Patent Application
20100129408
1. Field of the Invention
The present invention relates to a Klebsiella pneumoniae tonB deletion mutant and its producing methods. More particularly, the present tonB deletion mutant is used as an immunogenically effective amount of a live attenuated vaccine against the community-acquired pyogenic liver abscess.
2. Background of the Invention
Klebsiella pneumoniae belongs to the family of Enterobacteriaceae; which is Gram-negative, flagella-less and motionless bacterium with polysaccharide capsules. Strains of Klebsiella pneumoniae exist in the respiratory tract, digestive tract or urinary tract of healthy people. It's an opportunistic infectious pathogen. Infection results in pneumonia, urinary tract infections, or infections after surgical operation or intubation and the like. Klebsiella pneumoniae has become the predominant pathogen causing community-acquired pyogenic liver abscess (PLA) according to previous studies since 1986, which is an emerging endemic infectious disease particularly common in Asia and South Africa areas. Among the PLA patients, 10-13% combined with meningitis or endophthalmitis, and 40% to 78% of the patients had prior diabetes mellitus.
The cases of community-acquired PLA caused by Klebsiella pneumoniae have increased year by year from recent epidemiologic studies in Korea, Taiwan and USA. Characterization studies using Ribotyping and Pulsed-Field gel electrophoresis have excluded the homology of these Klebsiella pneumoniae strains. It's shown that PLA was not caused by one or a few specified epidemic strain but predominate a capsular subtype of K1. And magA (mucoviscosityassociated gene) is one of the genetic determinants of capsular serotype K1.
The known virulence factors of PLA-causing Klebsiella pneumoniae include not only K1 capsular associated antigens, magA, but also Allantoin metabolic gene, iron uptake gene kfu, and a regulatory gene for capsular exopolysaccharide synthesis, rmpA gene (regulator of the mucoid phenotype A). The general virulence factors include: (1) capsule, which prevents phagocytic destruction and complement serum lysis; and K1, K2 are the most virulent serotypes of the 77 recognized serotype strains; (2) lipopolysaccharide (endotoxin), elicits an immune reaction which is responsible for many of the harmful effects seen in septic shock, or provide serum resistance to avoid complement-mediated lysis; (3) adhesins, 5 types such as type 1, type 3, Klebsiella pneumoniae F-28 fimbriae, CF29K, and aggregative adhesion were identified, that are major factors in urinary tract and, respiratory tract infection since they facilitate the attachment of bacterium to the surface of host mucous; (4) siderophores, an iron chelating compound secreted by pathogenic bacteria to compete for iron with host iron-binding proteins since the concentration of free iron ions are very low in human bodies. Klebsiella pneumoniae acquires iron ion through synthesizing and secreting siderophores or hemophores to capture iron or heme, and using specialized outer membrane receptor to transport the irons. In addition, all siderophores, hemophores and outer membrane receptors shared the same characteristics: low similarity in N-terminal sequences, requiring a TonB complex which consists of 3 cytoplasmic membrane proteins: TonB, ExbB and ExbD. This TonB complex transduces energy to facilitate the iron internalization. The active transportation is coupled through gradients of concentration.
Klebsiella pneumoniae Studies toward community-acquired PLA or non-community PLA using transposon mutagenesis, full genome expression analysis and comparative genomic hybridization successfully identified virulence genes magA, allS and kfu. Further analysis by polymerase chain reaction (PCR) showed that these genes were mainly found in clinical isolates from patients of community-acquired PLA but not from non-community PLA. The induction of pathogenic genes of Klebsiella pneumoniae may be induced by environmental pressure during infection.
Recently, capsular serotype K1 was found to be the major virulence strain for Klebsiella pneumoniae causing PLA. And magA is one of the major components in capsular genetic determinants. Mutations in magA lost the exopolysaccharides and became avirulent. Therefore, the magA deletion mutant is not considered to be an ideal vaccine strain because of the lack of exopolysaccharides and immunogenicity.
