 and 48 hours. In addition, the middle ear fluids were recovered 24 and 48 hours after infection. Two-color FACS analysis was used to isolated bacteria that were expressing GFP from other cells and debris associated with the effusion. Following isolation, DNA sequence of the Haemophilus inserts 5′ of the gfpmut3 gene were determined and analyzed. In this manner, we identified genes that are up-regulated as NTHi sense and respond to the environment of the chinchilla middle ear during AOM. The following genes were identified and due to their up-regulation during NTHi infection, they may play a role in NTHi infection and virulence.
As described below in Example 7, following the DFI procedure described above and subsequent FACS analysis of gfp-expressing clones, 52 candidate clones containing potential in vivo-regulated promoters were isolated. The genes these clones control were categorized based upon general description and function within the cell and include general metabolic processes, environmental informational processing and membrane transport, membrane proteins and hypothetical proteins. Eight of these 52 clones contain sequences that are unique to NTHi strain 86-028NP. Importantly, 3 clones were isolated from independent screens in more than one animal thereby verifying the method of isolation.
In order to independently confirm the FACS data, we determined the relative expression of candidate genes by quantitative RT-PCR. The parent strain 86-028NP, was used for these studies. Thus, wild-type gene expression without the influence of plasmid copy number on gene regulation was analyzed, allowing for the indication of false-positive clone identification by FACS. Of the 44 candidate clones containing sequence similar to that identified in H. influenzae strain Rd, quantitative comparison of gene expression in vitro and in vivo confirmed up-regulated gene expression for twenty-six genes (60%) when NTHi respond to environmental cues present in the chinchilla middle ear. This analysis identified in vivo-regulated promoters which drive expression of genes involved in membrane transport, environmental informational processing, cellular metabolism, gene regulation, as well as hypothetical proteins with unknown function. (See Table 4 in Example 6).
Quantitative RT-PCR demonstrated a two-fold increase in lolA expression, enabling lipoprotein transport from the inner membrane to the outer membrane. Bacteria grow rapidly in the middle ear environment reaching 5.0×108 CFU NTHi ml middle ear fluid within 48 hours. The bacteria sense and respond to the environment, acquiring or synthesizing the necessary nutrients for growth and survival. The gene encoding the membrane component in ribose sugar transport, rbsC (SEQ ID NO: 619), showed a 5-fold increase in expression in vivo compared to cells growing in vitro. In addition, many genes involved in metabolic processes show a dramatic increase in gene expression in vivo compared to cells growing in vitro. These include a riboflavin synthesis gene, ribB (SEQ ID NO: 623), a purine nucleotide biosynthetic gene purE (SEQ ID NO: 621), ornithine carbamoyltransferase, arcB (SEQ ID NO: 625), involved in arginine degradation via the urea cycle and uxuA (SEQ ID NO: 627), encoding mannonate hydrolase, required for the uptake of D-glucuronate and transformation into glyceraldehyde 3-phosphate. In addition, but to a lesser degree, genes for histidine biosynthesis (hisB; SEQ ID NO: 615), DNA repair (radC; SEQ ID NO: 639) and a putative intracellular septation transmembrane protein (ispZ; SEQ ID NO: 637) were up-regulated.
Disulfide bond formation is important for folding and assembly of many secreted proteins in bacteria. In prokaryotes, DsbA and DsbB make up the oxidative pathway responsible for the formation of disulfides. DsbB reoxidizes DsbA, which donates disulfide bonds directly to unfolded polypeptides, and DsbB has been demonstrated to generate disulfides de novo from oxidized quinones (Collet and Bardwell, Mol. Microbiol., 44: 1-8, 2002). In H. influenzae strain Rd, DsbA is required for competence for transformation (Tomb, Proc. Natl. Acad. Sci. U.S.A., 89: 10252-10256, 1992). Herein, an approximate 3-fold increase in dsbB gene (SEQ ID NO: 629) transcription was demonstrated, illuminating an important role for disulfide interchange for NTHi growing in the middle ear environment.
Bacteria colonization of the middle ear, a noiuially sterile environment, results in a host inflammatory response and subsequent neutrophil infiltration. Bacteria have evolved numerous strategies to combat this host response. NTHi increase gene expression (4-fold) of ureH (SEQ ID NO:631), a homologue of a gene required for expression of active urease in Helicobacter, shown to be involved in acid tolerance (Young et al., J. Bacterol., 178: 6487-6495, 1996). Recently, it has been reported that urease activity may play a role in chronic Actinobacillus pleuropneumoniae infection by counteracting the decrease in pH occurring upon infection (Baltes et al., Infect. Inunun., 69: 472-478, 2000; Baltes et al., Infect. Immun., 69: 472-478, 2001; Bosse and MacInnes, Can. J. Vet. Res., 64: 145-150). A biotype analysis on NTHi isolates from middle ear effusions demonstrated that 87% are urease positive (DeMaria et al., J. Clin. Microbiol., 20: 1102-1104, 1984). However, the role of urease in NTHi virulence is unknown. Similarly, an increase in expression of a gene whose product demonstrates 88% sequence identity to a pyridoxine biosynthesis protein in S. pneumoniae and 60% homology to a putative singlet oxygen resistance protein that may function as an antioxidant. Phosphorylcholine (ChoP) has been implicated in the pathogenesis of NTHi (Weiser et al., Infect. Immun., 65: 943-950, 1997). NTHi modulates ChoP expression by phase variation, decorating the LOS on the cell surface. ChoP may contribute to NTHi persistence in the respiratory tract via decreased susceptibility to antimicrobial peptides (Lysecko et al., Infect. Immun., 68: 1664-1671, 2000) and alter the sensitivity to serum killing mediated by C-reactive protein (CRP) (Weiser et al., J. Exp. Med., 187: 631-640, 1998). The microenvironment of the nasopharynx and middle ear cavity may select for the ChoP+ phenotype, as ChoP+ strains show greater colonization of the chinchilla nasopharynx (Tong et al., Infect. Immun., 68: 4593-4597, 2000). Expression of the licC gene (SEQ ID NO: 633) was also increased. The licC gene encodes a phosphorylcholine cytidylyltransferase that plays a role in the biosynthesis of phosphorylcholine-derivatized LOS (Rock et al., J. Bacterol., 183: 4927-4931, 2001).
Also included among the in vivo-induced genes is a set whose products subsequently regulate gene expression or DNA replication. These genes include transcriptional regulation of glycerol metabolism by the glp repressor, glpR (SEQ ID NO: 643), the arginine repressor gene, argR (SEQ ID NO: 647), and the integration host factor (IHF) beta subunit, ihfB (SEQ ID NO: 645). IHF is a histone-like protein that binds DNA at specific sequences, an accessory factor involved in replication, site-specific recombination and transcription, altering the activity of a large number of operons (Goosen and van de Putte, Mol. Microbiol. 16: 1-7, 1995). In addition, CspD inhibits DNA replication during stationary phase-induced stress response in E. coli (Yamanaka et al., Mol. Microbiol., 39: 1572-1584, 2001) and the mukF (SEQ ID NO: 641) gene protein homologue contributes to a remodeling of the nucleiod structure into a more compact fog in prior to cell segregation (Sawitzke and Austin, Proc. Natl. Acad. Sci. U.S.A., 62: 1710-1718, 2000). The DFI strategy described herein also identified promoters induced in vivo for genes of unknown function. The hypothetical protein, HI0094, demonstrated an 8-fold increase in gene expression during early OM but its role remains unknown. HI1163 (SEQ ID NO: 651) showed 58% amino acid identity with the hypothetical YdiJ proteins, a putative oxidase, of E. coli.
A high-density transposon mutagenesis strategy was used to identify H. influenzae genes essential for growth on rich medium (Akerley et al., Proc. Natl. Acad. Sci. U.S.A., 99: 966-971, 2002). Six genes were identified in the screen described herein that are included in essential gene set described in Akerley' et al., supra. (hisB, lppB, lolA, ispZ, mukF and unknown HI0665). Recently genes of non-typeable H. influenzae that are expressed upon interaction with two human respiratory tract-derived epithelial cell lines have been identified. These genes included those involved in metabolic processes, stress responses, gene expression, cell envelope biosynthesis, DNA-related processes, cell division and ORF's encoding proteins of unknown function. (Ulsen et al., Mol. Microbiol., 45: 485-500, 2002). Similarly the stress response gene, cspD (SEQ ID NO: 649), genes involved in purine and riboflavin biosynthesis, and a protein of unknown function, vapA was identified in the screen described herein. Expression of vapA was detected in vitro, yet vapA gene expression increased two-fold in vivo. These unique approaches identified known genes that are upregulated in NTHi-induced OM and therefore are likely to play a role in NTHi infection and virulence; and may be potential candidates for vaccines and antisense therapies and other therapeutic methods of treatment of NTHi related disorders.
The DFI strategy resulted in the identification of promoters induced in vivo for genes of unknown function as well. The hypothetical protein, HI0094, demonstrated a 8-fold increase in gene expression during early OM but its role remains unknown. HI1163 (SEQ ID NO: 651) showed 58% amino acid identity with the hypothetical YdiJ proteins, a putative oxidase, of E. coli. Therefore, these hypothetical genes are likely to play a role in OM induced by NTHi infection.
The following examples illustrate the invention wherein Example 1 describes the sequence of the NTHi genome, Example 2 describes the identified contigs and initial gene discovery, Example 3 describes construction of the NTHi promoter trap library, Example 4 describes the analyses of 86-028NP derivatives expressing GFP, Example 5 demonstrates direct labelling of bacteria from middle ear fluids, Example 6 describes identification of promoters induced in vivo in acute otitis media, Example 7 describes identification of virulence-associated genes, Example 8 describes identification of unique NTHi gene sequences, Example 9 described the analysis of the complete NTHi 86-028NP genome, and Example 10 compares the genomic DNA sequences of NTHi, strain 86-028NP and H. influenzae serotype d, strain kw20
NTHi strain 86-028NP is a minimally passaged clinical isolate obtained from a pediatric patient who underwent tympanostomy and tube insertion for chronic OM at Columbus Children's Hospital. (Bakaletz et al. Infection and Immunity, 56(2): 331-335, 1988) The 86-028NP strain was deposited with the American Type Tissue Collection (Manassas, Va. 20108 USA) on Oct. 16, 2002 and assigned accession no. PTA-4764.
