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The autophagy pathway plays a crucial role in resistance to bacterial infection in the host. Salmonella enterica serovar Typhi (S. Typhi), a human restricted pathogen, causes a systemic infection known as typhoid fever. Vi capsular polysaccharide not only forms a physical barrier on the surface of S. Typhi but also serves as an important virulence factor. Here, the effects of Vi expression on autophagy of host cells were investigated. Vi was highly expressed in super optimal broth (SOB) medium for 8 h and in the early stage of macrophage infection. Strains of S. Typhi with a mutation in Vi capsule, ΔtviA and ΔvexE, were constructed and showed lower intracellular survival in macrophage THP-1 cells compared with wild type strain. Western blot, immunofluorescence and flow cytometry were used to detected the autophagy level of macrophages infected by Vi mutant and wild type strains, respectively. Autophagy receptor p62 protein level significantly decreased and LC3-II protein level significantly increased in Vi mutant strains compared with wild type strain, which indicated increased autophagy in macrophages infected by Vi mutant strains. The qRT-PCR and Western blot results showed that not Nod1, but Nod2 and Galectin-8 were up-regulated in Vi mutant strains. In summary, we propose that Vi capsule of S. Typhi decreased autophagy of macrophages to increase its survival in host cells by decreasing the expression of Nod2 and Galectin-8., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships at work that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Many bacterial pathogens, including the human exclusive pathogen Salmonella Typhi, express capsular polysaccharides as a crucial virulence factor. Here, through S. Typhi whole genome sequence analyses and functional studies, we found a list of single point mutations that make S. Typhi hypervirulent. We discovered a single point mutation in the Vi biosynthesis enzymes that control Vi polymerization or acetylation is enough to result in different capsule variants of S. Typhi. All variant strains are pathogenic, but the hyper Vi capsule variants are particularly hypervirulent, as demonstrated by the high morbidity and mortality rates observed in infected mice. The hypo Vi capsule variants have primarily been identified in Africa, whereas the hyper Vi capsule variants are distributed worldwide. Collectively, these studies increase awareness about the existence of different capsule variants of S. Typhi, establish a solid foundation for numerous future studies on S. Typhi capsule variants, and offer valuable insights into strategies to combat capsulated bacteria., (© 2024. The Author(s).)

Salmonella enterica serovar Typhi causes typhoid fever. It possesses a Vi antigen capsular polysaccharide coat that is important for virulence and is the basis of a current glycoconjugate vaccine. Vi antigen is also produced by environmental Bordetella isolates, while mammal-adapted Bordetella species (such as Bordetella bronchiseptica) produce a capsule of undetermined structure that cross-reacts with antibodies recognizing Vi antigen. The Vi antigen backbone is composed of poly-α-(1→4)-linked N-acetylgalactosaminuronic acid, modified with O-acetyl residues that are necessary for vaccine efficacy. Despite its biological and biotechnological importance, some central aspects of Vi antigen production are poorly understood. Here we demonstrate that TviE and TviD, two proteins encoded in the viaB (Vi antigen production) locus, interact and are the Vi antigen polymerase and O-acetyltransferase, respectively. Structural modeling and site-directed mutagenesis reveal that TviE is a GT4-family glycosyltransferase. While TviD has no identifiable homologs beyond Vi antigen systems in other bacteria, structural modeling suggests that it belongs to the large SGNH hydrolase family, which contains other O-acetyltransferases. Although TviD possesses an atypical catalytic triad, its O-acetyltransferase function was verified by antibody reactivity and 13 C NMR data for tviD-mutant polysaccharide. The B. bronchiseptica genetic locus predicts a mode of synthesis distinct from classical S. enterica Vi antigen production, but which still involves TviD and TviE homologs that are both active in a reconstituted S. Typhi system. These findings provide new insight into Vi antigen production and foundational information for the glycoengineering of Vi antigen production in heterologous bacteria., Competing Interests: Conflicts of interest The authors declare that they have conflicts of interest with the contents of this article., (Crown Copyright © 2021. Published by Elsevier Inc. All rights reserved.)

