Genetic Basis of Vi Antigen Expression in Salmonella paratyphi C

Analysis of hybrids formed in a cross between a Salmonella paratyphi C Hfr and an S. typhimurium recipient indicated that the structural genetic determinants of the S. paratyphi C Vi antigen are located closely adjacent to the mel determinant, between this marker and purA. A similar location was indicated for the structural genetic determinants of the S. typhi Vi antigen (the viaB locus) by the results of a mating in which a hybrid S. typhimurium Hfr bearing the S. typhi viaB determinants was used to transfer these genes to an S. typhimurium recipient. Mating experiments with a Vi-antigen-expressing S. typhi Hfr and an S. typhimurium hybrid recipient expressing the Vi antigen of S. paratyphi C yielded no recombinants in which loss of Vi antigen expression occurred, indicating that the chromosomal locus occupied by the genetic determinants of the S. paratyphi C Vi antigen is the same one at which, in S. typhi, the viaB genes reside. Introduction of a mutant S. typhi viaA gene into an S. typhimurium hybrid expressing the Vi antigen, as the consequence of prior receipt of the S. paratyphi C viaB determinants, resulted in that hybrid's loss of Vi antigen expression, demonstrating that the viaA determinant plays a role in Vi antigen expression in S. paratyphi C, as well as in S. typhi. Although the percentages of coinheritance of the viaB and mel determinants in the mating experiments suggested that their linkage is sufficiently close to allow cotransduction by P22, attempts to accomplish this with lysates prepared on S. typhimurium hybrids expressing either S. typhi or S. paratyphi C viaB determinants were not successful.     

Genetic regulation of variable Vi antigen expression in a strain of Citrobacter freundii.

Certain strains of the genus Citrobacter exhibit a variable expression of the Vi surface antigen that appears to involve a special mechanism for regulation of gene expression. Two nonlinked chromosomal loci, viaA and viaB, are known to determine nonvariable Vi antigen expression in strains of Salmonella. To confirm the presence of analogous loci in Citrobacter and to ascertain whether either of them is involved in variable Vi antigen expression in this organism, donor strains were constructed from Citrobacter freundii WR7004 and used to transfer their Vi antigen-determining genes to ViaA- and ViaB- Salmonella typhi recipient strains. Vi antigen expression in C. freundii was found to be controlled by loci analogous to the Salmonella via genes. S. typhi recipients of the C. freundii viaA+ genes were restored to the full, continuous expression of the Vi antigen normally seen in S. typhi. Thus, the C. freundii viaA genes appeared to play no role in the variable expression of the Vi antigen. In contrast, S. typhi recipients of the C. freundii viaB+ genes exhibited the rapid, reversible alternation between full Vi antigen expression and markedly reduced Vi antigen expression that was seen to occur in the C. freundii parent. The C. freundii viaB locus was thus identified as the one whose genes are regulated so as to produce variable Vi antigen expression. Genes determining another C. freundii surface antigen, the synthesis of which is not affected by the mechanism regulating Vi expression, were coinherited with the C. freundii viaB+ genes. An invertible, insertion sequence element located within the C. freundii viaB locus is proposed to account for the regulation of variable Vi antigen expression.     

Expression of Vi antigen in Escherichia coli K-12: characterization of ViaB from Citrobacter freundii and identity of ViaA with RcsB.

The Vi antigen in Salmonella typhi is stably expressed and may act to protect the strain against the defensive system of the host. Citrobacter freundii, not usually a common human pathogen, also expresses the Vi antigen but expresses it unstably, exhibiting a reversible transition between the Vi+ and Vi- states. Two widely separated chromosomal regions, ViaA and ViaB, are needed for Vi synthesis. Escherichia coli K-12 harboring a functional ViaB plasmid can also express Vi antigen, but the cloned ViaB sequence can only be stably maintained and expressed in recA hosts. Vi- derivatives arise either through IS1-like insertional events occurring in ViaB sequences or by chromosomal mutations at the ViaA region. P1vir mapping indicates that the ViaA mutations are located at min 47.75 on the E. coli chromosome. All the spontaneous viaA mutants isolated from E. coli and S. typhi were identified as rcsB mutants by complementation tests using plasmid pJB100. Introduction of rcsA::Tn10 into E. coli harboring functional ViaB sequences eliminates the expression of Vi antigen. These results indicate that Vi antigen synthesis is regulated by the same regulatory proteins involved in colanic acid synthesis in E. coli.     

