The PlcR Virulence Regulon of Bacillus cereus

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 Δ-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

Transcriptional Profiling of Bacillus anthracis Sterne (34F2) during Iron Starvation

Lack of available iron is one of many environmental challenges that a bacterium encounters during infection and adaptation to iron starvation is important for the pathogen to efficiently replicate within the host. Here we define the transcriptional response of B. anthracis Sterne (34F2) to iron depleted conditions. Genome-wide transcript analysis showed that B. anthracis undergoes considerable changes in gene expression during growth in iron-depleted media, including the regulation of known and candidate virulence factors. Two genes encoding putative internalin proteins were chosen for further study. Deletion of either gene (GBAA0552 or GBAA1340) resulted in attenuation in a murine model of infection. This attenuation was amplified in a double mutant strain. These data define the transcriptional changes induced during growth in low iron conditions and illustrate the potential of this dataset in the identification of putative virulence determinants for future study

IlsA, A Unique Surface Protein of Bacillus cereus Required for Iron Acquisition from Heme, Hemoglobin and Ferritin

The human opportunistic pathogen Bacillus cereus belongs to the B. cereus group that includes bacteria with a broad host spectrum. The ability of these bacteria to colonize diverse hosts is reliant on the presence of adaptation factors. Previously, an IVET strategy led to the identification of a novel B. cereus protein (IlsA, Iron-regulated leucine rich surface protein), which is specifically expressed in the insect host or under iron restrictive conditions in vitro. Here, we show that IlsA is localized on the surface of B. cereus and hence has the potential to interact with host proteins. We report that B. cereus uses hemoglobin, heme and ferritin, but not transferrin and lactoferrin. In addition, affinity tests revealed that IlsA interacts with both hemoglobin and ferritin. Furthermore, IlsA directly binds heme probably through the NEAT domain. Inactivation of ilsA drastically decreases the ability of B. cereus to grow in the presence of hemoglobin, heme and ferritin, indicating that IlsA is essential for iron acquisition from these iron sources. In addition, the ilsA mutant displays a reduction in growth and virulence in an insect model. Hence, our results indicate that IlsA is a key factor within a new iron acquisition system, playing an important role in the general virulence strategy adapted by B. cereus to colonize susceptible hosts

Pathogenic Bacillus anthracis in the progressive gene losses and gains in adaptive evolution

Background: Sequence mutations represent a driving force of adaptive evolution in bacterial pathogens. It is especially evident in reductive genome evolution where bacteria underwent lifestyles shifting from a free-living to a strictly intracellular or host-depending life. It resulted in loss of function mutations and/or the acquisition of virulence gene clusters. Bacillus anthracis shares a common soil bacterial ancestor with its closely related bacillus species but is the only obligate, causative agent of inhalation anthrax within the genus Bacillus. The anthrax-causing Bacillus anthracis experienced the similar lifestyle changes. We thus hypothesized that the bacterial pathogen would follow a compatible evolution path. Results: In this study, a cluster-based evolution scheme was devised to analyze genes that are gained by or lost from B. anthracis. The study detected gene losses/gains at two separate evolutionary stages. The stage I is when B. anthracis and its sister species within the Bacillus cereus group diverged from other species in genus Bacillus. The stage II is when B. anthracis differentiated from its two closest relatives: B. cereus and B. thuringiensis. Many genes gained at these stages are homologues of known pathogenic factors such those for internalin, B. anthracis-specific toxins and large groups of surface proteins and lipoproteins. Conclusion: The analysis presented here allowed us to portray a progressive evolutionary process during the lifestyle shift of B. anthracis, thus providing new insights into how B. anthracis had evolved and bore a promise of finding drug and vaccine targets for this strategically important pathogen

Whole-genome phylogenies of the family Bacillaceae and expansion of the sigma factor gene family in the Bacillus cereus species-group

<p>Abstract</p> <p>Background</p> <p>The <it>Bacillus cereus </it><it>sensu lato </it>group consists of six species (<it>B. anthracis</it>, <it>B. cereus</it>, <it>B. mycoides</it>, <it>B. pseudomycoides</it>, <it>B. thuringiensis</it>, and <it>B. weihenstephanensis</it>). While classical microbial taxonomy proposed these organisms as distinct species, newer molecular phylogenies and comparative genome sequencing suggests that these organisms should be classified as a single species (thus, we will refer to these organisms collectively as the <it>Bc </it>species-group). How do we account for the underlying similarity of these phenotypically diverse microbes? It has been established for some time that the most rapidly evolving and evolutionarily flexible portions of the bacterial genome are regulatory sequences and transcriptional networks. Other studies have suggested that the sigma factor gene family of these organisms has diverged and expanded significantly relative to their ancestors; sigma factors are those portions of the bacterial transcriptional apparatus that control RNA polymerase recognition for promoter selection. Thus, examining sigma factor divergence in these organisms would concurrently examine both regulatory sequences and transcriptional networks important for divergence. We began this examination by comparison to the sigma factor gene set of <it>B. subtilis</it>.</p> <p>Results</p> <p>Phylogenetic analysis of the <it>Bc </it>species-group utilizing 157 single-copy genes of the family <it>Bacillaceae </it>suggests that several taxonomic revisions of the genus <it>Bacillus </it>should be considered. Within the <it>Bc </it>species-group there is little indication that the currently recognized species form related sub-groupings, suggesting that they are members of the same species. The sigma factor gene family encoded by the <it>Bc </it>species-group appears to be the result of a dynamic gene-duplication and gene-loss process that in previous analyses underestimated the true heterogeneity of the sigma factor content in the <it>Bc </it>species-group.</p> <p>Conclusions</p> <p>Expansion of the sigma factor gene family appears to have preferentially occurred within the extracytoplasmic function (ECF) sigma factor genes, while the primary alternative (PA) sigma factor genes are, in general, highly conserved with those found in <it>B. subtilis</it>. Divergence of the sigma-controlled transcriptional regulons among various members of the <it>Bc </it>species-group likely has a major role in explaining the diversity of phenotypic characteristics seen in members of the <it>Bc </it>species-group.</p

