Patent Application
20170281740
The present invention relates to compounds, compositions, and methods for the serodiagnosis of salmon poisoning disease (Neorickettsia helminthoeca).
Salmon poisoning disease (SPD), an acute and often-fatal illness in wild and domestic canids, was first discovered in the 1800s when early settlers in Pacific Northwest noted their dogs becoming ill following ingestion of salmon (Philip, 1955). In 1950, a bacterial pathogen was implicated as the causative agent of SPD and named Neorickettsia helminthoeca, due to its biological similarity to the members of the family Rickettsiaceae and the novel invertebrate/helminth vector (Cordy and Gorham, 1950; Philip, 1955). N. helminthoeca exists in all life stages of the fluke Nanophyetus salmincola (Bennington and Pratt, 1960; Schlegel et al., 1968), which has a complicated digenetic life cycle involving both pleurocid fresh water snails (Oxyfrema silicula) and salmonid fish as intermediate hosts (Millemann and Knapp, 1970; Headley et al., 2011). Due to the limited geographic range of the vector and intermediate hosts, the distribution of SPD was thought to be limited to the northern Pacific coast. However, SPD cases have been confirmed in Southern California (this study) (Veterinary Practice News, 2009), Vancouver Island, Canada (Booth et al., 1984) and Maringa, Brazil using immunohistochemical, histopathological and molecular diagnostic techniques (Table 1) (Headley et al., 2004; Headley et al., 2006; Headley et al., 2011), though the vector and life cycle in these regions remain to be identified. The expansion of the geographic distribution of SPD where N. salmincola has not been documented suggests the potential adaptation of this organism to other trematode vectors.
While there is a large range of definitive hosts for the trematode, N. helminthoeca causes severe SPD in members of the Canidae family including dogs, foxes, and coyotes (Cordy and Gorham, 1950; Philip et al., 1954a; Philip et al., 1954b; Philip, 1955; Foreyt et al., 1987). Dogs most commonly acquire SPD when they eat raw or undercooked salmonid fish containing encysted trematodes injected with N. helminthoeca. Upon ingestion, the metacercariae stage of the trematode matures in the intestinal lumen for 5-8 days and releases the bacteria to be picked up by monocytes and macrophages in the intestinal wall. The exact mechanism of bacterial entry into these cells is not known, but morphological studies demonstrate the organism existing as clusters termed morulae or singly within a host cell-derived membrane vacuole in the cytoplasm of the canine host cell (Rikihisa et al., 1991). N. helminthoeca-infected cells travel throughout the circulation and accumulate in the thoracic and abdominal lymph nodes with the mesenteric and ileocecal lymph nodes being most commonly affected (Philip et al., 1954a; Philip, 1955; Headley et al., 2011). Symptoms begin with pyrexia (39.8-40.9° C.) that persists for 6-7 days and anorexia (Rikihisa et al., 1991). Dogs progress to vomiting and diarrhea that may or may not contain blood 4-6 days following development of a fever. Other symptoms include ocular discharge, weight loss, lethargy, and dehydration. If left untreated, death occurs 2-10 days after development of symptoms (Philip, 1955). Current therapies for SPD include fluid therapy, blood transfusions for hemorrhagic diarrhea, anti-helminthic praziquantel, and oral doxycycline or intravenous oxytetracycline. Affected individuals produce specific immunity to SPD following recovery from the disease (Philip et al., 1954a; Philip, 1955).
Neorickettsia species are obligatory intracellular α-proteobacteria that belong to the family Anaplasmataceae in the order Rickettsiales (Rikihisa et al., 2005). Neorickettsia spp. are the deepest branching lineage in the family Anaplasmataceae, whereas Anaplasma and Ehrlichia are sister genera that share a common ancestor with Wolbachia spp. (
Currently, only three pathogenic species of Neorickettsia, namely N. helminthoeca (type species), N. sennetsu (agent of human Sennetsu fever), and N. risticii (agent of Potomac horse fever) have been culture isolated and characterized in sufficient details with documented biological and medical significance (Table 1) (Rikihisa et al., 1991; Rikihisa et al., 2005). All of them are known to transmit from trematodes to monocytes/macrophages of mammals (dogs, humans, and horses, respectively) and cause severe, sometimes fatal illnesses (Table 1) (Rikihisa et al., 2005). In addition, the Stellantochasmus falcatus (SF) agent, which is closely related to N. risticii, was culture isolated from S. falcatus fluke encysting the grey mullet fish in Japan (Wen et al., 1996) and from fish in Oregon (Rikihisa et al., 2004). The initial 16S rRNA gene sequence-based phylogenetic analysis of N. helminthoeca revealed that the divergence of 16S rRNA sequences is around 5% between N. helminthoeca and N. risticii or N. sennetsu, whereas it is only 0.7% between N. risticii and N. sennetsu.
As endosymbionts of digenetic trematodes (parasitic flatworms or flukes), Neorickettsia species are abundant in nature and have been identified throughout the life cycle of the trematodes and the hosts of trematodes including the essential first intermediate host of snails, the second intermediate hosts such as fish and aquatic insects, and the definitive hosts such as mammals and birds wherein the trematodes sexually reproduce fertilized eggs (Cordy and Gorham, 1950; Philip et al., 1954a; Philip et al., 1954b; Philip, 1955; Foreyt et al., 1987; Gibson et al., 2005; Rikihisa et al., 2005; Gibson and Rikihisa, 2008; Greiman et al., 2016). Recent reports revealed more than 10 new genotypes of Neorickettsia in divergent digenean families throughout the world, including Asia, Africa, Australia, Americas, and even Antarctica (Ward et al., 2009; Tkach et al., 2012; Greiman et al., 2014; Greiman et al., 2017), suggesting a global distribution of Neorickettsia spp. Notably, a Neorickettsia sp. was found in the medically important trematode Fasciola hepatica (the liver fluke, fasciolosis disease agent) isolated from a sheep in Oregon US (McNulty et al., 2017). In addition, a related new species named Candidatus “Xenolissoclinum pacificiensis L6” was identified in the ascidian tunicate Lissoclinum patella, a marine chordate animal at the coast of Papua New Guinea (Kwan and Schmidt, 2013), implicating even boarder distribution of Neorickettsia-like bacteria among diverse invertebrates. To date, the complete genome sequences have been determined only for N. sennetsu (Dunning Hotopp et al., 2006) and N. risticii (Lin et al., 2009), and almost complete genome sequences were obtained for Neorickettsia endobacterium of F. hepatica (NFh) and Candidatus “X. pacificiensis” (Kwan and Schmidt, 2013; McNulty et al., 2017). The phylogenetic analysis based on 16S rRNA gene sequences suggests that NFh shares >99% identity with N. risticii and N. sennetsu, while Candidatus “X. pacificiensis” is distantly related to Neorickettsia spp. (
Because the mortality rate of SPD is >90% without rapid antibiotic treatment (Philip, 1955; Rikihisa et al., 1991), the current inefficient diagnostic method (fecal examination for parasite eggs and/or Romanowsky staining of lymph node aspirates), and the expansion of the geographic distribution of SPD, there remains a need for more rapid, sensitive, and specific serodiagnostic technique, as well as an effective vaccine.
As disclosed herein, the genome of N. helminthoeca Oregon consists of a small, single circular chromosome of 884,232 bp and encodes 37 RNA species and 774 proteins. Although N. helminthoeca has a very limited capacity to synthesize amino acids and lacks many metabolic pathways, it is capable of making all major vitamins, cofactors, and nucleotides, which may be beneficial to the trematode host. Like other members of the family Anaplasmataceae, helminthoeca lacks genes for lipopolysaccharide biosynthesis. However, peptidoglycan biosynthesis pathway is conserved, suggesting its mechanical strength and inflammatory potential. Genes potentially involved in the pathogenesis of N. helminthoeca were identified, including putative outer membrane proteins, two-component systems, type I and IV secretion systems, and putative transcriptional regulators. Five predicted major surface antigens P51, NSP-1/2/3, and SSA of N. helminthoeca were cloned and expressed and reactivity of both experimentally and naturally infected dog blood specimens to these antigens were evaluated. The result showed strong antigenicity. These findings provide the tools with which to design rapid and sensitive serodiagnostic methods and new prevention strategies for Salmon poisoning disease.
Therefore, disclosed is an immunogenic composition comprising one or more isolated Neorickettsia helminthoeca proteins, or immunogenic fragments or variants thereof, or a fusion protein containing same, and a pharmaceutically acceptable carrier, wherein said composition is capable of producing antibodies specific to N. helminthoeca in a subject to whom the immunogenic composition has been administered, and wherein the isolated N. helminthoeca protein is selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5.
In one aspect, disclosed herein is a method of preventing or inhibiting salmon poisoning disease (SPD) in a subject comprising:
administering to the subject an immunogenic composition comprising one or more isolated Neorickettsia helminthoeca proteins, or immunogenic fragments or variants thereof, or a fusion protein containing same, and a pharmaceutically acceptable carrier,
wherein said composition is administered in an amount effective to prevent or inhibit salmon poisoning disease (SPD), and
wherein the isolated N. helminthoeca protein is selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5.
In some embodiments, the isolated helminthoeca protein is SEQ ID NO:1. In some embodiments, the isolated N. helminthoeca protein is SEQ ID NO:2, In some embodiments, the isolated N. helminthoeca protein is SEQ ID NO:3. In some embodiments, the isolated N. helminthoeca protein is SEQ ID NO:4. In some embodiments, the isolated N. helminthoeca protein is SEQ ID NO:5.
In some embodiments, the subject is a member of the Canidae family
Also disclosed is a method for detecting Neorickettsia helminthoeca infection in a canine subject, comprising assaying a sample from the subject for antibodies specific for a N. helminthoeca protein selected from the group consisting of P51, NSP1, NSP2, NSP3, and SSA.
In some embodiments, the N. helminthoeca protein is P51. In some embodiments, the N. helminthoeca protein is NSP1. In some embodiments, the N. helminthoeca protein is NSP2. In some embodiments, the N. helminthoeca protein is NSP3. In some embodiments, the N. helminthoeca protein is SSA.
Further disclosed is a method of treating a Neorickettsia helminthoeca infection in a subject, comprising: assaying a sample from the subject for antibodies specific for a N. helminthoeca protein selected from the group consisting of P51, NSP1, NSP2, NSP3, and to SSA; and treating the subject for the Neorickettsia helminthoeca infection when antibodies specific for a N. helminthoeca protein selected from the group consisting of P51, NSP1, NSP2, NSP3, and SSA are present. In one embodiment, the subject is further treated with praziquantel, oral doxycycline, or intravenous oxytetracycline.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects described below.
GenBank Accession numbers and locus tag numbers for the 16S rRNA sequences are: N. helminthoeca Oregon, NZ13 CP007481/NHE_RS00195; N. risticii Illinois, NC13 013009.1/NRI_RS00185; N. sennetsu Miyayama, NC_007798.1/NSE13 RS00200; A. phagocytophilum HZ, NC_007797.1/APH13 RS03965; A. marginale Florida, NC_012026.1/AMF_RS06130; E. chaffeensis Arkansas, NC_007799.1/ECH_RS03785; E. canis Jake, NC_007354.1/ECAJ_RS00995; E. ruminantium Welgevonden, NC_005295.2/ERUM_RS01035; E. muris AS145, NC_023063.1/MR76_RS00900; Ehrlichia sp. HF, NZ_CP007474.1/EHF_RS03625; Wolbachia pipientis wMel, NC_002978.6/WD_RS05540; Wolbachia endosymbiont of Brugia malayi, NC_006833.1/WBM_RS02885; Rickettsia rickettsii str. R, L36217; Neorickettsia Endobacterium of Fasciola hepatica, LNGI01000001/AS219_00180; Candidatus “Xenolissoclinum pacificiensis L6”, AXCJ01000001/P857_926.