肠毒素源性定居因子CS6在减素伤寒沙门氏菌中表达及免疫原性的研究

将编码肠毒素源性大肠杆菌定居因子抗原ＣＳ６基因克隆到ｐＸＬ６７０,转化ａｓｄ基因突变的Ｅ．ｃｏｌｉ Ｘ６０９７,获得重组质粒ｐＳＳ６４,再将后者转化至减毒的△ａｒｏＡ、△ａｒｏＣ、△ａｓｄ伤寒沙门氏菌,构建了无药物抗性且稳定的大肠杆菌和伤寒双价菌苗候选株。小鼠腹腔免疫和攻击实验表明,该菌株对伤寒沙门氏菌毒株的攻击具有良好的保护作用。家兔免疫实验证明,该菌株能产生抗ＣＳ６和伤寒菌Ｖｉ抗原的血清抗体。     

Genomic cleavage map of Salmonella typhi Ty2.

The genomic cleavage map of Salmonella typhi Ty2, 4,780 kb in size, was determined through digestion of the genomic DNA with endonucleases and separation of the fragments by pulsed-field gel electrophoresis. The chromosome has 33, 26, 7, and 35 sites for the enzymes XbaI, BlnI, I-CeuI, and SpeI, respectively. The fragments were arranged around the chromosome through excision of fragments from the gel, redigestion with a second enzyme, and labelling with 32P, and reelectrophoresis and named in alphabetical order. Tn10 transposons inserted in 82 different genes of Salmonella typhimurium were transduced by phage P22 into S. typhi, and the location of Tn10, and thus of the gene, was mapped through the XbaI and BlnI sites of Tn10. All seven I-CeuI sites (in rrl genes for 23S rRNA) were conserved, and the gene order within the I-CeuI fragments resembles that of S. typhimurium LT2, but the order of I-CeuI fragments is rearranged from ABCDEFG in S. typhimurium LT2 to AGCEFDB in S. typhi. In addition, there is a 500-kb inversion which covers the terminus region. Comparisons of lengths of segments between genes showed that S. typhi has segments which differ in size from those in S. typhimurium. The viaB locus, for synthesis of the Vi antigen of S. typhi, was shown to be within a 118-kb loop (a segment of DNA with no homolog in most other Salmonella species) between mel and poxA on the chromosome.     

Vi-Suppressed wild strain Salmonella typhi cultured in high osmolarity is hyperinvasive toward epithelial cells and destructive of Peyer's patches

Salmonella typhi GIFU10007-3 which lost a viaB locus on its chromosome became highly invasive in our previous study. To investigate the phenomenon, we controlled Vi expression in wild strain S. typhi GIFU10007, and studied the invasive phenotype both in vitro and in vivo. When the wild strain of S. typhi was cultured in 300 mM NaCl containing Luria-Bertani broth (LBH), the expression of Vi antigen was suppressed, but secretion of invasion proteins (SipC, SipB and SipA) was increased. In this condition, wild strain S. typhi became highly invasive toward both epithelial cells and M cells of rat Peyer's patches. When GIFU10007 was cultured under conditions of high osmolarity, the bacteria disrupted Peyer's patches and induced massive bleeding in these structures only 20 min after inoculation into the ileal loop. In contrast, Vi-encapsulated wild strain GIFU10007 cultured under low osmolarity was not destructive, even after 60 min. To understand the role of the type III secretion system under conditions of high osmolarity, we knocked out the invA and sipC genes of both GIFU10007 and GIFU10007-3. Neither invA nor sipC mutants could invade epithelial cells or M cells in a high osmolarity environment. Our data show that the highly invasive phenotype was only expressed when the wild strain S. typhi was cultured under high osmolarity, which induced a state of Vi suppression, and in the presence of the type III secretion system.     

