Thomas Dubois

A cell–cell communication system regulates protease production during sporulation in bacteria of the Bacillus cereus group

In sporulating Bacillus, major processes like virulence gene expression and sporulation are regulated by communication systems involving signalling peptides and regulators of the RNPP family. We investigated the role of one such regulator, NprR, in bacteria of the Bacillus cereus group. We show that NprR is a transcriptional regulator whose activity depends on the NprX signalling peptide. In association with NprX, NprR activates the transcription of an extracellular protease gene (nprA) during the first stage of the sporulation process. The transcription start site of the nprA gene has been identified and the minimal region necessary for full activation has been characterized by promoter mutagenesis. We demonstrate that the NprX peptide is secreted, processed and then reimported within the bacterial cell. Once inside the cell, the mature form of NprX, presumably the SKPDIVG heptapeptide, directly binds to NprR allowing nprA transcription. Alignment of available NprR sequences from different species of the B. cereus group defines seven NprR clusters associated with seven NprX heptapeptide classes. This cell–cell communication system was found to be strain‐specific with a possible cross‐talk between some pherotypes. The phylogenic relationship between NprR and NprX suggests a coevolution of the regulatory protein and its signalling peptide.

Necrotrophism is a quorum-sensing-regulated lifestyle in Bacillus thuringiensis.

How pathogenic bacteria infect and kill their host is currently widely investigated. In comparison, the fate of pathogens after the death of their host receives less attention. We studied Bacillus thuringiensis (Bt) infection of an insect host, and show that NprR, a quorum sensor, is active after death of the insect and allows Bt to survive in the cadavers as vegetative cells. Transcriptomic analysis revealed that NprR regulates at least 41 genes, including many encoding degradative enzymes or proteins involved in the synthesis of a nonribosomal peptide named kurstakin. These degradative enzymes are essential in vitro to degrade several substrates and are specifically expressed after host death suggesting that Bt has an active necrotrophic lifestyle in the cadaver. We show that kurstakin is essential for Bt survival during necrotrophic development. It is required for swarming mobility and biofilm formation, presumably through a pore forming activity. A nprR deficient mutant does not develop necrotrophically and does not sporulate efficiently in the cadaver. We report that necrotrophism is a highly regulated mechanism essential for the Bt infectious cycle, contributing to spore spreading.

Integration of metabolism and virulence in Clostridium difficile

Synthesis of the major toxin proteins of the diarrheal pathogen, Clostridium difficile, is dependent on the activity of TcdR, an initiation (sigma) factor of RNA polymerase. The synthesis of TcdR and the activation of toxin gene expression are responsive to multiple components in the bacteriums nutritional environment, such as the presence of certain sugars, amino acids, and fatty acids. This review summarizes current knowledge about the mechanisms responsible for repression of toxin synthesis when glucose or branched-chain amino acids or proline are in excess and the pathways that lead to synthesis of butyrate, an activator of toxin synthesis. The regulatory proteins implicated in these mechanisms also play key roles in modulating bacterial metabolic pathways, suggesting that C. difficile pathogenesis is intimately connected to the bacteriums metabolic state.

Clostridium difficile: New Insights into the Evolution of the Pathogenicity Locus.

The major virulence factors of Clostridium difficile are toxins A and B. These toxins are encoded by tcdA and tcdB genes, which form a pathogenicity locus (PaLoc) together with three additional genes that have been implicated in regulation (tcdR and tcdC) and secretion (tcdE). To date, the PaLoc has always been found in the same location and is replaced in non-toxigenic strains by a highly conserved 75/115 bp non-coding region. Here, we show new types of C. difficile pathogenicity loci through the genome analysis of three atypical clinical strains and describe for the first time a variant strain producing only toxin A (A+B−). Importantly, we found that the PaLoc integration sites of these three strains are located in the genome far from the usual single known PaLoc integration site. These findings allowed us to propose a new model of PaLoc evolution in which two “Mono-Toxin PaLoc” sites are merged to generate a single “Bi-Toxin PaLoc”.

Structure, biosynthesis, and properties of kurstakins, nonribosomal lipopeptides from Bacillus spp.

A new family of lipopeptides produced by Bacillus thuringiensis, the kurstakins, was discovered in 2000 and considered as a biomarker of this species. Kurstakins are lipoheptapeptides displaying antifungal activities against Stachybotrys charatum. Recently, the biosynthesis mechanism, the regulation of this biosynthesis and the potential new properties of kurstakins were described in the literature. In addition, kurstakins were also detected in other species belonging to Bacillus genus such as Bacillus cereus. This mini-review gathers all the information about these promising bioactive molecules.

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.

Activity of the Bacillus thuringiensis NprR-NprX cell-cell communication system is co-ordinated to the physiological stage through a complex transcriptional regulation

NprR is a quorum sensor of the RNPP family found in bacteria of the Bacillus cereus group. In association with its cognate peptide NprX, NprR controls the expression of genes essential for survival and sporulation of Bacillus thuringiensis during its necrotrophic development in insects. Here, we report that the nprR–nprX genes are not autoregulated and are co‐transcribed from a σA‐dependent promoter (PA) located upstream from nprR. The transcription from PA starts at the onset of the stationary phase and is controlled by two transcriptional regulators: CodY and PlcR. The nutritional repressor CodY represses nprR–nprX transcription during the exponential growth phase and the quorum sensor PlcR activates nprR–nprX transcription at the onset of stationary phase. We show that nprX is also transcribed independently of nprR from two promoters, PH and PE, dependent on the sporulation‐specific sigma factors, σH and σE respectively. Both promoters ensure nprX transcription during late stationary phase while transcription from PA has decreased. These results show that the activity of the NprR–NprX quorum sensing system is tightly co‐ordinated to the physiological stage throughout the developmental process of the Bacillus.