The low- and middle-income countries bear the highest burden of typhoid fever in the world. India, along with other South Asian countries, has a significant incidence of typhoid fever among young children though there is a paucity of published data on community burden. In spite of the availability of Vi-polysaccharide (Vi-PS) and conjugated Vi-PS vaccines, these are not adequately utilized in India and in the neighbouring countries. To address many shortcomings of the unconjugated Vi-PS vaccines, typhoid conjugate vaccines (TCVs) are developed by conjugating Vi-PS with different carrier proteins. Three such vaccines using tetanus toxoid as a carrier protein are already licensed in India. Several other Vi-PS conjugates are currently in various stages of development. The current review provides an update on the existing and upcoming new TCVs along with a detailed discussion on the various issues involved with their clinical use and limitations., Competing Interests: None

The pathogenesis of Salmonella enterica serovar Typhi (S. Typhi), the cause of typhoid fever in humans, is mainly attributed to the acquisition of horizontally acquired DNA elements. Salmonella pathogenicity islands (SPIs) are indubitably the most important form of horizontally acquired DNA with respect to pathogenesis of this bacterium. The insertion or deletion of any of these transferrable SPIs may have impact on the virulence potential of S. Typhi. In this study, the virulence potential and genetic relatedness of 35 S. Typhi isolates, collected from 2004 to 2013 was determined by identification of SPI and non-SPI virulence factors through a combination of techniques including virulotyping, Whole Genome Sequencing (WGS), and Variable Number of Tandem Repeats (VNTR) profiling. In order to determine the virulence potential of local S. Typhi isolates, 56 virulence related genes were studied by PCR. These genes are located in the core as well as accessory genome (SPIs and plasmid). Major variations among studied virulence determinants were found in case of SPI-7 and SPI-10 associated genes. On the basis of presence of virulence related genes, the studied S. Typhi isolates from Pakistan were clustered into two virulotypes Vi-positive and Vi-negative. Interestingly, SPI-7 and SPI-10 were collectively absent or present in Vi-negative and Vi-positive strains, respectively. Two Vi-negative and 11 Vi-positive S. Typhi strains were also analyzed by whole genome sequencing (WGS) and their results supported the PCR results. Genetic diversity was tested by VNTR-based molecular typing. All 35 isolates were clustered into five groups. Overall, all Vi-negative isolates were placed in a single group (T5) whereas Vi-positive isolates were grouped into four types. Vi-negative and Vi-positive isolates were mutually exclusive. This is the first report on the comparative distribution of SPI and non-SPI related virulence genes in Vi-negative and Vi-positive S. Typhi isolates with an important finding that SPI-10 is absent in all Vi-negative isolates., Competing Interests: The authors have declared that no competing interests exist.

Typhoid is a major global disease. The causative agent, Salmonella enterica serovar Typhi (S. Typhi) has a capsular antigen called Vi antigen which is traditionally considered to be the main cause of virulence. All the current vaccines are based on Vi antigen. However, the realization of the fact that there are S. Typhi strains which lack Vi antigen but still exist naturally and can cause disease has stirred great scientific interest. It is also interesting to note that their relative prevalence is affected by climatic conditions. Now it is established that Vi positive and Vi negative S. Typhi have different modes of pathogenesis; and as recent studies suggest, different structure of polysaccharide antigens. This means that current vaccines are not effective against a significant number of S. Typhi strains which not only affect the success of vaccination programs but also help in rapid emergence of Vi negative S. Typhi due to natural selection. The focus should be on vaccines based on antigens which are universally present in all S. Typhi. One such candidate is O-specific polysaccharides (OSPs). Successful attempts have been made to prepare conjugate vaccines based on OSPs.

Vi capsular polysaccharide, a linear homopolymer of α-1,4-linked N-acetylgalactosaminuronate, is characteristically produced by Salmonella enterica serovar Typhi. The Vi capsule covers the surface of the producing bacteria and serves as an virulence factor via inhibition of complement-mediated killing and promoting resistance against phagocytosis. Furthermore, Vi also represents a predominant protective antigen and plays a key role in the development of vaccines against typhoid fever. Herein, we reviewed the latest advances associated with the Vi polysaccharide, from its synthesis and transport within bacterial cells, mechanisms involved in virulence, immunological characteristics, and applications in vaccine, as well as its purification and detection methods.

Unlabelled: Expression of capsular polysaccharides is a variable trait often associated with more-virulent forms of a bacterial species. For example, typhoid fever is caused by the capsulated Salmonella enterica serovar Typhi, while nontyphoidal Salmonella serovars associated with gastroenteritis are noncapsulated. Here we show that optimization of the immune evasive properties conferred by the virulence-associated (Vi) capsular polysaccharide involved an additional alteration to the cell envelope of S. Typhi, namely inactivation of the fepE gene, encoding the regulator of very-long O-antigen chains. Introduction of the capsule-encoding viaB locus into the nontyphoidal S. enterica serovar Typhimurium reduced complement deposition in vitro and intestinal inflammation in a mouse colitis model. However, both phenotypes were markedly enhanced when the viaB locus was introduced into an S. Typhimurium fepE mutant, which lacks very-long O-antigen chains. Collectively, these data suggest that during the evolution of the S. Typhi lineage, loss of very-long O-antigen chains by pseudogene formation was an adaptation to maximize the anti-inflammatory properties of the Vi capsular polysaccharide., Importance: Genomic comparison illustrates that acquisition of virulence factors by horizontal gene transfer is an important contributor to the evolution of enteric pathogens. Acquisition of complex virulence traits commonly involves horizontal transfer of a large gene cluster, and integration of the gene cluster into the host genome results in the formation of a pathogenicity island. Acquisition of the virulence-associated (Vi) capsular polysaccharide encoded by SPI7 (Salmonella pathogenicity island 7) was accompanied in the human-adapted Salmonella enterica serovar Typhi by inactivation of the fepE gene, encoding the regulator of very-long O-antigen chains. We show that the resulting loss of very-long O-antigen chains was an important mechanism for maximizing immune evasion mediated by the Vi capsular polysaccharide. These data suggest that successful incorporation of a capsular polysaccharide requires changes in the cell envelope of the hosting pathogen.

