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Salmonella typhi encodes a functional cytolethal distending toxin that is delivered into host cells by a bacterial-internalization pathway.

ABSTRACT: Many bacterial pathogens encode the cytolethal distending toxin (CDT), which causes host cells to arrest during their cell cycle by inflicting DNA damage. CDT is composed of three proteins, CdtA, CdtB, and CdtC. CdtB is the enzymatically active or A subunit, which possesses DNase I-like activity, whereas CdtA and CdtC function as heteromeric B subunits that mediate the delivery of CdtB into host cells. We show here that Salmonella enterica serovar Typhi encodes CDT activity, which depends on the function of a CdtB homologous protein. Remarkably, S. enterica serovar Typhi does not encode apparent homologs of CdtA or CdtC. Instead, we found that toxicity, as well as cdtB expression, requires bacterial internalization into host cells. We propose a pathway of toxin delivery in which bacterial internalization relieves the requirement for the functional equivalent of the B subunit of the CDT toxin.

SUBMITTER: Haghjoo E

PROVIDER: S-EPMC384795 | biostudies-literature | 2004 Mar

REPOSITORIES: biostudies-literature

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Salmonella typhi encodes a functional cytolethal distending toxin that is delivered into host cells by a bacterial-internalization pathway.

Haghjoo Erik E Galán Jorge E JE

Proceedings of the National Academy of Sciences of the United States of America 20040301 13

Many bacterial pathogens encode the cytolethal distending toxin (CDT), which causes host cells to arrest during their cell cycle by inflicting DNA damage. CDT is composed of three proteins, CdtA, CdtB, and CdtC. CdtB is the enzymatically active or A subunit, which possesses DNase I-like activity, whereas CdtA and CdtC function as heteromeric B subunits that mediate the delivery of CdtB into host cells. We show here that Salmonella enterica serovar Typhi encodes CDT activity, which depends on the ...[more]

PMID: 15070766

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Project description:BackgroundAggregatibacter actinomycetemcomitans, a periodontal pathogen, secretes a cytolethal distending toxin (AaCDT) that causes host cell cycle arrest and cell death. Although CDT could be an important virulence factor, it is unclear how it enters the nucleus to exert its cytotoxicity.ObjectiveTo investigate the mechanisms of AaCDT by genome-wide screening for host mutations that confer resistance to the catalytic subunit, AaCdtB, in a Saccharomyces cerevisiae model.MethodsWe transformed the yeast haploid deletion library, a collection of yeast mutants with single gene deletions of virtually all non-essential ORFs in the genome, with plasmids carrying galactose-inducible AaCdtB. Yeast mutants that showed resistance to AaCdtB were selected and rescreened by a spotting assay. AaCdtB expression was confirmed by western blot analysis; any mutants that showed no or weak expression of AaCdtB were omitted from the analysis. The lists of genes whose mutations confer resistance to AaCdtB were analyzed for Gene Ontology (GO) term enrichments. Localization of AaCdtB-EGFP was examined using fluorescent microscopy. Nuclear localization relative to EGFP control was calculated and compared to wild-type.ResultsOut of approximately 5,000 deletion mutants, we isolated 243 mutants that are resistant to AaCdtB. GO analyses indicated that genes associated with organic anion transport are significantly enriched (16 genes). Furthermore, several genes associated with the nucleus and endoplasmic reticulum (ER) were identified. Localization studies of AaCdtB, in mutants with the deletion of genes associated with the GO term organic anion transport, showed lower nuclear localization than wild-type. The results suggest that these genes may be required for AaCdtB translocation into the nucleus and its cytotoxicity.ConclusionThe genome-wide screen in the yeast deletion library allowed us to identify a large number of host genes required for AaCdtB cytotoxicity. Further investigation could lead to more insights into the mechanisms of CdtB intoxication.

Project description:Select nontyphoidal Salmonella enterica (NTS) serotypes were recently found to encode the Salmonella cytolethal distending toxin (S-CDT), an important virulence factor for serotype Typhi, the causative agent of typhoid fever. Using a PCR-based assay, we determined that among 21 NTS serotypes causing the majority of food-borne salmonellosis cases in the United States, genes encoding S-CDT are conserved in isolates representing serotypes Javiana, Montevideo, and Oranienburg but that among serotype Mississippi isolates, the presence of S-CDT-encoding genes is clade associated. HeLa cells infected with representative strains of these S-CDT-positive serotypes had a significantly higher proportion of cells arrested in the G2/M phase than HeLa cells infected with representative strains of S-CDT-negative serotypes Typhimurium, Newport, and Enteritidis. The G2/M cell cycle arrest was dependent on CdtB, the active subunit of S-CDT, as infection with isogenic ΔcdtB mutants abolished their ability to induce a G2/M cell cycle arrest. Infection with S-CDT-encoding serotypes was significantly associated with activation of the host cell's DNA damage response (DDR), a signaling cascade that is important for detecting and repairing damaged DNA. HeLa cell populations infected with S-CDT-positive serotypes had a significantly higher proportion of cells with DDR protein 53BP1 and γH2AX foci than cells infected with either S-CDT-negative serotypes or isogenic ΔcdtB strains. Intoxication with S-CDT occurred via autocrine and paracrine pathways, as uninfected HeLa cells among populations of infected cells also had an activated DDR. Overall, we show that S-CDT plays a significant role in the cellular outcome of infection with NTS serotypes. The recent discovery that multiple serotypes encode S-CDT, which was previously established as an important virulence factor for serotype Typhi, suggested that this toxin may also contribute to the outcome of infection with nontyphoidal serotypes. In this study, we demonstrate that at a cellular level, S-CDT significantly alters the outcome of infection by inducing DNA damage which is associated with a cell cycle arrest and activation of the host cell's DDR. Importantly, these results contribute valuable information for assessing the public health implications of S-CDT in infections with NTS serotypes. Our data suggest that infection with Salmonella strains that encode S-CDT has the potential to result in DNA damage, which may contribute to long-term sequelae.

Dataset Information

Salmonella typhi encodes a functional cytolethal distending toxin that is delivered into host cells by a bacterial-internalization pathway.

Publications

Salmonella typhi encodes a functional cytolethal distending toxin that is delivered into host cells by a bacterial-internalization pathway.

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