Time dynamics of the Bacillus cereus exoproteome are shaped by cellular oxidation.
ABSTRACT: At low density, Bacillus cereus cells release a large variety of proteins into the extracellular medium when cultivated in pH-regulated, glucose-containing minimal medium, either in the presence or absence of oxygen. The majority of these exoproteins are putative virulence factors, including toxin-related proteins. Here, B. cereus exoproteome time courses were monitored by nanoLC-MS/MS under low-oxidoreduction potential (ORP) anaerobiosis, high-ORP anaerobiosis, and aerobiosis, with a specific focus on oxidative-induced post-translational modifications of methionine residues. Principal component analysis (PCA) of the exoproteome dynamics indicated that toxin-related proteins were the most representative of the exoproteome changes, both in terms of protein abundance and their methionine sulfoxide (Met(O)) content. PCA also revealed an interesting interconnection between toxin-, metabolism-, and oxidative stress-related proteins, suggesting that the abundance level of toxin-related proteins, and their Met(O) content in the B. cereus exoproteome, reflected the cellular oxidation under both aerobiosis and anaerobiosis.
Project description:In contrast to Bacillus subtilis, the role of the two-component regulatory system ResDE has not yet been investigated in the facultative anaerobe Bacillus cereus. We examined the role of ResDE in the food-borne pathogen B. cereus F4430/73 by constructing resDE and resE mutants. Growth performances, glucose metabolism, and expression of hemolysin BL (Hbl) and nonhemolytic enterotoxin (Nhe) were analyzed in the three strains under distinct oxygenation and extracellular oxidoreduction potential (ORP) conditions. We show that growth and glucose metabolism were only moderately perturbed in both resDE and resE mutants under aerobiosis, microaerobiosis, and anaerobiosis generated under N(2) atmosphere (initial ORP = +45 mV). The major effects of resDE and resE mutations were observed under low-ORP anaerobic conditions generated under hydrogen atmosphere (iORP = -148 mV). These conditions normally favor enterotoxin production in the wild type. The resE mutation was more deleterious to the cells than the resDE mutation, causing growth limitation and strong deregulation of key catabolic genes. More importantly, the resE mutation abolished the production of enterotoxins under all of the conditions examined. The resDE mutation only decreased enterotoxin expression under anaerobiosis, with a more pronounced effect under low-ORP conditions. Thus, the ResDE system was found to exert major control on both fermentative growth and enterotoxin expression, and it is concluded that the ResDE system of B. cereus should be considered an anaerobic redox regulator. The data presented also provide evidence that the ResDE-dependent regulation of enterotoxins might function at least partially independently of the pleiotropic virulence gene regulator PlcR.
Project description:The facultative anaerobe, Bacillus cereus, causes diarrheal diseases in humans. Its ability to deal with oxygen availability is recognized to be critical for pathogenesis. The B. cereus genome comprises a gene encoding a protein with high similarities to the redox regulator, Rex, which is a central regulator of anaerobic metabolism in Bacillus subtilis and other Gram-positive bacteria. Here, we showed that B. cereus rex is monocistronic and down-regulated in the absence of oxygen. The protein encoded by rex is an authentic Rex transcriptional factor since its DNA binding activity depends on the NADH/NAD+ ratio. Rex deletion compromised the ability of B. cereus to cope with external oxidative stress under anaerobiosis while increasing B. cereus resistance against such stress under aerobiosis. The deletion of rex affects anaerobic fermentative and aerobic respiratory metabolism of B. cereus by decreasing and increasing, respectively, the carbon flux through the NADH-recycling lactate pathway. We compared both the cellular proteome and exoproteome of the wild-type and ?rex cells using a high throughput shotgun label-free quantitation approach and identified proteins that are under control of Rex-mediated regulation. Proteomics data have been deposited to the ProteomeXchange with identifier PXD000886. The data suggest that Rex regulates both the cross-talk between metabolic pathways that produce NADH and NADPH and toxinogenesis, especially in oxic conditions.
Project description:Helicobacter pylori colonizes the human stomach and is associated with an increased risk of gastric cancer and peptic ulcer disease. Analysis of H. pylori protein secretion is complicated by the occurrence of bacterial autolysis. In this study, we analyzed the exoproteome of H. pylori at multiple phases of bacterial growth and identified 74 proteins that are selectively released into the extracellular space. These include proteins known to cause alterations in host cells, antigenic proteins, and additional proteins that have not yet been studied in any detail. The composition of the H. pylori exoproteome is dependent on the phase of bacterial growth. For example, the proportional abundance of the vacuolating toxin VacA in culture supernatant is higher during late growth phases than early growth phases, whereas the proportional abundance of many other proteins is higher during early growth phases. We detected marked variation in the subcellular localization of putative secreted proteins within soluble and membrane fractions derived from intact bacteria. By providing a comprehensive view of the H. pylori exoproteome, these results provide new insights into the array of secreted H. pylori proteins that may cause alterations in the gastric environment.
