CalY is a major virulence factor and a biofilm matrix protein.

The extracellular biofilm matrix often contains a network of amyloid fibers which, in the human opportunistic pathogen Bacillus cereus, includes the two homologous proteins TasA and CalY. We show here, in the closely related entomopathogenic species Bacillus thuringiensis, that CalY also displays a second function. In the early stationary phase of planktonic cultures, CalY was located at the bacterial cell-surface, as shown by immunodetection. Deletion of calY revealed that this protein plays a major role in adhesion to HeLa epithelial cells, to the insect Galleria mellonella hemocytes and in the bacterial virulence against larvae of this insect, suggesting that CalY is a cell-surface adhesin. In mid-stationary phase and in biofilms, the location of CalY shifted from the cell surface to the extracellular medium, where it was found as fibers. The transcription study and the deletion of sipW suggested that CalY change of location is due to a delayed activity of the SipW signal peptidase. Using purified CalY, we found that the protein polymerization occurred only in the presence of cell-surface components. CalY is, therefore, a bifunctional protein, which switches from a cell-surface adhesin activity in early stationary phase, to the production of fibers in mid-stationary phase and in biofilms.

SinR controls enterotoxin expression in Bacillus thuringiensis biofilms.

The entomopathogen Bacillus thuringiensis produces dense biofilms under various conditions. Here, we report that the transition phase regulators Spo0A, AbrB and SinR control biofilm formation and swimming motility in B. thuringiensis, just as they control biofilm formation and swarming motility in the closely related saprophyte species B. subtilis. However, microarray analysis indicated that in B. thuringiensis, in contrast to B. subtilis, SinR does not control an eps operon involved in exopolysaccharides production, but regulates genes involved in the biosynthesis of the lipopeptide kurstakin. This lipopeptide is required for biofilm formation and was previously shown to be important for survival in the host cadaver (necrotrophism). Microarray analysis also revealed that the SinR regulon contains genes coding for the Hbl enterotoxin. Transcriptional fusion assays, Western blots and hemolysis assays confirmed that SinR controls Hbl expression, together with PlcR, the main virulence regulator in B. thuringiensis. We show that Hbl is expressed in a sustained way in a small subpopulation of the biofilm, whereas almost all the planktonic population transiently expresses Hbl. The gene coding for SinI, an antagonist of SinR, is expressed in the same biofilm subpopulation as hbl, suggesting that hbl transcription heterogeneity is SinI-dependent. B. thuringiensis and B. cereus are enteric bacteria which possibly form biofilms lining the host intestinal epithelium. Toxins produced in biofilms could therefore be delivered directly to the target tissue.

Growth-related variations in the Bacillus cereus secretome.

Using 2-DE, transcriptional gene fusions and cell cytotoxicity assays, we followed changes in the Bacillus cereus strain ATCC14579 secretome, gene expression and culture supernatant cytotoxicity from the end of the vegetative phase up to 5 h after entry into the stationary phase. The concentration of each of the 22 proteins in the culture supernatant was determined at various times. In addition, the stability of the proteins was studied. Fifteen of these proteins, including 14 members of the virulence regulon PlcR, were known or predicted to be secreted. All of the secreted proteins reached a maximum concentration during early stationary phase, but there were significant differences in the kinetics of their concentrations. The time courses of protein concentrations were in agreement with gene expression data, except for cytotoxin CytK, which was unstable, and for the metalloprotease InhA1. Supernatant cytoxicity also peaked in early stationary phase, and the kinetics of cytotoxicity paralleled the time course of concentration of the PlcR-controlled toxin, CytK. Our concomitant study of the time course of protein concentrations, gene expression and supernatant cytotoxicity reveals that the pathogenic potential of B. cereus peaks during the transition state. It also suggests that there is diversity in the regulation of gene expression within the PlcR regulon.

FlhA influences Bacillus thuringiensis PlcR-regulated gene transcription, protein production, and virulence.

Bacillus thuringiensis and Bacillus cereus are closely related. B. thuringiensis is well known for its entomopathogenic properties, principally due to the synthesis of plasmid-encoded crystal toxins. B. cereus appears to be an emerging opportunistic human pathogen. B. thuringiensis and B. cereus produce many putative virulence factors which are positively controlled by the pleiotropic transcriptional regulator PlcR. The inactivation of plcR decreases but does not abolish virulence, indicating that additional factors like flagella may contribute to pathogenicity. Therefore, we further analyzed a mutant (B. thuringiensis 407 Cry(-) DeltaflhA) previously described as being defective in flagellar apparatus assembly and in motility as well as in the production of hemolysin BL and phospholipases. A large picture of secreted proteins was obtained by two-dimensional electrophoresis analysis, which revealed that flagellar proteins are not secreted and that production of several virulence-associated factors is reduced in the flhA mutant. Moreover, we quantified the effect of FlhA on plcA and hblC gene transcription. The results show that the flhA mutation results in a significant reduction of plcA and hblC transcription. These results indicate that the transcription of several PlcR-regulated virulence factors is coordinated with the flagellar apparatus. Consistently, the flhA mutant also shows a strong decrease in cytotoxicity towards HeLa cells and in virulence against Galleria mellonella larvae following oral and intrahemocoelic inoculation. The decrease in virulence may be due to both a lack of flagella and a lower production of secreted factors. Hence, FlhA appears to be an essential virulence factor with a pleiotropic role.

