The quorum-sensing peptidic inhibitor rescues host immune system eradication: A novel infectivity mechanism.

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.

A Sporulation-Independent Way of Life for Bacillus thuringiensis in the Late Stages of an Infection.

The formation of endospores has been considered the unique survival and transmission mode of sporulating Firmicutes due to the exceptional resistance and persistence of this bacterial form. However, nonsporulated bacteria (Spo-) were reported at the early stages following the death of a host infected with Bacillus thuringiensis, an entomopathogenic sporulating bacterium. Here, we investigated the characteristics of the bacterial population in the late stages of an infection in the B. thuringiensis/Galleria mellonella infection model. Using fluorescent reporters and molecular markers coupled to flow cytometry, we demonstrated that the Spo- cells persist and constitute about half of the population 2 weeks post-infection (p.i.). Protein synthesis and growth recovery assays indicated that they are in a metabolically slowed-down state. These bacteria were extremely resistant to the insect cadaver environment, which did not support growth of in vitro-grown vegetative cells and spores. A transcriptomic analysis of this subpopulation at 7 days p.i. revealed a signature profile of this state, and the expression analysis of individual genes at the cell level showed that more bacteria mount an oxidative stress response as their survival time increases, in agreement with the increase of the free radical level in the host cadaver and in the number of reactive oxygen species (ROS)-producing bacteria. Altogether, these data show for the first time that nonsporulated bacteria are able to survive for a prolonged period of time in the context of an infection and indicate that they engage in a profound adaptation process that leads to their persistence in the host cadaver. IMPORTANCE Bacillus thuringiensis is an entomopathogenic bacterium widely used as a biopesticide. It belongs to the Bacillus cereus group, comprising the foodborne pathogen B. cereus sensu stricto and the anthrax agent Bacillus anthracis. Like other Firmicutes when they encounter harsh conditions, these Gram-positive bacteria can form dormant cells called spores. Due to its highly resistant nature, the spore was considered the unique mode of long-term survival, eclipsing any other form of persistence. Breaking this paradigm, we observed that B. thuringiensis was able to persist in its host cadaver in a nonsporulated form for at least 14 days. Our results show that these bacteria survived in the cadaver environment, which proved hostile for actively growing bacteria by engaging in a profound adaptation process. Studying this facet of the life cycle of a sporulating bacterium provides new fundamental knowledge and might lead to the development of strategies to combat sporulating pathogenic species.

Plasmid - Chromosome interplay in natural and non-natural hosts: global transcription study of three Bacillus cereus group strains carrying pCER270 plasmid.

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.

Key amino acids residues enhance the ability of CpcR to activate cry gene expression in Bacillus thuringiensis.

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 (P35) when introduced in the heterologous HD73- strain. It was shown that P35 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 P35 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.

Deletion of the novel gene mother cell lysis X results in Cry1Ac encapsulation in the Bacillus thuringiensis HD73.

The novel protein MclX (mother cell lysis X) in Bacillus thuringiensis subsp. kurstaki strain HD73 (B. thuringiensis HD73) was characterized in this work. MclX has no known domain and its gene deletion in HD73 resulted in Cry1Ac encapsulation in the mother cell and did not influence Cry1Ac protein production or insecticidal activity. In vitro cell wall hydrolysis experiments showed that MclX cannot hydrolyze the cell wall. In mclX deletion mutants, the expression of cwlC (which encodes a key cell wall hydrolase) was significantly decreased, as shown by the ß-galactosidase activity assay. MclX cannot directly bind to the cwlC promoter, based on the electrophoretic mobility shift assay (EMSA). The cwlC was reported to be regulated by σK and GerE. However, the transcriptional activities of sigK and gerE showed no difference between HD73 and the mclX deletion mutant. It is indicated that MclX influenced cwlC expression independently of σK or GerE, through a new pathway to regulate cwlC expression. mclX deletion could be a new approach for insecticidal protein encapsulation in Bacillus thuringiensis.

Expression of the Bacillus thuringiensis vip3A Insecticidal Toxin Gene Is Activated at the Onset of Stationary Phase by VipR, an Autoregulated Transcription Factor.

