Search

Subverting the host immune system is a major task for any given pathogen to assure its survival and proliferation. For the opportunistic human pathogen Bacillus cereus (Bc), immune evasion enables the establishment of potent infections. In various species of the Bc group, the pleiotropic regulator PlcR and its cognate cell--cell signaling peptide PapR7 regulate virulence gene expression in response to fluctuations in population density, i.e., a quorum-sensing (QS) system. However, how QS exerts its effects during infections and whether PlcR confers the immune evading ability remain unclear. Herein, we report how interception of the QS communication in Bc obliterates the ability to affect the host immune system. Here, we designed a peptide-based QS inhibitor that suppresses PlcR- dependent virulence factor expression and attenuates Bc infectivity in mouse models. We demonstrate that the QS peptidic inhibitor blocks host immune system--mediated eradication by reducing the expression of PlcR-regulated major toxins similarly to the profile that was observed for isogenic strains. Our findings provide evidence that Bc infectivity is regulated by QS circuit-mediated destruction of host immunity, thus reveal a interesting strategy to limit Bc virulence and enhance host defense. This peptidic quorum-quenching agent constitutes a readily accessible chemical tool for studying how other pathogen QS systems modulate host immunity and forms a basis for development of anti-infective therapeutics. [ABSTRACT FROM AUTHOR]

Cell differentiation within an isogenic population allows the specialisation of subpopulations and a division of labour. Bacillus thuringiensis is a spore‐forming bacterium that produces insecticidal crystal proteins (Cry proteins) in sporulating cells. We recently reported that strain B. thuringiensis LM1212 presents the unique ability to differentiate into two subpopulations during the stationary phase: spore‐formers and crystal‐producers. Here, we characterised the transcriptional regulator CpcR responsible for this differentiation and the expression of the cry genes. cpcR is located on a plasmid that also harbours cry genes. The alignment of LM1212 cry gene promoters revealed the presence of a conserved DNA sequence upstream from the −35 region. This presumed CpcR box was also found in the promoter of cpcR and we showed that cpcR transcription is positively autoregulated. Electrophoretic mobility shift assays suggested that CpcR directly controls the transcription of its target genes by binding to the CpcR box. We showed that CpcR was able to direct the production of a crystal consisting of a heterologous insecticidal Cry protein in non‐sporulating cells of a typical B. thuringiensis kurstaki strain. Moreover, the expression of cpcR induced a reduction in the sporulation of this B. thuringiensis strain, suggesting an interaction between CpcR and the sporulation regulatory networks. [ABSTRACT FROM AUTHOR]

The transport of peptides in microorganisms plays an important role in their physiology and behavior, both as a nutrient source and as a proxy to sense their environment. This latter function is evidenced in Gram-positive bacteria where cell-cell communication is mediated by small peptides. Here, we highlight the importance of the oligopeptide permease (Opp) systems in the various major processes controlled by signaling peptides, such as sporulation, virulence and conjugation. We underline that the functioning of these communication systems is tightly linked to the developmental status of the bacteria via the regulation of opp gene expression by transition phase regulators. [ABSTRACT FROM AUTHOR]

We explored quenching of the PlcR–PapR quorum-sensing system in Bacillus cereus. We generated PapR7-peptidic derivatives that inhibit this system and thus the production of virulence factors, reflected by a loss in hemolytic activity, without affecting bacterial growth. To our knowledge, these peptides represent the first potent synthetic inhibitors of quorum-sensing in B. cereus. [ABSTRACT FROM AUTHOR]

