Catherine Duport

Expanding the known repertoire of virulence factors produced by Bacillus cereus through early secretome profiling in three redox conditions

The pathogen Bacillus cereus causes diarrheal disease in humans. In the small intestine, B. cereus has to deal with anaerobiosis, low oxidoreduction potential, and carbohydrate limitation conditions. To gain insight into the virulence potential of low density B. cereus cells in such an environment, we cultured bacteria in low and high oxidoreduction potential anoxic conditions and in fully oxic conditions and compared their full secretomes. A unique pattern of proteins assigned to virulence factors was revealed. Among the 57 virulence-related factors, 31 were found for the first time in the B. cereus secretome. The putative fourth component of hemolysin BL (HblB′), enterotoxin FM, hemolysin II, and three new putative conserved enterotoxins were uncovered. Cross-comparison of the relative abundance of secreted proteins reveals that a restricted set comprising 19 proteins showed significant changes in response to redox condition changes. We complemented these results with transcriptomics data and confirmed the cytotoxicity of the B. cereus secretome toward Caco-2 human epithelial cells. Our data suggest that (i) the redox-dependent regulatory pathway may modulate the expression of a subset of virulence factors to ensure an appropriate response in a specific redox environment, and (ii) an early growth phase-dependent pathway could regulate the expression of several virulence factors, allowing B. cereus to infect a host whatever the redox conditions. This early growth phase-dependent pathway may function, at least partially, independently of the pleiotropic virulence gene regulator PlcR and may therefore be more specific to the B. cereus group.

Control of Enterotoxin Gene Expression in Bacillus cereus F4430/73 Involves the Redox-Sensitive ResDE Signal Transduction System

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.

The Redox Regulator Fnr Is Required for Fermentative Growth and Enterotoxin Synthesis in Bacillus cereus F4430/73

Glucose-grown cells of Bacillus cereus respond to anaerobiosis and low extracellular oxidoreduction potentials (ORP), notably by enhancing enterotoxin production. This response involves the ResDE two-component system. We searched the B. cereus genome for other redox response regulators potentially involved in this adaptive process, and we identified one gene encoding a protein predicted to have an amino acid sequence 58% identical (80% similar) to that of the Bacillus subtilis Fnr redox regulator. The fnr gene of the food-borne pathogen B. cereus F4430/73 has been cloned and partially characterized. We showed that fnr was up-regulated during anaerobic fermentation, especially when fermentation occurred at low ORP (under highly reducing conditions). The expression of fnr was down-regulated in the presence of O(2) and nitrate which, unlike fumarate, stimulated the respiratory pathways. The inactivation of B. cereus fnr abolished fermentative growth but only moderately affected aerobic and anaerobic nitrate respiratory growth. Analyses of glucose by-products and the transcription profiles of key catabolic genes confirmed the strong regulatory impact of Fnr on B. cereus fermentative pathways. More importantly, the fnr mutation strongly decreased the expression of PlcR-dependent hbl and nhe genes, leading to the absence of hemolysin BL (Hbl) and nonhemolytic enterotoxin (Nhe) secretion by the mutant. These data indicate that fnr is essential for both fermentation and toxinogenesis. The results also suggest that both Fnr and the ResDE two-component system belong to a redox regulatory pathway that functions at least partially independently of the pleiotropic virulence gene regulator PlcR to regulate enterotoxin gene expression.

Exoproteomics: exploring the world around biological systems

The term ‘exoproteome’ describes the protein content that can be found in the extracellular proximity of a given biological system. These proteins arise from cellular secretion, other protein export mechanisms or cell lysis, but only the most stable proteins in this environment will remain in abundance. It has been shown that these proteins reflect the physiological state of the cells in a given condition and are indicators of how living systems interact with their environments. High-throughput proteomic approaches based on a shotgun strategy, and high-resolution mass spectrometers, have modified the authors’ view of exoproteomes. In the present review, the authors describe how these new approaches should be exploited to obtain the maximum useful information from a sample, whatever its origin. The methodologies used for studying secretion from model cell lines derived from eukaryotic, multicellular organisms, virulence determinants of pathogens and environmental bacteria and their relationships with their habitats are illustrated with several examples. The implication of such data, in terms of proteogenomics and the discovery of novel protein functions, is discussed.