Control of Clostridium difficile Physiopathology in Response to Cysteine Availability

ABSTRACT The pathogenicity of Clostridium difficile is linked to its ability to produce two toxins: TcdA and TcdB. The level of toxin synthesis is influenced by environmental signals, such as phosphotransferase system (PTS) sugars, biotin, and amino acids, especially cysteine. To understand the molecular mechanisms of cysteine-dependent repression of toxin production, we reconstructed the sulfur metabolism pathways of C. difficile strain 630 in silico and validated some of them by testing C. difficile growth in the presence of various sulfur sources. High levels of sulfide and pyruvate were produced in the presence of 10 mM cysteine, indicating that cysteine is actively catabolized by cysteine desulfhydrases. Using a transcriptomic approach, we analyzed cysteine-dependent control of gene expression and showed that cysteine modulates the expression of genes involved in cysteine metabolism, amino acid biosynthesis, fermentation, energy metabolism, iron acquisition, and the stress response. Additionally, a sigma factor (SigL) and global regulators (CcpA, CodY, and Fur) were tested to elucidate their roles in the cysteine-dependent regulation of toxin production. Among these regulators, only sigL inactivation resulted in the derepression of toxin gene expression in the presence of cysteine. Interestingly, the sigL mutant produced less pyruvate and H2S than the wild-type strain. Unlike cysteine, the addition of 10 mM pyruvate to the medium for a short time during the growth of the wild-type and sigL mutant strains reduced expression of the toxin genes, indicating that cysteine-dependent repression of toxin production is mainly due to the accumulation of cysteine by-products during growth. Finally, we showed that the effect of pyruvate on toxin gene expression is mediated at least in part by the two-component system CD2602-CD2601.

Determinants of dietary diversity and the potential role of men in improving household nutrition in Tanzania

Good nutrition is a prerequisite for a healthy and active life, especially for agriculture-dependent households. However, diets in most households in Tanzania lack diversity because the intake of meat, poultry, fish, and vegetables and fruits is low. This study estimates factors influencing dietary diversity of the household, children under five years, and women using primary survey data. It qualitatively assesses male dietary patterns and men’s potential role in improving the nutritional status of the entire household. The findings show that the most consumed foods within the household are cereals, vegetables, oils and fats, spices, condiments and beverages. Children (d = 0.4; p<0.05) and women (d = 0.5; p<0.01) in female-headed households have low dietary diversity compared to those in male-headed households. Women and children access less diverse diets since 46% and 26%, achieved minimum dietary diversity respectively. Production of vegetables (coef. 0.34; p<0.05) play an important role in improving the dietary diversity of women. Gender (coef. 0.05; p<0.10) and education of the household head (coef. 0.02; p<0.01), food preparation and nutrition training (coef. 0.10; p<0.05) are important factors influencing dietary diversity of the members of a household. Results suggest that there is a need to support community-based programs to provide information on food and the importance of vegetables, their preparation, consumption and utilization to address food and nutrition challenges. Men can contribute towards improving household nutrition security by reducing consumption of food away from the home, especially during periods of food shortages. We recommend the use of complementary quantitative research to determine the patterns and dynamics of men’s dietary diversity and compare it with that of other household members.

Comparative evaluation of DNA extraction methods for amplification by qPCR of superficial vs intracellular DNA from Bacillus spores

This study was designed to assess the efficiency of eight extraction methods regarding their ability to release superficial (exogenous) and intracellular (endogenous) DNA from B. cereus spores for subsequent analysis by quantitative PCR (qPCR). B. cereus spore suspensions were subjected to both commercial DNA extraction kits and mechanical DNA extraction methods. The spores were observed by transmission electron microscopy to evaluate any damage caused during extraction. The efficiency of both extraction and purification were assessed using a qPCR assay targeting the bclA gene. Most of the extraction methods assessed, except the passage through the French press or the use of the QIAamp DNA Blood Mini kit without 95°C treatment, allowed the amplification of significant amounts of DNA. By using propidium monoazide, which is a photoreactive DNA-binding dye, the presence of non-negligible amounts of amplifiable DNA at the spore surface was highlighted. A further set of extraction assays was then performed on spores previously treated with PMA. The results of this study show that both superficial and intracellular spore DNA can be released by extraction methods to a greater or lesser extent and then further amplified by qPCR. The Precellys extraction allowed the detection of both intracellular and superficial DNA, the DNeasy Blood & Tissue kit the specific detection of intracellular DNA, while the Instagene kit detected only superficial DNA. Of the methods tested in this study, the Precellys extraction was the most efficient in terms of further DNA detection. SIGNIFICANCE AND IMPACT OF THE STUDY In order to verify the presence or absence of B. cereus spores in food or on surfaces in the food environment, the use of an efficient extraction method is required, followed by a qPCR analysis on the DNA released. Conversely, in order to quantify the population of Bacillus spores, any superficial DNA must be blocked, e.g. with PMA, prior to intracellular DNA extraction and further amplification.