Polysaccharides and their common proteinic carrier in the Vi antigens of Citrobacter ballerup and Salmonella typhi Ty2

Immunochemical analysis of Citrobacter ballerup and Salmonella typhi Ty2 showed that the strains share native and heat-resistant proteins that are, apparently, the carriers of a common polysaccharidic determinant present in their respective somatic antigens. After the classic acetic acid hydrolysis, the somatic antigen of C. ballerup reacted, in agar gel, against the homologous antiserum by two precipitation lines, one of which also precipitated against the anti S, typhi Ty2 serum; the hydrolysis of the S. typhi Ty2 somatic antigen demonstrated that, in addition to the 'O' polysaccharide, reacting against all the S. typhi antisera, it contains a polysaccharide that precipitated against the anti-C. ballerup serum. The elusiveness in the agglutinability of only freshly isolated bacterial authorizes some doubt concerning the responsibility of the antipolysaccharide antibodies in the agglutinating Vi sera; in order to induce anitpolysaccharides hyperimmunizations are needed while antiproteins are easily induced by short immunizations.     

Molecular cloning and expression in E. coli of a Salmonella typhi porin gene

Immunoscreening of a Salmonella typhi cosmid library in E. coli allowed the detection of clones producing a 36 kDa porin from S. typhi. The gene is efficiently expressed in an E. coli porin-less mutant and the protein is exported to the outer membrane envelope. Two clones which markedly differ in their level of expression have been isolated.     

Molecular cloning of the ViaB region of Salmonella typhi

The ViaB region required for Vi antigen production in Salmonella typhi was cloned. The plasmid pGBM124 containing a 14-kb S. typhi chromosomal DNA fragment conferred the ability to produce Vi antigen on Escherichia coli HB101 and ViaB-deleted S. typhi GIFU10007-3. Tn5 insertion analysis showed that the 14-kb DNA was split into three regions. Region 1 and region 2 are involved in the biosynthesis of Vi polysaccharide. Region 3 is involved in translocation of the Vi polysaccharide to the cell surface. Southern blot hybridization showed that regions 2 and 3 but not region 1, were considerably homologous to the DNA of Vi-positive Citrobacter freundii.     

宋内氏痢疾菌无毒株S7表达Vi抗原工程菌的构建

将含有编码Ｖｉ抗原ＶｉａＢ基因片断的质粒转导进入宋内氏痢疾菌无毒株Ｓ７中,组建了重组菌株Ｓ７Ｖｉ。质粒电泳图谱显示重组菌株Ｓ７Ｖｉ中存在被转入的外源质粒带。重组株的生化特性没有改变。菌体凝集及Ｖｉ抗血清标记的ＳＰＡ菌液凝集反应证明在重组株的菌体表面,同时表达了Ｖｉ抗原和宋内氏毒菌的Ｏ抗原。以５×１０~８ＣＦＵ、１０×１０~８ＣＦＵ的重组株免疫近交系的ＬＩＢＰ小鼠,免疫小鼠对宋内氏毒株Ｓ６３攻击的保护率为６０％至９０％,对伤寒毒株Ｔｙ２攻击的保护率为２５％至４０％。     

Protective efficacy and immunogenicity of Vi-porin conjugate against Salmonella typhi.

A conjugate vaccine against Salmonella typhi was prepared by covalently binding capsular polysaccharide (Vi) with porin, both isolated from S. typhi. First, Vi and porins were extracted. The Vi was purified from S. typhi Ty2. The purified Vi conformed to the requirements of the World Health Organization. Porins were purified from S. typhi 0901. The Vi was bound to the porins by a heterobifunctional cross-linking reagent, N-succinimidyl-3-(2-pyridyl dithio)-propionate (SPDP). After preparing the Vi-porin conjugate, its protective ability and immunogenicity were studied in mice following systemic immunization. The results showed that the conjugate is 6.5-fold more protective than Vi alone against S. typhi. The mice immunized with conjugate elicited higher anti-Vi antibody (IgG) levels (P < 0.01) than the mice immunized with Vi alone. Anti-porin antibodies were also induced by the conjugate. To study the mucosal immune responses, secretory IgA (sIgA) in the intestinal fluid was measured. Conjugate-immunized mice showed the induction of sIgA as compared to Vi alone. The results showed that when Vi is bound to porins, both isolated from same organism, the resultant conjugate induced both systemic and mucosal immune responses and provided better protection against S. typhi than Vi alone.     

