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Manipulation of Salmonella Typhi Gene Expression Impacts Innate Cell Responses in the Human Intestinal Mucosa.

ABSTRACT: Although immunity induced by typhoid fever is moderated and short-lived, typhoid vaccination with the attenuated Ty21a oral vaccine generates long-lasting protection rates reaching up to 92%. Thus, there are important differences on how wild-type Salmonella and typhoid vaccine strains stimulate host immunity. We hypothesize that vaccine strains with different mutations might affect gut inflammation and intestinal permeability by different mechanisms. To test this hypothesis, we used an in vitro organotypic model of the human intestinal mucosa composed of human intestinal epithelial cells, lymphocytes/monocytes, endothelial cells, and fibroblasts. We also used six Salmonella enterica serovar Typhi (S. Typhi) strains: the licensed Ty21a oral vaccine, four typhoid vaccine candidates (i.e., CVD 908, CVD 909, CVD 910, and CVD 915) and the wild-type Ty2 strain. We found that genetically engineered S. Typhi vaccine strains elicit differential host changes not only in the intestinal permeability and secretion of inflammatory cytokines, but also in the phenotype and activation pathways of innate cells. These changes were distinct from those elicited by the parent wild-type S. Typhi and depended on the genetic manipulation. In sum, these results emphasize the importance of carefully selecting specific manipulations of the Salmonella genome in the development of typhoid vaccines.

SUBMITTER: Salerno-Goncalves R

PROVIDER: S-EPMC6221971 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundAlthough zebrafish are commonly used to study intestinal mucosal immunity, no dedicated procedure for isolating immune cells from zebrafish intestines is currently available. A speedy and simple operating approach for preparing cell suspension from mucosa has been devised to better understanding of intestinal cellular immunity in zebrafish.Methods and resultsThe mucosal villi were separated away from the muscle layer by repeated blows. The complete deprivation of mucosa was done and evidenced by HE and qPCR results. Higher expression of both innate (mpeg1, mpx, and lck) and adaptive immune genes (zap70, blnk, foxp3a, and foxp3b) was revealed compared to cells obtained by typical mesh rubbing. The cytometric results also revealed that the tested operation group had a higher concentration and viability. Further, fluorescent-labelled immune cells from 3mo Tg(lyz:DsRED2), Tg(mpeg1:EGFP), Tg(Rag2:DsRED), and Tg(lck:EGFP), were isolated and evaluated for the proportion, and immune cells' type could be inferred from the expression of marker genes. The transcriptomic data demonstrated that the intestinal immune cell suspension made using the new technique was enriched in immune-related genes and pathways, including il17a/f, il22, cd59, and zap70, as well as pattern recognition receptor signaling and cytokine-cytokine receptor interaction. In addition, the low expression of DEG for the adherent and close junctions indicated less muscular contamination. Also, lower expression of gel-forming mucus-associated genes in the mucosal cell suspension was consistent with the current less viscous cell suspension. To apply and validate the developed manipulation, enteritis was induced by soybean meal diet, and immune cell suspensions were analyzed by flow cytometry and qPCR. The finding that in enteritis samples, there was inflammatory increase of neutrophils and macrophages, was in line with upregulated cytokines (il8 and il10) and cell markers (mpeg1 and mpx).ConclusionAs a result, the current work created a realistic technique for studying intestinal immune cells in zebrafish. The immune cells acquired may aid in further research and knowledge of intestinal illness at the cellular level.

Project description:Salmonella enterica serotype Typhi is a human-restricted intracellular pathogen and the cause of typhoid fever. Cellular immune responses are required to control and clear Salmonella infection. Despite this, there are limited data on cellular immune responses in humans infected with wild type S. Typhi.For this work, we used an automated approach to purify a subset of S. Typhi proteins identified in previous antibody-based immuno-affinity screens and antigens known to be expressed in vivo, including StaF-putative fimbrial protein-STY0202, StbB-fimbrial chaperone-STY0372, CsgF-involved in curli production-STY1177, CsgD- putative regulatory protein-STY1179, OppA-periplasmic oligopeptide binding protein precursor-STY1304, PagC-outer membrane invasion protein-STY1878, and conserved hypothetical protein-STY2195; we also generated and analyzed a crude membrane preparation of S. Typhi (MP). In comparison to samples collected from uninfected Bangladeshi and North American participants, we detected significant interferon-? responses in PBMCs stimulated with MP, StaF, StbB, CsgF, CsgD, OppA, STY2195, and PagC in patients bacteremic with S. Typhi in Bangladesh. The majority of interferon-? expressing T cells were CD4 cells, although CD8 responses also occurred. We also assessed cellular proliferation responses in bacteremic patients, and confirmed increased responses in infected individuals to MP, StaF, STY2195, and PagC in convalescent compared to acute phase samples and compared to controls. StaF is a fimbrial protein homologous to E. coli YadK, and contains a Pfam motif thought to be involved in cellular adhesion. PagC is expressed in vivo under the control of the virulence-associated PhoP-regulon required for intra-macrophage survival of Salmonella. STY2195 is a conserved hypothetical protein of unknown function.This is the first analysis of cellular immune responses to purified S. Typhi antigens in patients with typhoid fever. These results indicate that patients generate significant CD4 and CD8 interferon-? responses to specific S. Typhi antigens during typhoid fever, and that these responses are elevated at the time of clinical presentation. These observations suggest that an interferon-? based detection system could be used to diagnose individuals with typhoid fever during the acute stage of illness.

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Manipulation of Salmonella Typhi Gene Expression Impacts Innate Cell Responses in the Human Intestinal Mucosa.

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