The Gram-positive pathogen Bacillus cereus is able to grow in chains of rod-shaped cells, but the regulation of chaining remains largely unknown. Here, we observe that glucose-grown cells of B. cereus ATCC 14579 form longer chains than those grown in the absence of glucose during the late exponential and transition growth phases, and identify that the clhAB2 operon is required for this chain lengthening phenotype. The clhAB2 operon is specific to the B. cereus group (i.e., B. thuringiensis, B. anthracis and B. cereus) and encodes two membrane proteins of unknown function, which are homologous to the Staphylococcus aureus CidA and CidB proteins involved in cell death control within glucose-grown cells. A deletion mutant (ΔclhAB2) was constructed and our quantitative image analyses show that ΔclhAB2 cells formed abnormal short chains regardless of the presence of glucose. We also found that glucose-grown cells of ΔclhAB2 were significantly wider than wild-type cells (1.47 μm ±CI95% 0.04 vs 1.19 μm ±CI95% 0.03, respectively), suggesting an alteration of the bacterial cell wall. Remarkably, ΔclhAB2 cells showed accelerated autolysis under autolysis-inducing conditions, compared to wild-type cells. Overall, our data suggest that the B. cereus clhAB2 operon modulates peptidoglycan hydrolase activity, which is required for proper cell shape and chain length during cell growth, and down-regulates autolysin activity. Lastly, we studied the transcription of clhAB2 using a lacZ transcriptional reporter in wild-type, ccpA and codY deletion-mutant strains. We found that the global transcriptional regulatory protein CodY is required for the basal level of clhAB2 expression under all conditions tested, including the transition growth phase while CcpA, the major global carbon regulator, is needed for the high-level expression of clhAB2 in glucose-grown cells. [ABSTRACT FROM AUTHOR]

Bacteria use quorum sensing to coordinate adaptation properties, cell fate or commitment to sporulation. The infectious cycle of Bacillus thuringiensis in the insect host is a powerful model to investigate the role of quorum sensing in natural conditions. It is tuned by communication systems regulators belonging to the RNPP family and directly regulated by re-internalized signaling peptides. One such RNPP regulator, NprR, acts in the presence of its cognate signaling peptide NprX as a transcription factor, regulating a set of genes involved in the survival of these bacteria in the insect cadaver. Here, we demonstrate that, in the absence of NprX and independently of its transcriptional activator function, NprR negatively controls sporulation. NprR inhibits expression of Spo0A-regulated genes by preventing the KinA-dependent phosphorylation of the phosphotransferase Spo0F, thus delaying initiation of the sporulation process. This NprR function displays striking similarities with the Rap proteins, which also belong to the RNPP family, but are devoid of DNA-binding domain and indirectly control gene expression via protein-protein interactions in Bacilli. Conservation of the Rap residues directly interacting with Spo0F further suggests a common inhibition of the sporulation phosphorelay. The crystal structure of apo NprR confirms that NprR displays a highly flexible Rap-like structure. We propose a molecular regulatory mechanism in which key residues of the bifunctional regulator NprR are directly and alternatively involved in its two functions. NprX binding switches NprR from a dimeric inhibitor of sporulation to a tetrameric transcriptional activator involved in the necrotrophic lifestyle of B. thuringiensis. NprR thus tightly coordinates sporulation and necrotrophism, ensuring survival and dissemination of the bacteria during host infection. [ABSTRACT FROM AUTHOR]

Bacterial cell-cell communication or quorum sensing (QS) is a biological process commonly described as allowing bacteria belonging to a same pherotype to coordinate gene expression to cell density. In Gram-positive bacteria, cell-cell communication mainly relies on cytoplasmic sensors regulated by secreted and re-imported signaling peptides. The Bacillus quorum sensors Rap, NprR, and PlcR were previously identified as the first members of a new protein family called RNPP. Except for the Rap proteins, these RNPP regulators are transcription factors that directly regulate gene expression. QS regulates important biological functions in bacteria of the Bacillus cereus group. PlcR was first characterized as the main regulator of virulence in B. thuringiensis and B. cereus. More recently, the PlcR-like regulator PlcRa was characterized for its role in cysteine metabolism and in resistance to oxidative stress. The NprR regulator controls the necrotrophic properties allowing the bacteria to survive in the infected host. The Rap proteins negatively affect sporulation via their interaction with a phosphorelay protein involved in the activation of Spo0A, the master regulator of this differentiation pathway. In this review we aim at providing a complete picture of the QS systems that are sequentially activated during the lifecycle of B. cereus and B. thuringiensis in an insect model of infection. [ABSTRACT FROM AUTHOR]

