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Mechanisms of substrate recognition by a typhoid toxin secretion-associated muramidase.

ABSTRACT: Typhoid toxin is a virulence factor for the bacterial pathogen Salmonella Typhi, which causes typhoid fever in humans. After its synthesis by intracellular bacteria, typhoid toxin is secreted into the lumen of the Salmonella-containing vacuole by a secretion mechanism strictly dependent on TtsA, a specific muramidase that facilitates toxin transport through the peptidoglycan layer. Here we show that substrate recognition by TtsA depends on a discrete domain within its carboxy terminus, which targets the enzyme to the bacterial poles to recognize YcbB-edited peptidoglycan. Comparison of the atomic structures of TtsA bound to its substrate and that of a close homolog with different specificity identified specific determinants involved in substrate recognition. Combined with structure-guided mutagenesis and in vitro and in vivo crosslinking experiments, this study provides an unprecedented view of the mechanisms by which a muramidase recognizes its peptidoglycan substrate to facilitate protein secretion.

SUBMITTER: Geiger T

PROVIDER: S-EPMC6996933 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Mechanisms of substrate recognition by a typhoid toxin secretion-associated muramidase.

Geiger Tobias T Lara-Tejero Maria M Xiong Yong Y Galán Jorge E JE

eLife 20200120

Typhoid toxin is a virulence factor for the bacterial pathogen <i>Salmonella</i> Typhi, which causes typhoid fever in humans. After its synthesis by intracellular bacteria, typhoid toxin is secreted into the lumen of the <i>Salmonella</i>-containing vacuole by a secretion mechanism strictly dependent on TtsA, a specific muramidase that facilitates toxin transport through the peptidoglycan layer. Here we show that substrate recognition by TtsA depends on a discrete domain within its carboxy termi ...[more]

PMID: 31958059

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Project description:Typhoid fever caused by Salmonella enterica serovar Typhi (S.Typhi) continues to be a major problem, especially in developing countries. Due to the rapid emergence of multi-drug-resistant (MDR) strains, which limits the efficacy of conventional antibiotics as well as problems associated with the existing vaccines, efforts are being made to develop effective prophylactic agents. CdtB subunit of typhoid toxin was selected for assessing its vaccine potential due to its high conservation throughout the Typhi strains. In-vitro assessment of DNase activity of cloned and purified CdtB protein showed a significant decrease in the band intensity of DNA. The measure of metabolic activity and morphological alterations assessed using different cell lines in the presence of CdtB protein showed no significant signs of toxicity. These observations were further strengthened by cell cycle analysis, assessed by flow cytometry. Keeping these observations in mind, the immunoprotective potential of CdtB was assessed using S.Typhi induced mouse peritonitis model. A significant titer of IgG antibodies (>128000) against CdtB protein was recorded in the immunized mice by enzyme-linked immunosorbent assay (ELISA), which was also validated by immunoblotting. Active immunization with the protein protected 75% mice against a lethal dose of S.Typhi Ty2. The data indicated a significant (up to 5 log) reduction in the bacterial load in the spleen and liver of immunized-infected mice compared to control (unimmunized-infected) mice which might have resulted in the modulation of histoarchitecture of spleen and liver and the levels of cytokines (IL-6, TNF-α and IL-10) production; thereby indicating the effectiveness of the subunit. The observations deduced from the study give the proof of concept of immunogenic potential of protein. However, further studies involving the immunoreactivity of CdtB with the statistically significant number of sera samples obtained from the human patients would be helpful in establishing the relevance of CdtB protein in humans and for making the strategies to develop it as an effective vaccine candidate.

Dataset Information

Mechanisms of substrate recognition by a typhoid toxin secretion-associated muramidase.

Publications

Mechanisms of substrate recognition by a typhoid toxin secretion-associated muramidase.

Similar Datasets

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