Pathogen invasion-dependent tissue reservoirs and plasmid-encoded antibiotic degradation boost plasmid spread in the gut

Many plasmids encode antibiotic resistance genes. Through conjugation, plasmids can be rapidly disseminated. Previous work identified gut luminal donor/recipient blooms and tissue-lodged plasmid-bearing persister cells of the enteric pathogen; Salmonella enterica; serovar Typhimurium (; S; .Tm) that survive antibiotic therapy in host tissues, as factors promoting plasmid dissemination among Enterobacteriaceae. However, the buildup of tissue reservoirs and their contribution to plasmid spread await experimental demonstration. Here, we asked if re-seeding-plasmid acquisition-invasion cycles by; S; .Tm could serve to diversify tissue-lodged plasmid reservoirs, and thereby promote plasmid spread. Starting with intraperitoneal mouse infections, we demonstrate that; S; .Tm cells re-seeding the gut lumen initiate clonal expansion. Extended spectrum beta-lactamase (ESBL) plasmid-encoded gut luminal antibiotic degradation by donors can foster recipient survival under beta-lactam antibiotic treatment, enhancing transconjugant formation upon re-seeding.; S; .Tm transconjugants can subsequently re-enter host tissues introducing the new plasmid into the tissue-lodged reservoir. Population dynamics analyses pinpoint recipient migration into the gut lumen as rate-limiting for plasmid transfer dynamics in our model. Priority effects may be a limiting factor for reservoir formation in host tissues. Overall, our proof-of-principle data indicates that luminal antibiotic degradation and shuttling between the gut lumen and tissue-resident reservoirs can promote the accumulation and spread of plasmids within a host over time

The SseK effector proteins of Salmonella Typhimurium target host cell signaling proteins

© 2019 Joshua Patrick Mark NewsonPathogenic serovars of Salmonella are the causative agents of a variety of disease states, including typhoid fever, self-limiting gastroenteritis, and invasive bacteremia. To achieve infection, Salmonella relies on two type-three secretion systems (T3SS) to deliver distinct cohorts of effector proteins into host cells. These effector proteins interact with specific human proteins to subvert normal cellular processes, thus impairing the ability of host cells to respond to the invading bacteria. To date, more than 40 different effector proteins have been identified, though many remain poorly characterised. This thesis focused on the SseK family of effector proteins, which had a largely unknown molecular mechanism and role in Salmonella infection. The aim of this thesis was to identify the host proteins that are targeted by the SseK effectors in order to determine how these effectors contribute to Salmonella virulence. The SseK effectors show strong sequence similarity to NleB1, a unique T3SS effector of enteropathogenic E. coli, which functions as an arginine glycosyltransferase and catalyses the addition of N-acetylglucosamine (GlcNAc) to arginine residues of the mammalian signaling adaptors FADD and TRADD. Based on strong sequence homology to NleB1, we predicted that the SseK effectors would similarly catalyse arginine glycosylation. Here, we determined that SseK1 and SseK3, but not SseK2, also function as arginine glycosyltransferases. We showed that these effectors catalyse arginine glycosylation of different host proteins and appear to play different roles during infection. We developed a mass spectrometry-based strategy to enrich for arginine glycosylated peptides from host cells infected with Salmonella Typhimurium (S. Typhimurium). Using this approach, we identified the preferred substrate of SseK1 as the signaling adaptor TRADD, which participates in a range of innate immune signaling pathways. We also showed that overexpression of SseK1 broadens the range of glycosylated substrates, and that SseK1 was capable of glycosylating both mammalian and bacterial proteins under these conditions. Further, we identified the site of glycosylation within TRADD, and using a mutagenesis approach we showed that SseK1 is also capable of glycosylating secondary sites within TRADD. Collectively, these data show that the preferred substrate of SseK1 is TRADD, and highlight the importance of studying effectors in the natural context of infection. Next, we applied our strategy for enriching arginine glycosylated peptide to identify the substrates of SseK3. We identified the host signaling receptors TNFR1 and TRAILR as the preferred substrates of SseK3 during S. Typhimurium infection, and conducted a range of experiments to validate the glycosylation of these receptors and identify the specific residues that are modified. We also conducted preliminary analyses to explore the contribution of these glycosylation events to virulence in vivo. Together, the data presented in this thesis demonstrate that the S. Typhimurium effectors SseK1 and SseK3 function as arginine glycosyltransferases that target different innate immune signaling proteins during infection. We showed that SseK1 prefers the adaptor protein TRADD while SseK3 targets the signaling receptors TNFR1 and TRAILR. These observations provide new mechanisms by which Salmonella may manipulate innate immune signaling during infection