The Vegetative insecticidal protein Vip3A is produced by some Bacillus thuringiensis strains from the mid-log growth phase to sporulation. Although Vip3A is important for the entomopathogenicity of B. thuringiensis, the vip3A gene regulation is unknown. In the B. thuringiensis serovar kurstaki HD1 strain, vip3A is carried by the pBMB299 plasmid, which is absent in the closely related strain B. thuringiensis kurstaki HD73. Using a transcriptional fusion between the vip3A promoter and lacZ, we observed that the HD73 strain is unable to express vip3A. This result suggests that a specific regulator is required for vip3A expression. Assuming that the regulator gene is located on the same plasmid as vip3A, we transferred pBMB299 from the HD1 strain to the HD73 strain. We found that Vip3A was produced in the HD73 strain containing pBMB299, suggesting that the regulator gene is located on this plasmid. Using this heterologous host and promoter-lacZ transcription fusions, we showed that a specific regulator, VipR, is essential to activate vip3A expression at the onset of stationary phase. We demonstrated that vipR transcription is positively autoregulated and the determination of the vipR and vip3A promoters pinpointed a putative VipR target upstream from the Sigma A-specific -10 region of these two promoters. Surprisingly, this conserved sequence was also found upstream of cry1I and cry2 genes. Finally, we showed that vip3A and vipR expression is increased drastically in a Δspo0A mutant unable to initiate sporulation. In conclusion, we have characterized a novel regulator involved in the entomopathogenic potency of B. thuringiensis through a sporulation-independent pathway. IMPORTANCE The insecticidal properties of Bacillus thuringiensis are due mainly to Cry toxins which form a crystalline inclusion during sporulation. However, other proteins participate in the pathogenicity of the bacterium, notably, the Vip3A toxins that are produced from vegetative growth to sporulation. The VipR regulator that activates vip3A gene expression at the onset of stationary phase is positively autoregulated, and an analysis of the promoter region of the vip3A and vipR genes reveals the presence of a highly conserved DNA sequence. This possible VipR target sequence is also found upstream of the cry2A and cry1I genes, suggesting that Cry toxins can be produced before the bacteria enter sporulation. Such a result could allow us to better understand the role of Cry and Vip3A toxins during the B. thuringiensis infectious cycle in insects, in addition to the primary role of the Cry toxins in the toxemia caused by ingestion of crystals.

The Transcription Factor CpcR Determines Cell Fate by Modulating the Initiation of Sporulation in Bacillus thuringiensis.

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. IMPORTANCE Formation of spores is a paradigm for study of cell differentiation in prokaryotes. Sporulation initiation is governed by a gradual increase in the level and activity of the master regulator Spo0A. Spo0A is usually indirectly phosphorylated by a multicomponent phosphorelay, and modulation of this phosphorelay system is a critical aspect of Bacillus physiology. Though we know that this phosphorelay system is usually affected by two negative regulatory mechanisms, i.e., rap genes and spo0E family genes, the regulatory mechanisms controlling the transcription of these genes are poorly understood. Here, we report that the transcription factor CpcR positively regulates a spo0E family gene and that variations in spo0E expression can modulate the production of spores in B. thuringiensis. This work emphasizes the diversity in modes of sporulation and illustrates the diversity in the strategies employed by bacteria to control this differentiation pathway and ensure their survival.

The Fate of Bacteria of the Bacillus cereus Group in the Amoeba Environment.

The Bacillus cereus sensu lato group consists of several closely related species, including B. anthracis, B. cereus sensu stricto, and B. thuringiensis. Spores of these pathogenic bacteria are commonly found in the soil but evidence suggests that they are unable to grow in such a natural environment in the absence of nutrient input. Amoebas have been reported to be an amplifier for several species of pathogenic bacteria and their potential involvement to explain the large amount of B. thuringiensis and B. cereus spores in soil has been frequently proposed. Here, we studied the fate of Bacillus and amoebas when cultured together. We show that the virulence factors produced by B. thuringiensis and B. cereus do not affect the amoeba Acanthamoeba castellanii, which, on the contrary, can phagocytose and effectively digest vegetative Bacillus cells to grow and prevent the formation of cysts. Bacterial spores can germinate in the amoeba environment and the vegetative cells can then form chains or aggregates that appear to be less efficiently phagocyted by the amoeba. The use of transcriptional fusions between fluorescent reporter genes and stationary phase- and sporulation-specific promoters showed that the sporulation process occurs more efficiently in the presence of amoebas than in their absence. Moreover, our results showed the amoeba environment to promote spore germination and allow the bacteria to complete their developmental cycle. Overall, this study suggests that the amoeba-Bacillus interaction creates a virtuous circle in which each protagonist helps the other to develop.