In an effort to more broadly approach the identification of the virulence determinants in NTHi, the genome of the NTHi 86-028NP strain was sequenced to 3-fold coverage. Chromosomal DNA was prepared from strain 86-028NP using the Puregene protocol and sheared to 2-4 kb in size with a Hydroshear instalment (Gene Machines). The sheared DNA was ethanol-precipitated, end-repaired using a mixture of Klenow enzyme and T4 DNA polymerase, and size-selected by agarose gel electrophoresis to obtain 2-4 kb fragments as described in Chissoe et al. (Methods: a Companion to Methods of Enzymology 3: 55-65, 1991) and Sambrook et al. (Molecular Cloning: a Laboratory Manual, 2nd Ed. Cold Springs Harbor, N.Y., 1989). These fragments were cloned into vector pUC18 using the SmaI restriction site (phosphatase-treated) and transformed into E. coli XL-1 Blue, selecting for ampicillin resistance. Colonies that contain inserts were identified by blue/white screening on LB-Amp plates containing X-gal, and transferred into 96-deep well plates containing 1.5 ml of TB-Amp (TB=Terrific Broth) broth. The deep-well plate cultures were grown overnight (18-22 hours) at 37° C. Template preparation, sequencing and contig assembly were performed.
Automated template preparation was performed on the Beckman Biomek 2000 automated robotics workstation as described in Chissoe et al., (supra.) Briefly, each 96-deep well plate, containing the clones prepared above, was centrifuged to pellet the cells, the supernatant decanted, and the cells frozen (if necessary) at −20° C. Four 96-deep well blocks were placed on the Biomek table, and the liquid handling robot was used to prepare the template using an automated version of a typical SDS-NaOH lysis protocol as described in Chissoe et al., (supra.). The final ethanol-precipitated templates were each dissolved in 50 μl ddH2O, and used for DNA sequencing.
Sequencing reactions were run by re-arraying the templates (from 96-well plates) into 384-well plates, using the Robbins Hydra 96 robot. Cycle-sequencing reactions were run using PE Big-Dye™ terminators and universal primers (M13 forward and reverse), cleaned up over Sephadex G50 columns, and analyzed on a PE Biosystems 3700 capillary electrophoresis DNA sequencer according to the manufacturer's instructions. Sequencing reads (8219) were assembled into 576 contigs (SEQ ID NOS: 1-576 herein). The statistics for the 3-fold sequencing are shown in Table 2A. The total unique sequence in assembly 17 is 1.74 Mb.
Subsequently, 8-fold sequencing analysis of the NTHi genome was carried out. The 8-fold sequencing assembled the NTHi genome into 11 contigs. Contigs 5, 8, 9, 10, 12-18 are denoted as SEQ ID NOS: 675-685 herein. The statistics for the 8-fold sequencing are shown in Table 2B.
Seventy-five of the 88 contigs with length ≧5000 bp, identified with the 3-fold sequence analysis, show significant similarity via BLASTN to genes in H. influenzae strain Rd. To visualize the potential relationship between the gene order in H. influenzae strain 86-028NP and H. influenzae strain Rd, the 86-028NP three-fold contig set and the Rd gene set were bidirectionally compared using BLASTN. The results were plotted in gene-order verses contig space by sorting the contigs based on gene coordinates of the Rd genes hit, anchoring each contig at the smallest coordinate found as described in Ray et al., (Bioinformatics 17: 1105-12, 2001). Compared in this fashion, an incomplete assembly of a genome with identical gene order to a completely known genome would display a monotonically increasing stair-stepped form.
BLASTX was used to identify hits to sequences with homology to genes in the strain Rd genome as well as genes not found in H. influenzae strain Rd. Hits to strain Rd sequences were removed from the data set and the other hits summarized in Table 3A. The data are presented as follows: contig # (SEQ ID NO: #), column 1; E score for each hit, column 2; the name of the protein that had homology to a portion of the amino acid translation of the cited contig, column 3; the organism producing the homologue, column 4; and the Genbank protein identifier for each of the proteins cited in column 3, column 5; the corresponding nucleotides within the contig (referenced by SEQ ID NO:). In most instances, several homologues were identified but for clarity, the protein of greatest homology is cited in Table 3A.
The sequences for some of the genes listed in Table 3A were identified within the 8-fold sequencing of the NTHi genome. Table 3B lists the location of these genes within the 11 contigs, the full length open reading frame sequence (identified by SEQ ID NO:), the derived amino acid sequence encoded by the open reading frame and the gene with high homology identified by BLASTX (as listed in Table 3A).
To examine the relative short range gene arrangements in NTHi and the Rd strain, the gene order in two gene clusters that have been well-described were compared. First, the genes present in the hemagglutinating pilus (LKP) gene region were examined. (Mhlanga-Mutangadura et al., J Bacteriol. 180(17): 4693-703, 1998). The pilus gene cluster is located between the purE and pepN genes, only fragments of which are depicted in
A global analysis of the current assembly indicates that the gene content and order are similar to that in strain Rd. A more detailed analysis revealed that there are a substantial number of NTHi genes not previously seen in the Pasteurellaceae and some regions where the NTHi gene content and order is different than that seen in strain Rd. Thus, the current data suggest that the strain 86-028NP genome will contain a complex mosaic of Rd and non-Rd like features.
The DFI strategy also identified novel NTHi sequences that had increased gene expression. A list of these novel contig sequences that contain genes or gene fragments that have homology to ORFs in other organisms (primarily gram-negative bacteria) is set out in Table 3A. For example, the nucleotide sequence of contig 442 (SEQ ID NO: 442), nucleotides 1498-1845 are highly homologous to the sequences encoding amino acids 1-116 of H. influenzae strain Rd lipoprotein B (LppB). The gene is positioned between the stationary phase survival gene, surE, and a gene encoding a 43 kD antigenic outer membrane lipoprotein that is highly homologous to the recently identified bacterial lipoprotein, LppB/NlpD, which has been associated with virulence (Padmalayam et al., Infect. Immun., 68: 4972-4979, 2000). Recently, Zhang and coworkers demonstrated that nlpD and surE gene expression was induced during stationary phase of bacterial growth in Thermotoga maritima (Zhang et al., Structure (Camb), 9: 1095-1106, 2001). Therefore, under stress-induced conditions in the middle ear, this NTHi lipoprotein may be expressed.
Pasteurella
multocida
Pyrococcus
horikoshii
Pasteurella
multocida
Chlorobium
tepidum
Salmonella
enterica subsp.
enterica serovar
Typhi
Salmonella
enterica subsp.
enterica serovar
Typhi
Salmonella
enterica subsp.
enterica serovar
Typhi
Pasteurella
multocida
Paramecium
bursaria Chlorella
Vibrio cholerae
Pasteurella
multocida
Proteus mirabilis
Spirulina platensis
Neisseria
meningitidis
Salmonella
enterica subsp.
enterica serovar
Typhi
Pasteurella
multocida
Pectobacterium
carotovorum
Pasteurella
multocida
Haemophilus
aegyptius
Salmonella
typhimurium LT2
Salmonella
enterica subsp.
enterica serovar
Typhi
Escherichia coli
Escherichia coli
Borrelia
burgdorferi
Homo sapiens
Actinobacillus
pleuropneumoniae
Buchnera sp.
Escherichia coli
Pasteurella
multocida
Rickettsia conorii
Vibrio cholerae
Pasteurella
multocida
Neisseria
gonorrhoeae
Pasteurella
multocida
Vibrio cholerae
Caulobacter
crescentus
Mus musculus
Homo sapiens
Mesorhizobium
loti
Buchnera sp.
Campylobacter
jejuni
Ralstonia
solanacearum
Salmonella
enterica subsp.
enterica serovar
Typhi
Homo sapiens
Xylella fastidiosa
Pasteurella
multocida
Haemophilus
somnus
Haemophilus
somnus
Escherichia coli
Pasteurella
multocida
Salmonella
enterica subsp.
enterica serovar
Typhi
Salmonella
enterica subsp.
enterica serovar
Typhi
Pasteurella
multocida
Pasteurella
multocida
Pasteurella
multocida
Ralstonia
solanacearum
Escherichia coli
Pasteurella
multocida
Vibrio cholerae
Chlamydia
trachomatis
Agrobacterium
tumefaciens str.
Pasteurella
multocida
Salmonella
typhimurium
typhimurium LT2]
Salmonella
typhimurium
Xylella fastidiosa
Clostridium
acetobutylicum
Drosophila
melanogaster
Salmonella
typhimurium LT2,
typhimurium LT2
Pseudomonas
aeruginosa
Brevibacterium
epidermidis
Xylella fastidiosa
Salmonella
enterica subsp.
enterica serovar
Typhi
Synechococcus
Aquifex aeolicus
Ralstonia
solanacearum
Caulobacter
crescentus
Pasteurella
multocida
Vibrio cholerae
Listeria
monocytogenes
Salmonella
enterica subsp.
enterica serovar
Typhi
Salmonella
enterica subsp.
enterica serovar
Typhi
Haemophilus
influenzae
influenzae
Haemophilus
paragallinarum
Actinobacillus
actinomycetemco-
mitans
Actinobacillus
actinomycetemco-
mitans
Actinobacillus
actinomycetemco-
mitans
Pasteurella
multocida
Pasteurella
multocida
Neisseria
gonorrhoeae
Vibrio cholerae
Pasteurella
multocida
Salmonella
enterica subsp.
enterica serovar
Typhi
Pasteurella
multocida
Haemophilus
somnus
Salmonella
enterica subsp.
enterica serovar
Typhi
Listeria innocua
Escherichia coli
Haemophilus
aegyptius
Haemophilus
aegyptius
Haemophilus
influenzae biotype
aegyptius
Haemophilus
influenzae
Vibrio cholerae
Haemophilus
influenzae DL42
Haemophilus
influenzae
Streptococcus
pyogenes M1
Shewanella sp.