A comparative study of Bacillus cereus, Bacillus thuringiensis and Bacillus anthracis extracellular proteomes.

Bacillus cereus, Bacillus thuringiensis and Bacillus anthracis are closely related species that share a similar genetic background but occupy different ecological niches. Virulence plasmids bearing genes coding for toxins, may explain, at least partly, this specialization. We have compared by 2-DE in the early stationary phase of growth the extracellular proteomes of three strains of these species that have lost their virulence plasmids. Proteins expected to be secreted or to belong to the cell wall or to the cytosol were found in the three proteomes. For the cell wall and cytosolic proteins located in the extracellular space, the three proteomes were similar. Cytosolic proteins included enolase, GroEL, PdhB, PdhD, SodA and others. Cell surface proteins were mainly autolysins, proteases, nucleotidases and OppAs. In contrast, the secreted proteins profiles of B. cereus and B. thuringiensis were quite different from that of B. anthracis. B. cereus and B. thuringiensis extracellular proteomes both contained large amounts of secreted degradative enzymes and toxins, including nine proteases, three phospholipases, two haemolysins and several enterotoxins. Most of the genes encoding these enzymes and toxins are controlled by the transcriptional activator PlcR. The extracellular proteome of the pXO1-, pXO2- B. anthracis 9131 strain contained only one secreted protein: the metalloprotease InhA1, also found in the proteomes of the two other strains and possibly involved in antibacterial peptide degradation.

Two-dimensional electrophoresis analysis of the extracellular proteome of Bacillus cereus reveals the importance of the PlcR regulon.

Many virulence factors are secreted by the gram-positive, spore forming bacterium Bacillus cereus. Most of them are regulated by the transcriptional activator, PlcR, which is maximally expressed at the beginning of the stationary phase. We used a proteomic approach to study the impact of the PlcR regulon on the secreted proteins of B. cereus, by comparing the extracellular proteomes of strains ATCC 14579 and ATCC 14579 Delta plcR, in which plcR has been disrupted. Our study indicated that, quantitatively, most of the proteins secreted at the onset of the stationary phase are putative virulence factors, all of which are regulated, directly or indirectly, by PlcR. The inactivation of plcR abolished the secretion of some of these virulence factors, and strongly decreased that of others. The genes encoding proteins that are not secreted in the DeltaplcR mutant possessed a regulatory sequence, the PlcR box, upstream from their coding sequence. These proteins include collagenase, phospholipases, haemolysins, proteases and enterotoxins. Proteins for which the secretion was strongly decreased, but not abolished, in the DeltaplcR mutant did not display the PlcR box upstream from their genes. These proteins include flagellins and InhA2. InhA2 is a homologue of InhA, a Bacillus thuringiensis metalloprotease that specifically degrades antibacterial peptides. The mechanism by which PlcR affects the production of flagellins and InhA2 is not known.

The plcR regulon is involved in the opportunistic properties of Bacillus thuringiensis and Bacillus cereus in mice and insects.

Bacillus thuringiensis has been widely used for 40 years as a safe biopesticide for controlling agricultural pests and mosquitoes because it produces insecticidal crystal proteins. However, spores have also been shown to contribute to overall entomopathogenicity. Here, the opportunistic properties of acrystalliferous B. thuringiensis Cry(-) and Bacillus cereus strains were investigated in an insect species, Galleria mellonella, and in a mammal, BALB/c mice. In both animal models, the pathogenicity of the two bacterial species was similar. Mutant strains were constructed in which the plcR gene, encoding a pleiotropic regulator of extracellular factors, was disrupted. In larvae, co-ingestion of 10(6) spores of the parental strain with a sublethal concentration of Cry1C toxin caused 70% mortality whereas only 7% mortality was recorded if spores of the DeltaplcR mutant strain were used. In mice, nasal instillation of 10(8) spores of the parental strain caused 100% mortality whereas instillation with the same number of DeltaplcR strain spores caused much lower or no mortality. Similar effects were obtained if vegetative cells were used instead of spores. The cause of death is unknown and is unlikely to be due to actual growth of the bacteria in mice. The lesions caused by B. thuringiensis supernatant in infected mice suggested that haemolytic toxins were involved. The cytolytic properties of strains of B. thuringiensis and B. cereus, using sheep, horse and human erythrocytes and G. mellonella haemocytes, were therefore investigated. The level of cytolytic activity is highly reduced in DeltaplcR strains. Together, the results indicate that the pathogenicity of B. thuringiensis strain 407 and B. cereus strain ATCC 14579 is controlled by PlcR.