State-of-the-art production technologies for conjugate vaccines are complex, multi-step processes. An alternative approach to produce glycoconjugates is based on the bacterial N-linked protein glycosylation system first described in Campylobacter jejuni. The C. jejuni N-glycosylation system has been successfully transferred into Escherichia coli, enabling in vivo production of customized recombinant glycoproteins. However, some antigenic bacterial cell surface polysaccharides, like the Vi antigen of Salmonella enterica serovar Typhi, have not been reported to be accessible to the bacterial oligosaccharyltransferase PglB, hence hamper development of novel conjugate vaccines against typhoid fever. In this report, Vi-like polysaccharide structures that can be transferred by PglB were evaluated as typhoid vaccine components. A polysaccharide fulfilling these requirements was found in Escherichia coli serovar O121. Inactivation of the E. coli O121 O antigen cluster encoded gene wbqG resulted in expression of O polysaccharides reactive with antibodies raised against the Vi antigen. The structure of the recombinantly expressed mutant O polysaccharide was elucidated using a novel HPLC and mass spectrometry based method for purified undecaprenyl pyrophosphate (Und-PP) linked glycans, and the presence of epitopes also found in the Vi antigen was confirmed. The mutant O antigen structure was transferred to acceptor proteins using the bacterial N-glycosylation system, and immunogenicity of the resulting conjugates was evaluated in mice. The conjugate-induced antibodies reacted in an enzyme-linked immunosorbent assay with E. coli O121 LPS. One animal developed a significant rise in serum immunoglobulin anti-Vi titer upon immunization.

Introduction: Salmonella enterica serovar Typhi is the causative agent of typhoid fever with over 22 million cases and over 200,000 deaths reported annually. A vaccine is much needed for use in young children and the Novartis Vaccines Institute for Global Health (NVGH) is developing a conjugate vaccine which targets S. Typhi Vi capsular polysaccharide., Methodology: Here we describe a method suitable for industrial scale production of the Vi antigen based on expression by a Citrobacter line. We optimized the production of Vi by selecting a suitable Citrobacter strain (Citrobacter 328) that yields high and stable expression of Vi in chemically defined medium under industrial-scale fermentation conditions., Results: Vi-CRM197 made using Vi from Citrobacter 328 elicited high anti-Vi antibody levels in mice and rabbits., Conclusions: Citrobacter 328 is a suitable strain for production of Vi for conjugate anti-Typhi vaccines. Being a BSL-1 organism, which grows in defined medium and stably produces high yields of Vi, it offers excellent potential for safe production of inexpensive vaccines for populations at risk of typhoid fever.