Klebsiella pneumoniae vaccines using purified capsules of Klebsiella pneumoniae and other non-capsular compositions such as lipopolysaccharides, type 3 pilus, or even capsular gene deletion mutants were developed but with little success. Generally patients with PLA have been treated with catheter drainage and antibiotic therapy, but still showed a mortality of 10-40%. There is room for improvement since inactivated dead bacteria used in vaccine preparation limited the efficiency of antibody induction.
The present invention provides a novel Klebsiella pneumoniae tonB deletion mutant and a producing method based on the research of previous studies to improve the efficiency of antibody induction.
The objective of the present invention is to provide a Klebsiella pneumoniae tonB deletion mutant used as an immunogenically effective amount of a live attenuated vaccine. The tonB deletion mutant was stored in Bioresource Collection and Research Center (Food Industry Research and Development Institute, Hsinchu, Taiwan) with an accession number of BCRC 910405. The live attenuated vaccine used for immunization against Klebsiella pneumoniae caused PLA has no pathogenicity to BALB/c mouse after intraperitoneal injection.
Another object of the present invention is to provide a method for producing the Klebsiella pneumoniae tonB deletion mutant. The tonB deletion mutant contains exopolysaccharides K1 and serum resistance, and grows in a condition containing iron ions. And the live attenuated vaccine further comprises a pharmaceutical carrier, diluent, or excipient.
The abovementioned method for producing a Klebsiella pneumoniae tonB deletion mutant comprises the steps of: (a) constructing a plasmid containing a tonB gene of Klebsiella pneumoniae and flanking regions of the tonB gene using a first primer set and a second primer set; (b) digesting the plasmid with a restriction enzyme to form a restriction fragment containing the flanking regions of tonB gene, and ligating the restriction fragment with a vector containing a temperature-sensitive region, an antibiotic selection marker and a negative selection marker; (c) transforming the vector into a Klebsiella pneumoniae strain; and (d) screening the tonB deletion mutant by an antibiotic and a negative selection drug, and obtaining the tonB deletion mutant containing the directly ligated flanking regions without tonB gene, and no selection markers.
The first primer set of step (a) is SEQ ID NO: 43 and SEQ ID NO: 44, and the second primer set is SEQ ID NO: 45 and SEQ ID NO: 46, the plasmid is a TA plasmid. The restriction enzyme of step (b) is NotI, and the vector is a pKO3 plasmid containing a temperature-sensitive origin, an antibiotic selection marker and a negative selection marker. The screened tonB deletion mutant was proved to be not pathogenicity to BALB/c mouse after activity assay but could be used for immunization against PLA—causing Klebsiella pneumoniae to induce anti-EPS IgG.
The present invention is further explained in the following embodiment illustration and examples. Those examples below should not, however, be considered to limit the scope of the invention, it is contemplated that modifications will readily occur to those skilled in the art, which modifications will be within the spirit of the invention and the scope of the appended claims.
a. The genes of Yersinia HPI and iucABCDiutA distributed in Klebsiella pneumoniae genome.
b. The genes of iroA and hmuRSTUV distributed in Klebsiella pneumoniae genome.
a. Construction of the deletion mutants of irp2 and
iuc.
b. Construction of the deletion mutants of iroA and
kfu.
The present invention used polysaccharide-protein conjugate vaccine which is the most effective vaccine.
The known virulence factors of PLA-causing Klebsiella pneumoniae include capsular associated antigens, metabolic gene, iron uptake gene, and a regulatory gene for capsular exopolysaccharide synthesis. Among them, the gene involved in iron transport system, such as tonB, is important since iron can be used in anti-oxidation of the hosts. Therefore, several Klebsiella pneumoniae deletion mutants are constructed in the present invention to against PLA causing Klebsiella pneumoniae. The non-pathogenic infection tonB deletion mutant was screened as the major active ingredient of vaccine candidates because it can induce the immunity to the same pathogens.
Single dose of tonB deletion mutant was found to be enough for the BALB/c mice to generate immunity from the screening studies on deletion mutants of PLA causing Kiebsiella pneumonia in the present invention. This showed that the tonB deletion mutant is able to stimulate BALB/c mice to generate protective memory immune responses. The tonB deletion mutant would not be cleaned immediately by the immune responses of the hosts since it contains complete K1 capsular exopolysaccharides, serum resistance and also induces anti-EPS IgG production in comparison to magA mutants.