Abbreviations: GlcN,
GenBank Accession numbers: 151 proteins—N. helminthoeca Oregon, WP_051579521; N. sennetsu Miyayama, WP_011451642; N. sennetsu strain 11908, AAL79561; N. sennetsu Nakazaki, AAR23990; N. risticii Illinois, WP_015816118; N. risticii strain 90-12, AAB46982; Neorickettsia sp. SF agent, AAR23988.
NSP Proteins: N. helminthoeca Oregon—NSP1, WP_038560103; NSP2, WP_038560106; NSP3, WP_038560109; N. sennetsu Miyayama—NSP1, WP_011452245; NSP2, WP_011452246; NSP3, WP_011452248; N. risticii Illinois—NSP1, WP_015816683; NSP2, WP_015816684; NSP3, WP_015816686.
SSA Proteins: N. helminthoeca Oregon—SSA, WP_038560160; N. sennetsu Miyayama—SSA1, WP_011452276; SSA3, WP_011452279; N. risticii Illinois—SSA1, WP_015816716; SSA2, WP_015816703; SSA3, WP_015816717.
Domain abbreviations and functions: HTH, DNA-binding helix-turn helix domain; MerR, MerR family regulatory domain (DNA-binding, winged helix-turn-helix domain of about 70 residues present in the merR family of transcriptional regulators); Rrf2, Transcriptional regulator; Aminotran_5, Aminotransferase class V; EAL, EAL domain (diguanylate phosphodiesterase activity for degradation of a second messenger, cyclic di-GMP. Together with the GGDEF domain, EAL might be involved in regulating cell surface adhesiveness in bacteria); HD, HD domain (metal-dependent phosphohydrolases).
Disclosed herein are isolated polypeptides comprising an amino acid sequence corresponding to Neorickettsia helminthoeca (NH) proteins, or functional derivatives thereof.
In some embodiments, the polypeptide comprises an NH P51 protein, or an immunogenic fragment thereof. Therefore in some embodiments, the polypeptide comprises the amino acid sequence SEQ ID NO:1.
Neorickettsia helminthoeca Oregon P51 Protein Sequence:
In some embodiments, the polypeptide comprises an NH P51 functional derivative. In some embodiments, the polypeptide comprises an NH P51 variant. In some embodiments, the polypeptide comprises an NH P51 variant with an amino acid sequence which is at least 85% (for example, at least 85%, at least 90%, at least 95%) identical to SEQ ID NO:1.
In some embodiments, the polypeptide comprises an NH strain-specific antigen (SSA) protein, or an immunogenic fragment thereof. Therefore in some embodiments, the polypeptide comprises the amino acid sequence SEQ ID NO:2.
Neorickettsia helminthoeca Oregon SSA Protein Sequence:
In some embodiments, the polypeptide comprises an NH SSA functional derivative. In some embodiments, the polypeptide comprises an NH SSA variant. In some embodiments, the polypeptide comprises an NH SSA variant with an amino acid sequence which is at least 85% (for example, at least 85%, at least 90%, at least 95%) identical to SEQ ID NO:2.
In some embodiments, the polypeptide comprises a Neorickettsia helminthoeca surface protein 1 (NSP1) protein, or an immunogenic fragment thereof. Therefore in some embodiments, the polypeptide comprises the amino acid sequence SEQ ID NO:3.
Neorickettsia helminthoeca Oregon NSP1 Protein Sequence:
In some embodiments, the polypeptide comprises an NH NSP1 functional derivative. In some embodiments, the polypeptide comprises an NH NSP1 variant. In some embodiments, the polypeptide comprises an NH NSP1 variant with an amino acid sequence which is at least 85% (for example, at least 85%, at least 90%, at least 95%) identical to SEQ ID NO:3.
In some embodiments, the polypeptide comprises a Neorickettsia helminthoeca surface protein 2 (NSP2) protein, or an immunogenic fragment thereof. Therefore in some embodiments, the polypeptide comprises the amino acid sequence SEQ ID NO:4.
Neorickettsia helminthoeca Oregon NSP2 Protein Sequence:
In some embodiments, the polypeptide comprises an NH NSP2 functional derivative. In some embodiments, the polypeptide comprises an NH NSP2 variant. In some embodiments, the polypeptide comprises an NH NSP2 variant with an amino acid sequence which is at least 85% (for example, at least 85%, at least 90%, at least 95%) identical to SEQ ID NO:4.
In some embodiments, the polypeptide comprises a Neorickettsia helminthoeca surface protein 3 (NSP3) protein, or an immunogenic fragment thereof. Therefore in some embodiments, the polypeptide comprises the amino acid sequence SEQ ID NO:5.
Neorickettsia helminthoeca Oregon NSP3 Protein Sequence:
In some embodiments, the polypeptide comprises an NH NSP3 functional derivative. In some embodiments, the polypeptide comprises an NH NSP3 variant. In some embodiments, the polypeptide comprises an NH NSP3 variant with an amino acid sequence which is at least 85% (for example, at least 85%, at least 90%, at least 95%) identical to SEQ ID NO:5.
Also provided herein are functional derivatives of the NH proteins enumerated above. A “functional derivative” of an NH protein or peptide sequence is a molecule that possesses immunoreactivity to NH antibodies that is substantially similar to that of the corresponding NH protein or peptide, i.e. an “immunoreactive” functional derivative is a polypeptide that has a specific binding affinity for anti-N. helminthoeca antibodies.
The functional derivatives of an NH protein can be identified using any of a variety of routine assays for detecting peptide antigen-antibody complexes, the presence of which is an indicator of selective binding. Such assays include, without limitation, enzyme-linked immunosorbent assays (ELISA), radioimmunoassays, western blotting, enzyme immunoassays, fluorescence immunoassays, luminescent immunoassays and the like. Methods for detecting a complex between a peptide and an antibody, and thereby determining if the peptide is an “immunoreactive functional derivative” are well known to those skilled in the art and are described, for example, in ANTIBODIES: A LABORATORY MANUAL (Edward Harlow & David Lane, eds., Cold Spring Harbor Laboratory Press, 2.sup.nd ed. 1998a); and USING ANTIBODIES: A LABORATORY MANUAL: PORTABLE PROTOCOL No. I (Edward Harlow & David Lane, Cold Spring Harbor Laboratory Press, 1998b), which are hereby incorporated by reference in their entirety.
Thus, the terms “functional derivative” and “immunoreactive functional derivative” are used interchangeably and refer to peptides and proteins that can function in substantially the same manner as the NH proteins or peptides disclosed herein, and can be substituted for the N. helminthoeca proteins or peptides in the disclosed compositions and methods.
A “functional derivative” of a protein or peptide can contain post-translational modifications such as covalently linked carbohydrate, depending on the necessity of such modifications for the performance of a specific function. The term “functional derivative” is intended to include the immunoreactive “variants” and “fragments” of the NH proteins.
A “variant” of an NH protein refers to a molecule substantially similar in structure and immunoreactivity to the NH protein. Thus, provided that two molecules possess a common immunoactivity and can substitute for each other, they are considered “variants” as that term is used herein even if the composition or secondary, tertiary, or quaternary structure of one of the molecules is not identical to that found in the other, or if the amino acid or nucleotide sequence is not identical. Thus, in one embodiment, a variant refers to a protein whose amino acid sequence is similar to the amino acid sequences of a mature NH protein, hereinafter referred to as the reference amino acid sequence, but does not have 100% identity with the respective reference sequence. The variant protein has an altered sequence in which one or more of the amino acids in the reference sequence is deleted or substituted, or one or more amino acids are inserted into the sequence of the reference amino acid sequence. As a result of the alterations, the variant protein has an amino acid sequence which is at least 85%, 86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the reference sequence. For example, variant sequences which are at least 95% identical have no more than 5 alterations, i.e. any combination of deletions, insertions or substitutions, per 100 amino acids of the reference sequence. Percent identity is determined by comparing the amino acid sequence of the variant with the reference sequence using any available sequence alignment program. An example includes the MEGALIGN project in the DNA STAR program. Sequences are aligned for identity calculations using the method of the software basic local alignment search tool in the BLAST network service (the National Center for Biotechnology Information, Bethesda, Md.) which employs the method of Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. (1990) J. Mol. Biol. 215, 403-410. Identities are calculated by the Align program (DNAstar, Inc.) In all cases, internal gaps and amino acid insertions in the candidate sequence as aligned are not ignored when making the identity calculation.
Variants of the NH proteins can include nonconservative as well as conservative amino acid substitutions. A conservative substitution is one in which the substituted amino acid has similar structural or chemical properties with the corresponding amino acid in the reference sequence. By way of example, conservative amino acid substitutions involve substitution of one aliphatic or hydrophobic amino acids, e.g. alanine, valine, leucine and isoleucine, with another; substitution of one hydroxyl-containing amino acid, e.g. serine and threonine, with another; substitution of one acidic residue, e.g. glutamic acid or aspartic acid, with another; replacement of one amide-containing residue, e.g. asparagine and glutamine, with another; replacement of one aromatic residue, e.g. phenylalanine and tyrosine, with another; replacement of one basic residue, e.g. lysine, arginine and histidine, with another; and replacement of one small amino acid, e.g., alanine, serine, threonine, methionine, and glycine, with another.
The alterations are designed not to abolish the immunoreactivity of the variant NH protein with antibodies that bind to the reference protein. Guidance in determining which amino acid residues may be substituted, inserted or deleted without abolishing such immunoreactivity of the variant protein are found using computer programs well known in the art, for example, DNASTAR software.
Preparation of an NH protein variant in accordance herewith can be achieved by site-specific mutagenesis of DNA that encodes an earlier prepared variant or a nonvariant version of the protein. Site-specific mutagenesis allows the production of NH protein variants through the use of specific oligonucleotide sequences that encode the DNA sequence of the desired mutation. In general, the technique of site-specific mutagenesis is well known in the art, as exemplified by publications such as Adelman et al., DNA 2:183 (1983) and Ausubel et al. “Current Protocols in Molecular Biology”, J. Wiley & Sons, NY, N.Y., 1996. As will be appreciated, the site-specific mutagenesis technique can employ a phage vector that exists in both a single-stranded and double-stranded form. Typical vectors useful in site-directed mutagenesis include vectors such as the M13 phage, for example, as disclosed by Messing et al., Third Cleveland Symposium on Macromolecules and Recombinant DNA, Editor A. Walton, Elsevier, Amsterdam (1981). These phage are readily commercially available and their use is generally well known to those skilled in the art. Alternatively, plasmid vectors that contain a single-stranded phage origin of replication (Vieira et al., Meth. Enzymol. 153:3 (1987)) can be employed to obtain single-stranded DNA.
In general, site-directed mutagenesis in accordance herewith is performed by first obtaining a single-stranded vector that includes within its sequence a DNA sequence that encodes the relevant protein. An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically, for example, by the method of Crea et al., Proc. Natl. Acad. Sci. (USA) 75:5765 (1978). This primer is then annealed with the single-stranded protein-sequence-containing vector, and subjected to DNA-polymerizing enzymes such as E. coli polymerase I Klenow fragment, to complete the synthesis of the mutation-bearing strand. Thus, a mutated sequence and the second strand bears the desired mutation. This heteroduplex vector is then used to transform appropriate cells and clones are selected that include recombinant vectors bearing the mutated sequence arrangement. After such a clone is selected, the mutated protein region can be removed and placed in an appropriate vector for protein production, generally an expression vector of the type that can be employed for transformation of an appropriate host.