The aim of this study was to explore the role of SecDF in protein secretion in Bacillus cereus ATCC 14579 by in-depth characterization of a markerless secDF knock out mutant. Deletion of secDF resulted in pleiotropic effects characterized by a moderately slower growth rate, aberrant cell morphology, enhanced susceptibility to xenobiotics, reduced virulence and motility. Most toxins, including food poisoning-associated enterotoxins Nhe, Hbl, and cytotoxin K, as well as phospholipase C were less abundant in the secretome of the ΔsecDF mutant as determined by label-free mass spectrometry. Global transcriptome studies revealed profound transcriptional changes upon deletion of secDF indicating cell envelope stress. Interestingly, the addition of glucose enhanced the described phenotypes. This study shows that SecDF is an important part of the Sec-translocase mediating efficient secretion of virulence factors in the Gram-positive opportunistic pathogen B. cereus, and further supports the notion that B. cereus enterotoxins are secreted by the Sec-system. [ABSTRACT FROM AUTHOR]

Bacillus thuringiensis differs from the closely related Bacillus cereus group species by its ability to produce crystalline inclusions. The production of these crystals mainly results from the expression of the cry genes, from the stability of their transcripts and from the synthesis, accumulation and crystallization of large amounts of insecticidal Cry proteins. This process normally coincides with sporulation and is regulated by various factors operating at the transcriptional, post-transcriptional, metabolic and post-translational levels. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 σ A-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, σ H and σ E 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. [ABSTRACT FROM AUTHOR]

Summary 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. [ABSTRACT FROM AUTHOR]

PlcR is a Bacillus cereus transcriptional regulator, which activates gene expression by binding to a nucleotidic sequence called the 'PlcR box'. To build a list of all genes included in the PlcR regulon, a consensus sequence was identified by directed mutagenesis. The reference strain ATCC14579 sequenced genome was searched for occurrences of this consensus sequence to produce a virtual regulon. PlcR control of these genes was confirmed by comparing gene expression in the reference strain and its isogenic D-plcR strain using DNA microarrays, lacZ fusions and proteomics methods. The resulting list included 45 genes controlled by 28 PlcR boxes. Forty of the PlcR controlled proteins were exported, of which 22 were secreted in the extracellular medium and 18 were bound or attached to cell wall structures (membrane or peptidoglycan layer). The functions of these proteins were related to food supply (phospholipases, proteases, toxins), cell protection (bacteriocins, toxins, transporters, cell wall biogenesis) and environment-sensing (two-component sensors, chemotaxis proteins, GGDEF family regulators). Four genes coded for cytoplasmic regulators. The PlcR regulon appears to integrate a large range of environmental signals, including food deprivation and self cell-density, and regulate the transcription of genes designed to overcome obstacles that hinder B. cereus growth within the host: food supply, host barriers, host immune defenses, and competition with other bacterial species. PlcR appears to be a key component in the efficient adaptation of B. cereus to its host environment. [ABSTRACT FROM AUTHOR]

Gram-positive bacteria use a wealth of extracellular signaling peptides, so-called autoinducers, to regulate gene expression according to population densities. These "quorum sensing" systems control vital processes such as virulence, sporulation, and gene transfer. Using x-ray analysis, we determined the structure of PIcR, the major virulence regulator of the Bacillus cereus group, and obtained mechanistic insights into the effects of autoinducer binding. Our structural and phylogenetic analysis further suggests that all of those quorum sensors that bind directly to their autoinducer peptide derive from a common ancestor and form a single family (the RNPP family, for Rap/NprR/PlcR/PrgX) with conserved features. As a consequence, fundamentally different processes in different bacterial genera appear regulated by essentially the same autoinducer recognition mechanism. Our results shed light on virulence control by PlcR and elucidate origin and evolution of multicellular behavior in bacteria. [ABSTRACT FROM AUTHOR]