Background: Current recommendations are that HIV-infected persons should not be given live vaccines. We set out to assess potential toxicity of three live, attenuated oral vaccines (against rotavirus, typhoid and ETEC) in a phase 1 study., Methods: Two commercially available oral vaccines against rotavirus (Rotarix) and typhoid (Vivotif) and one candidate vaccine against Enterotoxigenic Escherichia coli (ACAM2017) were given to HIV seropositive (n=42) and HIV seronegative (n=59) adults. Gastrointestinal symptoms were sought actively by weekly interview up to 1 month of vaccination. In rotavirus vaccine recipients, intestinal biopsies were collected by endoscopy and evaluated for expression of IL-8 and pro-inflammatory cytokines., Results: No difference was observed between symptoms in HIV infected and HIV uninfected vaccinees, except for diarrhoea reported more than 7 days after the last dose of vaccine. If only diarrhoea episodes within 7 days of vaccination are included, diarrhoea was not more frequent in HIV seropositive than in HIV seronegative vaccinees (OR 6.7, 95% CI 1.2-67; P=0.09). However, if later episodes of diarrhoea are included, a significant increase in diarrhoea was demonstrated (OR 5.3, 95% CI 0.98-53; P=0.04). All episodes were mild and transient. IL-8 was slowly up-regulated over the week following vaccination (P=0.02), but IL-β, IFNγ or TNFα were not., Conclusions: No evidence was found of adverse events following administration of these three vaccines, except for late episodes of diarrhoea which may not be attributable to vaccination. Our data do not support the need for a prohibition on oral administration of live, attenuated vaccines to all HIV infected adults, though further work on severely immunocompromised adults and children are required., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The Vi capsular polysaccharide (CPS) of Salmonella enterica serovar Typhi, the cause of human typhoid, is important for infectivity and virulence. The Vi biosynthetic machinery is encoded within the viaB locus composed of 10 genes involved in regulation of expression (tviA), polymer synthesis (tviB-tviE), and cell surface localization of the CPS (vexA-vexE). We cloned the viaB locus from S. Typhi and transposon insertion mutants of individual viaB genes were characterized in Escherichia coli DH5α. Phenotype analysis of viaB mutants revealed that tviB, tviC, tviD and tviE are involved in Vi polymer synthesis. Furthermore, expression of tviB-tviE in E. coli DH5α directed the synthesis of cytoplasmic Vi antigen. Mutants of the ABC transporter genes vexBC and the polysaccharide copolymerase gene vexD accumulated the Vi polymer within the cytoplasm and productivity in these mutants was greatly reduced. In contrast, de novo synthesis of Vi polymer in the export deficient vexA mutant was comparable to wild-type cells, with drastic effects on cell stability. VexE mutant cells exported the Vi, but the CPS was not retained at the cell surface. The secreted polymer of a vexE mutant had different physical characteristics compared to the wild-type Vi.

The Vi capsular polysaccharide is a virulence-associated factor expressed by Salmonella enterica serotype Typhi but absent from virtually all other Salmonella serotypes. In order to study this determinant in vivo, we characterised a Vi-positive S. Typhimurium (C5.507 Vi(+)), harbouring the Salmonella pathogenicity island (SPI)-7, which encodes the Vi locus. S. Typhimurium C5.507 Vi(+) colonised and persisted in mice at similar levels compared to the parent strain, S. Typhimurium C5. However, the innate immune response to infection with C5.507 Vi(+) and SGB1, an isogenic derivative not expressing Vi, differed markedly. Infection with C5.507 Vi(+) resulted in a significant reduction in cellular trafficking of innate immune cells, including PMN and NK cells, compared to SGB1 Vi(-) infected animals. C5.507 Vi(+) infection stimulated reduced numbers of TNF-α, MIP-2 and perforin producing cells compared to SGB1 Vi(-). The modulating effect associated with Vi was not observed in MyD88(-/-) and was reduced in TLR4(-/-) mice. The presence of the Vi capsule also correlated with induction of the anti-inflammatory cytokine IL-10 in vivo, a factor that impacted on chemotaxis and the activation of immune cells in vitro.

Salmonella enterica serotype Typhi (S. Typhi) is a causative organism of typhoid fever. A number of Salmonella serovars express a capsular polysaccharide antigen known as Vi, the biosynthetic and export proteins of which are encoded within the viaB locus of Salmonella Pathogenicity Island -7 (SPI-7). SPI-7 is inserted between two partially duplicated copies of tRNA -pheU gene. We have investigated the frequency of viaB operon deletion and loss of SPI-7 due to storage of strains collected during the period 1987-2006 by PCR amplification of fliC (for confirmation of serotype Typhi), tviB (for status of viaB operon) and tRNA -pheU (for absence of SPI-7). All 111 isolates were observed with positive amplification of 495 bp amplicon for fliC. A total of 36 isolates were negative for Vi by agglutination while 39 were negative for viaB operon. Interestingly, 106 isolates were found to have SPI-7. The 5 SPI-7 negative isolates were isolated during recent years. Long-term storage and repeated culture had little or no effect on SPI-7, as none of the 18 isolates recovered from blood before 1997 lacked SPI-7. On the other hand, loss of viaB operon was directly proportional to duration of storage. Thus, it is proposed that stability of Vi gene is dependent on the presence of selection pressure.

Vi capsular polysaccharide is a major virulence determinant of the human typhoid- causing pathogen Salmonella typhi; it is absent in nontyphoidal Salmonella serovars. We show in this study that through its specific interaction with the membrane recognition complex containing the prohibitin family of molecules, Vi can inhibit the production of inflammatory cytokines from mononuclear phagocytes stimulated with Salmonella flagellin. Remarkably, Vi lost this anti-inflammatory capability and switched to a proinflammatory state when cell stimulations were performed in the presence of serum. The serum-transformed proinflammatory form of Vi induced secretion of cytokines from monocytes by specifically engaging TLR-2/TLR-1. The molecule responsible for bringing about this conversion of Vi from an anti-inflammatory to a proinflammatory form was serum-derived hemoglobin. Derivatives of Vi incapable of interacting with hemoglobin did not switch to a proinflammatory state in vitro or in vivo. These findings provide compelling evidence for a role of hemoglobin in transforming the anti-inflammatory S. typhi virulence polysaccharide into an immune activator.

