Project description:Bacterial type III protein secretion systems inject effector proteins into eukaryotic host cells in order to promote survival and colonization of Gram-negative pathogens and symbionts. Secretion across the bacterial cell envelope and injection into host cells is facilitated by a so-called needle complex. Its small hydrophobic export apparatus components SpaP and SpaR were shown to nucleate assembly of the needle complex and to form the central “cup” substructure of a Salmonella Typhimurium secretion system, however, the in vivo placement of these components in the needle complex and their function during the secretion process remained poorly defined. Here we show that a SpaP pentamer forms a 15 Å wide pore and present a detailed map of SpaP interactions with the export apparatus components SpaQ, SpaR, and SpaS. We further demonstrate the formation of a continuous conduit for substrate translocation and injection by intimate interactions of the periplasmic domains of SpaP and SpaR with the inner rod protein PrgJ, refine the current view of export apparatus assembly and consolidate transmembrane topology models for SpaP and SpaR.
Project description:affy_xoo_rice - affy_xoo_rice - The Bacterial Leaf Blight disease of rice is due to Xanthomonas oryzae pv. oryzae. As for many pathogenic bacteria, it relies on a type 3 secretion system that is devoted to the injection of type 3 effectors into the eukaryotic host cell. These proteins are meant to suppress host basal defense responses and/or mimic some host regulatory function promoting bacterial survey in the plant. We are interested in the functional analysis of a subgroup of Xoo T3Es, that are specialized in host cell transcriptome remodelling. These effectors, therefore called TAL for Transcription Activator-Like proteins (also named AvrBs3/PthA-like), are often key virulence factors essential to Xoo pathogenicity such as the effector protein Talc of african Xoo strain BAI3. Our goal is to understand its function during disease development, by identifying rice host genes that are being directly up- or down-regulated by Talc. To that end, we aim at performing Affymetrix transcriptomic analysis, comparing leaf samples of a susceptible rice line inoculated with Xoo to leaves challenged with a Talc-deficient mutant and water-treated leaves. Highly induced genes are likely to be Talc primary targets and therefore potentially good susceptibility gene candidates.-The goal of the experiment is to identify the rice genes up- or down-regulated by the type III effector Talc from Xoo African strain BAI3, upon the inoculation of susceptible rice leaves 24 hours post-infection. To that end, the experimental design includes the inoculation of Nipponbare rice leaves with the virulent Xoo strain BAI3, that will be compared to Nipponbare rice leaves inoculated with a talc K.O. mutant strain and water. Keywords: wt vs virulence mutant 9 arrays - rice
Project description:<p><em>Salmonella Typhimurium</em> establishes systemic infection by replicating in host macrophages. Here we show that macrophages infected with <em>S. Typhimurium</em> exhibit upregulated glycolysis and decreased serine synthesis, leading to accumulation of glycolytic intermediates. The effects on serine synthesis are mediated by bacterial protein SopE2, a type III secretion system (T3SS) effector encoded in pathogenicity island SPI-1. The changes in host metabolism promote intracellular replication of <em>S. Typhimurium</em> via two mechanisms: decreased glucose levels lead to upregulated bacterial uptake of 2- and 3-phosphoglycerate and phosphoenolpyruvate (carbon sources), while increased pyruvate and lactate levels induce upregulation of another pathogenicity island, SPI-2, known to encode virulence factors. Pharmacological or genetic inhibition of host glycolysis, activation of host serine synthesis, or deletion of either the bacterial transport or signal sensor systems for those host glycolytic intermediates impairs <em>S. Typhimurium</em> replication or virulence.</p>
Project description:SrfJ is an effector of the type III secretion systems of the Gram-negative intracellular pathogen Salmonella enterica serovar Typhimurium. To study the effects of this effector on global gene expression in host cells, we have infected murine RAW264.7 macrophages with two strains of Salmonella enterica serovar Typhimurium. The comparison between cells infected with the wild-type strain and cells infected with a srfJ mutant revealed a number of genes that are differentially expressed when SrfJ is present.
Project description:SrfJ is an effector of the type III secretion systems of the Gram-negative intracellular pathogen Salmonella enterica serovar Typhimurium. To study the effects of this effector on global gene expression in host cells, we have expressed SrfJ on human HeLa cells through transient transfection. The comparison with HeLa cells transfected with a plasmid not expressing SrfJ, revealed a number of genes that are differentially expressed when SrfJ is present.
Project description:Injection of effectors via a type III secretion system (T3SS) is an infection strategy shared by many Gram-negative bacterial pathogens. While individual T3SS effectors are well characterized, their network-level organization and the distinction between core and accessory effectors remain incompletely understood. Here, we systematically dissect the T3SS effector network from Citrobacter rodentium (CR), identifying a subset of 12 accessory effectors that, while dispensable for colonization, significantly alter infection outcomes. A strain lacking these effectors (CRM12) remained virulent in susceptible hosts yet resulted in reduced epithelial barrier damage, inflammation and immune cell infiltration in resistant mouse hosts. Deep proteomic analysis specifically targeting CR-attached colonic epithelial cells revealed that, despite lacking 39% of its effector repertoire, infection with CRM12 results in similar changes to global protein expression as seen in mice infected with the wild-type strain, though key regulators of barrier disruption were differentially expressed. Using a host model with impaired barrier repair, we confirmed that accessory effectors shape infection dynamics without significantly impacting virulence. This study thus refines the concept of core and accessory effectors, providing a basis for further studies into effector-driven host adaptation.
Project description:In the context of host-pathogen interactions, gram-negative bacterial virulence factors, such as effectors, may be transferred from bacterial to eukaryotic host cytoplasm by multicomponent Type III protein secretion systems (T3SSs). Central to Salmonella enterica serovar Typhimurium (S. Typhimurium) pathogenesis is the secretion of over 40 effectors by two T3SSs encoded within pathogenicity islands SPI-1 and SPI-2. These effectors manipulate miscellaneous host cellular processes, such as cytoskeleton organization and immune signaling pathways, thereby permitting host colonization and bacterial dissemination. Recent research on effector biology provided mechanistic insights for some effectors. However, for many effectors, clearly defined roles and host target repertoires—further clarifying effector interconnectivity and virulence networks—are yet to be uncovered. Here we demonstrate the utility of the recently described viral-like particle trapping technology Virotrap as an effective approach to catalogue S. Typhimurium effector-host protein complexes (EH-PCs). Mass spectrometry-based Virotrap analysis of the novel E3 ubiquitin ligase SspH2 previously shown to be implicated in modulating actin dynamics and immune signaling, exposed known host interactors PFN1 and -2 and several putative novel, interconnected host targets. Network analysis revealed an actin(-binding) cluster among the significantly enriched hits for SspH2, consistent with the known localization of the S-palmitoylated effector with actin cytoskeleton components in the host. We show that Virotrap complements the current state-of-the-art toolkit to study protein complexes and represents a valuable means to screen for effector host targets in a high-throughput manner, thereby bridging the knowledge gap between effector-host interplay and pathogenesis.
Project description:Xyloglucans are highly substituted polysaccharides found in the primary cell walls of all vascular plants, acting as a first barrier against pathogens. Herein, we revealed that the diverse and economically relevant Xanthomonas phytopathogens are endowed with an enzymatic system targeting xyloglucans. Furthermore, we demonstrate that the products generated by this system elicit the expression of multiple virulence factors including the type III secretion system, a membrane-embedded machinery to deliver effector proteins into the host cells. Together, these findings illuminate the intricate enzymatic apparatus employed by Xanthomonas to break down xyloglucans and connect this system to the pathogenesis through activating the expression of key virulence factors.