Some deletions and insertions, and substitutions are not expected to produce radical changes in the characteristics of NH proteins. However, when it is difficult to predict the exact effect of the substitution, deletion, or insertion in advance of doing so, one skilled in the art will appreciate that the effect will be evaluated by routine screening assays. For example, a variant typically is made by site-specific mutagenesis of the native encoding nucleic acid, expression of the variant nucleic acid in recombinant cell culture, and, optionally, purification from the cell culture, for example, by immunoaffinity adsorption to on a column (to absorb the variant by binding it to at least one remaining immune epitope). The activity of the cell lysate or purified variant is then screened in a suitable screening assay for the desired characteristic. For example, a change in the immunological character of the molecule, such as affinity for a given antibody, is measured by a competitive type immunoassay. Changes in immunomodulation activity are measured by the appropriate assay. Modifications of such protein properties as redox or thermal stability, hydrophobicity, susceptibility to proteolytic degradation or the tendency to aggregate with carriers or into multimers are assayed by methods well known to the ordinarily skilled artisan.
A “fragment” is an immunoreactive fragment of an NH protein that has a length of from about 6 amino acids to less than the full length NH protein and includes a sequence that contains at least 6 consecutive amino acids of a sequence of the NH protein. These fragments are collectively referred to herein as “NH peptides.” In some embodiments, the fragment has at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 consecutive amino acids of an NH protein sequence. The fragment can have a length of at most, e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, or 300 amino acids. In some embodiments, an immunoreactive fragment has from six to sixty amino acids, from six to fifty amino acids, from ten to fifty amino acids, from six to twenty amino acids, from eight to twenty amino acids, from ten to twenty amino acids, from twelve to twenty amino acids or from twelve to seventeen amino acids.
In some embodiments, the immunoreactive peptides are from six (6) amino acids up to less than the full length NH protein, and are antigenic, i.e. are recognized by mammalian immune systems effectively. For this purpose, the peptides comprise segments that are bacterial surface exposed, rather than bacterial cytoplasmic side-exposed or embedded within the lipid bilayer membrane. Such surface exposed regions of NH proteins can be identified using computer programs using algorithms that can predict the three dimensional structure of the NH proteins based on the hydrophobicity/hydrophilicity of the amino acid regions and the repeated β sheet model.
Also provided herein are fusion proteins in which a tag or one or more amino acids from a heterologous protein are added to the amino or carboxy terminus of the amino acid sequence of an NH protein or a functional derivative thereof. At least one of the proteins or peptides can be in a multimeric form. As used herein, the term “heterologous protein” means a protein derived from a source other than the N. helminthoeca gene, operationally linked to a N. helminthoeca protein or a functional derivative thereof, as disclosed in the present specification, to form a chimeric or fusion N. helminthoeca protein or peptide. Typically, such additions are made to stabilize the resulting fusion protein or to simplify purification of an expressed recombinant form of the corresponding NH protein, variant, or peptide. Such tags are known in the art. Representative examples of such tags include sequences which encode a series of histidine residues, the Herpes simplex glycoprotein D, or glutathione S-transferase. Such a chimeric or fusion protein can have a variety of lengths including, but not limited to, a length of at most 100 residues, at most 200 residues, at most 300 residues, at most 400 residues, at most 500 residues, at most 800 residues or at most 1000 residues. Non-limiting examples of chimeric N. helminthoeca proteins include fusions of N. helminthoeca protiens, or variants, or peptides: with immunogenic polypeptides, such as flagellin and cholera enterotoxin; with immunomodulatory polypeptides, such as IL-2 and B7-1; with tolerogenic polypeptides; with another N. helminthoeca protein, or variant, or peptide; and with synthetic sequences. Other examples include linking the NH protein, or variant or peptide with an indicator reagent, an amino acid spacer, an amino acid linker, a signal sequence, a stop transfer sequence, a transmembrane domain, a protein purification ligand or a combination of thereof. The fusion proteins can have similar or substantially similar immunoreactivity to NH antibodies as the NH proteins from which they derive.
The disclosed NH polypeptides can be used in a variety of procedures and methods, such as for the generation of antibodies, immunogenic compositions and vaccines; for use in identifying pharmaceutical compositions; for studying DNA/protein interaction; as well as for diagnostic and screening methods.
Also provided are compositions of matter comprising one or more NH proteins, their functional derivatives and/or NH fusion proteins. The isolated or purified polypeptide in such compositions can be in a multimeric form and can further include a carrier. The purified polypeptide can be linked to an indicator reagent, an amino acid spacer, an amino acid linker, a signal sequence, a stop transfer sequence, a transmembrane domain, a protein purification ligand, or a combination of these. Alternatively, one or more NH proteins or peptides may be linked together.
Also disclosed are polynucleotides encoding an NH protein, or variant thereof, disclosed herein.
In some embodiments, the polynucleotide encodes an NH P51 protein, or an immunogenic fragment thereof. Therefore in some embodiments, the polynucleotide comprises the nucleic acid sequence SEQ ID NO:6.
Neorickettsia helminthoeca Oregon P51 Gene Sequence:
In some embodiments, the polynucleotide encodes an NH P51 functional derivative. In some embodiments, the polynucleotide encodes an NH P51 variant. In some embodiments, the polynucleotide encodes an NH P51 variant with a nucleic acid sequence which is at least 85% (for example, at least 85%, at least 90%, at least 95%) identical to SEQ ID NO:6.
In some embodiments, the polynucleotide encodes an NH SSA protein, or an immunogenic fragment thereof. Therefore in some embodiments, the polynucleotide comprises the nucleic acid sequence SEQ D NO:7.
Neorickettsia helminthoeca Oregon SSA Gene Sequence:
In some embodiments, the polynucleotide encodes an NH SSA functional derivative. In some embodiments, the polynucleotide encodes an NH SSA variant. In some embodiments, the polynucleotide encodes an NH SSA variant with a nucleic acid sequence which is at least 85% (for example, at least 85%, at least 90%, at least 95%) identical to SEQ ID NO:7.
In some embodiments, the polynucleotide encodes an NH NSP1 protein, or an immunogenic fragment thereof. Therefore in some embodiments, the polynucleotide comprises the nucleic acid sequence SEQ ID NO:8.
Neorickettsia helminthoeca Oregon NSP1 Gene Sequence:
In some embodiments, the polynucleotide encodes an NH NSP1 functional derivative. In some embodiments, the polynucleotide encodes an NH NSP1 variant. In some embodiments, the polynucleotide encodes an NH NSP1 variant with a nucleic acid sequence which is at least 85% (for example, at least 85%, at least 90%, at least 95%) identical to SEQ ID NO:8.
In some embodiments, the polynucleotide encodes an NH NSP2 protein, or an immunogenic fragment thereof. Therefore in some embodiments, the polynucleotide comprises the nucleic acid sequence SEQ ID NO:9.
Neorickettsia helminthoeca Oregon NSP2 Gene Sequence:
In some embodiments, the polynucleotide encodes an NH NSP2 functional derivative. In some embodiments, the polynucleotide encodes an NH NSP2 variant. In some embodiments, the polynucleotide encodes an NH NSP2 variant with a nucleic acid sequence which is at least 85% (for example, at least 85%, at least 90%, at least 95%) identical to SEQ ID NO:9.
In some embodiments, the polynucleotide encodes an NH NSP3 protein, or an immunogenic fragment thereof. Therefore in some embodiments, the polynucleotide comprises the nucleic acid sequence SEQ ID NO:10.
Neorickettsia helminthoeca Oregon NSP3 Gene Sequence:
In some embodiments, the polynucleotide encodes an NH NSP3 functional derivative. In some embodiments, the polynucleotide encodes an NH NSP3 variant. In some embodiments, the polynucleotide encodes an NH NSP3 variant with a nucleic acid sequence which is at least 85% (for example, at least 85%, at least 90%, at least 95%) identical to SEQ ID NO:10.
Also disclosed are polynucleotides complementary to the disclosed nucleic acid sequences. Also disclosed are polynucleotides that can hybridize to a nucleic acid sequence disclosed herein under stringent hybridization conditions, or highly stringent hybridization conditions. It is understood that the polynucleotides encoding the NH polypeptides can have a different sequence than the nucleotide sequences disclosed herein due to the degeneracy of the genetic code. Thus, also included are the functional equivalents of the herein-described isolated polynucleotides and derivatives thereof. For example, the nucleic acid sequences can be altered by substitutions, additions or deletions that provide for functionally equivalent molecules. In addition, the polynucleotide can comprise a nucleotide sequence which results from the addition, deletion or substitution of at least one nucleotide to the 5′-end and/or the 3′-end of the disclosed nucleic acid segments, or a derivative thereof. Any polynucleotide can be used in this regard, provided that its addition, deletion or substitution does not substantially alter the amino acid sequence of the NH protein, or functional derivatives or fusion proteins thereof, encoded by the polynucleotide sequence. Moreover, the polynucleotide of the present invention can, as necessary, have restriction endonuclease recognition sites added to its 5′-end and/or 3′-end.
Further, it is possible to delete codons or to substitute one or more codons by codons other than degenerate codons to produce a structurally modified polypeptide, but one which has substantially the same utility or activity of the polypeptide produced by the unmodified nucleic acid molecule. As recognized in the art, the two polypeptides are functionally equivalent, as are the two nucleic acid molecules which give rise to their production, even though the differences between the nucleic acid molecules are not related to degeneracy of the genetic code.
The NH polynucleotides described herein are also useful for designing hybridization probes for isolating and identifying cDNA clones and genomic clones encoding the NH proteins, peptides or allelic forms thereof. Such hybridization techniques are known to those of skill in the art.
Therefore, in another embodiment, a nucleic acid probe is provided for the specific detection of the presence of one or more NH polynucleotides in a sample comprising the above-described isolated polynucleotides or at least a fragment thereof, which binds under stringent conditions, or highly stringent conditions, to NH polynucleotides.
The term “stringent conditions” as used herein is the binding which occurs within a range from about Tm 5° C. (5° C. below the melting temperature Tm of the probe) to about 20° C. to 25° C. below Tm. The term “highly stringent hybridization conditions” as used herein refers to conditions of: at least about 6×SSC and 1% SDS at 65° C., with a first wash for 10 minutes at about 42° C. with about 20% (v/v) formamide in 0.1×SSC, and with a subsequent wash with 0.2×SSC and 0.1% SDS at 65° C.
In some embodiments, the isolated nucleic acid probe consisting of 10 to 1000 nucleotides (for example: 10 to 500, 10 to 250, 10 to 100, 10 to 50, 10 to 35, 20 to 1000, 20 to 500, 20 to 250, 20 to 100, 20 to 50, or 20 to 35, etc.) which hybridizes preferentially to RNA or DNA of NH but not to RNA or DNA of non-NH organisms, wherein said nucleic acid probe is or is complementary to a nucleotide sequence consisting of at least 10 consecutive nucleotides, or 15, 20, 25, 30, 50, 100, 250, 500, 600, 700, 800, or 900 consecutive nucleotides, or along the entire length, of one or more of the NH polynucleotides described above.
Such hybridization probes can have a sequence which is at least 90%, 95%, 98%, 99% or 100% complementary with a sequence contained within the sense strand of a DNA molecule which encodes each of the NH proteins or with a sequence contained within its corresponding antisense strand. Such hybridization probes bind to the sense or antisense strand under stringent, or highly stringent, conditions.
The hybridization probes can be labeled by standard labeling techniques such as with a radiolabel, enzyme label, fluorescent label, biotin-avidin label, chemiluminescence, and the like. After hybridization, the probes can be visualized using known methods.