Isolation of stable aroA mutants of Salmonella typhi Ty2: properties and preliminary characterisation in mice

Summary Derivatives of the Salmonella typhi strain Ty2 carrying stable mutations in the aroA gene were isolated. The mutations were generated by transducing an aroA::Tn10 marker into Ty2 and selecting for derivatives which were tetracyline sensitive and dependent on aromatic compounds for growth. Isolates that did not revert to aromatic compound independence at a detectable frequency were obtained. An S. typhimurium derived aroA specific DNA probe was used to demonstrate the presence of DNA rearrangements in the aroA region of the chromosome of some of the S. typhi aroA mutants. Most of these isolates still expressed Vi antigen. Aromatic compound dependent mutants of S. typhi were less virulent in mice than S. typhi Ty2 following intraperitoneal challenge with bacteria suspended in mucin. Mice immunised with one of these mutants, named WBL85-1, were protected against a potentially lethal challenge of S. typhi Ty2.     

Complete nucleotide sequence and molecular characterization of ViaB region encoding Vi antigen in Salmonella typhi.

Plasmid pGBM124, which contains a 14-kb Salmonella typhi chromosomal DNA fragment capable of producing the Vi antigen in Escherichia coli HB101 and ViaB-deleted S. typhi GIFU 10007-3, was studied. We determined the complete nucleotide sequence of this fragment and found 11 open reading frames. Mutagenesis, subcloning, and complementation analysis showed that three genes (vipA, vipB, and vipC) are involved in biosynthesis of the Vi polysaccharide. The putative primary amino acid sequence suggests that both vipA and vipB encode the NAD- or NADP-dependent enzymes to synthesize the nucleotide sugar for the Vi polysaccharide. Five genes (vexA, vexB, vexC, vexD, and vexE) may be involved in translocation of the Vi polysaccharide. Proteins VexA, VexB, VexC, and VexD had moderate similarities to components of group II capsule transporters, and the VexC protein had a putative ATP-binding site. These data indicate that the transport system for the Vi polysaccharide belongs to the ATP-binding cassette transporters. By using the isogenic Vi+ and Vi- strains constructed in this study, we reconfirmed that the Vi antigen is necessary for the serum resistance of S. typhi.     

The Vi-capsule prevents Toll-like receptor 4 recognition of Salmonella

The viaB locus enables Salmonella enterica serotype Typhi to reduce Toll-like receptor (TLR) dependent cytokine production in tissue culture models. This DNA region contains genes involved in the regulation ( tviA ), biosynthesis ( tviBCDE ) and export ( vexABCDE ) of the Vi capsule. Expression of the Vi capsule in S.  Typhimurium, but not expression of the TviA regulatory protein, reduced tumour necrosis factor-alpha (TNF-α) and IL-6 production by murine bone-marrow derived macrophages. Production of TNF-α and IL-6 was dependent on expression of TLR4 as stimulation of macrophages from TLR4^−/− mice with S.  Typhimurium did not result in expression of these cytokines. Intraperitoneal infection of mice with S.  Typhimurium induced expression of TNF-α and inducible nitric oxide synthase (iNOS) in the liver. Introduction of the cloned viaB region into S.  Typhimurium reduced TNF-α and iNOS expression to levels observed after infection with a S.  Typhimurium msbB mutant. In contrast, no differences in TNF-α expression between the S.  Typhimurium wild type and strains expressing the Vi-capsule or carrying a mutation in msbB were observed after infection of TLR4^−/− mice. We conclude that the Vi capsule prevents both in vitro and in vivo recognition of S.  Typhimurium lipopolysaccharide by TLR4.     

Attenuated Salmonella enterica serovar typhi live vector with inducible chromosomal expression of the T7 RNA polymerase and its evaluation with reporter genes

Attenuated Salmonella strains with defined gene deletions have been extensively evaluated as suitable live carriers of passenger antigens. A number of strategies for antigen delivery by these strains have been attempted, ranging from plasmid-based to chromosomal integration systems. We report here the chromosomal integration of the T7 RNA polymerase gene (T7pol) in the attenuated strain Salmonella enterica serovar Typhi (Salmonella typhi) CVD908 (aroC(-), aroD(-)). The T7pol gene was amplified by PCR from Escherichia coli BL21(DE3) and cloned in the pNir3 plasmid under the control of the anaerobically inducible nirB promoter. Then it was subcloned in a pKTN701 derivative, suicide plasmid with the R6K ori, and flanked by the aroC gene. After evaluation of its functionality in E. coli SY327, the aroC-T7pol-aroC cassette was integrated into the aroC locus of S. typhi CVD908 by homologous recombination. The resulting strain, S. typhi CVD908-T7pol, was able to transcomplement two plasmids bearing the luc or the lacZ reporter genes controlled by the T7 promoter and produce luciferase and beta-galactosidase under anaerobic culture conditions. Therefore, an inducible system for recombinant antigen production in attenuated S. typhi was achieved.     

