Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Yersinia type three secretion system effectors cooperate to suppress the major immune pathways in primary human macrophages

ABSTRACT: Numerous bacterial pathogens utilize type 3 secretion systems that inject effector proteins into host immune cells to support their pathogenesis. We investigated the T3SS effectors (Yops) of Yersinia enterocolitica for their individual and combined effects on gene expression, inflammasome formation and Ca2+ signaling in primary human macrophages. The up- or down-regulation of thousands of macrophage genes by Yersinia inflammatory stimuli was effectively suppressed by YopP, which correlated with regulation of histone phosphorylation at H3S10ph, and YopM and YopQ systematically counteracting selected YopP transcriptional effects. Inflammasome formation was reduced by a combination of YopP and YopQ, but not by single effectors or other combinations of them. Intracellular Ca2+ transients were blocked by YopH alone. Thus, the T3SS effectors of Yersinia individually, synergistically or antagonistically suppress the major immune pathways of human macrophages to dampen the immune response.

INSTRUMENT(S): NextSeq 500

ORGANISM(S): Homo sapiens

SUBMITTER: Indra Bekere

PROVIDER: E-MTAB-14995 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Many species of pathogenic bacteria harbor critical plasmid-encoded virulence factors, and yet the regulation of plasmid replication is often poorly understood despite playing key role in plasmid-encoded gene expression. Human pathogenic Yersinia, including the plague agent Yersinia. pestis and its close relative Y. pseudotuberculosis, require the type III secretion system (T3SS) virulence factor to subvert host defense mechanisms and colonize host tissues. The Yersinia T3SS is encoded on the IncFII plasmid for Yersinia virulence (pYV). Several layers of gene regulation enables a large increase in expression of Yersinia T3SS genes at mammalian body temperature. Surprisingly, T3SS expression is also controlled at the level of gene dosage. The number of pYV molecules relative to the number of chromosomes per cell, referred to as plasmid copy number, increases with temperature. The ability to increase and maintain elevated pYV plasmid copy number, and therefore T3SS gene dosage, at 37˚C is important for Yersinia virulence. In addition, pYV is highly stable in Yersinia at all temperatures, despite being dispensable for growth outside the host. Yet how Yersinia reinforces elevated plasmid replication and plasmid stability remains unclear. In this study, we show that the chromosomal gene pcnB encoding the polyadenylase PAP I is required for regulation of pYV plasmid copy number (PCN), maintenance of pYV in the bacterial population outside the host, robust T3SS activity, and Yersinia virulence in a mouse infection model. Likewise, pcnB/PAP I is also required for robust expression of the Shigella flexneri virulence plasmid-encoded T3SS that, similar to Yersinia, is encoded on a virulence plasmid whose replication is regulated by sRNA. Furthermore, Yersinia and Shigella pcnB/PAP I is required for maintaining normal PCN of model antimicrobial resistance (AMR) plasmids whose replication is regulated by sRNA, thereby increasing antibiotic resistance by ten-fold. These data suggest that pcnB/PAP I contributes to the spread and stabilization of sRNA-regulated virulence and AMR plasmids in bacterial pathogens, and is essential in maintaining the gene dosage required to mediate plasmid-encoded traits. Importantly pcnB/PAP I has been bioinformatically identified in many species of bacteria despite being studied in only a few species to date. Our work highlights the potential importance of pcnB/PAP I in antibiotic resistance, and shows for the first time that pcnB/PAP I reinforces PCN andpromotes virulence plasmid stability in natural pathogenic hosts with a direct impact on bacterial virulence.

Project description:Many Gram-negative bacterial pathogens interact with mammalian cells by using type III secretion systems (T3SS) to inject virulence proteins directly into infected host cells. A subset of these injected protein ‘effectors’ are enzymes that modify the structure and inhibit the function of human proteins by catalyzing the addition of unusual post-translational modifications. T3SS effectors play essential roles in bacterial virulence and their modes of action have provided great insight into the functions and the components of the innate immune system. The E. coli and Citrobacter rodentium NleB effectors, as well as the Salmonella enterica SseK effectors are glycosyltransferases that modify host protein substrates with N-acetyl glucosamine (GlcNAc) on arginine residues. Arginine glycosylation is unusual because it occurs on the guanidinium groups of arginines, which are poor nucleophiles. This post-translational modification disrupts the normal functioning of host immune response proteins. T3SS effectors are chaperoned in the bacterium to keep the effectors partially unfolded and competent for secretion, as well as for targeting the effectors to the T3SS sorting platform. The chaperones are then stripped from their effector substrates at the sorting platform and the effectors are secreted in an unfolded conformation. T3SS effectors are thought to be inactive within the bacterium and fold into their active conformations after they are injected into host cells. While performing mass spectrometry experiments to identify glycosylation substrates of NleB orthologs, we unexpectedly observed that the E. coli glutathione synthetase (GshB) is glycosylated on an arginine residue (R256) by NleB. NleB glycosyltransferase activity is essential to C. rodentium survival in oxidative stress conditions because glycosylation of GshB results in enhanced glutathione production. These data represent, to our knowledge, the first intra-bacterial activity for a T3SS effector and show that effector activities thought to be restricted to host cell compartments additionally play important roles in regulating bacterial physiology.

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Yersinia type three secretion system effectors cooperate to suppress the major immune pathways in primary human macrophages

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