Salmonella enterica serotype Typhimurium elicits acute neutrophil influx in the human intestinal mucosa within 1 or 2 days after infection, resulting in inflammatory diarrhea. In contrast, no overt symptoms are observed within the first 1 or 2 weeks after infection with S. enterica serotype Typhi. Here we show that introduction of the capsule-encoding viaB locus of serotype Typhi reduced the ability of serotype Typhimurium to elicit acute intestinal inflammation in a streptomycin-pretreated mouse model. Serotype Typhimurium requires a functional invasion-associated type III secretion system (type III secretion system 1 [T3SS-1]) to elicit cecal inflammation within 48 h after infection of streptomycin-pretreated mice, and the presence of the viaB locus reduced its invasiveness for human intestinal epithelial cells in vitro. However, a reduced activity of T3SS-1 could not account for the ability of the viaB locus to attenuate cecal inflammation, because introduction of the viaB locus into an invasion-deficient serotype Typhimurium strain (invA mutant) resulted in a significant reduction of pathology and inflammatory cytokine expression in the cecum 5 days after infection of mice. We conclude that a T3SS-1-independent mechanism contributes to the ability of the viaB locus to reduce intestinal inflammation.

The synthesis of Vi polysaccharide, a major virulence determinant in Salmonella enterica serotype Typhi (S. Typhi), is under the control of two regulatory systems, ompR-envZ and rscB-rscC, which respond to changes in osmolarity. Some S. Typhi isolates exhibit over-expression of Vi polysaccharide, which masks clinical detection of LPS O-antigen. This variation in Vi polysaccharide and O-antigen display (VW variation) has been observed since the initial studies of S. Typhi. We have reported that the status of the rpoS gene is responsible for this phenomenon. We review the regulatory network of the Vi polysaccharide, linking osmolarity and RpoS expression. Also, we discuss how this may impact live attenuated Salmonella vaccine development.

Human infections with Salmonella enterica results in two major groups of diseases: gastroenteritis and typhoid fever. Clinical observations suggest that gastroenteritis, caused by non-typhoidal Salmonella serovars, is characterized by a massive neutrophil influx, which keeps the infection localized to the intestinal mucosa. In contrast, the absence of neutrophilic intestinal infiltrates in the acute phase of typhoid fever suggests a propensity for typhoidal Salmonella serovars (S. Typhi, S. Paratyphi A, S. Paratyphi B and S. Paratyphi C) to evade aspects of the innate immune response and cause a systemic infection. The fact that there are no virulence genes shared by typhoidal Salmonella serovars that are absent from non-typhoidal Salmonella serovars, suggests that this innate immune evasion is mediated by different mechanisms in different typhoidal serovars. This review discusses what is known about the clinical pathogenesis of typhoid fever.

The generation of complete genome sequences provides a blueprint that facilitates the genetic characterization of pathogens and their hosts. The genome of Salmonella enterica serovar Typhi (S. Typhi) harbors ~5 million base pairs encoding some 4000 genes, of which >200 are functionally inactive. Comparison of S. Typhi isolates from around the world indicates that they are highly related (clonal) and that they emerged from a single point of origin ~30,000-50,000 years ago. Evidence suggests that, as well as undergoing gene degradation, S. Typhi has also recently acquired genes, such as those encoding the Vi antigen, by horizontal transfer events.

Salmonella enterica serovar Typhi uses type IVB pili to facilitate eukaryotic cell invasion. Here, we compare environmental and genetic controls on pil operon transcription with those regulating viaB genes required for Vi antigen expression. Transcription of pil occurs only in the late logarithmic and stationary phases of bacterial growth while viaB expression occurs in the logarithmic growth phase. Expression of both viaB and pil was, however, optimal at 100 mM NaCl, and mutations in envZ/ompR, rcsB/rcsC, (but not rcsA), tviA, ihfB or fis affected transcription of both viaB and pil DNA. As both Vi antigen and Type IVB pili facilitate serovar Typhi invasion of human monocytes, an overlap of production controls is logical. It appears that Vi antigen synthesis precedes pilus production.