PleR is a pleiotropic regulator that activates the expression of genes encoding various virulence factors, such as phospholipases C, proteases and hemolysins, in Bacillus thuringiensis and Bacillus cereus. Here we show that the activation mechanism is under the control of a small peptide: PapR. The papR gene belongs to the PlcR regulon and is located 70 bp downstream from plcR. It encodes a 48-amino-acid peptide. Disruption of the papR gene abolished expression of the PleR regulon, resulting in a large decrease in hemolysis and virulence in insect larvae. We demonstrated that the PapR polypeptide was secreted, then reimported via the oligopeptide permease Opp. Once inside the cell, a processed form of PapR, presumably a pentapeptide, activated the PlcR regulon by allowing PlcR to bind to its DNA target. This activating mechanism was found to be strain specific, with this specificity determined by the first residue of the pentapeptide. [ABSTRACT FROM AUTHOR]

Bacillus thuringiensis is a bacterium capable of differentiating into a spore, a dormant and highly resistant cellular form. During the sporulation process, this bacterium produces insecticidal toxins in the form of a crystal inclusion, usually in the sporulating cell. We previously reported that the B. thuringiensis LM1212 strain can differentiate into two distinct subpopulations of sporeformers and crystal producers and that this division-of-labor phenotype provides the bacterium with a fitness advantage in competition with a typical B. thuringiensis strain. The transcription factor CpcR was characterized as the regulator responsible for this phenotype. Here, we examined how CpcR interacts with the sporulation network to control the cell differentiation. We found that the sporulation process was inhibited prior to polar septum formation and that Spo0A activity was impaired in the presence of cpcR in strain LM1212. Using bioinformatics and genetic tools, we identified a gene positively controlled by CpcR encoding a putative phosphatase of the Spo0E family known to specifically dephosphorylate phosphorylated Spo0A (Spo0A-P). We showed that this protein (called Spo0E1) is a negative regulator of sporulation and that variations in spo0E1 expression can modulate the production of spores. Using fluorescent reporters to follow gene expression at the single-cell level, we correlated expression of cpcR and sporulation genes to the formation of the two differentiated subpopulations. [ABSTRACT FROM AUTHOR]

Highlights: [•] B. thuringiensis haemolysin HlyII is a virulence factor responsible for insect death. [•] We show that hlyII gene regulation depends on the regulator Fur, independently of HlyIIR. [•] We show that hlyII is repressed by iron in vitro and during insect infection. [•] HlyII may induce host cell death to allow bacteria to gain access to iron. [ABSTRACT FROM AUTHOR]

The Bacillus cereus group comprises genetically related Gram-positive spore-forming bacteria that colonize a wide range of ecological niches and hosts. Despite their high degree of genome conservation, extrachromosomal genetic material diverges between these species. The discriminating properties of the B. cereus group strains are mainly due to plasmid-borne toxins, reflecting the importance of horizontal gene transfers in bacterial evolution and species definition. To investigate how a newly acquired megaplasmid can impact the transcriptome of its host, we transferred the pCER270 from the emetic B. cereus strains to phylogenetically distant B. cereus group strains. RNA-sequencing experiments allowed us to determine the transcriptional influence of the plasmid on host gene expression and the impact of the host genomic background on the pCER270 gene expression. Our results show a transcriptional cross-regulation between the megaplasmid and the host genome. pCER270 impacted carbohydrate metabolism and sporulation genes expression, with a higher effect in the natural host of the plasmid, suggesting a role of the plasmid in the adaptation of the carrying strain to its environment. In addition, the host genomes also modulated the expression of pCER270 genes. Altogether, these results provide an example of the involvement of megaplasmids in the emergence of new pathogenic strains. [ABSTRACT FROM AUTHOR]

Typical Bacillus thuringiensis (Bt) produces one or more parasporal crystals composed of insecticidal Cry proteins during the sporulation, and the parasporal crystals and spores are produced from the same cell. Strain Bt LM1212 is different from typical Bt strains in that its crystals and spores are produced in different cells. Previous studies have found that the cell differentiation process of Bt LM1212 is related to the transcription factor CpcR which activates the cry -gene promoters. In addition, CpcR could activate the Bt LM1212 cry35-like gene promoter (P 35) when introduced in the heterologous HD73- strain. It was shown that P 35 was only activated in non-sporulating cells. In this study, the peptidic sequences of CpcR homologous proteins found in other strains of the Bacillus cereus group were used as references to identify two key amino acid sites for CpcR activity. The function of these amino acids was investigated by measuring P 35 activation by CpcR in strain HD73-. These results will lay a foundation for the optimization of the insecticidal protein expression system in non-sporulating cells. [ABSTRACT FROM AUTHOR]