Bacillus
halodurans
Ralstonia
solanacearum
Homo sapiens
Deinococcus
radiodurans
Pasteurella
multocida
Haemophilus
somnus
Haemophilus
somnus
Haemophilus
somnus
Escherichia coli
Neisseria
meningitidis
Eubacterium
acidaminophilum
Pseudomonas
aeruginosa
Pasteurella
multocida
Pseudomonas
aeruginosa
Pasteurella
multocida
Escherichia coli
Salmonella
typhimurium LT2
Haemophilus
influenzae
Haemophilus
parahaemolyticus
Leishmania major
Neisseria
meningitidis
Pseudomonas
aeruginosa
Helicobacter
pylori 26695
Salmonella
typhimurium LT2
Xylella fastidiosa
Escherichia coli
Pasteurella
multocida
Salmonella
typhimurium LT2
Escherichia coli
Pediococcus
pentosaceus
Escherichia coli
Escherichia coli
Bacillus
halodurans
Caenorhabditis
elegans
Mesorhizobium
loti
Escherichia coli
Clostridium
perfringens
Salmonella
enterica subsp.
enterica serovar
Typhi
Xylella fastidiosa
Listeria innocua
Halobacterium sp.
Yersinia
enterocolitica
Pasteurella
multocida
Streptomyces
avermitilis
Escherichia coli
Yersinia
enterocolitica
Pasteurella
multocida
Escherichia coli
Mesorhizobium
loti
Coxiella burnetii
Salmonella
enterica subsp.
enterica serovar
Typhi
Bacillus
halodurans
Staphylocoecus
aureus subsp.
aureus Mu50
Homo sapiens
Xylella fastidiosa
Haemophiius
Salmonella
typhimurium LT2
Haemophilus
Haemophilus
influenzae N182
Salmonella
enterica
Mycoplasma
pulmonis
Neisseria
gonorrhoeae
Neisseria
meningitidis
Pasteurella
multocida
Pasteurella
multocida
Haemophilus
influenzae Tn106
Haemophilus
influenzae
Haemophilus
influenzae, type b,
Haemophilus
influenzae Tn106
Deinococcus
radiodurans
Vibrio cholerae
Pasteurella
multocida
Pasteurella
multocida
Escherichia coli
Pseudomonas
aeruginosa
Listeria innocua
Pectobacterium
carotovorum
Yersinia pestis
Thermoplasma
acidophilum
Pasteurella
multocida
Nephroselmis
olivacea
Brevibacterium
epidermidis
Saccharomyces
cerevisiae
Haemophilus
influenzae
Vibrio cholerae
Salmonella
enterica subsp.
enterica serovar
Typhi
Pasteurella
multocida
Salmonella
typhimurium
Staphylococcus
aureus subsp.
aureus Mu50
Pasteurella
multocida
Escherichia coli
Ureaplasma
urealyticum
Ralstonia
solanacearum
Haemophilus
somnus
Pseudomonas
putida
Pseudomonas
putida
Haemophilus
influenzae
Haemophilus
influenzae
Actinobacillus
actinomycetemco-
mitans
Synechocystis sp.
Staphylococcus
aureus subsp.
aureus Mu50
Burkholderia
pseudomallei
Synechocystis sp.
Salmonella
enterica subsp.
enterica serovar
Typhi
Haemophilus
Haemophilus
influenzae HI689
Neisseria
meningitidis
Salmonella
enterica subsp.
enterica serovar
Typhi
Mesorhizobium
loti
Salmonella
enterica subsp.
enterica serovar
Typhi
Salmonella
enterica subsp.
enterica serovar
Typhi
Pasteurella
multocida
Salmonella
enterica subsp.
enterica serovar
Typhi
Neisseria
meningitidis
Arabidopsis
thaliana
Pasteurella
multocida
Xylella fastidiosa
Neisseria
gonorrhoeae
Neisseria
meningitidis
Neisseria
meningitidis
Neisseria
meningitidis
Pasteurella
multocida
Zymomonas
mobilis
Clostridium
acetobutylicum
Staphylococcus
aureus
Actinobacillus
actinomycetemco-
mitans
Helicobacter
pylori J99
Acinetobacter
calcoaceticus
Pasteurella
multocida
Yersinia pestis
Xylella fastidiosa
Lactococcus lactis
Haemophilus
influenzae (strain
Pasteurella
multocida
Vibrio cholerae
Vibrio cholerae
Deinococcus
radiodurans
Ureaplasma
urealyticum
Haemophilus
influenzae
Pasteurella
multocida
Vibrio cholerae
Yersinia pestis
Pasteurella
multocida
Campylobacter
jejuni
Escherichia coli
Neisseria
meningitidis
Mesorhizobium
loti
Listeria innocua
Mesorhizobium
loti
Salmonella
enterica subsp.
enterica serovar
Typhi
Haemophilus
influenzae
Escherichia coli
Rickettsia conorii
Pasteurella
multocida
Escherichia coli
Pasteurella
multocida
Yersinia pestis
Vibrio cholerae
Pasteurella
multocida
Pasteurella
multocida
Salmonella
enterica subsp.
enterica serovar
Typhi
Ralstonia
solanacearum
Chlamydia
muridarum
Vibrio cholerae
Legionella
pneumophila
Salmonella
typhimurium LT2
Neisseria
meningitidis
Deinococcus
radiodurans
Escherichia coli
Buchnera sp.
Actinobacillus
pleuropneumoniae
Pasteurella
multocida
Chlorobium
tepidum
Neisseria
meningitidis
Pasteurella
multocida
Pectobacterium
carotovorum
Sinorhizobium
meliloti
Escherichia coli
Salmonella
enterica subsp.
enterica serovar
Typhi
Haemophilus
influenzae
Haemophilus
influenzae
Haemophilus
influenzae
Pasteurella
multocida
Pasteurella
multocida
Yersinia pestis
Pasteurella
multocida
Xylella fastidiosa
Deinococcus
radiodurans
Haemophilus
parainfluenzae
Vibrio cholerae
Haemophilus
influenzae
Bacillus firmus
Pasteurella
multocida
Pasteurella
multocida
Vibrio cholerae
Yersinia pestis
Neisseria
meningitidis
Haemophilus
influenzae HK715
Schizosaccharo-
myces pombe
Haemophilus
Salmonella
enterica subsp.
enterica serovar
Typhi
Salmonella
typhimurium LT2,
Neisseria
meningitidis
Escherichia coli
Neisseria
meningitidis
Haemophilus
Haemophilus
Haemophilus
influenzae biotype
aegyptius
Actinobacillus
actinomycetemco-
mitans
Salmonella
enterica subsp.
enterica serovar
Typhi
Xylella fastidiosa
Haemophilus
influenzae
Pasteurella
multocida
Escherichia coli
Neisseria
meningitidis
Haemophilus
influenzae (strain
Haemophilus
influenzae (isolate
Haemophilus
influenzae
Haemophilus
influenzae
Drosophila
melanogaster
Chlamydia
muridarum (strain
Pyrococcus
horikoshii
Sulfolobus
tokodaii
Chlamydophila
pneumoniae
Gallus gallus
Salmonella
enterica subsp.
enterica serovar
Typhi
Salmonella
enterica subsp.
enterica serovar
Typhi
Pasteurella
multocida
Salmonella
typhimurium LT2
Salmonella
enterica subsp.
enterica serovar
Typhi
Vibrio cholerae
Haemophilus
influenzae
Legionella
pneumophila
Pasteurella
multocida
Pasteurella
multocida
Helicobacter
pylori J99
Campylobacter
jejuni
Escherichia coli
Pasteurella
multocida
Thermoplasma
acidophilum
To identify potential virulence determinants of NTHi, bacterial gene expression was monitored by differential fluorescence induction (DFI) during early disease progression in one specific anatomical niche of a chinchilla model of NTHi-induced otitis media (OM). Genomic DNA fragments from NTHi strain 86-028NP were cloned upstream of the promoterless gfpmut3 gene using a promoter trap library. Plasmid pGZRS39A, a derivative of pGZRS-1 isolated from Actinobacillus pleuropneumoniae, is an A. pleuropneumoniae-Escherichia coli shuttle vector. This plasmid contains the origin of replication from A. pleuropneumoniae, the lacZα gene from pUC19 and the kanamycin resistance gene from Tn903. (West et al., Genes, 160: 81-86, 1995).
The promoter trap vector was constructed by cloning the GTP mutant gfpmut3 gene, as a BamHI to EcoRI fragment into pGZRS-39A to form pRSM2167. This mutant GTP gene contains two amino acid changes, S65G and S72A, that enhance fluorescence emission when excited at 488 nm. This mutant also has high solubility and fast kinetics of chromophore formation (Cormack et al., Gene, 173: 33-38, 1996). This plasmid was transformed by electroporation into NTHi strain 86-028NP, generating the parent-plasmid strain 86-028NP/pRSM2169.
Random genomic DNA fragments (described in Example 1) were prepared for ligation into the promoter probe vector. Genomic DNA was isolated from strain 86-028NP using the Puregene DNA isolation kit (Gentra Systems, Minneapolis, Minn.) according to the manufacturer's protocol. Due to restriction barriers, it was necessary to isolate the plasmid DNA and use this for the library generation. The isolated DNA was partially digested with Sau3AI (NEB, Beverly, Mass.; 0.25 units/μg DNA) for 1 hour at 37° C., separated by gel electrophoresis and DNA fragments 0.5-1.5 kb in size were recovered using the Qiagen gel extraction kit. For vector preparation, pRSM2167 was isolated from an overnight culture using the Wizard Plus Maxiprep DNA purification system (Promega, Madison Wis.) according to the manufacturer's protocol.
