Optimal response to quorum-sensing signals varies in different host environments with different pathogen group size (article)

This is the final version. Available on open access from the American Society for Microbiology via the DOI in this recordData availability: Experimental data on expression, bacterial densities, and relative fitness supporting this publication are openly available from the University of Exeter’s institutional repository at https://doi.org/10.24378/exe.1843The 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.Natural Environment Research Council (NERC)Biotechnology and Biological Sciences Research Council (BBSRC)French National Research Institute for Agriculture, Food and Environment (INRAE

Deciphering Morphological determinants of the helix-shaped Leptospira

Leptospira spp. are thin, highly motile, slow-growing spirochetes that can be distinguished from other bacteria on the basis of their unique helical shape. Defining the mechanisms by which these bacteria generate and maintain this atypical morphology should greatly enhance our understanding of the fundamental physiology of these pathogens. In this study, we showed that peptidoglycan sacculi from Leptospira spp. retain the helical shape of intact cells. Interestingly, the distribution of muropeptides was different from that in the Escherichia coli model, indicating that specific enzymes might be active on the peptidoglycan macromolecule. We could alter the shape of Leptospira biflexa with the broad-spectrum β-lactam antibiotic penicillin G and with amdinocillin and aztreonam, which are β-lactams that preferentially target penicillin-binding protein 2 (PBP2) and PBP3, respectively, in some species. Although genetic manipulations of Leptospira spp. are scarce, we were able to obtain mutants with alterations in genes encoding PBPs, including PBP3. Loss of this protein resulted in cell elongation. We also generated an L. biflexa strain that conditionally expresses MreB. Loss of the MreB function was correlated with morphological abnormalities such as a localized increased diameter and heterogeneous length. A prolonged depletion of MreB resulted in cell lysis, suggesting that this protein is essential. These findings indicate that important aspects of leptospiral cell morphology are determined by the cytoskeleton and the murein layer, thus providing a starting point for a better understanding of the morphogenesis in these atypical bacteria.This work was supported by the Institut Pasteur, Paris, France, and the French Ministry of Research ANR-08-MIE-018. M.A.D.P. was supported by Ministry of Education and Science, Spain (MEC, BFU2006-04574) and Fundación Ramón Areces.Peer Reviewe

CodY Regulates the Activity of the Virulence Quorum Sensor PlcR by Controlling the Import of the Signaling Peptide PapR in Bacillus thuringiensis

In Gram-positive bacteria, cell–cell communication mainly relies on cytoplasmic sensors of the RNPP family. Activity of these regulators depends on their binding to secreted signaling peptides that are imported into the cell. These quorum sensing regulators control important biological functions in bacteria of the Bacillus cereus group, such as virulence and necrotrophism. The RNPP quorum sensor PlcR, in complex with its cognate signaling peptide PapR, is the main regulator of virulence in B. cereus and Bacillus thuringiensis(Bt). Recent reports have shown that the global stationary phase regulator CodY, involved in adaptation to nutritional limitation, is required for the expression of virulence genes belonging to the PlcR regulon. However, the mechanism underlying this regulation was not described. Using genetics and proteomics approaches, we showed that CodY regulates the expression of the virulence genes through the import of PapR. We report that CodY positively controls the production of the proteins that compose the oligopeptide permease OppABCDF, and of several other Opp-like proteins. It was previously shown that the pore components of this oligopeptide permease, OppBCDF, were required for the import of PapR. However, the role of OppA, the substrate-binding protein (SBP), was not investigated. Here, we demonstrated that OppA is not the only SBP involved in the recognition of PapR, and that several other OppA-like proteins can allow the import of this peptide. Altogether, these data complete our model of quorum sensing during the lifecycle of Bt and indicate that RNPPs integrate environmental conditions, as well as cell density, to coordinate the behavior of the bacteria throughout growt

The Social Biology of Quorum Sensing in a Naturalistic Host Pathogen System.

SummaryMany microorganisms cooperate by secreting products that are commonly available to neighboring cells. These “public goods” include autoinduced, quorum-sensing (QS) molecules and the virulence factors activated by these signals [1–4]. Public goods cooperation is exploitable by cheaters, cells that avoid the costs of production but gain an advantage by freeloading on the products of others [5–8]. QS signals and responses can be cooperative under artificial laboratory conditions [1–4, 9], but it remains unclear whether QS is cooperative in nature: little is known about the frequency of cheaters in natural populations [10, 11], and cheaters may do poorly because of the importance of QS in major transcriptional networks [12–14]. Here, we investigate the cooperative nature of QS in a natural system: the Gram-positive insect pathogen Bacillus thuringiensis and the larvae of the diamondback moth, Plutella xylostella. Although we find evidence of cooperation, QS null mutants are not effective cheats in vivo and cannot outcompete wild-type strains. We show that spatial structure limits mutant fitness and that well-separated microcolonies occur in vivo because of the strong population bottlenecks occurring during natural infection. We argue that spatial structure and low densities are the norm in early-stage infections, and this can explain why QS cheaters are rare in B. thuringiensis and its relatives [10]. These results contrast with earlier experiments describing the high fitness of Gram-negative QS cheaters and suggest that QS suppression (“quorum quenching”) can be clinically effective without having negative impacts on the evolution of virulence