The original Klebsiella pneumoniae strain of the tonB mutant was screened and isolated from the blood of the PLA patient in National Taiwan University Hospital (NTUH) with an accession number of NTUH-K2044. The whole genome size is 5.2×106 bases, including about 5093 open reading frames (ORFs), a GC ratio of 57.68% and a plasmid pK2044 (large plasmid) of 2.24×105 bases, 299 ORFs, a GC ratio of 50.17%.
All the containers for NTUH-K2044 RNA extraction were washed by a solution of 0.1% DEPC to prevent the degradation of RNase in the air.
Overnight culture of NTUH-K2044 Klebsiella pneumoniae were cultured in 1 ml of LB media and 1 ml of diluted healthy human blood at 4% to 8%, or in 2 ml of LB for the control group. The cells were grown at 37° C. for 3-4 h to log phase, then centrifuged at 14000 g, 4° C. for 2 h. The supernatant was removed and the cell pellet was washed twice with 0.1% DEPC ddH2O. The cells were centrifuged again for 2 h then the supernatant was removed. Each ml of the cell pellet solution was resuspended into 0.25 ml of lysis buffer and then boiled at 100° C. for 5 min till solution was clear. The RNA of the strain was isolated with RNeasy Mini Kit (Qiagen, cat. No. 74106) and residual DNA was removed with RNase-Free DNase Set (cat. No. 79254) after cool down. RNA concentration and purity was determined using agarose electrophoresis and spectrophotometrically at OD260/280.
Microarray hybridization was used to analyze the expression level of extracted RNA from NTUH-K2044. To 5 μl of the RNA, 7.5 μl of random hexamer primer and 14.75 μl of DEPC-dH2O were added and heated at 70° C. for 3 min, followed by addition of 12.5 μl of 5× First RT buffer, 0.1 M DTT, and 2.5 μl each of dATP, dCTP, dGTP, and dTTP (in the concentration of 25 μl), 6 μl of biotin-16-dUTP (25 mM), 1.5 μl of RNaseOut™ Ribonuclease and 5 μl of Superscript II reverse transcriptase. The reverse transcription-PCR (RT-PCR) was carried out at 25° C. for 10 min, 42° C. for 90 min and 94° C. for 5 min. The biotin-16-dUTP could then be incorporated into the first strand of cDNA. This biotin-16-dUTP cDNA can be used as labeled probe after incubated with 6.9 μl NaOH (3M) at 50° C. for 30 min to remove the residual RNA and neutralized with 6.9 μl acetic acid (3M) at 50° C. for 30 min. The labeled probe can be precipitated with ethanol.
The microarray filter was incubated with 2 ml of hybridization buffer at 63° C. for 1.5-2 h. One ml of the hybridization buffer containing the labeled probe was added onto the surface of filter and incubated at 63° C. for 16-18 h. The filter was washed with 2×SSC and 0.1% SDS twice at room temperature (evenly shaking) for 5 min, with a final wash of 0.1×SSC and 0.1% SDS three times at 63° C. for 15 min.
Color development was performed after the microarray filter was treated in the following order: incubating with 2 ml of blocking buffer with shaking at room temperature for 1 h, incubating with 2 ml of reaction buffer at room temperature for 1 h, washing with PBST buffer for 4 times at room temperature for 5 min each, reacting with 1.5 ml of NBT/BCIP buffer at room temperature for 20-40 min, and stopping the reaction with water. The color developed in the filter was scanned in a scanner and stored in the computer followed by quantification of the images with NIH ImageJ software. The 23SrRNA was served as the internal control.