Project description:During aerobic respiratory growth, Bacillus cereus is exposed to continuously reactive oxidant, produced by partially reduced forms of molecular oxygen, known as reactive oxygen species (ROS). The sulfur-containing amino acid, methionine (Met), is particularly susceptible to ROS. The major oxidation products, methionine sulfoxides, can be readily repaired by methionine sulfoxide reductases, which reduce methionine sulfoxides [Met(O)] back to methionine. Here, we show that methionine sulfoxide reductase AB (MsrAB) regulates the Met(O) content of both the cellular proteome and exoproteome of B. cereus in a growth phase-dependent manner. Disruption of msrAB leads to metabolism changes resulting in enhanced export of Met(O) proteins at the late exponential growth phase and enhanced degradation of exoproteins. This suggests that B. cereus can modulate its capacity and specificity for protein export/secretion through the growth phase-dependent expression of msrAB. Our results also show that cytoplasmic MsrAB recycles Met residues in enterotoxins, which are major virulence factors in B. cereus.
Project description:Cellular proteomes and exoproteomes are dynamic, allowing pathogens to respond to environmental conditions to sustain growth and virulence. Bacillus cereus is an important food-borne pathogen causing intoxication via emetic toxin and/or multiple protein exotoxins. Here, we compared the dynamics of the cellular proteome and exoproteome of emetic B. cereus cells grown at low (16 °C) and high (30 °C) temperature. Tandem mass spectrometry (MS/MS)-based shotgun proteomics analysis identified 2063 cellular proteins and 900 extracellular proteins. Hierarchical clustering following principal component analysis indicated that in B. cereus the abundance of a subset of these proteins-including cold-stress responders, and exotoxins non-hemolytic enterotoxin (NHE) and hemolysin I (cereolysin O (CLO))-decreased at low temperature, and that this subset governs the dynamics of the cellular proteome. NHE, and to a lesser extent CLO, also contributed significantly to exoproteome dynamics; with decreased abundances in the low-temperature exoproteome, especially in late growth stages. Our data therefore indicate that B. cereus may reduce its production of secreted protein toxins to maintain appropriate proteome dynamics, perhaps using catabolite repression to conserve energy for growth in cold-stress conditions, at the expense of virulence.
Project description:Helicobacter pylori infection and a high salt diet are each risk factors for gastric cancer. In this study, we tested the hypothesis that environmental salt concentration influences the composition of the H. pylori exoproteome. H. pylori was cultured in media containing varying concentrations of sodium chloride, and aliquots were fractionated and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). We identified proteins that were selectively released into the extracellular space, and we identified selectively released proteins that were differentially abundant in culture supernatants, depending on the environmental salt concentration. We also used RNA-seq analysis to identify genes that were differentially expressed in response to environmental salt concentration. The salt-responsive proteins identified by proteomic analysis and salt-responsive genes identified by RNA-seq analysis were mostly non-concordant, but the secreted toxin VacA was salt-responsive in both analyses. Western blot analysis confirmed that VacA levels in the culture supernatant were increased in response to high salt conditions, and quantitative RT-qPCR experiments confirmed that vacA transcription was upregulated in response to high salt conditions. These results indicate that environmental salt concentration influences the composition of the H. pylori exoproteome, which could contribute to the increased risk of gastric cancer associated with a high salt diet. SIGNIFICANCE: Helicobacter pylori-induced alterations in the gastric mucosa have been attributed, at least in part, to the actions of secreted H. pylori proteins. In this study, we show that H. pylori growth in high salt concentrations leads to increased levels of a secreted VacA toxin. Salt-induced alterations in the composition of the H. pylori exoproteome is relevant to the increased risk of gastric cancer associated with consumption of a high salt diet.
Project description:Pathogenesis hinges on successful colonization of the gastrointestinal (GI) tract by pathogenic facultative anaerobes. The GI tract is a carbohydrate-limited environment with varying oxygen availability and oxidoreduction potential (ORP). How pathogenic bacteria are able to adapt and grow in these varying conditions remains a key fundamental question. Here, we designed a system biology-inspired approach to pinpoint the key regulators allowing Bacillus cereus to survive and grow efficiently under low ORP anoxic conditions mimicking those encountered in the intestinal lumen. We assessed the proteome components using high throughput nanoLC-MS/MS techniques, reconstituted the main metabolic circuits, constructed ?ohrA and ?ohrR mutants, and analyzed the impacts of ohrA and ohrR disruptions by a novel round of shotgun proteomics. Our study revealed that OhrR and OhrA are crucial to the successful adaptation of B. cereus to the GI tract environment. Specifically, we showed that B. cereus restricts its fermentative growth under low ORP anaerobiosis and sustains efficient aerobic respiratory metabolism, motility, and stress response via OhrRA-dependent proteome remodeling. Finally, our results introduced a new adaptive strategy where facultative anaerobes prefer to restrict their fermentative potential for a long term benefit.