Genomic Subtraction Identifies Salmonella typhimurium Prophages, F-Related Plasmid Sequences, and a Novel Fimbrial Operon, stf, Which Are Absent in Salmonella typhi

Salmonella typhimurium causes systemic and fatal infection in inbred mice, while the related serotype Salmonella typhi is avirulent for mammals other than humans. In order to identify genes from the virulent strain S. typhimurium ATCC 14028 that are absent in S. typhi Ty2, and therefore might be involved in S. typhimurium mouse virulence, a PCR-supported genomic subtractive hybridization procedure was employed. We have identified a novel putative fimbrial operon, stfACDEFG, located at centisome 5 of the S. typhimurium chromosome, which is absent in S. typhi, Salmonella arizonae, and Salmonella bongori but was detected in several other Salmonella serotypes. The fimbrial genes represent a genomic insertion in S. typhimurium compared to the respective region between fhuB and hemL in Escherichia coli K-12. In addition, the subtraction procedure yielded F plasmid-related sequences from the S. typhimurium virulence plasmid, a number of DNA fragments representing parts of lambdoid prophages and putative sugar transporters, and several fragments with unknown sequences. The majority of subtracted chromosomal sequences map to three distinct locations, around centisomes 5, 27, and 57.     

Proteomics analysis of the causative agent of typhoid fever

Typhoid fever is a potentially fatal disease caused by the bacterial pathogen Salmonella enterica serotype Typhi ( S. typhi). S. typhi infection is a complex process that involves numerous bacterially encoded virulence determinants, and these are thought to confer both stringent human host specificity and a high mortality rate. In the present study, we used a liquid chromatography-mass spectrometry (LC-MS)-based proteomics strategy to investigate the proteome of logarithmic, stationary phase, and low pH/low magnesium (MgM) S. typhi cultures. This represents the first large-scale comprehensive characterization of the S. typhi proteome. Our analysis identified a total of 2066 S. typhi proteins. In an effort to identify putative S. typhi-specific virulence factors, we then compared our S. typhi results to those obtained in a previously published study of the S. typhimurium proteome under similar conditions ( Adkins, J. N. et al. Mol. Cell. Proteomics 2006, 5, 1450-1461 ). Comparative proteomics analysis of S. typhi strain Ty2 and S. typhimurium strain LT2 revealed a subset of highly expressed proteins unique to S. typhi that were exclusively detected under conditions that are thought to mimic the infective state in macrophage cells. These proteins included CdtB, HlyE, and gene products of t0142, t1108, t1109, t1476, and t1602. The differential expression of T1108, T1476, and HlyE was confirmed by Western blot analysis. When our observations are taken together with the current literature, they suggest that this subset of proteins may play a role in S. typhi pathogenesis and human host specificity.     

Evaluation of phoP and rpoS mutants of Salmonella enterica serovar Typhi as attenuated typhoid vaccine candidates: virulence and protective immune responses in intranasally immunized mice

The attenuation and immunoenhancing effects of rpoS and phoP Salmonella enterica serovar strain Typhi (Salmonella typhi) mutants have not been compared. Here, three S. typhi deletion mutants (phoP, rpoS, and rpoS-phoP double mutant) are constructed and these mutants are characterized with respect to invasiveness, virulence, and protective immune response compared with wild-type Ty2. It was found that phoP and phoP-rpoS deletion mutants are less invasive to HT-29 cells than the wild-type Ty2 and the rpoS single-deleted strain. The LD(50) of immunized mice was higher for phoP than for rpoS mutants, and the highest for the phoP-rpoS double mutant. In addition, all S. typhi mutants showed an increase in the specific serum IgG levels and T-cell-mediated immunity, and showed equal protection abilities against a wild-type Ty2 challenge after two rounds of immunization in BALB/c mice. It is concluded that phoP genes appear to play a more important role than rpoS genes in both cellular invasion and virulence of S. typhi, but not in immunogenicity in mice. Furthermore, the data indicate that the phoP-rpoS double mutant may show promise as a candidate for an attenuated typhoid vaccine.