Expanding the repertoire of protein disulfide catalyzing machineries : functions of the SCS proteins in Salmonella enterica serovar Typhimurium

Abstract

Salmonella enterica serovar Typhimurium is a foodborne pathogen that makes both humans and animals sick. The pathogen blooms during gut inflammation owing to its capability to utilize the products (tetrathionate, nitrate) generated in the inflamed gut for anaerobic respiration. Suppression of copper sensitivity (Scs) proteins were first identified with the function of conferring copper tolerance in Salmonella. Previous work found that the production of ScsB and ScsC was upregulated more than 30-fold under anaerobic tetrathionate conditions. However, the functions of Scs proteins under anaerobic tetrathionate conditions are yet to be revealed. In this thesis, I employed a series of proteome-based approaches to identify potential substrates and binding partners of ScsC. First, comparative proteomics analysis of S. Typhimurium wild type (WT) and ΔscsBC under anaerobic tetrathionate conditions was performed. The expression of 59 proteins was found to be altered in ΔscsBC relative to the WT strain, and RT-qPCR analysis displayed that ΔscsBC resulted in reduced expression of STM14_4612. Second, a glutathione S-transferase (GST) pull-down assay following interactome analysis was conducted, identifying 163 proteins, most of which are involved in energy production using alternative electron acceptors. Third, a photo-crosslinking approach coupled with mass spectrometry was applied to capture weak protein-protein interactions, identifying 65 cysteine-containing periplasmic and membrane proteins mainly involved in transport processes and cellular respiration. Sixteen proteins were identified in at least two of these proteome-based analyses. Next, bacterial two-hybrid, GST pull-down assay, and physiological studies were utilized to investigate the functional interaction of five candidate proteins (TtrA, TtrB, DmsA, NapA, and STM14_4612) with ScsC. Bacterial two-hybrid and GST pull-down assays showed that ScsC interacts with TtrAB which are key enzymes that sustain anaerobic tetrathionate respiration. Truncation studies revealed that ScsC interacts with the Fe-S cluster binding regions of TtrA (34–154 amino acid region) and TtrB (34–100 amino acid region). UV-Vis spectroscopy and ferene assay demonstrated that TtrA contains an Fe-S cluster with approximately three iron atoms. Furthermore, ΔscsC cells displayed reduced growth capability and decreased tetrathionate consumption under high concentrations (> 80 mM) of tetrathionate. Transmission electron microscopy analysis revealed that S. Typhimurium 14028S cells lacking scsC exhibited aggregates and outflow of cellular components under anaerobic tetrathionate conditions. ScsC also interacts with two TtrA-paralogous proteins, DmsA and NapA, which are required for anaerobic DMSO and nitrate respiration, respectively. Truncation studies showed that ScsC interacts with the Fe-S cluster binding regions of DmsA (45-118 amino acid region) and NapA (32-95 amino acid region). During anaerobic nitrate respiration, ΔscsC cells exhibited reduced cell density in the stationary phase under high concentrations (> 60 mM) of nitrate. Bacterial two-hybrid and GST pull-down assays also revealed that ScsC interacts with STM14_4612, which is involved in anaerobic H2O2 respiration. Truncation studies demonstrated that ScsC interacts with the 28-224 amino acid region of STM14_4612, which contains a heme-binding domain. Collectively, these studies identified the substrates profiles and interaction partners of a new member (ScsC) of the protein disulfide catalyzing protein family and demonstrated its functional roles in the anaerobic respiration in S. Typhimurium. (499 words)