Vi antigen, the virulence factor of Salmonella typhi, has been used clinically as a molecular vaccine. TviB and TviC are two enzymes involved in the formation of Vi antigen, a linear polymer consisting of alpha-1,4-linked N-acetylgalactosaminuronate. Protein sequence analysis suggests that TviB is a dehydrogenase and TviC is an epimerase. Both enzymes are expected to be NAD(+) dependent. In order to verify their functions, TviB and TviC were cloned, expressed in Escherichia coli, and characterized. The C-terminal His(6)-tagged TviB protein, purified from soluble cell fractions in the presence of 10 mM DTT, shows UDP-N-acetylglucosamine 6-dehydrogenase activity and is capable of catalyzing the conversion of UDP-N-acetylglucosamine (UDP-GlcNAc) to UDP-N-acetylglucosaminuronic acid (UDP-GlcNAcA) with a k(cat) value of 15.5 +/- 1.0 min(-)(1). The K(m) values of TviB for UDP-GlcNAc and NAD(+) are 77 +/- 9 microM and 276 +/- 52 microM, respectively. TviC, purified as C-terminal hexahistidine-tagged protein, shows UDP-GlcNAcA 4-epimerase and UDP-N-acetylgalactosamine (UDP-GalNAc) 4-epimerase activities. The K(m) values of TviC for UDP-GlcNAcA and UDP-N-acetylgalactosaminuronic acid (UDP-GalNAcA) are 20 +/- 1 microM and 42 +/- 2 microM, respectively. The k(cat) value for the conversion of UDP-GlcNAcA to UDP-GalNAcA is 56.8 +/- 0.5 min(-)(1), while that for the reverse reaction is 39.1 +/- 0.6 min(-)(1). These results show that the biosynthesis of Vi antigen is initiated by the TviB-catalyzed oxidation of UDP-GlcNAc to UDP-GalNAc, followed by the TviC-catalyzed epimerization at C-4 to form UDP-GalNAcA, which serves as the building block for the formation of Vi polymer. These results set the stage for future in vitro biosynthesis of Vi antigen. These enzymes may also be drug targets to inhibit Vi antigen production.

The synthesis and transportation proteins of the Vi capsular polysaccharide of Salmonella enterica serovar Typhi (serovar Typhi) are encoded by the viaB operon, which resides on a 134-kb pathogenicity island known as SPI-7. In recent years, Vi-negative strains of serovar Typhi have been reported in regions where typhoid fever is endemic. However, because Vi negativity can arise during in vitro passage, the clinical significance of Vi-negative serovar Typhi is not clear. To investigate the loss of Vi expression at the genetic level, 60 stored strains of serovar Typhi from the Faisalabad region of Pakistan were analyzed by PCR for the presence of SPI-7 and two genes essential for Vi production: tviA and tviB. Nine of the sixty strains analyzed (15%) tested negative for both tviA and tviB; only two of these strains lacked SPI-7. In order to investigate whether this phenomenon occurred in vivo, blood samples from patients with the clinical symptoms of typhoid fever were also investigated. Of 48 blood samples tested, 42 tested positive by fliC PCR for serovar Typhi; 4 of these were negative for tviA and tviB. Three of these samples tested positive for SPI-7. These results demonstrate that viaB-negative, SPI-7-positive serovar Typhi is naturally occurring and can be detected by PCR in the peripheral blood of typhoid patients in this region. The method described here can be used to monitor the incidence of Vi-negative serovar Typhi in regions where the Vi vaccine is used.

Vi polysaccharide and iron-regulated outer-membrane proteins (IROMPs) were extracted and purified from the standard strain of Salmonella typhi, Ty2. These were then conjugated by chemical coupling using the carbodimide method. Vi-IROMP conjugate was tested for its ability to protect against colonization by S. typhi in different organs. Mice immunized with 2.5 microg Vi-IROMP conjugate showed the most protection, as the least bacterial colonization of spleen, liver and Peyer's patches was observed. Peritoneal macrophages obtained from conjugate-treated mice phagocytosed bacteria efficiently. Circulating antibodies and the delayed type hypersensitivity response elucidated by mouse foot-pad swelling was significantly higher in conjugate-treated animals. These results clearly demonstrate that an IROMP and polysaccharide conjugate of S. typhi prepared from the same strain has the potential to protect animals against challenge.

The large pathogenicity island (SPI7) of Salmonella enterica serovar Typhi is a 133,477-bp segment of DNA flanked by two 52-bp direct repeats overlapping the pheU (phenylalanyl-tRNA) gene, contains 151 potential open reading frames, and includes the viaB operon involved in the synthesis of Vi antigen. Some clinical isolates of S. enterica serovar Typhi are missing the entire SPI7, due to its precise excision; these strains have lost the ability to produce Vi antigen, are resistant to phage Vi-II, and invade a human epithelial cell line more rapidly. Excision of SPI7 occurs spontaneously in a clinical isolate of S. enterica serovar Typhi when it is grown in the laboratory, leaves an intact copy of the pheU gene at its novel join point, and results in the same three phenotypic consequences. SPI7 is an unstable genetic element, probably an intermediate in the pathway of lateral transfer of such pathogenicity islands among enteric gram-negative bacteria.