Plasmid DNA was linearized by BamHI digestion and 5′ phosphate groups removed by treatment with calf intestinal alkaline phosphatase (CIAP; GibcoBRL Life Technologies). Genomic DNA fragments were ligated with the linearized, phosphatase-treated vector and electroporated into competent NTHi strain 86-028NP prepared for electroporation according to a modified protocol (Mitchell et al., Nucleic Acids Res., 19: 3625-3628, 1991). When plasmid DNA was electroporated back into NTHi strain 86-028NP, transformation efficiency was improved by one-thousand fold. Briefly, cells were grown to an OD600=0.3 in sBHI (brain heart infusion) broth at 37° C., 220 rpm. Cells were chilled on ice for 30 minutes and subsequently washed with an equal volume of 0.5×SG (1×SG: 15% glycerol, 272 mM sucrose) at 4° C. Washes were repeated a total of three times. Subsequently, the cells were diluted in 1×SG to a 100× concentrated volume. The cells were electroporated using the BioRad Gene Pulser II set at 200 ohms, 2.5 kV and 25 μF and then diluted in 1 ml prewarmed sBHI, incubated for 2 hours at 37° C., 5% CO2 and plated on chocolate agar for overnight growth of transformants.
Transformants were selected and frozen in pools of 1000 clones in skim milk containing 20% glycerol (vol/vol). A 68,000 member gfp promoter probe library was generated. Using the probability calculation of Clarke and Carbon (Cell, 9: 91-99, 1976), to achieve a 99% probability of having a given DNA sequence represented in a library of 300 bp fragments of strain 86-028NP DNA (1.8×106 bp/genome), a library of 27,629 clones was needed. Therefore the present library represents 2.5 fold coverage of the 86-028NP genome.
In order to assess the quality of the library, fifty clones were selected at random, grown overnight on chocolate agar and the plasmids were isolated and insert DNA sequenced. A majority (64%) of the selected clones had insert sizes ranging between 200 and 500 bp while 32% exceeded 500 bp. The majority of inserts showed homology to unique H. influenzae strain Rd open reading frames (ORFs), and 15 clones had sequence unique to strain 86-028NP DNA. Of those clones with homology to strain Rd, 60% were in the correct orientation, 36% of which contained sequence upstream an ORF. Although a majority of clones had an insert size less than 500 bp, no correlation was found between small insert size and increased GFP expression. In fact four clones exhibited slight to moderate fluorescence in vitro, 3 of which had insert sizes between 200-500 base pairs and one had an insert that was greater than 700 base pairs.
A fraction of the library (approximately 1000 clones) was grown on chocolate agar, harvested in PBS and analyzed by flow cytometry for GFP fluorescence. Compared to strain 86-028NP/pRSM2169 that contains the promoter trap vector without insert DNA, the pool of library clones displays an increased fluorescence intensity. Thus, the library contains clones with promoters at varying levels of activity.
In order to establish the FACS parameters necessary to identify and sort gfp-expressing bacteria, a panel of isolates demonstrating varying levels of gfp expression was utilized. Background fluorescence was assessed using strain 86-028NP/pRSM2169 (negative control), therefore any observed fluorescence would be due to the lacZ promoter driving gfp expression. However, this strain does not produce detectable levels of GFP and in fact, does not demonstrate increased fluorescence when compared to the parent strain 86-028NP. A high-level gfp-expressing isolate was generated by cloning a 500 bp fragment containing the strong promoter for outer membrane protein P2 expression into SalI-BamHI digested pRSM2167. This plasmid was transformed into 86-028NP by electroporation, generating the high-level gfp expressing strain 86-028NP/pRSM2211 (highly fluorescent control). This strain demonstrated an approximate 100 fold increase in GFP fluorescence compared to strain 86-028NP/pRSM2169. An intermediate fluorescent derivative clone, 86-028NP/pKMM4B5 (intermediate fluorescent control), was isolated by FACS analysis and used both in preliminary experiments and as a control for cell sorting. The DNA fragment containing a promoter driving gfp expression in vitro is unique to strain 86-028NP, having no known homology to DNA of other organisms. This clone exhibits an approximate 10 fold increase in fluorescence compared to strain 86-028NP/pRSM2169.
The control strains were resuspended from growth on chocolate agar and labeled with cross-reactive Phycoprobe R-PE anti-human IgG (H+L) antibody (10 μg/ml in 100 μl PBS; Biomeda Corp) for 30 minutes at 4° C. Following three successive washes to remove unbound antibody, bacteria were resuspended in 300 μl DPBS for FACS analysis. These control preparations were used to set the appropriate size and fluorescence gates using a Coulter Epics Elite flow cytometer (Coulter Corp.) equipped with an argon laser emitting at 488 nm. Bacteria were gated for size based on log forward angle and side scatter detection and for sorting by FITC/PE labeling of bacteria. Sorted cells were collected into cold sBHI and plated on chocolate agar. After overnight growth, cells were collected for a secondary round of infection or were individually selected and grown overnight, screened by individual clone for fluorescence when grown in vitro, and frozen in skim milk containing 20% (vol/vol) glycerol prior to plasmid isolation and sequencing of insert DNA. Sorting efficiency of control strains was confirmed using a Coulter EPICS flow cytometer (Coulter Corp.).
Many plasmids were segregated rapidly in vitro in the absence of antibiotic selection. Thus, in order to assess whether the promoter trap vector used here was prone to this event, a single colony of strain 86-028NP/pRSM2211 (highly fluorescent control) was isolated on chocolate agar and passaged 20 times in the absence of antibiotic selection. No significant decrease in fluorescence intensity was observed when compared to bacteria grown in the presence of antibiotic. In addition, the plasmid is maintained in the absence of antibiotic selection in vivo. Similar bacterial counts were observed when bacteria-containing middle ear fluids collected from a chinchilla were plated on chocolate agar with or without kanamycin. These data demonstrate that the promoter trap vector was stably maintained in the absence of antibiotic selection.
In addition to problems with plasmid stability, early studies on the use of GFP as a reporter to study host-pathogen interactions demonstrated that GFP could be continuously synthesized as a cytoplasmic protein with low toxicity, having minimal effects on the bacterial cell-surface dynamics (Chalfie et al., Science, 263: 802-805, 1994). The construction of a high level gfp-expressing derivative allowed the assessment of the GFP toxicity on NTHi. Growth curves of both the wild-type strain (86-028NP) and the high GFP producing strain 86-028NP/pRSM2211 were compared when grown under similar conditions. The growth rates were similar, indicating that GFP expression was not toxic to the cells.
The 86-028NP gfp-expressing derivatives were used to define the parameters for efficient cell sorting. Strain 86-028NP/pRSM2169 was mixed with the intermediate gfp-expressing derivative, strain 86-028NP/pKMM4B5, at a 100:1 ratio, simulating the in vivo environment that is expected to contain a small percentage of gfp-expressing clones relative to the total bacterial population. This mixture was subjected to FACS analysis, collecting the 1.8% most fluorescent population and the 52% least fluorescent population. Flow cytometric analysis of the sorted populations revealed an enrichment of strain 86-028NP/pKMM4B5 to 65% of the bacterial population, a phenomenon that was not observed when sorting on the negative population. Subsequent rounds of sorting would be expected to further enrich for this intermediate fluorescent population. The inability to decrease the amount of fluorescent bacteria in the negative sort was attributed to the size of the gate set for negative sorting. GFP-negative cells were enriched by gating on the 10% least fluorescent population.
A similar strategy (as described in Example 5) was applied to sort fluorescent clones from effusions obtained from the chinchilla middle ear during AOM. Our ability to use differential fluorescence induction (DFI) in vivo was dependent upon our ability to sort gfp-expressing bacteria from non-fluorescent bacteria, fluorescent and non-fluorescent cellular debris, and eukaryotic cells.
Healthy adult chinchillas (Chinchilla lanigera) with no evidence of middle ear infection by either otoscopy or tympanometry were used to screen the library for promoter activity in vivo. Two pools of the NTHi/pRSM2169 library (1000 clones each) were grown overnight on chocolate agar containing kanamycin. The library was combined and diluted in cold 10 mM sterile PBS to 3.3×106 CFU/ml and 300 μl (1.0×106 CFU; 500 CFU/clone) was used to inoculate the left and the right chinchilla transbullar cavity (2000 clones/ear). OM development was monitored by video otoscopy and tympanometry at 24 and 48 hours. The bacteria multiplied in the middle ear cavity, reaching a concentration 500 times the inoculum dose by 48 hours as expected (Bakaletz et al., Infect. Immunity 67: 2746-62, 1999). This bacterial adaptation to the host environment results in an inflammatory response, indicated by erythema, vessel dilation and bulging of the tympanic membrane, infiltration of polymorphonuclear cells (PMN's), and accumulation of fluid in the middle ear cavity as observed by otoscopy and microscopic examination of recovered effusions. Twenty-four and 48 hours later, middle ear fluids were retrieved by epitympanic tap, and prepared for FACS.
It is important to note that this analysis was limited to those bacteria recoverable in the middle ear fluid. In some cases it was necessary to lavage the middle ear cavity to collect the bacteria for FACS analysis. Thus, this analysis includes genes up-regulated when NTHi are loosely adherent to mucosae. NTHi has been observed to form a biofilm in the middle ear cavity in a chinchilla model of OM (Erhlich et aL, JAMA, 287: 1710-5, 2002). Since the protocols described herein select for clones recovered from the planktonic population, it is not expected to recover those clones in which genes are up-regulated when the bacteria are associated with mucosal biofilms. Homogenization of middle ear mucosae and subsequent bacterial cell isolation however, would enable us to recover these clones. It is also possible that some GFP-expressing clones were recovered in the effusion, yet were adherent to eukaryotic cells present in the effusion as exfoliated cells, or in aggregates. These bacteria are difficult to recover from the effusion without compromising the sorting efficiency. Therefore the middle ear fluids were treated with a mucolytic agent, then centrifuged to remove large aggregates and eukaryotic cells and prior to labeling.