Immune Inhibitor A Metalloproteases Contribute to Virulence in Bacillus Endophthalmitis.

Endophthalmitis is a devastating infection that can cause blindness. Over half of Bacillus endophthalmitis cases result in significant loss of useful vision. Bacillus produces many virulence factors that may contribute to retinal damage and robust inflammation. We analyzed Bacillus immune inhibitor A (InhA) metalloproteases in the context of this disease, hypothesizing that InhAs contribute to Bacillus intraocular virulence and inflammation. We analyzed phenotypes and infectivity of wild-type (WT), InhA1-deficient (ΔinhA1), InhA2-deficient (ΔinhA2), or InhA1, A2, and A3-deficient (ΔinhA1-3) Bacillus thuringiensis. In vitro analysis of growth, proteolysis, and cytotoxicity were compared. WT and InhA mutants were similarly cytotoxic to retinal cells. The ΔinhA1 and ΔinhA2 mutants entered log-phase growth earlier than WT B. thuringiensis. Proteolysis by the ΔinhA1-3 mutant was decreased, but this strain grew similar to WT in vitro. Experimental endophthalmitis was initiated by intravitreally infecting C57BL/6J mice with 200 CFU of WT B. thuringiensis or InhA mutants. Eyes were analyzed for intraocular Bacillus and myeloperoxidase concentrations, retinal function loss, and gross histological changes. Eyes infected with the ΔinhA1 or ΔinhA2 mutant strains contained greater numbers of bacteria than eyes infected with WT throughout the infection course. Eyes infected with single mutants had inflammation and retinal function loss similar to eyes infected with the WT strain. Eyes infected with the ΔinhA1-3 mutant cleared the infection. Quantitative real-time PCR (qRT-PCR) results suggested that there may be compensatory expression of the other InhAs in the single InhA mutant. These results indicate that together, the InhA metalloproteases contribute to the severity of infection and inflammation in Bacillus endophthalmitis.

Massive Integration of Planktonic Cells within a Developing Biofilm.

During biofilm growth, the coexistence of planktonic and sessile cells can lead to dynamic exchanges between the two populations. We have monitored the fate of these populations in glass tube assays, where the Bacillus thuringiensis 407 strain produces a floating pellicle. Time-lapse spectrophotometric measurement methods revealed that the planktonic population grew until the pellicle started to be produced. Thereafter, the planktonic population decreased rapidly down to a value close to zero while the biofilm was in continuous growth, showing no dispersal until 120 h of culture. We found that this decrease was induced by the presence of the pellicle, but did not occur when oxygen availability was limited, suggesting that it was independent of cell death or cell sedimentation and that the entire planktonic population has integrated the biofilm. To follow the distribution of recruited planktonic cells within the pellicle, we tagged planktonic cells with GFP and sessile cells with mCherry. Fluorescence binocular microscopy observations revealed that planktonic cells, injected through a 24-h-aged pellicle, were found only in specific areas of the biofilm, where the density of sessile cells was low, showing that spatial heterogeneity can occur between recruited cells and sessile cells in a monospecies biofilm.

Long inverted repeats around the chromosome replication terminus in the model strain Bacillus thuringiensis serovar israelensis BGSC 4Q7.

Bacillus thuringiensis serovar israelensis is the most widely used natural biopesticide against mosquito larvae worldwide. Its lineage has been actively studied and a plasmid-free strain, B. thuringiensis serovar israelensis BGSC 4Q7 (4Q7), has been produced. Previous sequencing of the genome of this strain has revealed the persistent presence of a 235 kb extrachromosomal element, pBtic235, which has been shown to be an inducible prophage, although three putative chromosomal prophages have been lost. Moreover, a 492 kb region, potentially including the standard replication terminus, has also been deleted in the 4Q7 strain, indicating an absence of essential genes in this area. We reanalysed the genome coverage distribution of reads for the previously sequenced variant strain, and sequenced two independently maintained samples of the 4Q7 strain. A 553 kb area, close to the 492 kb deletion, was found to be duplicated. This duplication presumably restored the equal sizes of the replichores, and a balanced functioning of replication termination. An analysis of genome assembly graphs revealed a transient association of the host chromosome with the pBtic235 element. This association may play a functional role in the replication of the bacterial chromosome, and the termination of this process in particular. The genome-restructuring events detected may modify the genetic status of cytotoxic or haemolytic toxins, potentially influencing strain virulence. Twelve of the single-nucleotide variants identified in 4Q7 were probably due to the procedure used for strain construction or were present in the precursor of this strain. No sequence variants were found in pBtic235, but the distribution of the corresponding 4Q7 reads indicates a significant difference from counterparts in natural B. thuringiensis serovar israelensis strains, suggesting a duplication or over-replication in 4Q7. Thus, the 4Q7 strain is not a pure plasmid-less offshoot, but a highly genetically modified derivative of its natural ancestor. In addition to potentially influencing virulence, genome-restructuring events can modify the replication termination machinery. These findings have potential implications for the conclusions of virulence studies on 4Q7 as a model, but they also raise interesting fundamental questions about the functioning of the Bacillus genome.

