Project description:Identification of human proteins targeted by Citrobacter secreted effectors by affinity purification followed by mass spectrometry.
Project description:Strains of Salmonella utilise two distinct type three secretion systems to deliver effector proteins directly into host cells. The Salmonella effectors SseK1 and SseK3 are arginine glycosyltransferases that modify mammalian death domain containing proteins with N-acetyl glucosamine (GlcNAc) when overexpressed ectopically or as recombinant protein fusions. Here, we combined Arg-GlcNAc glycopeptide immunoprecipitation and mass spectrometry to identify host proteins GlcNAcylated by endogenous levels of SseK1 and SseK3 during Salmonella infection. We observed that SseK1 modified the mammalian signaling protein TRADD, but not FADD as previously reported. Overexpression of SseK1 greatly broadened substrate specificity, while ectopic co-expression of SseK1 and TRADD increased the range of modified arginine residues within the death domain of TRADD. In contrast, endogenous levels of SseK3 resulted in modification of the death domains of receptors of the mammalian TNF superfamily, TNFR1 and TRAILR, at residues Arg376 and Arg293 respectively. Structural studies on SseK3 showed that the enzyme displays a classic GT-A glycosyltransferase fold and binds UDP-GlcNAc in a narrow and deep cleft with the GlcNAc facing the surface. Together our data suggests that Salmonellae carrying sseK1 and sseK3 employ the glycosyltransferase effectors to antagonise different components of death receptor signaling.
Project description:Strains of Salmonella utilise two distinct type three secretion systems to deliver effector proteins directly into host cells. The Salmonella effector SseK3 is a arginine glycosyltransferase that modify mammalian proteins with N-acetyl glucosamine (GlcNAc). Here, we used Arg-GlcNAc glycopeptide immunoprecipitation and mass spectrometry appraoch to identify host proteins GlcNAcylated by endogenous levels of SseK3 during Salmonella infection. Focusing on the insoluble proteome we demostrate multiple typically insoluble proteins are targetted by SseK3 during infections. Surpisingly unlike SseK1 and the EPEC homologue NleB1 which target death domain contain proteins SseK3 appears to modify a number of proteins lacking death domains. These results demostrate that under endogenous infection non-death domain containing proteins can be modified by Salmonella.
Project description:Comparison of Campylobacter proteome in MH media with and without deoxycholic acid, in presence of FBS or after being exposed to INT 407 and Caco2 intestinal epithelial cells.
Project description:Utilized sensitive, high throughput multiplexed ion mobility-mass spectrometry (IM-MS) to characterize the serum proteome of tuberculosis patients prior to and at 8 weeks of antibiotic treatment. Goal is to identify a serum protein signature indicative of treatment effect.
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:Many bacterial infections are hard to treat and tend to relapse, possibly due to the presence of antibiotic-tolerant persisters that are considered dormant cells when bacteria are grown in laboratory medium. Non-growing persisters also form following uptake of Salmonella by macrophages but their nature is little understood. Here we show that Salmonella persisters arising during macrophage infection maintain an active state. Persisters reprogram macrophages by means of effectors secreted by the SPI-2 Type 3 Secretion System (T3SS), thereby dampening pro-inflammatory innate immune responses and inducing anti-inflammatory macrophage polarisation. Reprogramming allows non-growing Salmonella to survive for extended periods in their host. Persisters undermining the host immune defences might confer an advantage to the pathogen during relapse once the antibiotic pressure is relieved.