Salmonella enterica serovar Typhi has a 134-kb island of DNA identified as salmonella pathogenicity island 7 (SPI7), inserted between pheU and 'pheU (truncated), two genes for tRNA(Phe). SPI7 has genes for Vi exopolysaccharide, for type IVB pili, for putative conjugal transfer, and for sopE bacteriophage. Pulsed-field gel electrophoresis following digestion with the endonuclease I-CeuI, using DNA from a set of 120 wild-type strains of serovar Typhi assembled from several sources, identified eight strains in which the I-CeuI G fragment, which contains SPI7, had a large deletion. In addition, agglutination tests with Vi antiserum and phage typing with Vi phages show that all eight strains are Vi negative. We therefore tested these strains for deletion of SPI7 by multiplex PCR, by microarray analysis, and by sequencing of PCR amplicons. Data show that seven of the eight strains are precise deletions of SPI7: a primer pair flanking SPI7 results in a PCR amplicon containing a single pheU gene; microarrays show that all SPI7 genes are deleted. Two of the strains produce amplicons which have A derived from pheU at bp 27, while five have C derived from 'pheU at this position; thus, the position of the crossover which results in the deletion can be inferred. The deletion in the eighth strain, TYT1669, removes 175 kb with junction points in genes STY4465 and STY4664; the left junction of SPI7 and adjacent genes, as well as part of SPI7 including the viaB operon for Vi exopolysaccharide, was removed, while the right junction of SPI7 was retained. We propose that these deletions occurred during storage following isolation.

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.

The incidence of typhoid fever remains unacceptably high in developing countries. Because Salmonella typhi is disseminated by carriers, there is an urgent need to increase the rate of detection of carriers and to decrease the risk they pose to their communities. In urban areas where sewage disposal is lacking or inadequate, public water supplies are contaminated and typhoid fever is common. The contamination of food by carriers is the second commonest route of infection. Water purification processes lead to a rapid decline in the incidence of the disease; thus, many developing countries hope to develop pure water supplies for all citizens by the end of this century. Until this important public health goal is achieved, the use of vaccine, especially in children, could cause a significant decrease in the incidence of typhoid fever. A new oral attenuated vaccine promises to be effective and safe.

Adhesins contained in the preparation of Vi-antigen have been found to enhance its immunogenic and protective properties. In the preparations of Vi-antigen obtained from Salmonella typhi and Citrobacter freundii the presence of two antigenic determinants has been revealed. One of them is associated with the Vi-receptor and the other determinant, with adhesin. Both determinants take part in the protection of mice from Salmonella infection.

The viaB locus coding for the Vi antigen of Salmonella typhi Ty2 was cloned on a 40.6-kilobase fragment into the cosmid vector pHC79. The live, oral, attenuated Vi-negative S. typhi Ty21a vaccine strain was transformed with the recombinant cosmid encoding the viaB locus. Homologous recombination of the viaB locus into the chromosome of S. typhi Ty21a was induced by UV irradiation, and Vi-positive recombinants were selected in the presence of D-cycloserine. One such isolate, termed WR4103, contained no plasmids or the attendant antibiotic resistance markers and expressed the Vi antigen stably. Vi antigen extracted from WR4103 was immunologically indistinguishable from Vi antigen purified from S. typhi Ty2. The only detectable difference between Ty21a and WR4103 was in the production of Vi antigen. The mean lethal doses of Ty21a and WR4103 for mice were nearly identical. Immunization of mice with WR4103 engendered a Vi antibody response and afforded complete protection against fatal infection with virulent S. typhi Ty2. Thus, S. typhi WR4103 may serve as an improved oral vaccine for protection against typhoid fever.

The role of the Vi antigen, the capsular polysaccharide of Salmonella typhi, in the pathogenesis of and immunity to typhoid fever remains the subject of controversy. Vi-positive S. typhi resist phagocytosis and the action of serum complement, both of which actions are initiated by antibodies to Vi antigen. Both the laboratory potency in mice and the clinical effectiveness of whole-cell vaccines were related to their content of immunogenic Vi antigen. A Vi polysaccharide used for immunizing humans against experimental challenge with S. typhi failed to prevent typhoid fever; experimental conditions used to prepare this ineffective Vi antigen were shown to denature it and to reduce its immunogenicity. Assay of serum antibodies to Vi antigen with purified Vi antigen is a reliable method for diagnosis of typhoid fever and asymptomatic carriage of S. typhi. Vi polysaccharides prepared by modern techniques passed the requirements for meningococcal polysaccharide vaccines and had approximately 13 times the protective activity in the mouse potency assay as did the US Standard 6A whole-cell typhoid vaccine.