Insects are the largest group of animals on earth. Like mammals, virus, fungi, bacteria and parasites infect them. Several tissue barriers and defense mechanisms are common for vertebrates and invertebrates. Therefore some insects, notably the fly Drosophila and the caterpillar Galleria mellonella, have been used as models to study host–pathogen interactions for several insect and mammal pathogens. They are excellent tools to identify pathogen determinants and host tissue cell responses. We focus here on the comparison of effectors used by two different groups of bacterial insect pathogens to accomplish the infection process in their lepidopteran larval host: Bacillus thuringiensis and the nematode-associated bacteria, Photorhabdus and Xenorhabdus. The comparison reveals similarities in function and expression profiles for some genes, which suggest that such factors are conserved during evolution in order to attack the tissue encountered during the infection process. [ABSTRACT FROM AUTHOR]

ClpP and ClpC are subunits of the Clp ATP-dependent protease, which is ubiquitous among prokaryotic and eukaryotic organisms. The role of these proteins in stress tolerance, stationary-phase adaptive responses, and virulence in many bacterial species has been demonstrated. Based on the amino acid sequences of the Bacillus subtilis clpC and clpP genes, we identified one clpC gene and two clpP genes (designated clpP1 and clpP2) in Bacillus thuringiensis. Predicted proteins ClpP1 and ClpP2 have approximately 88 and 67% amino acid sequence identity with ClpP of B. subtilis, respectively. Inactivation of clpC in B. thuringiensis impaired sporulation efficiency. The clpP1 and clpP2 mutants were both slightly susceptible to salt stress, whereas disruption of clpP2 negatively affected sporulation and abolished motility. Virulence of the clp mutants was assessed by injecting bacteria into the hemocoel of Bombyx mori larvae. The clpP1 mutant displayed attenuated virulence, which appeared to be related to its inability to grow at low temperature (25°C), suggesting an essential role for ClpP1 in tolerance of low temperature. Microscopic examination of clpP1 mutant cells grown at 25°C showed altered bacterial division, with ceils remaining attached after septum formation. Analysis of lacZ transcriptional fusions showed that clpP1 was expressed at 25 and 37°C during the entire growth cycle. In contrast, clpP2 was expressed at 37°C but not at 25°C, suggesting that ClpP2 cannot compensate for the absence of ClpP1 in the clpP1 mutant cells at low temperature. Our study demonstrates that ClpP1 and ClpP2 control distinct cellular regulatory pathways in B. thuringiensis. [ABSTRACT FROM AUTHOR]

The virulence of Bacillus cereus requires that bacteria have the capacity to colonize their host, degrade specific tissues, and circumvent the host immune system. To study this aspect of pathogenesis, we focused on three metalloproteases, InhA1, InhA2, and InhA3, which share more than 66% identity. The expression of these metalloprotease genes was assessed by transcriptional fusions with a lacZ reporter gene. The expression profiles suggest a complementary time course of InhA production. Indeed, the genes are simultaneously expressed but are oppositely controlled during stationary phase. We constructed single and multiple inhA mutants and assessed the bacterial locations of the proteins as well as their individual or additive roles in macrophage escape and toxicity, antibacterial-peptide cleavage, and virulence. InhA1, a major component of the spore exosporium, is the only InhA metalloprotease involved in bacterial escape from macrophages. A mutant lacking inhA1, inhA2, and inhA3 shows a strong decrease in the level of virulence for insects. Taken together, these results show that the InhA metalloproteases of B. cereus are important virulence factors that may allow the bacteria to counteract the host immune system. [ABSTRACT FROM AUTHOR]