In some embodiments, a nucleic acid probe is immobilized on a solid support. Examples of such solid supports include, but are not limited to, plastics such as polycarbonate, complex carbohydrates such as agarose and sepharose, and acrylic resins, such as polyacrylamide and latex beads. Techniques for coupling nucleic acid probes to such solid supports are well known in the art.
NH polynucleotides disclosed herein are also useful for designing primers for polymerase chain reaction (PCR), a technique useful for obtaining large quantities of cDNA molecules that encode the NH polypeptides. PCR primers can also be used for diagnostic purposes. Thus, also included are oligonucleotides that are used as primers in polymerase chain reaction (PCR) technologies to amplify transcripts of the genes which encode the NH polypeptides, or portions of such transcripts. In some examples, the primers comprise a minimum of about 12 to 15 nucleotides and a maximum of about 30 to 35 nucleotides. The primers can have a G+C content of 40% or greater. Such oligonucleotides are at least 98% complementary with a portion of the DNA strand, i.e., the sense strand, which encodes the NH protein, or a portion of its corresponding antisense strand. In some embodiments, the primer has at least 99% complementarity, or 100% complementarity, with such sense strand or its corresponding antisense strand. Primers which have 100% complementarity with the antisense strand of a double-stranded DNA molecule encoding an NH protein have a sequence which is identical to a sequence contained within the sense strand.
One skilled in the art can readily design such probes and primers based on the sequences disclosed herein using methods of computer alignment and sequence analysis known in the art (see, for example, Molecular Cloning: A Laboratory Manual, second edition, edited by Sambrook, Fritsch, & Maniatis, Cold Spring Harbor Laboratory, 1989).
The primers herein are selected to be “substantially” complementary to different strands of a particular target DNA sequence. This means that the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5′ end of the primer, with the remainder of the primer sequence being complementary to the strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementary with the sequence or hybridize therewith and thereby form the template for the synthesis of the extension product.
Also disclosed are methods for diagnosing a canine subject with Neorickettsia helminthoeca infection using the disclosed polypeptides to detect antibodies specific for Neorickettsia helminthoeca in a sample from the subject. For example, the sample can be a blood, serum, or plasma sample containing antibodies. Immunodetection methods can be used to assay for the presence of antibodies that specifically bind an NH protein or peptide disclosed herein.
The method can involve contacting the sample with one or more Neorickettsia helminthoeca polypeptides, as described herein, under conditions that allow polypeptide/antibody complexes to form; and assaying for the formation of a complex between antibodies in the test sample and the one or NH polypeptides. Accordingly, detecting the formation of such a complex is an indication that antibodies specific for Neorickettsia helminthoeca are present in the test sample.
The steps of various useful immunodetection methods have been described in the scientific literature, such as, e.g., Maggio et al., Enzyme-Immunoassay, (1987) and Nakamura, et al., Enzyme Immunoassays: Heterogeneous and Homogeneous Systems, Handbook of Experimental Immunology, Vol. 1: Immunochemistry, 27.1-27.20 (1986), each of which is incorporated herein by reference in its entirety and specifically for its teaching regarding immunodetection methods. Immunoassays, in their most simple and direct sense, are binding assays involving binding between antibodies and antigen. Many types and formats of immunoassays are known and all are suitable for detecting the disclosed biomarkers. Examples of immunoassays are enzyme linked immunosorbent assays (ELISAs), radioimmunoassays (RIA), radioimmune precipitation assays (RIPA), immunobead capture assays, Western blotting, dot blotting, gel-shift assays, Flow cytometry, protein arrays, multiplexed bead arrays, magnetic capture, in vivo imaging, fluorescence resonance energy transfer (FRET), and fluorescence recovery/localization after photobleaching (FRAP/FLAP).
Also disclosed are immunogenic compositions comprising one or more of the disclosed Neorickettsia helminthoeca proteins, or immunogenic fragments and variants thereof, or a fusion protein containing same, collectively referred to herein as an “immunogenic NH polypeptide” and a pharmaceutically acceptable carrier.
The immunogenic NH polypeptides, as used herein, comprise an epitope-bearing portion of an NH protein. An immunogenic NH polypeptide is a polypeptide that is capable of producing antibodies with a specific binding affinity to N. helminthoeca in a subject to whom the immunogenic composition has been administered.
Also disclosed is a vaccine comprising an immunogenic NH polypeptide, together with a pharmaceutically acceptable diluent, carrier, or excipient, wherein the immunogenic NH polypeptide is present in an amount effective to elicit a beneficial immune response in a subject to NH. The immunogenic NH polypeptide may be obtained as described above and using methods well known in the art.
In another embodiment, the present invention relates to a vaccine comprising an NH nucleic acid (e.g., DNA) or a segment thereof (e.g., a segment encoding an immunogenic NH polypeptide) together with a pharmaceutically acceptable diluent, carrier, or excipient, wherein the nucleic acid is present in an amount effective to elicit, in a subject, a beneficial immune response to NH. The NH nucleic acid may be obtained as described above and using methods well known in the art.
In a further embodiment, the present invention relates to a method of producing an immune response which recognizes NH in a host, comprising administering to the host one or more of the above-described immunogenic NH polypeptides.
In some embodiments, the host or subject to be protected is a member of the Canidae family including domestic dogs, foxes, and coyotes.
Also disclosed is a method of preventing or inhibiting salmon poisoning disease (SPD) in a subject comprising administering to the subject the above-described vaccine, wherein the vaccine is administered in an amount effective to prevent or inhibit SPD. The vaccine of the invention is used in an amount effective depending on the route of administration. Although intra-nasal, subcutaneous or intramuscular routes of administration are suitable, the vaccine of the present invention can also be administered by an oral, intraperitoneal or intravenous route. One skilled in the art will appreciate that the amounts to be administered for any particular treatment protocol can be readily determined without undue experimentation. Suitable amounts are within the range of 2 μg of the NH vaccine per kg body weight to 100 micrograms per kg body weight (preferably, 2 μg to 50 μg, 2 μg to 25 μg, 5 μg to 50 μg, or 5 μg to 10 μg).
Examples of vaccine formulations including antigen amounts, route of administration and addition of adjuvants can be found in Kensil, Therapeutic Drug Carrier Systems 13:1-55 (1996), Livingston et al., Vaccine 12:1275 (1994), and Powell et al., AIDS RES, Human Retroviruses 10:5105 (1994). The disclosed vaccine may be employed in such forms as capsules, liquid solutions, suspensions or elixirs for oral administration, or sterile liquid forms such as solutions or suspensions. Any inert carrier may be used, such as saline, phosphate-buffered saline, or any such carrier in which the vaccine has suitable solubility properties. The vaccines may be in the form of single dose preparations or in multi-dose flasks which can be used for mass vaccination programs. Reference is made to Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., Osol (ed.) (1980); and New Trends and Developments in Vaccines, Voller et al (eds), University Park Press, Baltimore, Md. (1978), for methods of preparing and using vaccines.
The disclosed vaccines may further comprise adjuvants which enhance production of antibodies and immune cells. Such adjuvants include, but are not limited to, various oil formulations such as Freund's complete adjuvant (CFA), the dipeptide known as MDP, saponins (ex, QS-21, U.S. Pat. No. 5,047,540), aluminum hydroxide, or lymphatic cytokines. Freund's adjuvant is an emulsion of mineral oil and water which is mixed with the immunogenic substance. Although Freund's adjuvant is powerful, it is usually not administered to humans. Instead, the adjuvant alum (aluminum hydroxide) may be used for administration to a human. Vaccine may be absorbed onto the aluminum hydroxide from which it is slowly released after injection. The vaccine may also be encapsulated within liposomes according to Fullerton, U.S. Pat. No. 4,235,877.
In some embodiments, disclosed herein is a method of detecting an infection with N. helminthoeca in a Canidae patient comprising the steps of:
(a) providing a serum sample from the patient;
(b) providing an isolated or purified N. helminthoeca protein selected from the group consisting of P51, NSP1, NSP2, NSP3, and SSA;
(c) contacting the serum sample with the isolated or purified N. helminthoeca protein; and
(d) assaying for the formation of a complex between antibodies in the serum sample and the isolated or purified N. helminthoeca protein, wherein formation of said complex is indicative of infection with N. helminthoeca.
In some embodiments, disclosed herein is a method of detecting an infection with N. helminthoeca in a Canidae patient comprising the steps of:
(a) providing a serum sample from the patient;
(b) providing one or more antibodies that specifically bind to a N. helminthoeca polypeptide, wherein the N. helminthoeca polypeptide is selected from the group consisting of P51, NSP1, NSP2, NSP3, and SSA;
(c) contacting the serum sample with the one or more antibodies; and
(d) assaying for the formation of a complex between N. helminthoeca proteins in the serum sample and the one or more antibodies, wherein formation of said complex is indicative of infection with N. helminthoeca.
In some embodiments, disclosed herein is a method of detecting N. helminthoeca polypeptides in a test sample comprising
In some embodiments, the one or more antibodies are monoclonal antibodies, polyclonal antibodies, Fab fragments, Fab′ fragments, Fab′-SH fragments, F(ab′)2 fragments, Fv fragments, or single chain antibodies.
In some embodiments, disclosed herein is a method of detecting antibodies specific for N. helminthoeca comprising:
In some embodiments, the one or more isolated N. helminthoeca polypeptides is at least 85% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 (or a functional derivative thereof).
In some embodiments, the one or more isolated N. helminthoeca polypeptides comprises an immunoreactive fragment that has a length of from 6 amino acids to less than the full length of the N. helminthoeca protein and comprises 6 or more consecutive amino acids of an amino acid sequence that is set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and/or SEQ ID NO: 5.
In some embodiments, disclosed herein is an isolated or purified outer membrane protein of N. helminthoeca, a variant of said outer membrane protein, or an immunogenic fragment of said outer membrane protein, wherein said outer membrane protein is P51, NSP1, NSP2, NSP3, SSA, or a fragment thereof.
In some embodiments, disclosed herein is an expression vector for transformation of a host cell, said vector comprising an isolated polynucleotide that encodes an outer membrane protein of N. helminthoeca, a variant of said outer membrane protein, or an immunogenic fragment of said outer membrane protein, wherein said outer membrane protein is P51, NSP1, NSP2, NSP3, SSA, or a fragment thereof. In some embodiments, disclosed herein is a host cell comprising the expression vector comprising an isolated polynucleotide that encodes an outer membrane protein of N. helminthoeca, a variant of said outer membrane protein, or an immunogenic fragment of said outer membrane protein, wherein said outer membrane protein is P51, NSP1, NSP2 NSP3, SSA, or a fragment thereof.
In some embodiments, disclosed herein is an isolated outer membrane protein of N. helminthoeca consisting of a sequence that is at least 85% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. In some embodiments, to disclosed herein is an isolated outer membrane protein of N. helminthoeca consisting of a sequence that is at least 90% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. In some embodiments, disclosed herein is an isolated outer membrane protein of N. helminthoeca consisting of a sequence that is at least 95% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ NO: 5.
In some embodiments, disclosed herein is an isolated outer membrane protein of claim 1, wherein the polypeptide contains an immunoreactive fragment that is 6 or more consecutive amino acids from the following sequences: (1) SEQ ID NO: 1; (2) SEQ ID NO: 2; (3) SEQ ID NO: 3; (4) SEQ ID NO: 4; (5) SEQ ID NO: 5; or any combination of the sequences (1)-(5).
In some embodiments, disclosed herein is a kit for detecting N. helminthoeca in a subject, said kit comprising an N. helminthoeca protein, an antigenic fragment of an N. helminthoeca protein, or both; wherein the N. helminthoeca protein is selected from the group consisting of P51, NSP1, NSP2, NSP3, and SSA. In some embodiments the kit further comprises a biomolecule for detecting interaction between the N. helminthoeca protein reagent and antibodies in a bodily sample of the animal.