The Alternative Sigma Factor SigB Is Required for the Pathogenicity of Bacillus thuringiensis.

To adapt to changing and potentially hostile environments, bacteria can activate the transcription of genes under the control of alternative sigma factors, such as SigB, a master regulator of the general stress response in several Gram-positive species. Bacillus thuringiensis is a Gram-positive spore-forming invertebrate pathogen whose life cycle includes a variety of environments, including plants and the insect hemocoel or gut. Here, we assessed the role of SigB during the infectious cycle of B. thuringiensis in a Galleria mellonella insect model. We used a fluorescent reporter coupled to flow cytometry and showed that SigB was activated in vivo We also showed that the pathogenicity of the ΔsigB mutant was severely affected when inoculated via the oral route, suggesting that SigB is critical for B. thuringiensis adaptation to the gut environment of the insect. We could not detect an effect of the sigB deletion on the survival of the bacteria or on their sporulation efficiency in the cadavers. However, the gene encoding the pleiotropic regulator Spo0A was upregulated in the ΔsigB mutant cells during the infectious process.IMPORTANCE Pathogenic bacteria often need to transition between different ecosystems, and their ability to cope with such variations is critical for their survival. Several Gram-positive species have developed an adaptive response mediated by the general stress response alternative sigma factor SigB. In order to understand the ecophysiological role of this regulator in Bacillus thuringiensis, an entomopathogenic bacterium widely used as a biopesticide, we sought to examine the fate of a ΔsigB mutant during its life cycle in the natural setting of an insect larva. This allowed us, in particular, to show that SigB was activated during infection and that it was required for the pathogenicity of B. thuringiensis via the oral route of infection.

Rap-Phr Systems from Plasmids pAW63 and pHT8-1 Act Together To Regulate Sporulation in the Bacillus thuringiensis Serovar kurstaki HD73 Strain.

Bacillus thuringiensis is a Gram-positive spore-forming bacterium pathogenic to various insect species. This property is due to the Cry toxins encoded by plasmid genes and mostly produced during sporulation. B. thuringiensis contains a remarkable number of extrachromosomal DNA molecules and a great number of plasmid rap-phr genes. Rap-Phr quorum-sensing systems regulate different bacterial processes, notably the commitment to sporulation in Bacillus species. Rap proteins are quorum sensors acting as phosphatases on Spo0F, an intermediate of the sporulation phosphorelay, and are inhibited by Phr peptides that function as signaling molecules. In this study, we characterize the Rap63-Phr63 system encoded by the pAW63 plasmid from the B. thuringiensis serovar kurstaki HD73 strain. Rap63 has moderate activity on sporulation and is inhibited by the Phr63 peptide. The rap63-phr63 genes are cotranscribed, and the phr63 gene is also transcribed from a σH-specific promoter. We show that Rap63-Phr63 regulates sporulation together with the Rap8-Phr8 system harbored by plasmid pHT8_1 of the HD73 strain. Interestingly, the deletion of both phr63 and phr8 genes in the same strain has a greater negative effect on sporulation than the sum of the loss of each phr gene. Despite the similarities in the Phr8 and Phr63 sequences, there is no cross talk between the two systems. Our results suggest a synergism of these two Rap-Phr systems in the regulation of the sporulation of B. thuringiensis at the end of the infectious cycle in insects, thus pointing out the roles of the plasmids in the fitness of the bacterium.IMPORTANCE The life cycle of Bacillus thuringiensis in insect larvae is regulated by quorum-sensing systems of the RNPP family. After the toxemia caused by Cry insecticidal toxins, the sequential activation of these systems allows the bacterium to trigger first a state of virulence (regulated by PlcR-PapR) and then a necrotrophic lifestyle (regulated by NprR-NprX); ultimately, sporulation is controlled by the Rap-Phr systems. Our study describes a new rap-phr operon carried by a B. thuringiensis plasmid and shows that the Rap protein has a moderate effect on sporulation. However, this system, in combination with another plasmidic rap-phr operon, provides effective control of sporulation when the bacteria develop in the cadavers of infected insect larvae. Overall, this study highlights the important adaptive role of the plasmid Rap-Phr systems in the developmental fate of B. thuringiensis and its survival within its ecological niche.