The internalization and intracellular multiplication, i.e., the invasiveness, of Salmonella typhi strains recently isolated from typhoid fever patients were confirmed in HeLa cell monolayers. When stained with Giemsa solution, intracellular bacteria were 0.6 X 1.2 micron in size and stained purple, whereas extracellular bacteria associated or not with the HeLa cell surface were 1.0 X 3.0 micron and stained deep blue. Strain GIFU 10007 was internalized into 23% of the HeLa cells within 10 min after inoculation. About 90% of the HeLa cells were infected after 24 hr incubation in kanamycin (KM)-containing medium. Intracellular multiplication of the challenge organism was verified by a large number of intracellular bacteria after 24 hr incubation in KM-containing medium by both light-microscopy of the Giemsa stained preparation and viable counts of intracellular bacteria. The viable counts of strain 10007 showed an increase of more than 40-fold within 24 hr after inoculation, whereas in the four other less or non-infective strains, recovery of viable bacteria was poor or nil. Strains which were highly invasive usually failed to show strong adhesion. The contribution of Vi antigen to the internalization of challenge organisms was not proved. Infective strains, when killed by formalin were still adhesive, but were not internalized. The same strains, when killed by boiling, were neither adhesive nor internalized. From these findings it was concluded that the internalization and multiplication of infective S. typhi strains in cultured HeLa cells should be regarded as an invasion rather than phagocytosis by host cells, and such invasiveness could be an indicator to estimate the virulence of S. typhi strains.

The virulence of Salmonella typhi is associated with the presence of the Vi antigen. Mechanisms of Vi antigen virulence were examined in vitro. The Vi antigen-containing strain Quailes was significantly (P less than 0.025) more resistant to lysis by nonimmune serum than S. typhi 0901, which does not have the Vi antigen, and resulted in less activation of complement by the alternative pathway (P less than 0.05). Human polymorphonuclear leukocytes (PMNs) ingested strain Quailes significantly (P less than 0.01) more slowly and less completely than strain 0901 as assessed by three measures of phagocytic rate. In contrast to prior reports, the Vi antigen did not prevent an oxidative burst, measured by O2- production, chemiluminescence, and O2 consumption. The extent of the oxidative burst correlated directly and closely with the rate of phagocytosis. When the rate of PMN phagocytosis for both strains was equalized by opsonizing strain 0901 with 1% and strain Quailes with a 3% concentration of serum, the PMN oxidative burst was equal. C3 binding to strain Quailes was significantly (P less than 0.005) less than to strain 0901. Hence the Vi antigen inhibited phagocytosis by preventing C3b binding and solely as a consequence of this induced a lesser PMN oxidative burst. Furthermore, strain Quailes was significantly (P less than 0.025) less susceptible to killing by H2O2 than strain 0901. To ensure that these observations were a consequence of the Vi antigen and not other strain differences, another pair of S. typhi with and without the Vi antigen were similarly compared, and the results were the same as with strains Quailes and 0901. Strains 0901 and Quailes were killed by PMNs from a patient with chronic granulomatous disease but more slowly than by normal PMNs, indicating that S. typhi is susceptible to nonoxidative killing.

This study was undertaken to identify a DNA sequence that could be used to facilitate the diagnostic identification of Salmonella typhi, the causative agent of typhoid fever. All virulent S. typhi strains encode a relatively unique capsular antigen termed the virulence (Vi) antigen. Two distinct genetic loci, viaA and viaB, are involved in the synthesis of this antigen. The structural genes, located at viaB, were considered as a possible specific DNA probe. The viaB locus, contained in a recombinant cosmid, was subcloned to various plasmid vectors for this purpose. Selected viaB-region DNA fragments were then analyzed for specificity in DNA colony hybridization reactions with more than 170 strains representing a variety of enteric bacteria. An 8.6-kilobase EcoRI fragment was highly specific for the viaB gene region and was considered a good hybridization probe. This DNA probe should prove useful in rapid diagnostic assays set up to detect S. typhi in mixed bacterial samples (e.g., stools) within a few hours of specimen collection.

The release of Vi antigens from three clinical isolates of Salmonella typhi was measured by a Vi-specific monoclonal antibody. Large quantities of Vi antigens were detected in the culture supernates from all three strains using either passive latex agglutination or rocket immunoelectrophoresis. Vi antigens were also detected in broth cultures of S. typhi containing about 10(5) cells/ml using the sandwich enzyme linked immunosorbent assay. The significance of this finding in relationship to the virulence and the diagnosis of S. typhi was discussed.