In some embodiments, disclosed herein is a kit for detecting N. helminthoeca in a subject, said kit comprising an N. helminthoeca protein, an antigenic fragment of an N. helminthoeca protein, or both; wherein the N. helminthoeca protein is selected from the group consisting of P51, NSP1, NSP2, NSP3, and SSA.
In some embodiments, disclosed herein is a reagent kit for detecting infection with N. helminthoeca in a subject comprising one or more antibodies that specifically bind to a N. helminthoeca polypeptide, wherein the N. helminthoeca polypeptide is selected from the group consisting of P51, NSP1, NSP2, NSP3, and SSA.
A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.
The following examples are set forth below to illustrate the compounds, compositions, methods and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative compounds, compositions, methods and results. These examples are not intended to exclude equivalents and variations of the present invention which are apparent to one skilled in the art.
Neoricketts helminthoeca, a type species of the genus Neorickettsia, is an endosymbiont of digenetic trematodes of veterinary importance. Upon ingestion of salmonid fish parasitized with infected trematodes, canids develop salmon poisoning disease (SPD), an acute febrile illness that is particularly severe and often fatal in dogs without adequate treatment. The complete genome sequence of N. helminthoeca was determined and analyzed: a single small circular chromosome of 884,232 bp encoding 774 potential proteins. N. helminthoeca is unable to synthesize lipopolysaccharides and most amino acids, but is capable of synthesizing vitamins, cofactors, nucleotides, and bacterioferdtin. N. helminthoeca is, however, distinct from majority of the family Anaplasmataceae to which it belongs, as it encodes nearly all enzymes required for peptidoglycan biosynthesis, suggesting its structural hardiness and inflammatory potential. Using sera from dogs that were experimentally infected by feeding with parasitized fish or naturally infected in Southern California, western blotting analysis revealed that among five predicted N. helminthoeca outer membrane proteins, P51 and strain-variable surface antigen were uniformly recognized. These results aid in understanding pathogenesis, prevalence of N. helminthoeca infection among trematodes, canids, and potentially other animals in nature to develop effective SPD diagnostic and preventive measures. Recent progresses in large-scale genome sequencing have been uncovering broad distribution of Neorickettsia spp., the comparative genomics will facilitate understanding of biology and the natural history of these elusive environmental bacteria.
N. helminthoeca can stably be continuously cultured in a DH82 canine macrophage cell line for up to 3 months with inoculation of infected DH82 cells inducing a more severe form of the disease in dogs. This advancement has allowed for the investigation of genetic and antigenic properties of N. helminthoeca and clarification of its relationship to other members of the family Anaplasmataceae leading to reclassification of N. helminthoeca, N. risticii, N. sennetsu, and SF agent into their own clade (Table 1). In this study, experiments were conducted to synthesize the whole N. helminthoeca bacterial genome, determine, clone, and purify antigenic outer membrane proteins (OMPs), probe these recombinant OMPs using experimentally and clinically SPD infected dog sera, and determine specific highly antigenic, surface exposed regions of these outer membrane proteins that are phylogenetically divergent from species closely related to N. helminthoeca, namely N. risticii and N. sennetsu.
In this example, three results were sought: (1) determine the complete genome of N. helminthoeca and compare with closely-related N. risticii and N. sennetsu genomes; (2) determine, clone, and purify putative immunodominant major outer membrane proteins (OMPs); and (3) test immunoreactivity of these recombinant OMPs using sera from dogs that were experimentally or naturally infected with N. helminthoeca.
General Features of the Genome
The genome of N. helminthoeca Oregon consists of a single double-stranded circular chromosome spanning 884,232 bp, which is similar to those of N. risticii (Lin et al., 2009) and N. sennetsu (Dunning Hotopp et al., 2006) (Table 2), and smaller than those of other members in the family Anaplasmataceae (approximately 1.0-1.5 Mbp) (Dunning Hotopp et al., 2006). G+C content of N. helminthoeca genome is 41.7% (Table 2), which is similar to those of other Neorickettsia and Anaplasma spp., but greater than those (approximately 30%) of Ehrlichia spp. and Wolbachia spp. (Dunning Hotopp et al., 2006). The replication origin of N. helminthoeca (
The N. helminthoeca genome encodes one copy each of the 5S, 16S, and 23S rRNA genes, which are separated in 2 loci with the 5S and 23S rRNA genes forming an operon (
With 827 protein- and RNA-coding genes (
Comparison of Genomic Contents Among Neorickettsia Species
Previous studies have shown that Anaplasma spp. and Ehrlichia spp. have a single large-scale symmetrical inversion (X-alignment) near the replication origin, which is possibly mediated by duplicated rho genes (Dunning Hotopp et al., 2006; Frutos et al., 2007; Nene and Kole, 2009). In addition, Anaplasma and Wolbachia spp. have extensive genomic rearrangement throughout the genome (Wu et al., 2004; Dunning Hotopp et al., 2006). However, the synteny is highly conserved and such genomic rearrangements or a large scale inversion are not detected among N. helminthoeca, N. sennetsu, and N. risticii (
In order to compare the genomic contents among Neorickettsia spp., 2- and 3-way comparisons were performed using reciprocal B
The three Neorickettsia spp. are transmitted by distinct trematodes and cause severe diseases at high mortality in different mammalian hosts (Table 1) (Cordes et al., 1986; Dutta et al., 1988; Rikihisa et al., 1991; Rikihisa et al., 2004; Rikihisa et al., 2005; Gibson and Rikihisa, 2008; Lin et al., 2009). We, therefore, analyzed the species-specific genes based on the 2- and 3-way comparisons. There are 89 species-specific proteins in N. helminthoeca as compared to 23 and 28 in N. risticii and N. sennetsu, respectively (Tables 6-8). Of the genes unique to N. helminthoeca, more than half of them (50/89 ORFS) are hypothetical proteins without assigned functions (Table 6). Among the N. helminthoeca-specific proteins with assigned functions, ˜38% (15/39 ORFs) are involved in peptidoglycan biosynthesis that are absent in N. risticii and N. sennetsu (Table 6 and
Metabolism
Except for peptidoglycan biosynthesis, most metabolic pathways, transcription, translation, and regulatory functions, are highly conserved in N. helminthoeca compared to N. sennetsu and N. risticii (summarized in
Central metabolic pathways. Analysis of the metabolic pathways based on Kyoto Encyclopedia of Genes and Genomes (KEGG, http://www.kegg.jp) and BioCyc (http://biocyc.org/) indicates that, similar to other members in the family Anaplasmataceae, N. helminthoeca encodes pathways for aerobic respiration, including the tricarboxylic acid (TCA) cycle and the electron transport chain, but it is unable to use glucose, fructose, or fatty acids directly as a carbon or energy source, since essential enzymes for the utilization of these substrates such as hexokinases, the first enzyme in the glycolysis pathway that converts glucose to glucose-6-phosphate, and pyruvate kinase that converts phosphoenolpyruvate to pyruvate, are not identified (
Amino acids, nucleotides, fatty acids, and cofactor biosynthesis. Like other Neorickettsia, Ehrlichia, and Anaplasma spp. (Dunning Hotopp et al., 2006; Lin et al., 2009), N. helminthoeca synthesizes very limited amino acids including alanine, aspartate, glycine, glutamate, and glutamine (
Similar to all other sequenced members of Anaplasmataceae (Dunning Hotopp et al., 2006), N. helminthoeca encodes a nonoxidative pentose-phosphate pathway that utilizes glyceraldehyde-3-phosphate to produce pentose for nucleotide and cofactor biosynthesis. Accordingly, N. helminthoeca encodes complete pathways for de novo purine and pyrimidine biosynthesis, and is capable of synthesizing most vitamins or cofactors, such as biotin, folate, FAD, NAD, and protoheme (
Transporters and porins. To compensate for the incomplete biosynthesis or metabolic pathways, the N. helminthoeca genome encodes several orthologs involved in cytoplasmic membrane transport systems that can supply the necessary amino acids, metabolites, and ions, as analyzed by TransAAP (Transporter Automatic Annotation Pipeline, http://www.membranetransport.org/) (
Gram-negative bacteria also express porins spanning their outer membranes that enable the transport of hydrophilic and large molecules, such as amino acids, sugars, and other nutrients (Nikaido, 2003). Similar to other members of the Anaplasmataceae that have limited capabilities of amino acids biosynthesis, intermediary metabolism, and glycolysis, nutrient uptake in these bacteria necessitates pores or channels in the bacterial outer membrane (Huang et al., 2007; Kumagai et al., 2008; Gibson et al., 2010). Previous studies have determined that the major outer membrane proteins, including A. phagocytophilum P44s (Huang et al., 2007), E. chaffeensis P28/OMP-1F (Kumagai et al., 2008), and N. sennetsu P51 (Gibson et al., 2010), possess porin activities as determined by a proteoliposome swelling assay, which allow the diffusion of L-glutamine, the monosaccharides arabinose and glucose, the disaccharide sucrose, and even the tetrasaccharide stachyose. N. helminthoeca encodes a P51 protein (NHE_RS00965) that shares 60% amino acid sequence similarity with N. sennetsu P51 protein (
DNA, RNA, protein synthesis, and DNA repair, N. helminthoeca encodes proteins necessary for DNA replication, RNA synthesis and degradation, and ribosomal proteins. Although N. helminthoeca encodes proteins required for homologous recombination, including RecA/RecF (but not RecBCD) pathways (Lin et al., 2006) and RuvABC complexes for Holliday junction recombination as other members of the family Anaplasmataceae (Table 12), it has the least amount of enzymes involved in DNA repair compared to other members of the family Anaplasmataceae including N. sennetsu and N. risticii (7 in N. helminthoeca vs. 9 in N. sennetsu, 12 in E. chaffeensis, and 13 in A. phagocytophilum, Table 12) (Dunning Hotopp et al, 2006; Lin et al., 2009). N. helminthoeca lacks most genes required for mismatch repair, nucleotide excision repair (NER, such as uvrABC for UV-induced DNA damage), various glycosylases for base excision repair (BER), and DNA photolyases, which is an alternative mechanism to repair UV-damaged DNA identified in E. chaffeensis, A. phagocytophilum, and N. risticii (Dunning Hotopp et al., 2006; Lin et al., 2009).
Pathogenesis
Although SPD was recognized more than two centuries ago, the causative agent N. helminthoeca was only stably cultured in canine cell line in 1990 (Rikihisa et al., 1991), and there are little information available regarding the molecular determinants of N. helminthoeca to invade and cause severe disease in canine hosts. Here, genes and pathways were analyzed that are potentially involved in N. helminthoeca pathogenesis, including protein secretion systems, two-component/one-component regulatory systems, N. helminthoeca-specific genes, and putative membrane proteins or lipoproteins.