Chinchilla middle ear fluids were diluted, if necessary, to 250 μl with sterile saline. An equal volume of N-acetyl-L-cysteine (0.5%; w/v) in DPBS (pH 7.4) was added for 5 minutes at room temperature as a mucolytic agent (Miyamoto and Bakaletz, Microb. Pathog., 21: 343-356 1996). Fluids were centrifuged (300×g, 5 min) to remove cellular debris, red blood cells and inflammatory cells, and supernatants containing bacteria were transferred to a fresh tube. Bacteria were incubated with chinchilla antiserum (1:50 dilution) directed against a whole OMP preparation, derived from NTHi strain 86-028NP, for 45 minutes at 4° C., pelleted by centrifugation (2000×g, 5 min) and washed twice with cold DPBS containing 0.05% bovine serum albumin. Bacteria were subsequently labeled with cross-reactive phycoprobe R-PE anti-human IgG (H+L) antibody (10 μg/ml in 100 μl PBS; Biomeda Corp) for 30 minutes at 4° C. Following three successive washes to remove unbound antibody, cells were resuspended in 300 μl DPBS for FACS analysis.
H. influenzae 86-028NP transformed with the promoter trap library was grown overnight on chocolate agar. To select against those clones containing promoters that expressed gfp in vitro, the library was subjected to one round of FACS analysis (as described in Example 6), collecting only those clones expressing low-level amounts of GFP. These clones were pooled and used to inoculate the chinchilla middle ear transbullarly. Following 24 and 48 hours of infection, bacteria-containing effusions were removed by epitympanic tap. Bacteria were indirectly labeled with R-PE-labeled antibody and subjected to FACS analysis by gating on fluorescently tagged bacteria but sorting for those that were also expressing. These clones were used to reinfect animals for further enrichment. Following the final round of sorting, single colony isolates were screened in vitro for lack of fluorescence.
Those clones isolated by FACS analysis (positive for GFP fluorescence in vivo), which did not emit fluorescence in vitro were prepared for plasmid isolation and identification of insert DNA sequence. These clones were grown overnight on chocolate agar plates containing kanamycin and prepared for plasmid isolation using the Qiaprep Miniprep Kit (Qiagen) according to the manufacturer's protocol. Plasmid insert DNA was sequenced using the primer 5′-TGCCCATTAACATCACCATCTA-3′ (SEQ ID NO: 588) that is complementary to the gfpmut3 gene and downstream of the insert DNA. Sequencing reactions were performed using the ABI prism BigDye® terminator cycle sequencing ready reaction kit (Applied Biosystems) according to manufacturer's protocol using a GeneAmp PCR System 9700 (Applied Biosystems). The sequences were then purified by passage through sephadex G-50 in a 96-well multiscreen HV plate (Millipore) and subsequently analyzed on an ABI Prism 3100 DNA analyzer (Applied Biosystems).
Insert sequences were compared to the complete annotated sequence of H. influenzae strain Rd. Those inserts with no nucleotide homology to strain Rd were subsequently analyzed using the BLASTN and BLASTX algorithms. Further sequence analysis was performed with DNASTAR (Madison, Wis.). Inserts in the correct orientation and containing sequence 5′ to a predicted ORF contained a putative promoter that was preferentially active when the NTHi bacteria were in the chinchilla middle ear.
Fifty-two clones with putative promoters that were regulated in vivo were isolated. Of the 44 candidate clones containing sequence similar to that identified in H. influenzae strain Rd, quantitative comparison of gene expression in vitro and in vivo confirmed up-regulated gene expression for twenty-six genes (60%) when NTHi respond to environmental cues present in the chinchilla middle ear and these genes are summarized in Table 4A below. The in vivo-regulated promoters driving expression of genes are predicted to be involved in membrane transport, environmental informational processing, cellular metabolism, gene regulation, as well as hypothetical proteins with unknown function.
In order to confirm the induction of putative promoter candidates in vivo, the relative amount of messenger RNA expression was compared when NTHi strain 86-028NP was grown in vitro to mid-log phase or in vivo for 48 hours. The RNA was isolated using TRIzol LS reagent (Gibco Life Technologies) according to the manufacturer's protocol. DNA was removed from the RNA preparation using DNA-free kit (Ambion) according to the manufacturer's protocol. DNase I treated RNA samples were purified by passage through a Qiagen RNeasy column. RNA purity and integrity was assessed by 260/280 nm spectrophotometer readings and on the Agilent 2100 Bioanalyzer (Agilent Technologies), respectively.
In order to independently confirm the FACS data, we determined the relative expression of candidate genes by quantitative RT-PCR. The parent strain 86-028NP, was used for these studies. Real-time quantitative RT-PCR using the one-step QuantiTect SYBR Green RT-PCR kit (Qiagen) assessed transcription levels according to the manufacture's instructions. Briefly, using primers generated to an open reading frame downstream of the putative in vivo-induced promoters identified by FACS analysis, gene-specific mRNA was reverse transcribed and amplified by RT-PCR on the ABI Prism 7700 sequence detection system (Applied Biosystems). The amount of product was calculated using a standard curve generated to known amounts of bacterial genomic DNA (102-107 genomic copies DNA) by amplifying a fragment of the gyrase (gyr) gene. Controls were analyzed in parallel to verify the absence of DNA in the RNA preparation (−RT control) as well as the absence of primer dimers in control samples lacking template RNA. In addition, RT-PCR products were analyzed by gel electrophoresis and, in all cases, a single product was observed at the appropriate base pair size. Amounts of bacterial RNA between samples were normalized relative to gyr expression, shown to be constitutively expressed under various growth conditions that we tested in vitro. Known amounts of bacterial genomic DNA (102-107 genomic copies DNA) were used to generate a standard curve for RT-PCR quantitation by amplifying a fragment of the gyrase (gyr) gene. Gyrase is constitutively expressed in vitro under various growth conditions and was therefore used to normalize total bacterial RNA levels between samples. Relative gene expression in vivo was compared to that of gene expression in vitro and data expressed as fold-increase are summarized in Table 4A.
The 8-fold sequencing of the NTHi genome identified the full length open reading frames for the majority of genes listed in Table 4A. Table 4B provides the full length nucleotide sequence within the NTHi genome and the corresponding amino acid sequence. The fold induction of the gene due to environmental cues present in the chinchilla middle ear and the product or function of the gene are repeated in Table 4B for convenience.
In many bacterial species, a subset of virulence-associated genes is regulated by errors in replication of short repeats. These repeats may be 5′ to a gene or in the coding sequence, and their presence is an indication of controlled expression of the gene, which indicates association with virulence. Addition or deletion of a repeat results in the expression or of lack of expression of the particular virulence determinant.
The NTHi H. influenzae strain 86-028NP contig set was queried for short oligonucleotide repeats. The region surrounding the repeats was analyzed to identify the gene(s) associated with the repeat. Table 5 lists the identified repeats and the ORF (identified by BLAST) associated with each repeat.
Further sequence analysis has identified the full length nucleotide sequence of the virulence-associated genes and the corresponding amino acid sequences encoded by the ORF. The derived amino acid sequences are highly homologous to the listed Genbank sequence.
Genes associated with NTHi virulence were also identified by comparing the level of expression of the gene when the NTHi bacterium was infecting a tissue verses the level of expression of the same gene when the NTHi was grown on artificial laboratory media. These novel genes were identified using the promoter trap techniques described above in Examples 4-6, and subsequently comparisons with the known Rd genome demonstrated these genes are unique to NTHi strain 86-028NP.
The DNA sequence identified using this screening procedure are set forth as SEQ ID NOS: 577-580. These sequences did not contain genes or gene fragments that have homologues in the H. influenzae Rd. genome sequence. Even though these are completely novel sequences, due to their expression level during NTHi infection in the chinchilla middle ear, it is likely that expression of these genes are involved in NTHi virulence.
Library Construction:
Chromosomal DNA was prepared from strain 86-028NP using Puregene reagents (Gentra Systems, Minneapolis, Minn.). For the initial shotgun sequencing of the genome, 1 to 2 kb and 2 to 4 kb libraries of genomic DNA were constructed in pUC18 as previously described (Munson et al., Infect Immun 72:3002-10, 2004). For the scaffolding library, genomic DNA was manually sheared into a mean fragment size of 40 kb using a Hamilton syringe. After end repair, fragments were fractionated using a 0.7% low melting temperature agarose gel. Fragments larger than 30 kb were excised and an in-gel ligation to pEpiFOS-5 was performed. The ligation mixture recovered from the gel was packaged into Lambda phage, in vitro, and used to transfect EPI100 cells (Epicentre, Madison, Wis.).
Sequencing:
For the shotgun portion of the sequencing, cycle-sequencing reactions were run using PE Big-Dye™ terminators and universal primers (M13 forward and reverse) as previously described (Munson et al., Infect Immun 72:3002-10, 2004). To end-sequence the scaffolding library, plasmid was first purified using a R.E.A.L. Prep 96 Plasmid Kit (QIAGEN Inc., Valencia, Calif.), then amplified using a TempliPhi™ DNA Amplification Kit (Amersham Biosciences Corp., Piscataway, N.J.) before running reactions using PE Big-Dye™ terminators and pEpiFOS-5 forward and reverse sequencing primers (Epicentre, Madison, Wis.). The reactions for the clean-up portions of the project were run using PE Big-Dye™ terminators and custom primers (Integrated DNA Technologies, Coralville, Iowa). Excess dye terminators were removed with Sephadex G50 columns in 96-well format and sequence determined on either an ABI 3700 or an ABI 3100 capillary electrophoresis DNA sequencer (Applied Biosystems, Foster City, Calif.).
