S. Typhimurium sseJ gene decreases the S. Typhi cytotoxicity toward cultured epithelial cells

<p>Abstract</p> <p>Background</p> <p><it>Salmonella enterica </it>serovar Typhi and Typhimurium are closely related serovars as indicated by >96% DNA sequence identity between shared genes. Nevertheless, <it>S</it>. Typhi is a strictly human-specific pathogen causing a systemic disease, typhoid fever. In contrast, <it>S</it>. Typhimurium is a broad host range pathogen causing only a self-limited gastroenteritis in immunocompetent humans. We hypothesize that these differences have arisen because some genes are unique to each serovar either gained by horizontal gene transfer or by the loss of gene activity due to mutation, such as pseudogenes. <it>S</it>. Typhi has 5% of genes as pseudogenes, much more than <it>S</it>. Typhimurium which contains 1%. As a consequence, <it>S</it>. Typhi lacks several protein effectors implicated in invasion, proliferation and/or translocation by the type III secretion system that are fully functional proteins in <it>S</it>. Typhimurium. SseJ, one of these effectors, corresponds to an acyltransferase/lipase that participates in SCV biogenesis in human epithelial cell lines and is needed for full virulence of <it>S</it>. Typhimurium. In <it>S</it>. Typhi, <it>sseJ </it>is a pseudogene. Therefore, we suggest that <it>sseJ </it>inactivation in <it>S</it>. Typhi has an important role in the development of the systemic infection.</p> <p>Results</p> <p>We investigated whether the <it>S</it>. Typhi <it>trans</it>-complemented with the functional <it>sseJ </it>gene from <it>S</it>. Typhimurium (STM) affects the cytotoxicity toward cultured cell lines. It was found that <it>S</it>. Typhi harbouring <it>sseJ_STM</it>presents a similar cytotoxicity level and intracellular retention/proliferation of cultured epithelial cells (HT-29 or HEp-2) as wild type <it>S</it>. Typhimurium. These phenotypes are significantly different from wild type <it>S</it>. Typhi</p> <p>Conclusions</p> <p>Based on our results we conclude that the mutation that inactivate the <it>sseJ </it>gene in <it>S</it>. Typhi resulted in evident changes in the behaviour of bacteria in contact with eukaryotic cells, plausibly contributing to the <it>S</it>. Typhi adaptation to the systemic infection in humans.</p

stg fimbrial operon from S. Typhi STH2370 contributes to association and cell disruption of epithelial and macrophage-like cells

BACKGROUND: Salmonella enterica serovar Typhi (S. Typhi) stg operon, encoding a chaperone/usher fimbria (CU), contributes to an increased adherence to human epithelial cells. However, one report suggests that the presence of the Stg fimbria impairs the monocyte-bacteria association, as deduced by the lower level of invasion to macrophage-like cells observed when the stg fimbrial cluster was overexpressed. Nevertheless, since other CU fimbrial structures increase the entry of S. Typhi into macrophages, and considering that transcriptomic analyses revealed that stg operon is indeed expressed in macrophages, we reassessed the role of the stg operon in the interaction between S. Typhi strain STH2370 and human cells, including macrophage-like cells and mononuclear cells directly taken from human peripheral blood. RESULTS: We compared S. Typhi STH2370 WT, a Chilean clinical strain, and the S. Typhi STH2370 Astg mutant with respect to association and invasion using epithelial and macrophage-like cells. We observed that deletion of stg operon reduced the association and invasion of S. Typhi, in both cellular types. The presence of the cloned stg operon restored the WT phenotype in all the cases. Moreover, we compared Salmonella enterica sv. Typhimurium 14028s (S. Typhimurium, a serovar lacking stg operon) and S. Typhimurium heterologously expressing S. Typhi stg. We found that the latter presents an increased cell disruption of polarized epithelial cells and an increased association in both epithelial and macrophage-like cells. CONCLUSIONS: S. Typhi stg operon encodes a functional adhesin that participates in the interaction bacteria-eukary-otic cells, including epithelial cells and macrophages-like cells. The phenotypes associated to stg operon include increased association and consequent invasion in bacteria-eukaryotic cells, and cell disruption

Insertions of mini-Tn10 transposon T-POP in Salmonella enterica sv. typhi.

We have mutagenized a clinical strain of Salmonella enterica sv. typhi with mini-transposon Tn10dTet (T-POP) to obtain conditional lethal (tetracycline-dependent) mutants with T-POP insertions upstream of essential genes. Generalized transducing phage P22 was used to introduce T-POP from a S. typhimurium donor into a S. typhi recipient. Chromosomal DNA was purified from the mutagenized donor strains, fragmented, and then electroporated into S. typhi to backcross the original T-POP insertions. Four tetracycline-dependent mutants with two distinct terminal phenotypes were found among 1700 mutants with T-POP insertions. When grown in the absence of tetracycline, two of the four tetracycline-dependent mutants arrest at a late stage in the cell cycle, can be rescued by outgrowth in media with tetracycline, and define a reversible checkpoint late in the cell cycle. One of these insertions creates an operon fusion with a gene, yqgF, that is conserved among gram-negative bacteria and likely encodes an essential Holliday junction resolvase. T-POP insertions can be used not only to identify essential S. typhi genes but also to reveal novel phenotypes resulting from the depletion of their products

The Salmonella enterica Serovar Typhi tsx Gene, Encoding a Nucleoside-Specific Porin, Is Essential for Prototrophic Growth in the Absence of Nucleosides

The Salmonella enterica serovar Typhi tsx gene encodes a porin that facilitates the import of nucleosides. When serovar Typhi is grown under anaerobic conditions, Tsx is among the outer membrane proteins whose expression increases dramatically. This increase in expression is due, at least in part, to increased transcription and is dependent on Fnr but not on ArcA. A mutant derivative of serovar Typhi strain STH2370 with a deletion of the tsx gene is an auxotroph that requires either adenosine or thymidine for growth on minimal medium. In contrast, an otherwise isogenic nupG nupC double mutant, defective in the inner membrane nucleoside permeases, is a prototroph. Because anaerobic growth enhances the virulence of serovar Typhi in vitro, we assessed the role that the tsx gene plays in pathogenicity and found that the serovar Typhi STH2370 Δtsx mutant is defective in survival within human macrophage-like U937 cells. To understand why the Δtsx mutant is an auxotroph, we selected for insertions of minitransposon T-POP in the Δtsx genetic background that restored prototrophy. One T-POP insertion that suppressed the Δtsx mutation in the presence of the inducer tetracycline was located upstream of the pyrD gene. The results of reverse transcription-PCR analysis showed that addition of the inducer decreased the rate of pyrD transcription. These results suggest that the Tsx porin and the balance of products of the tsx and pyrD genes play critical roles in membrane assembly and integrity and thus in the virulence of serovar Typhi

Precise Excision of the Large Pathogenicity Island, SPI7, in Salmonella enterica Serovar Typhi

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