Protein secretion systems. Two major pathways exist to secrete proteins across the cytoplasmic membrane in bacteria. The general Secretion route, termed Sec-pathway, catalyzes the transmembrane translocation of proteins in their unfolded conformation, whereupon they fold into their native structure at the trans-side of the membrane (Natale et al., 2008). All major components for the Sec-dependent pathway are identified, including signal recognition particle (SRP) protein, SRP-docking protein FtsY, the cytosolic protein-export chaperone SecB, peripheral associated ATP-dependent motor protein SecA, membrane-embedded protein conducting channel SecYEG, periplasmic protein YajC that involved in preprotein translocase activity, and the membrane complex SecDF that enhances proton motive force (
Twin-arginine translocation (Tat)-pathway, which consists of the TatA, TatB, and TatC proteins, can transport folded proteins across the bacterial cytoplasmic membrane by recognizing N-terminal signal peptides harboring a distinctive twin-arginine motif (Lee et al., 2006; Sargent et al., 2006). All genes encoding Tat apparatus are identified in the N. helminthoeca genome (tatA/NHE_RS02000, tatB/NHE_RS02160, and tatC/NHE_RS00490) (
Extracellular secretion of various virulence factors across the bacterial cell envelope is one of the major mechanisms by which pathogenic bacteria alter host cell functions, thus enhancing survival of the bacteria and damaging hosts. At least six distinct extracellular protein secretion systems, referred to as type I-VI secretion systems (T1SS-T6SS) (Papanikou et al., 2007; Costa et al., 2015), have been classified in Gram-negative bacteria that secrete effector molecules across two lipid bilayers and the periplasm. Except for T2SS, all double-membrane-spanning secretion systems (T1SS, T3SS, T4SS and T6SS) use a one-step mechanism to transport substrates directly from the bacterial cytoplasm into the extracellular space or into a target cell (Costa et al., 2015). Bioinfomatic analysis shows that, similar to all other sequenced members of the family Anaplasmataceae, N. helminthoeca genome encodes both T1SS and T4SS for secretion of proteins across the membranes, but it lacks homologs of T2SS, T3SS, T5SS, or T6SS components (
T4SS can translocate bacterial effector molecules into host cells, thus often plays a key role in pathogenesis of Gram-negative host-associated bacteria (Cascales and Christie, 2003; Backert and Meyer, 2006; Gillespie et al., 2010; Christie et al., 2014). In several intracellular bacteria including the family Anaplasmataceae such as E. chaffeensis and A. phagocytophilum, the T4SS is critical for survival and replication inside host cells, by inducing autophagy for nutrient acquisition and inhibition of host cell apoptosis (Niu et al.; 2006; Lin et al., 2007; Niu et al., 2010; Liu et al., 2012; Niu et al., 2012; Lin et al., 2016). In the N. helminthoeca genome, a T4SS encoded by virB/D genes distributed in four separate loci was identified. The organization of virB/D gene clusters is conserved among Neorickettsia spp. as with other Anaplasmataceae, with duplicated genes of virB4, virB8, and virB9, and multiple copies of virB2 and virB6 genes (Tables 5 and 10).
Subcellular fractionation and functional studies have demonstrated that VirB2 is the major pilus component of T4SS extracellular filaments (Cascales and Christie, 2003; Backert and Meyer, 2006). A previous study has confirmed that N. risticii VirB2 was localized at the opposite poles on the bacterial surface (Lin et al., 2009), suggesting that VirB2 might serve as secretion channels for the T4SS apparatus like that of Agrobacterium (Cascales and Christie, 2003), and play critical roles in mediating the interaction with host cells. Analysis of N. helminthoeca genome reveals three copies of virB2 upstream of virB4, whereas N. risticii and N. sennetsu encode two virB2 genes (Table 10) (Lin et al., 2009). Alignment of VirB2 protein sequences indicates that VirB2s of Neorickettsia spp. are closely related to those of other α-proteobacteria like Rickettsia, Agrobacterium, and Caulobacter, but are phylogenetically distinct from VirB2s of E. chaffeensis and A. phagocytophilum that form a separate clade (
Two-component regulatory systems. Two-component regulatory systems (TCRS) are signal transduction systems that allow bacteria to sense and respond rapidly to changing environmental conditions (Mitrophanov and Groisman, 2008; Wuichet et al., 2010). TCRS consists of a sensor histidine protein kinase that responds to specific signals, and a cognate response regulator. Phosphorylation of a response regulator by a cognate histidine kinase changes the biochemical properties of its output domain, which can participate in DNA binding and transcriptional control, perform enzymatic activities, bind RNA, or engage in protein—protein interactions (Gao et al., 2007). TCRS plays a key role in controlling virulence responses in a wide variety of bacterial pathogens (Dorman et al., 2001; Mitrophanov and. Groisman, 2008), including E. chaffeensis and A. phagocytophilum in the family Anaplasmataceae, which encode three pairs of TCRS, including CckA/CtrA, PleC/PleD, and NtrX/NtrY (Cheng et al., 2006; Kumagai et al., 2006; Cheng et al., 2011; Kumagai et al., 2011).
Computational analysis reveals that the three sequenced Neorickettsia spp. encode two pairs of TCRS: CckA/CtrA and PleC/PleD (Table 10). The histidine kinase CckA/response regulator CtrA pair, identified only in α-proteobacteria, also have been demonstrated to coordinate multiple cell cycle events at the transcriptional level in E. chaffeensis to regulate bacterial developmental cycle (Cheng et al., 2011). Different from Ehrlichia and Anaplasma, the three Neorickettsia spp. encode two copies of PleC histidine kinase (NHE_RS00035/NHE_RS02255, Tables 5 and 10) and a one-component signal transduction protein, an EAL domain protein (NHE_RS01830) (
One-component regulatory systems and transcriptional regulations. One-component regulatory systems consist of a single protein containing both input and output domains, but lack the phospho-transfer domains of TCRS, and carry out signaling events in prokaryotes (Ulrich et al., 2005; Ulrich and Zhulin, 2007, 2010). This study found that compared to Ehrlichia and Anaplasma, the three Neorickettsia spp. encode more proteins in one-component systems (indicated by asterisks in
Perhaps due to the relatively homeostatic intracellular environment of the eukaryotic host cells, members of the order Rickettsiales and Chlamydiaceae have a small number of transcriptional regulators. N. helminthoeca as all other members of the family Anaplasmataceae encodes only two sigma factors: the essential RNA polymerase sigma-70 factor (RpoD, RHE_RS01300) responsible for most RNA synthesis in exponentially growing cells, and sigma-32 factor (RpoH, NHE_RS01445) responsible for expression from heat shock promoters.
N. helminthoeca encodes a putative transcriptional regulator NhxR (N. helminthoeca expression regulator), a 12.5-kDa DNA binding protein (NHE_RS00155) that has 90% amino acid identity with N. risticii NrxR (NRI_RS00145) and N. sennetsu, NsxR (NSE_RS00160). NhxR homologs, A. phagocytophilum ApxR and E. chaffeensis EcxR have shown to regulate the expression of P44 outer membrane proteins and the T4SS, respectively (Wang et al., 2007b; Wang et al., 2007a; Cheng et al., 2008). The other putative transcriptional regulator Tr1 (NHE_RS00915) is homologous to A. phagocytophilum and E. chaffeensis Tr1, which is regulated by ApxR in A. phagocytophilum and located at the upstream of the tandem genes encoding the major outer membrane proteins (OMPs), like Omp-1/Msp-2/P44 expression loci in A. phagocytophilum (Lin et al., 2004) or P28/Omp-1 gene clusters in E. chaffeensis (Ohashi et al., 2001; Wang et al., 2007a; Rikihisa, 2010). However, Tr1 in N. helminthoeca, N. risticii, or N. sennetsu is not located at upstream of any of genes encoding the major OMPs of N. helminthoeca including P51, SSA, or NSPs (Table 4).
The present study identified several other N. helminthoeca DNA-binding regulators, which are conserved in N. risticii and N. sennetsu (
Ankyrin domain proteins. Ankyrin-repeat domains (Ank), found predominantly in eukaryotic proteins, are known to mediate protein-protein interactions involved in a multitude of host processes, including cytoskeletal motility, tumor suppression, and transcriptional regulation (Bennett and Baines, 2001; Mosavi et al., 2004). Compared to free-living bacteria, Ank proteins are enriched in facultative and obligate intracellular bacteria of eukaryotes (Jernigan and Bordenstein, 2014). Several studies have shown that the ankyrin repeat-containing protein AnkA of A. phagocytophilum is secreted into host cells by the T4SS and plays an important role in facilitating intracellular infection by activating the Abl-1 protein tyrosine kinase, interacting with the host tyrosine phosphatase SHP-1, or regulation of host cell transcription (Udo et al., 2007; Lin et al., 2007; Garcia-Garcia et al., 2009). In E. chaffeensis, AnkA homolog Ank200 is translocated into the host cell nucleus though a T1SS-dependent manner, and binds to Alu elements and numerous host proteins (Zhu et al., 2009; Wakeel et al., 2011). Four ankyrin-repeat containing proteins were identified in the N. helminthoeca genome (4 in N. risticii and 3 in N. sennetsu) (Table 10). Phylogenetic analysis indicated that N. helminthoeca encodes one Ank protein (NHE_RS00105) that is clustered with E. chaffeensis T1SS substrate Ank200 (11.6% amino acid similarities) (Wakeel et al., 2011) and less related to A. phagocytophilum T4SS substrate AnkA (8.6% amino acid similarities) (Lin et al., 2007) (
Iron uptake and storage. Iron is an essential element for almost all living organisms, and serves as a cofactor in key metabolic processes including energy generation, electron transport, and DNA synthesis (Skaar, 2010). This study found that the three Neorickettsia spp., E. chaffeensis, and A. phagocytophilum encode proteins for iron transport across inner membranes, including periplasmic Fe3+-binding protein FbpA (NHE_RS00045), cytoplasmic membrane permease component FbpB (NHE_RS01265), and cytoplasmic ABC transporter FbpC (PotC, NHE_RS01995) (Table 5). However, homologs to known bacterial siderophore and outer membrane receptors for iron or chelated iron are not identified in these bacteria, suggesting that they might use a unique system to bind and uptake iron from their host. Infection of N. risticii, N. sennetsu, and E. chaffeensis, but not A. phagocytophilum, are inhibited by an intracellular labile iron chelator deferoxamine (Park and Rikihisa, 1992; Barnewall and Rikihisa, 1994; Barnewall et al., 1999), suggesting that these bacteria may utilize different iron-uptake system to obtain iron from the host. Unlike E. chaffeensis and A. phagocytophilum, current analysis found that the three Neorickettsia spp. encode a bacterioferritin (NHE_RS01470) (Table 5, under role category “Transport and binding proteins”), which can capture soluble but potentially toxic Fe2+ by compartmentalizing it in the form of a bioavailable ferric mineral inside the protein's hollow cavity. In the family Anaplasmataceae, bacterioferritin is also found in the Wolbachia endosymbiont of insects or nematode (Kremer et al., 2009). This could be due to differences in their life cycle and invertebrate host: the entire life cycles of Neorickettsia and Wolbachia spp. are within trematodes, insects, or nematodes with limited labile iron pools, whereas Ehrlichia and Anaplasma live within mammalian blood cells and tick vectors fed on blood rich in iron (
Cell Wall Components
Lipopolysaccharide and peptidoglycan. N. helminthoeca lacks all genes encoding lipopolysaccharide (LPS) biosynthesis pathway including lipid A (the core component of LPS) as other sequenced members of the family Anaplasmataceae (Lin and Rikihisa, 2003; Dunning Hotopp et al., 2006; Lin et al., 2009), including the recently sequenced NFh (McNulty et al., 2017). Although few genes involved in LPS biosynthesis were identified in the draft genome of Candidatus “X. pacificiensis”, it was not expected to possess a functional LPS biosynthesis pathway (Kwan and Schmidt, 2013).