Referring to
All the cloned fragments with higher gene expression levels than the control group were selected as candidates of deletion mutants. The method for producing a Klebsiella pneumoniae tonB deletion mutant comprises the steps of: (a) constructing a plasmid containing a tonB gene of Klebsiella pneumoniae and flanking regions of the tonB gene using a first primer set and a second primer set; (b) digesting the plasmid with a restriction enzyme to form a restriction fragment containing the flanking regions of tonB gene, and ligating the restriction fragment with a vector containing a temperature-sensitive region, an antibiotic selection marker and a negative selection marker; (c) transforming the vector from step (b) into a Klebsiella pneumoniae strain; and (d) screening the tonB deletion mutant by an antibiotic and a negative selection drug, and obtaining the tonB deletion mutant containing the directly ligated flanking regions without tonB gene, and no selection markers. The abovementioned primer sets were listed in Table 1:
Primer sets listed in the above table and PCR performed in step (a) were used as reference for gene deletion to detect the distribution of gene fragments such as Yersinia HPI, iucABCDiutA, iroA and hmuRSTUV in community acquired PLA or non-community acquired PLA. The cycling program consisted of one denaturation step of 3 min at 96° C. and 30 cycles of 30 s at 96° C., 15 s at 52° C., and 1 min at 74° C., followed by 10 min at 72° C. Referring to
Referring to irp2 and
iuc,
iroA and
kfu were constructed. Unmarked deletion method is one of the several methods used to construct the deletion mutants in the invention. The targeted deletion genes such as
irp2,
iuc,
iroA and
kfu in dotted lines were the few examples shown in
tonB,
irp2 iuc iroA and
irp2 iuc iroA kfu can further be included.
The tonB deletion mutant was taken as an example. Primers tonB-FR (SEQ ID NO: 43) and tonB-RR (SEQ ID NO: 44) were used to synthesize a complete tonB gene through PCR, including 1 kb of the upstream and downstream flanking regions in step (a). This gene fragment was ligated into a plasmid such as TA. A reverse PCR was performed using the resulting plasmid as a template, plus the primers tonB-Fout (SEQ ID NO: 45) and tonB-Rout (SEQ ID NO: 46, LA-Taq polymerase to yield products. The A-tail of the 3′-end was removed from the products using T4 DNA polymerase (NEB) and phosphate was attached to the 5′-end of the products using polynucleotide kinase (NEB) for self-ligation.
The restriction enzymes used to digest the tonB gene and the flanking regions from the TA cloning vector in Step (b) including but not limited to NotI, the vector containing temperature-sensitive region, an antibiotic selection marker and a negative selection marker including but not limited to pKO3, the antibiotic selection marker including but not limited to kanamycin, the negative selection marker including but not limited to sacB (encoding levansucrase).
Electroporation can be used in step (c) to transform the flanking regions of tonB gene into the NTUH-K2044 strain.
A tonB deletion mutant containing the directly ligated flanking regions without tonB gene was screened by the antibiotic and the negative selection marker in step (d), which contained no selection marker in the genome. The sequence of mutant was confirmed with PCR and sequence analysis. The negative selection marker was sucrose in step (d).
The functional analysis was carried out with trans-complementation, cross-feeding assay, growth study, String test, Serum resistance assays, serotyping, animal study, Enzyme-Linked Immunosorbent Assay (ELISA) and immunoblotting.
The growth curves of the NTUH-K2044 strain and each deletion mutants (irp2, iuc, or iroA single mutants, irp2 iuc iroA triple mutant and irp2 iuc iroA kfu quadruple mutant) were shown in
‡R, resistance.
The ΔtonB deletion mutant was shown to remain hyperviscous, K1 capsular exopolysaccharides, and serum resistance according to Table 2. Animal studies were carried out to test the possibility for mutants as vaccine candidates. Five-week-old female BALB/c mice were obtained from National Laboratory Animal Center. The BALB/c mice were infected intraperitoneally (IP) or intragastrically (IG) with Klebsiella pneumoniae NTIH-K2044 mutants including irp2 iuc iroA triple mutant,
irp2 iuc iroA kfu quadruple mutant,
tonB mutant,
magA mutant (not able to synthesize K1 capsular exopolysaccharide) at the dose of 1×103 CFU to
irp2 iuc iroA triple mutant,
irp2 iuc iroA kfu quadruple mutant, and 1×104 CFU or 1×105 CFU to
tonB mutant, and 1×106 CFU to
magA mutant; saline was used in the control group. After 4 weeks, immunized and non-immunized control BALB/c mice were challenged with 1×103 CFU of NTUH-K2044 (greater than the wild-type LD50 value). The challenged mice were monitored for another 4 weeks. The pathogenicity was analyzed with Kaplan-Meier analysis (SPSS 12th ed.) and a log-rank test; P<0.05 was considered to be statistically significant.
