Exploring the adaptation mechanisms during anaerobic tetrathionate respiration in salmonella enterica serovar typhimurium

Abstract

Salmonella enterica serovar Typhimurium is a foodborne pathogen that infects both animals and humans and causes acute intestinal inflammation. During its infection of the hosts, S. Typhimurium is challenged with various physiological stresses including low oxygen tension, host derived antimicrobial peptides, reactive oxygen species, etc. To adapt to these environments, the pathogen often alters global protein expression profiles and activates various stress response and virulence proteins. Tetrathionate broth has long been utilized to selectively enrich Salmonella from clinical samples. Recent studies show that the metabolic interaction of microbiota and host immune response led to the production of tetrathionate in the inflamed gut. The capability of S. Typhimurium to respire tetrathionate provides significant selective advantages to outgrow under this condition. However, how S. Typhimurium adapts to this metabolic status remains largely unknown. In this thesis, I present a high-throughput study to investigate the functional proteomics of S. Typhimurium ATCC 14028s in response to tetrathionate respiration. Expression of 126 proteins is found to be altered during tetrathionate respiration compared to that during aerobic respiration. Among them, 23 proteins are down-regulated and 103 proteins are up-regulated, including the tetrathionate reductase TtrAB and thiosulphate reductase PhsA known to be upregulated under this condition. I subsequently examined two classes of these proteins which have not been indicated to be involved in tetrathionate metabolism previously but are up-regulated in our analysis: ScsBC and AcrD. My study showed that expression of ScsBC, a disulfide bond formation system, is up-regulated 35-fold during tetrathionate respiration. Deletion of scsBC leads to increased expression of the primary disulfide bond formation system DsbA and DsbC. This led me to hypothesize that protein disulfide bond formation is defected during anaerobic tetrathionate respiration, presumably due to the production of sulfide during this metabolic process. Indeed, LC-MS/MS identified Cys S-sulfhydration in four proteins, HisJ, STM3650, GlpQ, and OppA in S. Typhimurium cells undergo tetrathionate respiration. Phenotypic studies revealed that scsBC leads to decreased viability of the bacterium, increased sensitivity to copper (Cu), reduced swarming motility, and reduced secretion of SPI-1 effector SipB. In an in vivo assay, scsBC is also found to be necessary for the survival of S. Typhimurium in the intestine of C. elegans host, suggesting that ScsBC is important for the physiology and virulence of S. Typhimurium under this metabolic condition. In the next section, I show that tetrathionate respiration leads to 2-fold up-regulation of the multidrug efflux pump AcrD. Mutagenesis analysis revealed that this up-regulation is mediated by the envelope stress response regulators BaeSR and CpxAR, which are activated by the sulfide signal. Expression of acrD is further induced in ΔscsBC cells. Physiological analysis showed that ΔacrD results in increased sensitivity to aminoglycoside antibiotics and SDS. Expression of AcrD is also necessary for the survival of S. Typhimurium in J774A.1 macrophages. Taken together, my studies reveal two novel adaptation strategies S. Typhimurium utilizes to adapt to the physiologically relevant tetrathionate respiration condition. These studies advanced our understandings on the virulence and drug resistance development during bacterial stress adaptation.