Interestingly, nearly all genes involved in peptidoglycan biosynthesis are identified in N. helminthoeca, A. marginale, and Wolbachia wMel (endosymbiont of insect Drosophila melanogaster) or wBm (endosymbiont of nematode Brugia malayi) in the family Anaplasmataceae. On the contrary, only a very limited numbers of genes in peptidoglycan biosynthesis are present in the genomes of N. risticii, N. sennetsu, E. chaffeenis, E. ruminantium, and A. phagocytophilum (
Analysis of N. helminthoeca genome suggests that it can perform de novo synthesis of
The incorporation of anhydromuropeptide subunits into the murein sacculus requires multiple enzymes like MtgA, MrcA/B, FtsI (PbpB), PbpC, MrdA (Pbp2), MrdB, DacF, Pal, MreB/C (Vollmer and Bertsche, 2008; Gillespie et al., 2010); however, only 3 genes encoding MrdA, FtsI (PbpB), and DacC were identified in N. helminthoeca (
However, it is possible that N. helminthoeca can still produce precursors or components of peptidoglycan. Since several peptidoglycan components are potent stimulants for innate immunity and anti-microbial responses in host immune defensive cells (Dziarski, 2003; Guan and Mariuzza, 2007; Sukhithasri et al., 2013), the presence of these components in N. helminthoeca could elicit anti-microbial and inflammatory activities in leukocytes, and may account for the high acute mortality of SPD (Philip, 1955; Rikihisa et al., 1991) compared to less severe or chronic infections caused by other Neorickettsia, Ehrlichia, or Anaplasma spp. that lack peptidoglycan biosynthesis genes.
Lipoproteins and putative outer membrane proteins. A previous study indicates that E. chaffeensis expresses mature lipoproteins on the bacterial surface, which induced delayed-type hypersensitivity reaction in dogs (Huang et al., 2008). This study found N. helminthoeca, like other sequenced members of the family Anaplasmataceae, encodes all three lipoprotein-processing enzymes (Lgt, LspA, and Lnt) (Table 13) (Gupta and Wu, 1991; Paetzel et al., 2002). Computational analysis with LipoP 1.0 (http://www.cbs.dtu.dk/services/LipoP) (Juncker et 2003) identified thirteen putative lipoproteins in N. helminthoeca (Table 13), which may also be involved in pathogenesis and immune response in infected canids as in E. chaffeensis (Huang et al., 2008). Homologs of several N. helminthoeca lipoproteins are also identified as lipoproteins in N. risticii, including OmpA, CBS domain protein and VirB6 family proteins (Table 5 and 14) (Lin et al., 2009).
Computational analysis using the pSort-B algorithm predicted only four outer membrane proteins, two of which (BamD lipoprotein and beta-barrel OMP BamA, also called Omp85/YaeT), are part of the beta-barrel assembly machinery (BAM) and essential for the folding and insertion of outer membrane proteins of Gram-negative bacteria (Surana et al., 2004) (Table 4). Unlike Ehrlichia and Anaplasma spp. that encode a diverse members of the OMP-1/P28/MSP2/P44 outer member superfamily proteins (Pfam01617), Neorickettsia spp. encode only one group of putative outer surface proteins that falls into this PFAM family (Dunning Hotopp et al., 2006). This group of proteins consists of three N. helminthoeca surface proteins (NSP1/2/3), which are approximately 30 kDa in mass and likely surface-exposed based on their similarities to Ehrlichia P28/Omp-1 (Ohashi et al., 1998a; Ohashi et al., 2001), A. phagocytophilum P44 (Zhi et al, 1998), and N. risticii/N. sennetsu NSPs (Gibson et al., 2010; Gibson et al., 2011) (
In addition to NSP family OMPs, several studies have identified additional sets of potential surface proteins in other Neorickettsia spp., which include a 51-kDa protein (P51) and Neorickettsia strain-specific antigens (SSA) (Biswas et al., 1998; Vemulapalli et al., 1998; Rikihisa et al., 2004; Lin et al., 2009; Gibson et al., 2010; Gibson et al., 2011). P51 belongs to an ortholog cluster (cluster 409) that exists in all Rickettsiales (Dunning Hotopp et al., 2006), and is highly conserved among all sequenced Neorickettsia spp. including N. helminthoeca (NHE_RS00965) and the SF agent (Rikihisa et al., 2004) (
Strain-specific antigens (SSAs), proteins of ˜50 kDa with extensive intramolecular repeats, have been reported to be a protective antigen of N. risticii against homologous challenge (Biswas et al., 1998; Dutta et al., 1998). Unlike N. risticii or N. sennetsu that encodes two to three tandem genes of nonidentical SSAs, N. helminthoeca only encodes one SSA protein (NHE_RS03855, 35 kDa) (
Immunoreactivities of putative outer membrane proteins. Except for Candidatus“X. pacificiensis” that maintains many genes involved in flagella assembly like hook, ring, and rod (Kwan and Schmidt, 2013), all members of the family Anaplasmataceae lack LPS, capsule, flagella, or common pili (Dunning Hotopp et al., 2006). In agreement with previous electron microscope images (Rikihisa et al., 1991), analysis of N. helminthoeca genome indicates that it did not produce a type 4 pili. Therefore, outer membrane proteins play critical roles in bacterium-host cell interactions and induce strong humoral immune responses (Rikihisa et al., 1992; Rikihisa et al., 1994; Ohashi et al., 1998b; Zhi et al., 1998; Rikihisa et al., 2004; Gibson et al., 2011). Analysis of infection-induced immune reactions to outer membrane proteins provide tools to determine prevalence of N. helminthoeca exposure/infection among various species of animals, and provide novel rapid immunodiagnostic methods and protective vaccines for SPD as disclosed herein.
To elucidate immune reactions of SPD dog sera to P51, NSP1/2/3, and SSA, these proteins were cloned into the pET-33b(+) expression vector, and recombinant proteins were purified from transformed E. coli (
A previous study showed that sera from N. helminthoeca-infected dogs, N. sennetsu-infected horse, N. risticii-infected horses, or E. canis-infected dogs cross-reacted with other species but with at least 16-fold lower than those for homologous antigens by immunofluorescence assay (Rikihisa, 1991b; Rikihisa. et al., 1991). This study also showed that approximately 78-80 kDa and 64 kDa proteins were the major antigens shared by N. helminthoeca, N. risticii, N. sennetsu, and E. canis (Rikihisa, 1991b) (
Despite expansion of DNA sequences of Neorickettsia spp. in various trematode species worldwide, biology and natural history have been best studied in N. helminthoeca, the type species of the genus Neorickettsia. In this study, the complete genome sequence of N. helminthoeca was determined and analyzed, providing a valuable resource necessary for understanding the metabolism of N. helminthoeca and its digenean host associations, the evolution and phylogeny among Neorickettsia spp., potential virulence factors of N. helminthoeca, pathogenic mechanisms of SPD, and environmental spreading of N. helminthoeca and trematodes infection in nature. Comparative genomics data of three Neorickettsia spp. of known biological significance is expected to help elucidating biology of other Neorickettsia spp. in the environment.
As SPD progression is rapid, and the case fatality rate is quite high, prevention and early diagnosis of SPD are critical. The serological assay based on defined outer membrane protein antigens is simple, consistent, specific, objective, and convenient, thus helps generating epidemiological information on N. helminthoeca exposure among various wild and domestic animals to raise awareness of SPD. Similar to bats that are the definitive hosts of Acanthatrium oregonense trematodes, the vector of N. risticii transmission (Gibson et al., 2005; Gibson and Rikihisa, 2008), the definitive hosts of N. helminthoeca-infected trematodes in nature are likely asymptomatic, but have antibodies against N. helminthoeca.
Furthermore, these recombinant proteins are used herein in a simple and rapid serodiagnostic test for SPD in dogs. The limitation of the assay is, as in any other serologic assays, false negative results at early stages of infection and in immunosuppressed dogs. Clinical diagnosis is used to determine sensitivity and specificity of the test using a larger number of well-defined canine specimens from broader geographic regions. For this and understanding the pathogenesis and canine immune responses in SPD, culture isolation of additional N. helminthoeca strains is desirable. Characterization of the antigenic surface proteins of N. helminthoeca provides valuable information for the development of rapid, sensitive, and specific serodiagnostic approaches or preventive vaccines for SPD as disclosed herein.
Organisms Culture, Bacteria Purification, and DNA Preparation.
N. helminthoeca Oregon strain, which was previously isolated from dog NH1 fed with fluke N. salmincola-infested salmon kidneys (Rikihisa et al, 1991), was cultured in DH82 cells from the frozen cell stock in Dulbecco's minimal essential medium supplemented with 10% fetal bovine serum and 2 mM
Sequencing and Annotation.
Indexed Illumina mate pair libraries were prepared following the mate pair library v2 sample preparation guide (Illumina, San Diego, Calif.), with two modifications. First, the shearing was performed with the Covaris E210 (Covaris, Wobad, Mass.). The DNA was purified between enzymatic reactions and the size selection of the library was performed with AMPure XT beads (Beckman Coulter Genomics, Danvers, Mass.).
Illumina non-Truseq paired end genomic DNA libraries were constructed using the KAPA library preparation kit (Kapa Biosystems, Woburn, Mass.). DNA was fragmented with the Covaris E210. Then libraries were prepared using a modified version of manufacturer's protocol. The DNA was purified between enzymatic reactions and the size selection of the library was performed with AMPure XT beads (Beckman Coulter Genomics, Danvers, Mass.). For indexed samples the PCR amplification step was performed with primers containing a six nucleotide index sequence.
Concentration and fragment size of libraries were determined using the DNA High Sensitivity Assay on the LabChip GX (Perkin Elmer, Waltham, Mass.) and qPCR using the KAPA Library Quantification Kit (Complete, Universal) (Kapa Biosystems, Woburn, Mass.). The mate pair library was sequenced on an Illumina HiSeq 2500 (Illumina, San Diego, Calif.) while the paired end library was sequenced on an illumina MiSeq (Illumina, San Diego, Calif.).
DNA samples for PacBio sequencing were sheared to 8 khp using the Covaris gTube (Woburn, Mass.). Sequencing libraries were constructed and prepared for sequencing using the DNA Template Prep Kit 2.0 (3 kbp-10 khp) and the DNA/Polymerase Binding Kit 2.0 (Pacific Biosciences. Menlo Park, Calif.). Libraries were loaded onto v2 SMRT Cells, and sequenced with the DNA Sequencing Kit 2.0 (Pacific Biosciences).
Five assemblies were generated with various combinations of the data and assembly algorithms: (1) Celera Assembler v7.0 of only PacBio data, (2) Celera Assembler v7.0 of to PacBio data with correction using Illumina paired end data, (3) HGAP assembly of only PacBio data, (4) MaSuRCA 1.9.2 assembly of Illumina paired end data subsampled to 50× coverage, and (5) MaSuRCA 1.9.2 assembly of Illumina paired end data subsampled to 80× coverage. The first assembly was the optimal assembly, namely the one generated with Cetera Assembler v7.0 with only the PacBio data. The data set was subsampled to ˜22× coverage of the longest reads using an 8 kbp minimum read length cutoff, with the remainder of the reads used for the error correction step. The resulting single-contig assembly totaled ˜89.4 Kbp with 41.68% GC-content. The genome was trimmed to remove overlapping sequences, oriented, circularized, and rotated to the predicted origin of replication. Annotation for this finalized genome assembly was generated using the IGS prokaryotic annotation pipeline (Galens et al., 2011) and deposited in GenBank (accession number NZ_CP007481.1).
Bioinformatic Analysis.