Single mutants including irp2,
iuc,
iroA, and
kfu were shown to have the same pathogenicity with the wild type NTIH-K2044 (LD50<1×102 CFU) after IP infection. However, the mice survived after injection of the dose of 1×103 CFU to
irp2 iuc iroA triple mutant or
irp2 iuc iroA kfu quadruple mutant (LD50 of 1.3×104 CFU for irp2 iuc iroA triple mutant and 5.5×104 CFU for irp2 iuc iroA kfu quadruple mutant). In addition, there was no significant difference for the pathogenicity between the
irp2 iuc iroA triple mutant and the wild type strain after IG feeding (LD50 of 5.6×105 CFU for irp2 iuc iroA triple mutant and 1×105 CFU for the wild-type strain). Referring to
irp2 iuc iroA kfu quadruple mutant was even lower than the wild type strain or the
kfu mutant after IG feeding (LD50 of >1×107 CFU for irp2 iuc iroA kfu quadruple mutant and LD50 of 6.3×106 CFU for
kfu mutant).
tonB mutant was found to have no pathogenicity in mice after 28 days observation and the survival rate was 100%.
Analysis of the genomic sequence of Klebsiella pneumoniae NTUH-K2044 identified ten putative iron transport systems, and 7 of them were TonB dependent, such as Yersinia HPI, iucABCDiutA, and iroA. The tonB deletion mutant was unable to grow in iron-deficient media, whereas complementation of the tonB deletion mutants restored growth in iron-restricted media. The tonB deletion mutant was therefore confirmed to be defective in iron uptake. In comparison with the parental NTUH-K2044 strain, the tonB deletion mutant formed smaller colonies on either blood agar or LB media. IP inoculation of BALB/c mice with the tonB deletion mutant resulted in a less efficient spread in the body than IP injection of wild type strain at the same dose. A string test was examined in these deletion mutants and revealed that the tonB deletion mutant remained hyperviscous. There was no significant difference in CPS antigen serotyping using double immunodiffusion and serum sensitivity assays between the tonB mutant and the wild type NTUH-K2044 strain. Therefore, the immunogenicity was regarded the same for both strains.
Referring to irp2 iuc iroA,
irp2 iuc iroA kfu, and
tonB were compared with non-immunized BALB/c mice. These attenuated strains of each mutant were injected into BALB/c mice respectively to get immunity, followed by challenging with the lethal dose of NTUH-K2044. Four weeks later, all tonB deletion mutant-immunized BALB/c mice survived without any symptoms of disease, whereas 75% of the non-immunized control BALD/c mice or magA mutant-immunized BALB/c mice died within 5 days of infection. The survival rates of
irp2 iuc iroA and
irp2 iuc iroA kfu-mutant immunized BALB/c mice were lower than that of tonB mutant-immunized BALB/c mice. ELISA analysis showed that BALB/c mice immunized with the tonB mutant had EPS serum immunoglobulin G (anti-EPS IgG) responses against NTUH-K2044 strain, while control mice or magA mutant-immunized mice did not.
From the description and results of the abovementioned animal studies, the tonB deletion mutant was shown to be a potential live vaccine since the strain could survive in human blood and has the ability to synthesize K1 capsular exopolysaccharides. Therefore the tonB deletion strain can be prepared as live vaccine in large scale, resulting in induction of anti-EPS antibody in human and immediate protect against the Klebsiella pneumoniae—causing community acquired PLA. The tonB deletion mutant was stored in Bioresource Collection and Research Center (Food industry Research and Development Institute, Hsinchu, Taiwan) on Oct. 30, 2008 with an accession number of BCRC910405.
| Number | Date | Country | Kind |
|---|---|---|---|
| 097145798 | Nov 2008 | TW | national |