Optimal Response to Quorum-Sensing Signals Varies in Different Host Environments with Different Pathogen Group Size.

The persistence of genetic variation in master regulators of gene expression, such as quorum-sensing systems, is hard to explain. Here, we investigated two alternative hypotheses for the prevalence of polymorphic quorum sensing in Gram-positive bacteria, i.e., the use of different signal/receptor pairs ('pherotypes') to regulate the same functions. First, social interactions between pherotypes or 'facultative cheating' may favor rare variants that exploit the signals of others. Second, different pherotypes may increase fitness in different environments. We evaluated these hypotheses in the invertebrate pathogen Bacillus thuringiensis, using three pherotypes expressed in a common genetic background. Facultative cheating could occur in well-mixed host homogenates provided there was minimal cross talk between competing pherotypes. However, facultative cheating did not occur when spatial structure was increased in static cultures or in naturalistic oral infections, where common pherotypes had higher fitness. There was clear support for environment-dependent fitness; pherotypes varied in responsiveness to signals and in mean competitive fitness. Notably, competitive fitness varied with group size. In contrast to typical social evolution models of quorum sensing which predict higher response to signal at larger group size, the pherotype with highest responsiveness to signals performed best in smaller hosts where infections have a lower pathogen group size. In this system, low signal abundance appears to limit fitness in hosts, while the optimal level of response to signals varies in different host environments.IMPORTANCE Quorum sensing describes the ability of microbes to alter gene regulation according to their local population size. Some successful theory suggests that this is a form of cooperation, namely, investment in shared products is only worthwhile if there are sufficient bacteria making the same product. This theory can explain the genetic diversity in these signaling systems in Gram-positive bacteria, such as Bacillus and Staphylococcus sp. The possible advantages gained by rare genotypes (which can exploit the products of their more common neighbors) could explain why different genotypes can coexist. We show that while these social interactions can occur in simple laboratory experiments, they do not occur in naturalistic infections using an invertebrate pathogen, Bacillus thuringiensis Instead, our results suggest that different genotypes are adapted to differently sized hosts. Overall, social models are not easily applied to this system, implying that a different explanation for this form of quorum sensing is required.

The signaling peptide PapR is required for the activity of the quorum-sensor PlcRa in Bacillus thuringiensis.

The transcriptional regulator PlcR, its cognate cell-cell signaling heptapeptide PapR7, and the oligopeptide permease OppABCDF, required for PapR7 import, form a quorum-sensing system that controls the expression of virulence factors in Bacillus cereus and Bacillus thuringiensis species. In B. cereus strain ATCC 14579, the transcriptional regulator PlcRa activates the expression of abrB2 gene, which encodes an AbrB-like transcriptional regulator involved in cysteine biosynthesis. PlcRa is a structural homolog of PlcR: in particular, its C-terminal TPR peptide-binding domain could be similarly arranged as in PlcR. The signaling peptide of PlcRa is not known. As PlcRa is a PlcR-like protein, the cognate PapR7 peptide (ADLPFEF) is a relevant candidate to act as a signaling peptide for PlcRa activation. Also, the putative PapRa7 peptide (CSIPYEY), encoded by the papRa gene adjacent to the plcRa gene, is a relevant candidate as addition of synthetic PapRa7 induces a dose-dependent increase of abrB2 expression. To address the issue of peptide selectivity of PlcRa, the role of PapR and PapRa peptides in PlcRa activity was investigated in B. thuringiensis 407 strain, by genetic and functional complementation analyses. A transcriptional fusion between the promoter of abrB2 and lacZ was used to monitor the PlcRa activity in various genetic backgrounds. We demonstrated that PapR was necessary and sufficient for PlcRa activity. We showed that synthetic PapRs from pherogroups II, III and IV and synthetic PapRa7 were able to trigger abrB2 expression, suggesting that PlcRa is less selective than PlcR. Lastly, the mode of binding of PlcRa was addressed using an in silico approach. Overall, we report a new role for PapR as a signaling peptide for PlcRa activity and show a functional link between PlcR and PlcRa regulons in B. thuringiensis.