The 16S rRNA, NSP, P51, and SSA proteins were aligned with their Neorickettsia orthologs using CLUSTALW (Thompson et al., 1994) as implemented in BioEdit 7.2.5 (Hall, 1999) resulting in 1522 nt, 326 aa, 516 aa, and 578 aa alignments, respectively. A phylogenetic tree was inferred from the 16S rRNA alignment using RAxML v.7.3.0 (Stamatakis et al., 2005) with the GTRGAMMA model, specifically “RAxMLHPC -f a -m GTRGAMMA -p12345-x12345-N autoMRE -n T20”. The DIRE-based bootstopping criterion was not met, resulting in the use of 1000 bootstraps. For the protein alignments, the best-fit model of amino acid substitution was determined for each alignment separately with ProtTest3.2 (Darriba et al., 2011), with all 15 models of protein evolution tested in addition to the +G parameter. WAG+G was determined to be the best model for NSP and SSA while JTT was determined to be the best model for P51. Phylogenetic trees were inferred from the NSP and SSA alignments using RAxML v.7.3.0 (Stamatakis et al., 2005) with the best model, specifically “RAxMLHPC -f a -m PROTGAMMAWAG -p12345 -x 12345 -N autoMRE -n T20”. The MRE-based bootstopping criterion was met at 350 replicates for NSP and SSA. Phylogenetic trees were inferred from the P51 alignment using RAxML v.7.3.0 (Stamatakis et al., 2005) with the best model, specifically “RAxMLHPC -f a -m PROTCATJTT -p12345 -x12345 -N autoMRE -n T20”. The MRE-based bootstopping criterion was met at 50 replicates for P51. All trees and bootstrap values were visualized in Dendroscope v3.5.7.
The GC-skew was calculated as (C−G)/(C+G) in windows of 500 bp with step size of 250 bp along the chromosome. Synteny plots between Neorickettsia spp. were generated using MUMmer 3 program with default parameters (Delcher et al., 2002). Protein ortholog clusters among Neorickettsia spp., and N. helminthoeca-specific genes compared to other related organisms were determined by using reciprocal B
Metabolic pathways and transporters were compared across genomes using (1) the ortholog clusters generated with reciprocal B
Cloning, Expression, and Western Blot Analysis of Putative N. helminthoeca Outer Membrane Proteins.
Full-length p51, nsp1/2/3, and ssa genes without the signal peptide sequence were PCR amplified from N. helminthoeca genomic DNA, using specific primers (Table 15) and cloned into the pET-33b(+) vector (Novagen, Billerica). The plasmids were amplified by transformation into Escherichia coli PX5α cells (Protein Express, Inc. Cincinnati, Ohio), and the inserts were confirmed by sequencing. The plasmids were transformed into E. coli BL21(DE3) (Protein Express), and the expression of recombinant proteins was induced with 1 mM isopropyl β-d-thiogalactopyranoside. E. coli was sonicated for a total of 5 min (15 s pulse with 45 s interval) on ice, and the pellet containing recombinant protein was washed with 1% Triton X-100 in sodium phosphate buffer (SPB: 50 mM sodium phosphate, pH 8.0, 0.3 M NaCl). Recombinant proteins were denatured and solubilized with 6 M urea in SPB (for P51, SSA, and NSP2/3), or 6M Guanidine HCl in SPB (for NSP1) at 4° C. for 1 hr. Proteins were purified on a HisPur Cobalt Affinity resin (Pierce, Rockford, Ill.) and dialyzed using Buffer A (50 mM KCl, 100 mM NaCl, 50 mM Tris-HCl, pH 8.0) containing decreasing concentrations of urea (3 M, 1 M, then 0 M). Protein concentrations were determined by BCA assay (Pierce).
Bacterial lysates of purified N. risticii or N. helminthoeca, and recombinant NSP1/2/3, SSA, and P51 were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis as described previously (Lin et al., 2002). Gels were stained using GelCode Blue (Pierce), and the immuno-reactivities of these recombinant proteins were determined by western blot analysis using SPD dog sera against N. helminthoeca or horse anti-N. risticii serum as a negative control at 1:400 dilutions. Defined SPD dog sera against N. helminthoeca were obtained from dogs orally fed by fluke N. salmincola-infested salmon kidneys infected with N. helminthoeca, and sera collected at day 13 and 15 post exposure with IFA titers at 1:640 (NH1) and 1:1,280 (NH3), respectively (Rikihisa. et al., 1991). Clinical dog sera tested positive for N. helminthoeca-infection were received from southern California (“M” sera—IFA titer 1:80, from Dana Point, Calif. In 2012; “D” sera—PCR-positive for N. helminthoeca 16S rRNA gene, from Aliso Viejo, Calif. In 2010). Horse anti-N. risticii serum (Pony 19) was collected from a pony inoculated intravenously with N. risticii-infected U-937 cells (IFA titer 1:640) (Rikihisa et al., 1988). Reacting bands were detected with Horseradish peroxidase (HRP)-conjugated goat anti-dog (KPL Gaithersburg, Md.) or anti-horse (Jackson Immuno Research, West Grove, Pa.) secondary antibodies, and visualized with enhanced chemiluminescence (ECL) by incubating the membranes with LumiGLO™ chemiluminescent reagent (Pierce). Images were captured using an LAS3000 image documentation system (FUJIFILM Medical Systems USA, Stamford, Conn.).
GenBank Accession Numbers and Abbreviations of Bacteria.
N. helminthoeca Oregon (NHO), NZ_CP007481.1 (this example); N. risticii Illinois (NRI), NC_013009.1; N. sennetsu Miyayama (NSE), NC_007798.1; A. phagocytophilum HZ (APH), NC_007797.1; A. marginale Florida (AMA), NC_012026.1; E. chaffeensis Arkansas (ECH), NC_007799.1; E. canis Jake (ECA), NC_007354.1; E. ruminantium Welgevonden (ERU), NC_005295.2; E. muris AS145 (EMU), NC_023063.1; Ehrlichia sp. HF (EHF), NZ_CP007474.1; Wolbachia pipientis (wMel, Wolbachia endosymbiont of Drosophila melanoga), NC_002978.6; Wolbachia endosymbiont of Brugia malayi (wBm), NC_006833.1; Neorickettsia endobacterium of Fasciola hepatica (NFh), NZ _LNGI00000000, Candidatus Xenolissoclinum pacificiensis L6, AXCJ00000000.
Lin, M., Zhu, M. X., and Rikihisa, Y. (2002) Rapid activation of protein tyrosine kinase and phospholipase C-gamma2 and increase in cytosolic free calcium are required by Ehrlichia chaffeensis for internalization and growth in THP-1 cells. Infect Human 70: 889-898.
Lin, M., den Dulk-Ras, A., Hooykaas, P. J., and Rikihisa, Y. (2007) Anaplasma phagocytophilum AnkA secreted by type IV secretion system is tyrosine phosphorylated by Abl-1 to facilitate infection. Cell Microbiol 9: 2644-2657.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
N. helminthoeca
Nanophyetus
salmincola in
silicula) and
N. risticii
Acanthatrium
oregonense in
virginica) and
N. sennetsu
Sennetsu
1Transmission mode: all Neorickettsia spp. are transstadially and vertically transmitted through generations of trematodes.
1Abbreviations: NHO, N. helminthoeca Oregon (data obtained from in this study); NRI, N. risticii Illinois (Lin et al., 2009); NSE, N. sennetsu Miyayama (Dunning Hotopp et al., 2006).
2Percent coding includes tRNA, rRNA, small RNA, and all protein-coding genes.
1Abbreviations: NHO, N. helminthoeca Oregon; NRI, N. risticii Illinois; NSE, N. sennetsu Miyayama.
2Proteins conserved among three Neorickettsia spp. and specific to N. helminthoeca are based on 3-way comparison analysis by BlastP (E < e−10).
3
N. helminthoeca encodes nearly complete pathways for peptidoglycan biosynthesis.
4Certain proteins are assigned to multiple role categories.
1Location of outer member proteins is predicted by the pSort-B algorithm (http://psort.org/psortb). Other putative OMPs (P51, NSP1/2/3, and SSA) are determined by homology searches to N. risticii and N. sennetsu protein database using BLASTP.
1Ortholog clusters were constructed using reciprocal BLASTP algorithm with E-value <1e−10, and grouped by functional role categories. The protein name and role category of the ortholog cluster are based on those from N. helminthoeca genome.
N. helminthoeca-specific proteins compared to N. sennetsu and N. risticii1
Bacillus muralis
Wolbachia sp. of
Drosophila simulans
Campylobacter
ureolyticus (ε-
Paracoccus
tibetensis (α-
Magnetospirillum
marisnigri (α-
Ca. Neoehrlichia
lotoris (α-
Wolbachia sp. of
Cimex lectularius
Wolbachia sp. of
Cimex lectularius
Thermodesulfovibrio
Ca. Pelagibacter sp.
Bacillus bataviensis
Crenothrix
polyspora (γ-
Robiginitomaculum
antarcticum (α-
Caedibacter
varicaedens (α-
Anaplasma
marginale (α-
Prochlorococcus
marinus
Ralstonia
solanacearum (β-
Wolbachia pipientis
Caedibacter
varicaedens (α-
Vibrio halioticoli (γ-
Desulfovibrio
vulgaris (δ-
Ca. Neoehrlichia
lotoris (α-
1
N. helminthoeca-specific proteins were identified by comparison with N. sennetsu and N. risticii protein databases using BLASTP algorithm with E-value <1e−10.
2
N. helminthoeca-specific proteins were blasted against NCBI protein database NR excluding Neorickettsia spp. with E-value <1e−10. The species, class, and E-value of the top matches to the N. helminthoeca proteins were listed. Blank fields, no matches were identified based on the search criteria.
N. risticii-specific proteins compared to N. helminthoeca and N. sennetsu 1
1
N. risticii-specific proteins were identified by comparison with N. helminthoeca and N. sennetsu protein databases using Blastp algorithm with E-value <1e−10.
N. sennetsu-specific proteins compared to N. helminthoeca and N. risticii 1
1
N. sennetsu-specific proteins were identified by comparison with N. helminthoeca and N. risticii protein databases using BLASTP algorithm with E-value <1e−10.
+ 3
− 5
1 Abbreviations: ECH, Ehrlichia chaffeensis Arkansas; APH, Anaplasma phagocytophilum HZ; NSE, N. sennetsu Miyayama; NRI, N. risticii Illinois; NHO. N. helminthoeca Oregon.
2 Biosynthesis for these AAs in N. helminthoeca are converted from other AAs or metabolic intermediates.
3 Only partial enzymes are identified in Arginine biosynthesis pathway in APH.
4 Ech and APH can convert Pro to Glu through PutA (bifunctional proline dehydrogenase/pyrroline-5-carboxylate dehydrogenase). All Anaplasmataceae can convert Gln to Glu by CarA/B (carbamoyl phosphate synthase) or GS/PH (bifunctional glutamate synthase subunit beta/2-polyprenylphenol hydroxylase).
5
N. helminthoeca encodes complete pathways to synthesize meso- 2,6-diaminopimelate (mDAP) from L-Asp, but lacks diaminopimelate decarboxylase (LysA) at the last step to produce lysine.
6 ECH and APH can synthesize CoA from pantothenate, however, all Neorickettsia spp. can only convert 4′-phosphopantetheine to CoA.
1 Numbers inside parentheses indicate the copy numbers of the genes; otehrwise, only a single copy is present. Abbreviations: NHO, N. helminthoeca Oregon; NRI, N. risticii Illinois; NSE, N. sennetsu Miyayama.
2 All Neorickettsia spp. encodes two copies of sensor histidine kindase PleC.
1 Abbreviations: ECH, Ehrlichia chaffeensis Arkansas; APH, Anaplasma phagocytophilum HZ; NSE, N. sennetsu Miyayama; NRI, N. risticii Illinois; NHO, N. helminthoeca Oregon.
2 Proteins are truncations due to an internal mutation.
This application claims the benefit of U.S. Provisional Patent Application Ser. No. 62/316,254 filed Mar. 31, 2016, the disclosure of which is expressly incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 62316254 | Mar 2016 | US |