Genome Closure:
Paired end-sequences from the scaffolding library and PCR were used to order the contigs and to add sequence in areas of low sequence coverage. Paired custom primers (Integrated DNA Technologies, Coralville, Iowa) were designed to bind at the ends of each contig as well as regions flanking areas of low sequence coverage. The intervening regions were amplified with a standard PCR protocol as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (Third Edition). Cold Spring Harbor Laboratory Press., 2001 using Taq polymerase (Roche Diagnostics, Indianapolis, Ind.) and sequenced on both strands. Ribosomal RNA operons and the HMW gene clusters were completely sequenced using clones from the scaffolding library as templates.
Assembly:
Phred/Phrap was used for data assembly, employing the default assembly parameters (Ewing et al., Genome Res 8:186-94, 1998; Ewing et al., Genome Res 8:175-85, 1988; Gordon et al., Genome Res 8:195-202, 1998) as described in Munson et al., Infect Immun 72:3002-10, 2004. Assemblies were checked using the paired-end sequence data from 507 clones using the Seqman II program from the DNASTAR suite.
Data Analyses:
Coding regions were identified using Glimmer2 (v2.13) trained on the set of 1178 longest ORFs identified by the Glimmer2 long-orfs program (Delcher et al., Nucleic Acids Res 27:4636-41, 1999). Automated annotation by similarity was done by searching the Glimmer ORF set against the strain Rd proteome, the SwissProt database, the NCBI COGs database, and the KEGG database. The strain Rd database was compared bi-directionally with the strain 86-028NP ORF set using tricross to determine high-confidence regions of similarity and to produce the dotplot comparison of genome organization Ray et al., Bioinformatics 17:1105-12, 2001).
The automatically predicted annotation information was further manually curated using Artemis (Rodriguez et al., Infect Immun 71:1635-42, 2003) for visualization and demarcation of genomic regions of interest, and a custom FileMaker Pro database was generated which was then used to apply manual revisions and archive data related to the functional assignment. FASTA analyses were used for the primary automated comparisons. The strong synteny between the strain 80-028NP and strain Rd genomes allowed assignment of a function to the majority of the genes automatically, with similarity held to 90% or better at the amino acid level for matching. The near one-to-one mapping from the strain 86-028NP genome to the strain Rd genome was confirmed by assembly of the strain Rd ORFs onto the strain 86-028NP genome sequence, and the reverse assembly of the strain 86-028NP ORFs onto the Rd genome, using the SeqMan program with the assembly criteria of 80% identity at the nucleotide level.
Manual BLAST analyses were used to explore the potential function of ORFs that did not show strong similarity to known genes. Manual curation of the automatic assignments was carried out to conform annotations to the current literature and repair the few places where the automated algorithm was easily led astray (notably the HMW gene clusters, the hemoglobin-binding proteins and the hsd gene clusters, whose high family similarity confounds automated assignment).
The tRNA genes were identified by tRNAscan-SE v1.11 (Lowe et al., Nucleic Acids Res 25:955-64, 1997). The rRNA operons were identified based on 16, 23 and 5S rRNA similarity with strain Rd, and the CLUSTALW alignment of the neighborhoods containing these genes to determine the boundaries of the semi-conserved regions.
The complete genomic DNA sequence of the H. influenzae, strain 86-028NP, is set out as SEQ ID NO: 772. The open reading frames within the genomic DNA are set out as SEQ ID NOS: 773-2593 and are described in Table 6. The resulting gene products are set out as SEQ ID NOS: 2581-4414 and are described in Table 7. The “c” preceding the nucleotides of the open reading frames indicates the open reading frame is complementary to the 5′ to 3′ sequence set out as SEQ ID NO: 722. The unique NTHi genes are marked in Table 6. The genome sequence will be available from GenBank as Accession number CP000057, which is incorporated by reference herein in its entirety.
The following open reading frames are defined as pseudogenes in Table 6 because these nucleotide sequences contain a frameshift or a stop codon within the sequence: SEQ ID NO: 822 (kdgK), SEQ ID NO: 928, SEQ ID NO: 994 (bolA), SEQ ID NO: 276 (adhC) SEQ ID NO: 1102 (dusC), SEQ ID NO: 1121 (merP), SEQ ID NO: 1135, SEQ ID NO:1236, SEQ ID NO: 1254, SEQ ID NO: 1376 (lic2C), SEQ ID NO: 1431 (hgpD), SEQ ID NO: 1502, SEQ ID NO:1505 (ppx), SEQ ID NO: 1523 (hgpC), SEQ ID NO: 1585 (lex2A), SEQ ID NO: 1637 SEQ ID NO: 1713, SEQ ID NO: 1856 (mod), SEQ ID NO: 1899, SEQ ID NO: 2006, SEQ ID NO: 2080, SEQ ID NO: 2155, SEQ ID NO: 2202, SEQ ID NO:2257, SEQ ID NO: 2331, SEQ ID NO:2345, SEQ ID NO: 2365, SEQ ID NO: 2555 (metE) and SEQ ID NO:2563 (pmi). The hgpD, hgpC, lex2A and NTHI1769 genes contain contingency repeats that cause the sequence to shift in and out of the reading frame and the resulting amino acid sequences (SEQ ID NOS: 3242, 3332 and 4142) are partial translation. These contingency repeats regulate gene expression in the H. influenzae and therefore the genes encoding these proteins will be correctly translated after insertion or deletion of 1 or more repeats
The genomic sequence of strain 86-028NP contains 1,913,428 bp. This is approximately 4 percent larger than the strain Rd genome (1,830,137 bp) (Fleischmann et al., Science 269:496-512, 1995). There are also a larger number of genes in strain 86-028NP: 1942 compared to 1743 in strain Rd. The gene complement was compared to that of strain Rd using the Seqman program in the DNASTAR suite. With 80% identity at the nucleotide level as a cutoff value, 285 ORFS were identified in the 86-028NP genome that were absent from the strain Rd genome and 167 ORFs were identified in the strain Rd genome that are absent from the strain 86-028NP genome.
Strain 86-028NP, like strain Rd, has six ribosomal operons. Using tRNAscan-SE v1.11, 58 tRNA genes were identified in the strain 86-028NP genome, representing the 20 common amino acids. The tRNA-Glu, tRNA-Ala and tRNA-Ile genes were located in spacer regions between the 16S and 23S ribosomal RNA genes. A tRNA gene containing the UCA anticodon was also identified. This anticodon corresponds to an opal stop codon and is typically associated with an opal-suppressing tRNA that incorporates selenocysteine. The tRNA is adjacent to two genes encoding selB (NTHI0836), a Sec tRNA specific elongation factor, and selA (NTHI0835), the enzyme that converts serine to dehydroalanine preparatory to forming selenocysteine by incorporation of selenium (Forchhammer et al., Nature 342:453-6, 1989) The selD gene (NTHI0297), encoding selenophosphate synthetase was also identified. The importance of this selenocysteine system is evidenced by the coding sequence for the alpha subunit of formate dehydrogenase (NTHI0007) containing an inframe TGA stop codon that is presumably read as a selenocysteine codon. The inframe TGA stop codon was previously noted in the current annotation of the strain Rd formate dehydrogenase gene (GenPept Accession P46448).
A gross comparison between the genomes involving analysis of the gene order of strain 86-028NP and that of strain Rd reveals a single major rearrangement in the form of a large inversion. This 471 kb inversion represents almost 25% of the strain 86-028NP genome and is bounded by NTHI1391, and NTHI1394 (homologues of HI1218 and HI1645 respectively) and by NTHI11949 and NTHI1950 (homologues of HI1219 and HI1647 respectively). HI1219 and HI1646 are partially duplicated genes in strain Rd annotated as cmkA and cmkB (cytidylate kinases). One cmk gene (NTHI1949) is present in strain 86-028NP with a small cmk-like fragment between NTHI1391 and NTHI1394. Several clones from the scaffolding library overlap each end of the inversion in the 86-028NP genome validating our assembly. Within this large inversion are several insertions, the largest of which are approximately 13 kb, 27 kb and 51 kb in size. These regions contain predominantly hypothetical and conserved hypothetical genes as well as a number of homologues of phage genes. For example, the 27 kb insertion contains remnants of HP1- and HP2-like phage genes. The largest insert is bounded by homologues of integrase genes. In strain Rd, a mu-like phage is localized to this region (Morgan et al., J Mol Biol 317:337-59, 2002) This phage is not present in the strain 86-028NP genome. Also within the large inverted region is a 21 kb inversion that restores synteny with the Rd genome.
In addition to the large inversion, strain 86-028NP has other regions of divergence from co-linearity with the strain Rd genome. These include 9 regions greater than 5 kb, which contain sequences with no apparent homology to DNA that is present in strain Rd. Two of these regions contain the HMW adhesins that are discussed below. Hypothetical genes predominate in six of the unique regions. The ninth region is approximately 56 kb in size. It lies between NTHI0100 and NTHI0165. BLASTn analysis indicated that genes in this region, designated ICEHin86-028NP, have high homology to genes in the H. influenzae type b plasmid, ICEHin1056 (Mohd-Zain et al., J Bacteriol 186:8114-22, 2004). ICEHin1056 is a member of an extended family of genomic islands that are defined by a series of common core genes (Mohd-Zain et al., J Bacteriol 186:8114-22, 2004). ICEHin86-028NP possesses homologues of 45 ICEHin1056 ORFs. These include ORFs near the 5′ end of ICEHin86-028NP, including the defined core genes, that primarily encode proteins with putative roles in plasmid replication and conjugation and ORFs near the 3′ end that primarily encode conserved hypothetical proteins with motifs that suggest that they may be either membrane associated or exported. Notably, ICEHin86-028NP lacks the genes encoding proteins involved in tetracycline, chloramphenicol and β-lactam resistance found in ICEHin1056. Scattered within ICEHin86-028NP are a transposase, resolvases, and a putative integrase regulator suggesting that ICEHin86-028NP is a composite element derived from several mobile genetic elements.