The stationary phase regulator CpcR activates cry gene expression in non-sporulating cells of Bacillus thuringiensis.

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.

The oligopeptide ABC-importers are essential communication channels in Gram-positive bacteria.

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.

Elucidating the Hot Spot Residues of Quorum Sensing Peptidic Autoinducer PapR by Multiple Amino Acid Replacements.

The quorum sensing (QS) system of Bacillus cereus, an opportunistic human pathogen, utilizes the autoinducing PapR peptide signal that mediates the activation of the pleiotropic virulence regulator PlcR. A set of synthetic 7-mer PapR-derived peptides (PapR7; ADLPFEF) have been shown to inhibit efficiently the PlcR regulon activity and the production of virulence factors, reflected by a loss in hemolytic activity without affecting bacterial growth. Interestingly, these first potent synthetic inhibitors involved D-amino acid or alanine replacements of three amino acids; proline, glutamic acid, and phenylalanine of the heptapeptide PapR. To better understand the role of these three positions in PlcR activity, we report herein the second generation design, synthesis, and characterization of PapR7-derived combinations, alternate double and triple alanine and D-amino acids replacement at these positions. Our findings generate a new set of non-native PapR7-derived peptides that inhibit the PlcR regulon activity and the production of virulence factors. Using the amino acids substitution strategy, we revealed the role of proline and glutamic acid on PlcR regulon activation. Moreover, we demonstrated that the D-Glutamic acid substitution was crucial for the design of stronger PlcR antagonists. These peptides represent potent synthetic inhibitors of B. cereus QS and constitute new and readily accessible chemical tools for the study of the PlcR system. Our method might be applied to other quorum sensing systems to design new anti-virulence agents.

Diversity of the Rap-Phr quorum-sensing systems in the Bacillus cereus group.

Bacteria of the Bacillus cereus group colonize several ecological niches and infect different hosts. Bacillus cereus, a ubiquitous species causing food poisoning, Bacillus thuringiensis, an entomopathogen, and Bacillus anthracis, which is highly pathogenic to mammals, are the most important species of this group. These species are closely related genetically, and their specific toxins are encoded by plasmids. The infectious cycle of B. thuringiensis in its insect host is regulated by quorum-sensing systems from the RNPP family. Among them, the Rap-Phr systems, which are well-described in Bacillus subtilis, regulate essential processes, such as sporulation. Given the importance of these systems, we performed a global in silico analysis to investigate their prevalence, distribution, diversity and their role in sporulation in B. cereus group species. The rap-phr genes were identified in all selected strains with 30% located on plasmids, predominantly in B. thuringiensis. Despite a high variability in their sequences, there is a remarkable association between closely related strains and their Rap-Phr profile. Based on the key residues involved in RapH phosphatase activity, we predicted that 32% of the Rap proteins could regulate sporulation by preventing the phosphorylation of Spo0F. These Rap are preferentially located on plasmids and mostly related to B. thuringiensis. The predictions were partially validated by in vivo sporulation experiments suggesting that the residues linked to the phosphatase function are necessary but not sufficient to predict this activity. The wide distribution and diversity of Rap-Phr systems could strictly control the commitment to sporulation and then improve the adaptation capacities of the bacteria to environmental changes.

The Bacillus cereus Group: Bacillus Species with Pathogenic Potential.

The Bacillus cereus group includes several Bacillus species with closely related phylogeny. The most well-studied members of the group, B. anthracis, B. cereus, and B. thuringiensis, are known for their pathogenic potential. Here, we present the historical rationale for speciation and discuss shared and unique features of these bacteria. Aspects of cell morphology and physiology, and genome sequence similarity and gene synteny support close evolutionary relationships for these three species. For many strains, distinct differences in virulence factor synthesis provide facile means for species assignment. B. anthracis is the causative agent of anthrax. Some B. cereus strains are commonly recognized as food poisoning agents, but strains can also cause localized wound and eye infections as well as systemic disease. Certain B. thuringiensis strains are entomopathogens and have been commercialized for use as biopesticides, while some strains have been reported to cause infection in immunocompromised individuals. In this article we compare and contrast B. anthracis, B. cereus, and B. thuringiensis, including ecology, cell structure and development, virulence attributes, gene regulation and genetic exchange systems, and experimental models of disease.