We performed a proteomic survey of Salmonella enterica serovar Typhimurium during infection of host epithelial cells. Our data reveal substantial metabolic reshuffling of Salmonella in the host in addition to severe degeneration of bacterial flagella and chemotaxis systems. The large-scale quantitative data allowed us to chart an overview of intracellular Salmonella carbon metabolism. Notably, we found preferential utilization of glycolysis, the pentose phosphate pathway, mixed acid fermentation, and nucleotide metabolism. In contrast, the tricarboxylic acid (TCA) cycle and aerobic and anaerobic respiration pathways were largely repressed. Furthermore, inactivation of glycolysis and purine biosynthesis led to severe growth defects, indicating important roles in intracellular Salmonella replication. In summary, we exploited quantitative proteomics for rational design of follow-up genetic studies and identified pathways important for bacterial fitness within host cells.Keywords: bacterial infection; mass spectrometry; proteomics; Salmonella metabolism;

Elucidation of molecular mechanisms underlying host-pathogen interactions is important for control and treatment of infectious diseases worldwide. Within the last decade, mass spectrometry (MS)-based proteomics has become a powerful and effective approach to better understand complex and dynamic host-pathogen interactions at the protein level. Herein we will review the recent progress in proteomic analyses towards bacterial infection of their mammalian host with a particular focus on enteric pathogens. Large-scale studies of dynamic proteomic alterations during infection will be discussed from the perspective of both pathogenic bacteria and host cells.

•Trypsin autolysis contributes to the bulk of chemical noise in in-gel digestion.•10-fold less trypsin affords comparable performance of digestion.•Reduced trypsin usage substantially cuts the cost of enzyme expenditure.•Less trypsin leads to improved S/N and up to 30% more protein identifications.Pre-fractionation by gel electrophoresis is often combined with liquid chromatography–mass spectrometry (LC–MS) for large-scale profiling of complex protein samples. An essential component of this widely applied proteomic platform is in-gel protein digestion. In nearly two decades of practicing this approach, an extremely high level of trypsin has been utilized due to the consideration of slow enzyme diffusion into the gel matrix. Here we report that trypsin autolysis products contribute to the bulk of chemical noise in in-gel digestion and remarkably we found evidence that the amount of trypsin can be slashed by an order of magnitude with comparable digestion performance. By revising perhaps the most critical element of this decade-old digestion protocol, the proteomics community relying on gel separation prior to LC–MS analysis will benefit instantly from much lowered cost due to enzyme expenditure. More importantly, substantially reduced chemical noise (i.e., trypsin self-cleavage products) as a result of less enzyme usage translates into more protein identifications when limited amounts of samples are the interest of interrogation.Biological significanceIn-gel digestion is one of the most widely used methods in proteomics. An exceedingly high level of trypsin has been utilized due to the consideration of slow enzyme diffusion into the gel matrix. This requirement has been faithfully kept in nearly two decades of practicing this approach. Here we report that trypsin concentration can be slashed by at least an order of magnitude while still providing comparable digestion performance. Thus the proteomics community relying on gel separation prior to LC–MS analysis will benefit instantly from much lowered enzyme cost. More importantly, substantially reduced chemical noise (i.e., trypsin autolysis products) due to less enzyme usage translates into ~ 30% more protein identifications when limited amounts of protein samples are analyzed.

•A global survey of Salmonella proteome under oxidative stress•Induction of most known antioxidant proteins and DNA repair machinery•Up-regulation of iron acquisition systems to promote bacterial survival•Inhibition of Salmonella type III secretion system and bacterial virulenceSalmonella Typhimurium is a bacterial pathogen that can cause widespread gastroenteritis. Salmonella encounters reactive oxygen species both under free-living conditions and within their mammalian host during infection. To study its response to oxidative stress, we performed the first large-scale proteomic profiling of Salmonella upon exposure to H2O2. Among 1600 detected proteins, 83 proteins showed significantly altered abundance. Interestingly, only a subset of known antioxidants was induced, likely due to distinct regulatory mechanisms. In addition, we found elevation of several Salmonella acquired phage products with potential contribution to DNA repair under oxidative stress. Furthermore, we observed robust induction of iron-uptake systems and disruption of these pathways led to bacterial survival defects under H2O2 challenge. Importantly, this work is the first to report that oxidative stress severely repressed the Salmonella type III secretion system (T3SS), reducing its virulence.Biological significanceSalmonella, a Gram-negative bacterial pathogen, encounters reactive oxygen species (ROS) both endogenously and exogenously. To better understand its response to oxidative stress, we performed the first large-scale profiling of Salmonella protein expression upon H2O2 treatment. Among 1600 quantified proteins, the abundance of 116 proteins was altered significantly. Notably, iron acquisition systems were induced to promote bacterial survival under oxidative stress. Furthermore, we are the first to report that oxidative stress severely repressed Salmonella type III secretion system and hence reduced its virulence. We believe that these findings will not only help us better understand the molecular mechanisms that Salmonella has evolved to counteract ROS but also the global impact of oxidative stress on bacterial physiology.

Replacement of the native heme cofactor by manganese protoporphyrin IX (MnPPIX) to reconstitute manganese myoglobin (MnIIIMb) is an important approach to investigate the reactivity of the Mn center inside protein scaffolds. However, unlike the Mn porphyrin synthetic model compounds, MnPPIX reconstituted myoglobins (MnIIIMb) have no reactivity in the epoxidation of styrene using H2O2, which was attributed to the low reactivity of the MnIVO intermediate after homocleavage of the O–O bond in manganese peroxide. To address this issue, we herein chose Oxone® (2KHSO5·KHSO4·K2SO4), a well-known oxidant undergoing O–O bond heterocleavage. After screening 7 mutants and wild-type MnIIIMb, we found that the L29H/F43H mutant could generate a new species ([MnIVO]+˙), tentatively assigned by using UV-vis and EPR spectra, through heterocleavage of the O–O bond. Computational docking showed hydrogen bonds between three distal histidines (H64, L29H and F43H) and anions, which increase the binding affinity to persulfate. With Oxone® as the oxidant, MnIIIMb (L29H/F43H) showed the highest reactivity toward the epoxidation of styrene, different from that with the H2O2 oxidant. This work demonstrates the first example of MnPPIX reconstituted Mb which could catalyze styrene epoxidation and provides new insights to further explore the reactivity of the Mn center in protein scaffolds.