Restricting Fermentative Potential by Proteome Remodeling AN ADAPTIVE STRATEGY EVIDENCED IN BACILLUS CEREUS

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 ΔohrA and ΔohrR 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.

ApoFnr binds as a monomer to promoters regulating the expression of enterotoxin genes of Bacillus cereus.

Bacillus cereus Fnr is a member of the Crp/Fnr (cyclic AMP-binding protein/fumarate nitrate reduction regulatory protein) family of helix-turn-helix transcriptional regulators. It is essential for the expression of hbl and nhe enterotoxin genes independently of the oxygen tension in the environment. We studied aerobic Fnr binding to target sites in promoters regulating the expression of enterotoxin genes. B. cereus Fnr was overexpressed and purified as either a C-terminal His-tagged (Fnr(His)) fusion protein or an N-terminal fusion protein tagged with the Strep-tag (IBA BioTAGnology) ((Strep)Fnr). Both recombinant Fnr proteins were produced as apoforms (clusterless) and occurred as mixtures of monomers and oligomers in solution. However, apoFnr(His) was mainly monomeric, while apo(Strep)Fnr was mainly oligomeric, suggesting that the His-tagged C-terminal extremity may interfere with oligomerization. The oligomeric state of apo(Strep)Fnr was dithiothreitol sensitive, underlining the importance of a disulfide bridge for apoFnr oligomerization. Electrophoretic mobility shift assays showed that monomeric apoFnr, but not oligomeric apoFnr, bound to specific sequences located in the promoter regions of the enterotoxin regulators fnr, resDE, and plcR and the structural genes hbl and nhe. The question of whether apoFnr binding is regulated in vivo by redox-dependent oligomerization is discussed.

ResDE-Dependent Regulation of Enterotoxin Gene Expression in Bacillus cereus: Evidence for Multiple Modes of Binding for ResD and Interaction with Fnr

In the food-borne pathogen Bacillus cereus F4430/73, the production of major virulence factors hemolysin BL (Hbl) and nonhemolytic enterotoxin (Nhe) is regulated through complex mechanisms. The two-component regulatory system ResDE is involved in the activation of hbl and nhe transcription. Here, the response regulator ResD and the sensor kinase ResE were overexpressed and purified, and autophosphorylation of ResE and transphosphorylation of ResD by ResE were demonstrated in vitro. ResD is mainly monomeric in solution, regardless of its phosphorylation state. ResD was shown to interact directly with promoter regions (p) of the enterotoxin regulator genes resDE, fnr, and plcR and the enterotoxin structural genes nhe and hbl, but with different affinities. Binding of ResD to pplcR, pnhe, and phbl was not dependent on the ResD phosphorylation status. In contrast, ResD phosphorylation significantly increased interactions between ResD and presDE and pfnr. Taken together, these results showed that phosphorylation of ResD results in a different target expression pattern. Furthermore, ResD and the redox activator Fnr were found to physically interact and simultaneously bind their target DNAs. We propose that unphosphorylated ResD acts as an antiactivator of Fnr, while phosphorylated ResD acts as a coactivator of Fnr. Finally, our findings represent the first molecular evidence of the role of ResDE as a sentinel system capable of sensing redox changes and coordinating a response that modulates B. cereus virulence.

Fnr mediates carbohydrate-dependent regulation of catabolic and enterotoxin genes in Bacillus cereus F4430/73

We had previously demonstrated that Fnr is required for fermentative growth and oxic production of hemolysin BL (Hbl) and non-hemolytic enterotoxin (Nhe) in the food-borne pathogen Bacillus cereus F4430/73. In the present work, the regulatory impact of Fnr on microaerobic growth and enterotoxin production in response to carbohydrates was examined using glucose, fructose, sucrose or a glucose-fructose mixture as carbon and energy sources. Growth parameters, byproduct spectra and transcription levels of catabolic and enterotoxin genes were analyzed in a strain lacking Fnr in comparison to the parental F4430/73 strain. The results showed that B. cereus prefers glucose to other carbohydrates for microaerobic growth, and that lacking of Fnr less strongly affected the respiro-fermentative catabolism of glucose than fructose and, to a lesser extent than sucrose. In addition, lacking of Fnr strongly decreased expression of hbl and nhe genes, leading to the absence of Hbl and low production of Nhe independently of the carbohydrate used as carbon source. We conclude that Fnr is an important element for carbon source regulation in B. cereus F4430/73 and that the regulation of enterotoxin gene expression in response to carbohydrates may represent one aspect of overall catabolite control mediated by Fnr.