Molecular basis for group-specific activation of the virulence regulator PlcR by PapR heptapeptides

The transcriptional regulator PlcR and its cognate cell–cell signalling peptide PapR form a quorum-sensing system that controls the expression of extra-cellular virulence factors in various species of the Bacillus cereus group. PlcR and PapR alleles are clustered into four groups defining four pherotypes. However, the molecular basis for group specificity remains elusive, largely because the biologically relevant PapR form is not known. Here, we show that the in vivo active form of PapR is the C-terminal heptapeptide of the precursor, and not the pentapeptide, as previously suggested. Combining genetic complementation, anisotropy assays and structural analysis we provide a detailed functional and structural explanation for the group specificity of the PlcR–PapR quorum-sensing system. We further show that the C-terminal helix of the PlcR regulatory domain, specifically the 278 residue, in conjunction with the N-terminal residues of the PapR heptapeptide determines this system specificity. Variability in the specificity-encoding regions of plcR and papR genes suggests that selection and evolution of quorum-sensing systems play a major role in adaptation and ecology of Bacilli

FcpB Is a Surface Filament Protein of the Endoflagellum Required for the Motility of the Spirochete Leptospira

International audienceThe spirochete endoflagellum is a unique motility apparatus among bacteria. Despite its critical importance for pathogenesis, the full composition of the flagellum remains to be determined. We have recently reported that FcpA is a novel flagellar protein and a major component of the sheath of the filament of the spirochete Leptospira. By screening a library of random transposon mutants in the spirochete Leptospira biflexa, we found a motility-deficient mutant harboring a disruption in a hypothetical gene of unknown function. Here, we show that this gene encodes a surface component of the endoflagellar filament and is required for typical hook- and spiral-shaped ends of the cell body, coiled structure of the endoflagella, and high velocity phenotype. We therefore named the gene fcpB for flagellar-coiling protein B. fcpB is conserved in all members of the Leptospira genus, but not present in other organisms including other spirochetes. Complementation of the fcpB− mutant restored the wild-type morphology and motility phenotypes. Immunoblotting with anti-FcpA and anti-FcpB antisera and cryo-electron microscopy of the filament indicated that FcpB assembled onto the surface of the sheath of the filament and mostly located on the outer (convex) side of the coiled filament. We provide evidence that FcpB, together with FcpA, are Leptospira-specific novel components of the sheath of the filament, key determinants of the coiled and asymmetric structure of the endoflagella and are essential for high velocity. Defining the components of the endoflagella and their functions in these atypical bacteria should greatly enhance our understanding of the mechanisms by which these bacteria produce motility

Production, Secretion and Biological Activity of Bacillus cereus Enterotoxins

Bacillus cereus behaves as an opportunistic pathogen frequently causing gastrointestinal diseases, and it is increasingly recognized to be responsible for severe local or systemic infections. Pathogenicity of B. cereus mainly relies on the secretion of a wide array of toxins and enzymes and also on the ability to undergo swarming differentiation in response to surface-sensing. In this report, the pathogenicity exerted by B. cereus toxins is described with particular attention to the regulatory mechanisms of production and secretion of HBL, Nhe and CytK enterotoxins

Targeting the Replication Initiator of the Second Vibrio Chromosome: Towards Generation of Vibrionaceae-Specific Antimicrobial Agents

The Vibrionaceae is comprised of numerous aquatic species and includes several human pathogens, such as Vibrio cholerae, the cause of cholera. All organisms in this family have two chromosomes, and replication of the smaller one depends on rctB, a gene that is restricted to the Vibrionaceae. Given the increasing prevalence of multi-drug resistance in pathogenic vibrios, there is a need for new targets and drugs to combat these pathogens. Here, we carried out a high throughput cell-based screen to find small molecule inhibitors of RctB. We identified a compound that blocked growth of an E. coli strain bearing an rctB-dependent plasmid but did not influence growth of E. coli lacking this plasmid. This compound, designated vibrepin, had potent cidal activity against V. cholerae and inhibited the growth of all vibrio species tested. Vibrepin blocked RctB oriCII unwinding, apparently by promoting formation of large non-functional RctB complexes. Although vibrepin also appears to have targets other than RctB, our findings suggest that RctB is an attractive target for generation of novel antibiotics that only block growth of vibrios. Vibrio-specific agents, unlike antibiotics currently used in clinical practice, will not engender resistance in the normal human flora or in non-vibrio environmental microorganisms