ICEHin1506 has a sequence designated as an attP site 5′ the first gene. In strain 86-028NP, a perfect copy of this attP site is present 5′ to NTHI0101 and a copy of this attP site, with a single nucleotide change, is present 3′ of NTHI0164. The attP sites are implicated in the incorporation of mobile genetic elements into bacterial chromosomes to form genomic islands, possibly suggesting a mechanism by which this large section of genetic material became integrated into the strain 86-028NP genome (Dimopoulou et al., Antimicrob Agents Chemother 46:1602-3, 2002). ICEHin86-028NP has a G+C content of 39%, lower than any of the other related genomic islands and close to strain 86-028NP's overall genome G+C content of 38%. This implies a long-term genomic association for this element. The presence of this element with its complement of genes homologous to those in ICEHin1506 (Dimopoulou et al., Antimicrob Agents Chemother 46:1602-3, 2002) which are thought to encode membrane-associated and secreted proteins may have important implications for the virulence of strain 86-028NP.
Several members of the Pasteurellaceae including Haemophilus ducreyi, Pasteurella multocida and Actinobacillus actinomycetemcomitans produce well characterized protein toxins. In contrast, H. influenzae does not appear to produce protein toxins and genes encoding putative protein toxins were not identified in the strain 86-028NP genome. In H. influenzae, the genes encoding glycosyltransferases responsible for endotoxin biosynthesis and genes encoding proteins that give the bacteria enhanced “fitness” during the process of infection have generally been considered virulence determinants. These genes include those that encode adhesins, the heme and haemoglobin binding proteins as well as the genes that encode proteins that protect against oxidative stress.
Contingency Genes
H. influenzae has a limited number of two-component regulatory systems and other global regulators. Moxon and co-workers have argued that loci termed “simple contingency loci” provide an alternative mechanism for regulating gene expression, thus increasing the fitness of an organism by contributing to that organism's ability to rapidly respond to changing environmental conditions. These loci contain short tandem sequence repeats either within, or 5′ to, a coding region. During DNA replication, addition or loss of a repeat within a reading frame results in an alteration in the reading frame. When localized 5′ to a coding region, addition or loss of a repeat results in a change in promoter activity (Bayliss et al., Clin Invest 107:657-62, 2001). Loci containing simple sequence repeats have been studied extensively in H. influenzae, for example (Hood et al., Proc Nall Acad Sci USA 93:11121-5, 1996). Several of the loci described in the following sections as phase variable contain simple sequence repeats.
Adhesins
Strain 86-028NP possesses a number of genes which encode products that primarily function in adherence to host cells (Table 8). One of these, the outer membrane protein P5, has previously been identified and its function carefully dissected (Jiang et al., Infect Immun 67:187-92, 1999; Kennedy et al., Infect Immun 68:2756-65, 2000; Novotny et al., J Immunol 171:1978-83, 2003; Novotny et al., Infect Immun 68:2119-28, 2000; Novotny et al., Vaccine 20:3590-7, 2002; Sirakova et al., Infect Immun 62:2002-20, 1994). Strain 86-028NP possesses a gene cluster containing four genes that are homologues of pilABCD from strain Rd, Actinobacillus pleuropneumoniae and P. multocida (Bakaletz et al., Infect Immun 73:1635-4, 2005; Doughty et al., Vet Microbiol 72:79-90, 2000; Ruffolo et al., Infect Immun 65:339-43, 1997 Stevenson et al., Vet Microbiol 92:121-34, 2003). These genes together with the comE gene and genes yet to be identified encode a type IV pilus that has a role in adherence of strain 86-028NP to nasopharyngeal tissues (Kennedy et al., Infect. Immun., 68: 2756-2765, 2000).
Strain 86-028NP possesses two high molecular weight (HMW) adhesin gene clusters that are absent in strain Rd. The high molecular weight adhesins were first characterized in NTHi, strain 12, which has two HMW gene clusters, each encoding three proteins (HMWA, HMWB and HMWC). HMWA is the structural component of the adhesin, HMWB has a role in trans-membrane translocation, while HMWC is required for glycosylation of HMWA (Barenkamp et al., Infect Immun 60:1302-13, 1992; Barenkamp et al., Infect Immun 62:3320-8; 1994; Grass et al., Mol Microbiol 48:737-51, 2003; St Geme et al., Mol Microbiol 27:617-30, 1998). Similarly, strain 86-028NP's two HMW gene clusters contain homologues of the hmwA, B and C genes in the same gene context as in strain 12 (Buscher et al., J Bacterial 186:4209-17, 2004). The HMW1A and HMW2A proteins from strain 86-028NP are 72% identical, with the major area of divergence, including a 41 amino acid insertion in HMW2A, toward the C-termini. The paired HMWB and HMWC proteins from strain 86-028NP are 99% identical, respectively. The sequence ATCTTTC is repeated 17 times upstream of hmw1A and 23 times upstream of hmw2A. In strain 12, 16 repeats of this sequence are found 5′ of each hmw gene cluster (Barenkamp et al., Infect Immun 60:1302-13, 1992).
Hap is an autotransported protein with a domain homologous to the catalytic domain of IgA1 proteases. The NTHI0354 gene encodes a protein with 83% identity to Hap from the NTHi strain N187 (St Geme et al., Mol Microbiol 14:217-3, 1994). Strain 86-028NP, along with other NTHi strains that possess HMW1 and HMW2, lacks the gene encoding Hia, another Haemophilus adhesin (Barenkamp et al., Mol Microbiol 19:1215-23, 1996). Strain 86-028NP also lacks the hif gene cluster, encoding the hemagglutinating pilus as we previously reported (Munson et al., Infect Immuzz 72:3002-10, 2004).
In Tables 8, 9 and 10, the “NTHI number” refers to the locus tag number within the NTHi, strain 86-028NP genome as indicated at the Microbial-Pathogenesis H. influenzae 86028 NP web site and in Genbank Accession No. CP000057. The HI number” refers to the corresponding locus tag number in the TIGR (The Institute for genomic redearc
Lipooligosaccharide Synthesis [See [Page 13 of Original Application]
The structure, biosynthesis and role in virulence of H. influenzae lipooligosaccharide (LOS) has been studied extensively. Table 9 contains a list of genes involved in lipooligosaccharide biosynthesis. Strain 86-028NP has the full complement of genes required to synthesize the heptose-Kdo-Lipid A portion of LOS. The lgtF and lpsA genes encode glycosyltransferases that add glucose, and glucose or galactose, to heptose residues 1 and 3, respectively. Both of these genes are present in the strain 86-028NP genome, therefore it is likely that carbohydrate chains can be extended from the heptose 1 and heptose 3 residues of the strain 86-028NP LOS (Hood et al., Microbiology 150:2089-97, 2004). In the serotype b strain RM153, the lic2C gene encodes a glucosyltransferase that adds glucose to heptose 2 (Hood et al., Microbiology 150:2089-97, 2004). In the strain 86-028NP genome, this gene contains a frame shift. The phase variable lic2A and licA genes, encoding a galactosyltranferase and choline kinase, respectively, are present in the strain 86-028NP genome (High et al., Mol Microbiol 9:1275-82, 1993; Hood et al., Glycobiology 11:957-67, 2001; Weiser et aL, Infect Immun 65:943-50, 1997). The lex2B gene which encodes a glucosyltransferase in the serotype b strain DL42, as well as a number of other serotypeable strains, is present in the strain 86-028NP genome (Griffin et al., Microbiology 149:3165-75, 2003; Jarosik et al., Infect Immun 62:4861-7, 1994). Five-prime to the lex2B gene in strain DL42 is the short phase variable lex2A gene. In strain 86-028NP, this gene is out-of-frame compared to the DL42 sequence (Genbank Accession U05670), due to the loss of one tetranucleotide repeat and a 5 bp deletion. Recently, Hood and co-workers described a locus in strain Rd, designated hmg, that contains HI0866 through H10874 (Hood et al., J Bacteriol 186:7429-39, 2004). With the exception of a homologue of rmlB, these genes are absent from the strain 86-028NP genome. This includes the siaA gene which encodes a sialyltransferase recently shown to be important in biofilm formation in NTHI strain 2019 (Greiner et al., Infect Immun 72:4249-60, 2004; Jones et al., J Biol Chem 277:14598-611, 2002). Two copies of a homologue of the lic3A gene, encoding an alternative sialyltransferase, were identified in the strain 86-028NP genome (Hood et al., Mol Microbiol 39:341-50, 2001; Jones et al., J Biol Chem 277:14598-611, 2002), as well as a copy of the lsgB gene that encodes another sialyltransferase Jones et al., J Biol Chem 277:14598-611, 2002).
Iron Acquisition
H. influenzae strains have an absolute requirement for either heme or iron, together with protophorphyrin IX (PPIX), the immediate precursor of heme (Evans et al., J Med Microbiol 7:359-65, 1974; White et al., J Bacteriol 85:842-50, 1963). Table 10 contains a list of genes involved in iron acquisition. Three haemoglobin and haemoglobin-haptoglobin binding proteins HgpA, HgpB and HgpC, were identified in H. influenzae type b, strain HI689 (Jin et al., Microbiology 145 (Pt 4):905-14, 1999; Morton et al., Infect Immun 67:2729-39, 1999; Ren et al., Infect Immun 66:4733-41, 1998). In strain HI689, these genes have CCAA tetranucleotide repeats and are known to be regulated by slip-strand mispairing. Two of these genes are present in strain 86-028NP. They both contain CCAA repeats; the hgpB gene is in-frame while the hgpC gene is out-of-frame. The derived amino acid sequence of a third gene that contains CCAA repeats is 45% identical to hgpA. We have designated this gene hgpD. This gene is out-of-frame. Homologues of the hxuABC genes of H. influenzae type b that encode heme and heme-hemopexin complexes (Cope et al., Infect Immun 69:2353-63, 2001; Cope et al., Infect Immun 66:4511-6, 1998; Cope et al., J Bacteriol 177:2644-53, 1995) as well as a homologue of the hemR receptor were identified. Strain 86-028NP also has the gene encoding the heme-binding lipoprotein HbpA (Heath et al., Pediatr Infect Dis J20:300-5, 2001). Downstream of hbpA is NTHI1022, a hypothetical gene whose product is a member of COG0748, a cluster that includes putative heme utilization proteins. A homologue of the hup gene, recently identified in H. influenzae type b, that encodes a general heme utilization protein, was also identified (Morton et al., Microbiology 150:3923-33, 2004).