OhrRA functions as a redox-responsive system controlling toxinogenesis in Bacillus cereus

UNLABELLED Bacillus cereus OhrR is a member of the subgroup of the MarR (multiple antibiotic resistance) family of transcriptional regulators that use a cysteine-based redox sensing mechanism. OhrA is a thiol-dependent, peroxidase-like protein. The dual OhrRA system triggers B. cereus adaptation in response to redox changes, such as those encountered in the environment of the gastrointestinal tract. Here, we investigated the role of OhrRA in toxinogenesis. Comparative shotgun analysis of exoproteomes from ∆ohrA, ∆ohrR and wild-type cells revealed significant changes in the abundance levels of toxin-related proteins depending on the extracellular redox potential. We complemented these data by measuring the DNA binding activity of reduced and oxidized recombinant OhrR on toxin and putative toxin promoter regions. Furthermore, transcriptomic data and OhrRA-dependent, antiproliferative activity of the B. cereus exoproteome on Caco-2 human epithelial cells were recorded. The results indicate that OhrR controlled toxin gene expression directly or indirectly in a redox- and toxin-dependent manner, and may function as a repressor or an activator. Moreover, we found that OhrR restricts toxin-dependent antiproliferative activity of the B. cereus exoproteome whatever the growth conditions, while the restrictive impact of OhrA occurs only under low ORP anoxic conditions. BIOLOGICAL SIGNIFICANCE B. cereus is a notorious foodborne pathogen which causes gastroenteritis. Fatal and severe cases have been reported. The pathogenicity of B. cereus is intimately associated with the production of epithelial cell-destructive toxins in the small intestine. The small intestine poses several challenges for a pathogen because it is sliced into various niches with different oxygen concentrations and different redox potentials. We recently showed that the organic hydroperoxide resistance OhrRA system was crucial to the successful adaptation of B. cereus to extreme redox environments such as those encountered in the lumen (high reducing anoxic environment) and on the intestinal epithelium (transient oxic environment). Here we provide evidence that this bacterial system is a major virulence determinant in B. cereus in that it coordinates toxinogenesis in a redox dependent manner. Specifically, our comparative exoproteomic analyses reveal that OhrR strongly restricts B. cereus toxinogenesis under high reducing anoxic conditions while OhrA boosts toxinogenesis. Based on exoproteomic analyses, we further examined the role of OhrR and found that it functions as a redox-dependent transcriptional regulator of toxin and putative toxin genes. These findings provide novel insights into the weapons used by B. cereus to control its toxinogenic potential and, as a result its toxicity against human epithelial cells.

Lactate dehydrogenase A promotes communication between carbohydrate catabolism and virulence in Bacillus cereus

The diarrheal potential of a Bacillus cereus strain is essentially dictated by the amount of secreted nonhemolytic enterotoxin (Nhe). Expression of genes encoding Nhe is regulated by several factors, including the metabolic state of the cells. To identify metabolic sensors that could promote communication between central metabolism and nhe expression, we compared four strains of the B. cereus group in terms of metabolic and nhe expression capacities. We performed growth performance measurements, metabolite analysis, and mRNA measurements of strains F4430/73, F4810/72, F837/76, and PA cultured under anoxic and fully oxic conditions. The results showed that expression levels of nhe and ldhA, which encodes lactate dehydrogenase A (LdhA), were correlated in both aerobically and anaerobically grown cells. We examined the role of LdhA in the F4430/73 strain by constructing an ldhA mutant. The ldhA mutation was more deleterious to anaerobically grown cells than to aerobically grown cells, causing growth limitation and strong deregulation of key fermentative genes. More importantly, the ldhA mutation downregulated enterotoxin gene expression under both anaerobiosis and aerobiosis, with a more pronounced effect under anaerobiosis. Therefore, LdhA was found to exert a major control on both fermentative growth and enterotoxin expression, and it is concluded that there is a direct link between fermentative metabolism and virulence in B. cereus. The data presented also provide evidence that LdhA-dependent regulation of enterotoxin gene expression is oxygen independent. This study is the first report to describe a role of a fermentative enzyme in virulence in B. cereus.