ABSTRACT
Bacillus cereus is a ubiquitous bacterial pathogen increasingly reported to be the causative agent of foodborne infections and intoxications. Since the enterotoxins linked to the diarrheal form of food poising are foremost produced in the human intestine, the toxic potential of enteropathogenic B. cereus strains is difficult to predict from studies carried out under routine cultivation procedures. In this study, toxigenic properties of a panel of strains (n = 19) of diverse origin were compared using cell culture medium pre-incubated with CaCo-2 cells to mimic intestinal growth conditions. Shortly after contact of the bacteria with the simulated host environment, enterotoxin gene expression was activated and total protein secretion of all strains was accelerated. Although the signal stimulating enterotoxin production still needs to be elucidated, it could be shown that it originated from the CaCo-2 cells. Overall, our study demonstrates that the currently used methods in B. cereus diagnostics, based on standard culture medium, are not allowing a conclusive prediction of the potential health risk related to a certain strain. Thus, these methods should be complemented by cultivation procedures that are simulating intestinal host conditions.
ABSTRACT
Bacillus cereus is a food contaminant with greatly varying enteropathogenic potential. Almost all known strains harbor the genes for at least one of the three enterotoxins Nhe, Hbl, and CytK. While some strains show no cytotoxicity, others have caused outbreaks, in rare cases even with lethal outcome. The reason for these differences in cytotoxicity is unknown. To gain insight into the origin of enterotoxin expression heterogeneity in different strains, the architecture and role of 5' intergenic regions (5' IGRs) upstream of the nhe and hbl operons was investigated. In silico comparison of 142 strains of all seven phylogenetic groups of B. cereus sensu lato proved the presence of long 5' IGRs upstream of the nheABC and hblCDAB operons, which harbor recognition sites for several transcriptional regulators, including the virulence regulator PlcR, redox regulators ResD and Fnr, the nutrient-sensitive regulator CodY as well as the master regulator for biofilm formation SinR. By determining transcription start sites, unusually long 5' untranslated regions (5' UTRs) upstream of the nhe and hbl start codons were identified, which are not present upstream of cytK-1 and cytK-2. Promoter fusions lacking various parts of the nhe and hbl 5' UTR in B. cereus INRA C3 showed that the entire 331 bp 5' UTR of nhe is necessary for full promoter activity, while the presence of the complete 606 bp hbl 5' UTR lowers promoter activity. Repression was caused by a 268 bp sequence directly upstream of the hbl transcription start. Luciferase activity of reporter strains containing nhe and hbl 5' IGR lux fusions provided evidence that toxin gene transcription is upregulated by the depletion of free amino acids. Electrophoretic mobility shift assays showed that the branched-chain amino acid sensing regulator CodY binds to both nhe and hbl 5' UTR downstream of the promoter, potentially acting as a nutrient-responsive roadblock repressor of toxin gene transcription. PlcR binding sites are highly conserved among all B. cereus sensu lato strains, indicating that this regulator does not significantly contribute to the heterogeneity in virulence potentials. The CodY recognition sites are far less conserved, perhaps conferring varying strengths of CodY binding, which might modulate toxin synthesis in a strain-specific manner.
ABSTRACT
In recent years Bacillus cereus has gained increasing importance as a food poisoning pathogen. It is the eponymous member of the B. cereus sensu lato group that consists of eight closely related species showing impressive diversity of their pathogenicity. The high variability of cytotoxicity and the complex regulatory network of enterotoxin expression have complicated efforts to predict the toxic potential of new B. cereus isolates. In this study, comprehensive analyses of enterotoxin gene sequences, transcription, toxin secretion and cytotoxicity were performed. For the first time, these parameters were compared in a whole set of B. cereus strains representing isolates of different origin (food or food poisoning outbreaks) and of different toxic potential (enteropathogenic and apathogenic) to elucidate potential starting points of strain-specific differential toxicity. While toxin gene sequences were highly conserved and did not allow for differentiation between high and low toxicity strains, comparison of nheB and hblD enterotoxin gene transcription and Nhe and Hbl protein titers revealed not only strain-specific differences but also incongruence between toxin gene transcripts and toxin protein levels. With one exception all strains showed comparable capability of protein secretion and so far, no secretion patterns specific for high and low toxicity strains were identified. These results indicate that enterotoxin expression is more complex than expected, possibly involving the orchestrated interplay of different transcriptional regulator proteins, as well as posttranscriptional and posttranslational regulatory mechanisms plus additional influences of environmental conditions.