In addition to the heme transport systems, iron transport systems were also identified. The hitABC genes encode the FbpABC proteins respectively, members of a highly specific ferric iron ABC transport system that was elegantly characterized by complementing a siderophore-deficient E. coli strain with the hitABC genes cloned from an H. influenzae type b strain (Anderson et al., J Bacteriol 186:6220-9, 2004). Transferrin-binding proteins 1 and 2 encoded by tbpAB (Gray-Owen et al., Infect Immun 63:1201-10, 1995; Gray-Owen et al., Infect Immun 63:3809-15, 1995) as well as genes designated hfeABCD that are homologues of an ABC transport system involved in iron uptake, originally characterized in Yersinia pestis (Bearden et al., J Bacteriol 180:1135-47, 1998) were identified. This latter gene cluster is also present in strain Rd. NTHI2035 encodes a putative homologue of the NRAMP family of Mn2+ and Fe2+ transporters (Richer et al., J Mol Evol 57:363-7, 2003).
As noted above, H. influenzae can use iron, together with PPIX, as a source of heme for growth in vitro. The hemH gene encoding ferrochelatase, which catalyzes the incorporation of iron into PPIX (Schlor et al., Infect Immun 68:3007-9, 2000), was identified. The gene encoding the global regulator, Fur, was also identified (Andrews et al., FEMS Microbiol Rev 27:215-37, 2003; Smoot et al., J Med Microbiol 48:629-3, 1999).
Oxidative Stress
Although necessary for growth, the active acquisition of iron can have deleterious effects on bacterial cells. Through the Fenton reaction, iron can react with hydrogen peroxide and generate highly reactive hydroxyl radicals. These products have profound effects, including lipid peroxidation and damage to both iron-containing enzymes and DNA (Imlay, Annu Rev Microbiol 57:395-418, 2003). The best-known defense system against hydroxyl radicals consists of superoxide dismutase A and B which convert highly reactive superoxide to hydrogen peroxide, which is then converted, by catalase, into water and oxygen (Demple, Annu Rev Genet 25:315-37, 1991). Strains 86-028NP and Rd contain the sodA gene (NTHI1251), but lack the sodB gene. Both strains also possess a catalase gene hktE (NTHI1099) (Bishai et al., J Bacteriol 176:2914-21, 1994), the oxyR gene (NTHI0704) encoding a primary regulator of genes involved in protection against oxidative stress (Maciver & Hansen, Infect Immun 64:4618-29, 1996; Pomposiello et aL, Trends Biotechnol 19:109-14, 2001) and the gene encoding a chimeric peroxidase termed Prx/Grx that has a glutathione-dependent role in protection against small alkyl hydroperoxides (Pauwels et al., J Biol Chenz 278:16658-66, 2003; Vergauwen et al., J Bacteriol 185:5555-62, 2003; Vergauwen et al., J Bacteriol 185:1572-81, 2003). We previously identified NTHI0212, a gene encoding a homologue of the P. multocida peroxiredoxin, TsaA that is absent in strain Rd (Munson et al., Infect Immun 72:3002-10, 2004). Strain 86-028NP, however, lacks AhpF, a dedicated alkyl hydroperoxide reductase known to be involved in the reduction of TsaA in Salmonella (Poole et al., Biochemistry 39:6602-15, 2000). Further protection against oxidative stress may be afforded by the ferritin-like proteins encoded by the finA and ftnB (NTHI1773 and NTHI1772, respectively) genes. Over-expression of these proteins were shown to protect an iron overloaded E. coli fur mutant against oxidative damage Touati et al., J Bacteriol 177:2305-14, 1995). A conserved hypothetical gene, NTHI1817, encodes a protein with homology to a DNA-binding ferritin-like protein. This is a member of the Dps family of non-specific DNA binding proteins, which in S. enterica have roles in protection against oxidative stress, both in the presence of iron and during phagocytosis, and are important for virulence in a murine model of Salmonella infection (Halsey et al., Infect Immun 72:1155-8, 2004) In E. coli, Dps was shown to preferentially bind iron that had been oxidized by hydrogen peroxide, thus having an important role in abrogating the production of hydroxyl radicals generated via the Fenton reaction (Zhao et al., J Biol Chem 277:27689-96, 2002).
Secretion
In addition to the Sec system, strain 86-028NP has genes that encode the TatA, B and C proteins, cytoplasmic membrane-associated proteins that are involved in a Sec-independent transport of proteins with twin arginines in their signal peptides (NTHI0279, NTHI0280 and NTHI0282) (Bolhuis et al., J Biol Chem 276:20213-9, 2001; Yen et al., Arch Microbiol 177:441-50, 2002). As previously reported, strain 86-028NP possesses NTHI0585, the gene encoding the autotransported protein Lav (Munson et al., Infect Immun 72:3002-10, 2004). This protein is absent in strain Rd, present in Neisseria and appears, within Haemophilus, to be restricted to pathogenic strains (Davis et al., J Bacteriol 183:4626-35, 2001). Strain 86-028NP also has the gene encoding an IgA protease (NTHI1164) (Poulsen et al J Bacteriol 174:2913-21, 1992), and as noted above, the gene encoding the Hap adhesin. Both are proteins of the autotransporter class. As described above, the HMW adhesins are members of the two-partner secretion pathway group of proteins.
Outer Membrane Proteins
A number of outer membrane protein (OMP) encoding genes have been identified by homology to those in other Haemophilus isolates. These include the major OMPs that were all originally identified in H. influenzae type b; the surface expressed P1 (NTHI0522), the porin P2 (NTHI0225), the phosphomonoesterase and heme transporter P4 (NTHI0816), the adhesin P5 (NTHI1332) and the lipoprotein P6 (NTHI0501). Strain 86-028NP also shares a number of minor OMPs with other Haemophilus strains. These include D15 and the transferrin binding proteins from H. influenzae type b, as well as a homologue of OMP26, which was identified in NTHi strain 289 (Munson et al., Infect Immun 56:2235-42, 1988; Munson et al., Infect Immun 49:544-9, 1985; Munson et al., J Clin Invest 72:677-84, 1983; Reidl et al., J Exp Med 183:621-9, Reilly et al., J Bacteriol 181:6797-805, 1999; Reilly et al., FEBS Lett 494:19-23, 2001). All have subsequently been characterized in NTHi strains and analyzed as potential vaccine candidates (Poolman et al., Vaccine 19 Suppl 1:S108-15, 2000; Murphy et al. Curr Opin Infect Dis 16:129-34, 2003; McMichael et al., Curr Opin Investig Drugs 4:953-8, 2003 Cripps et al. Immunol Cell Biol 81:46-51, 2003; Bakaletz et al. Ann Otol Rhinol Laryngol Suppl 188:82-94, 2002).
Restriction Enzymes Systems:
Strain 86-028NP lacks the HindII and HindIII type II restriction systems (Fleischmann et al., Science 269:496-512. 80, 1995; Nwankwo et al., Gene 150:75-80. 104, 1994, Smith, & Marley. Methods Enzynzol 65:104-8, 1980). In contrast, genes encoding the HaeII system that was originally identified in H. aegyptius (Slatko et al., Gene 74:45-50, 1988) are present in the strain 86-028NP genome but absent in strain Rd. Both strain 86-028NP and strain Rd have Hsd type restriction systems encoding a methytransferase (HsdM), a sequence recognition protein (HsdS) and a restriction enzyme (HsdR) (Roberts et al., Nucleic Acids Res 31:1805-12, 2003). These genes are adjacent in the strain Rd genome (HI1285-H11287). The 86-028NP genome contains 3 hsd-like loci that each contain 4 genes. One hsd system is encoded by NTHI1838-NTHI1843. In this gene cluster, NTHI1841 encodes a hypothetical protein. A second hsd-like locus is encoded by NTHI0314-NTHI0318. In this gene cluster, NTHI0316 encodes a putative anticodon nuclease. This hsd-like system may be similar to the prr system in E. coli (Tyndall et al., J Mol Biol 237:266-74, 1994). A third hsd-locus is encoded by NTHI0188-NTHI0193. In this gene cluster, NTHI0190 encodes a predicted transcriptional regulator with a helix-turn-helix domain.
This application is a divisional of U.S. application Ser. No. 13/612,176 (now U.S. Pat. No. 8,652,773) filed Sep. 12, 2012 which is a divisional of U.S. application Ser. No. 11/1917,368 (now U.S. Pat. No. 8,283,114) filed Jan. 20, 2011 which is a national phase filing under 35 U.S.C. §371 of International Application No. PCT/US2006/1023428, which claims priority to U.S. Provisional Application No. 60/691,214, filed Jun. 16, 2005 and U.S. patent application Ser. No. 10/795,159 filed Mar. 5, 2004 (now U.S. Pat. No. 7,241,867), which claims priority to U.S. Provisional Application No. 60/1453,134 filed Mar. 6, 2003, all of which are incorporated by reference herein in its entirety.
| Number | Name | Date | Kind |
|---|---|---|---|
| 6797274 | Ruelle | Sep 2004 | B1 |
| Number | Date | Country |
|---|---|---|
| WO-9902457 | May 1999 | WO |
| WO-0234768 | May 2002 | WO |
| WO-2004078949 | Sep 2004 | WO |
| WO-2005111066 | Nov 2005 | WO |
| Entry |
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| Number | Date | Country | |
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| 20140127221 A1 | May 2014 | US |
| Number | Date | Country | |
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| 60691214 | Jun 2005 | US |
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