A comprehensive transcriptome and immune-gene repertoire of the lepidopteran model host Galleria mellonella

<p>Abstract</p> <p>Background</p> <p>The larvae of the greater wax moth <it>Galleria mellonella </it>are increasingly used (i) as mini-hosts to study pathogenesis and virulence factors of prominent bacterial and fungal human pathogens, (ii) as a whole-animal high throughput infection system for testing pathogen mutant libraries, and (iii) as a reliable host model to evaluate the efficacy of antibiotics against human pathogens. In order to compensate for the lack of genomic information in <it>Galleria</it>, we subjected the transcriptome of different developmental stages and immune-challenged larvae to next generation sequencing.</p> <p>Results</p> <p>We performed a <it>Galleria </it>transcriptome characterization on the Roche 454-FLX platform combined with traditional Sanger sequencing to obtain a comprehensive transcriptome. To maximize sequence diversity, we pooled RNA extracted from different developmental stages, larval tissues including hemocytes, and from immune-challenged larvae and normalized the cDNA pool. We generated a total of 789,105 pyrosequencing and 12,032 high-quality Sanger EST sequences which clustered into 18,690 contigs with an average length of 1,132 bases. Approximately 40% of the ESTs were significantly similar (<it>E </it>≤ e^-03) to proteins of other insects, of which 45% have a reported function. We identified a large number of genes encoding proteins with established functions in immunity related sensing of microbial signatures and signaling, as well as effector molecules such as antimicrobial peptides and inhibitors of microbial proteinases. In addition, we found genes known as mediators of melanization or contributing to stress responses. Using the transcriptomic data, we identified hemolymph peptides and proteins induced upon immune challenge by 2D-gelelectrophoresis combined with mass spectrometric analysis.</p> <p>Conclusion</p> <p>Here, we have developed extensive transcriptomic resources for <it>Galleria</it>. The data obtained is rich in gene transcripts related to immunity, expanding remarkably our knowledge about immune and stress-inducible genes in <it>Galleria </it>and providing the complete sequences of genes whose primary structure have only partially been characterized using proteomic methods. The generated data provide for the first time access to the genetic architecture of immunity in this model host, allowing us to elucidate the molecular mechanisms underlying pathogen and parasite response and detailed analyses of both its immune responses against human pathogens, and its coevolution with entomopathogens.</p

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

The Secret Life of the Anthrax Agent Bacillus anthracis: Bacteriophage-Mediated Ecological Adaptations

Ecological and genetic factors that govern the occurrence and persistence of anthrax reservoirs in the environment are obscure. A central tenet, based on limited and often conflicting studies, has long held that growing or vegetative forms of Bacillus anthracis survive poorly outside the mammalian host and must sporulate to survive in the environment. Here, we present evidence of a more dynamic lifecycle, whereby interactions with bacterial viruses, or bacteriophages, elicit phenotypic alterations in B. anthracis and the emergence of infected derivatives, or lysogens, with dramatically altered survival capabilities. Using both laboratory and environmental B. anthracis strains, we show that lysogeny can block or promote sporulation depending on the phage, induce exopolysaccharide expression and biofilm formation, and enable the long-term colonization of both an artificial soil environment and the intestinal tract of the invertebrate redworm, Eisenia fetida. All of the B. anthracis lysogens existed in a pseudolysogenic-like state in both the soil and worm gut, shedding phages that could in turn infect non-lysogenic B. anthracis recipients and confer survival phenotypes in those environments. Finally, the mechanism behind several phenotypic changes was found to require phage-encoded bacterial sigma factors and the expression of at least one host-encoded protein predicted to be involved in the colonization of invertebrate intestines. The results here demonstrate that during its environmental phase, bacteriophages provide B. anthracis with alternatives to sporulation that involve the activation of soil-survival and endosymbiotic capabilities

Comparative analysis of the virulence of invertebrate and mammalian pathogenic bacteria in the oral insect infection model <em>Galleria mellonella</em>

International audienceInfection of Galleria mellonella by feeding a mixture of Bacillus thuringiensis spores or vegetative bacteria in association with the toxin Cry] C results in high levels of larval mortality. Under these conditions the toxin or bacteria have minimal effects on the larva when inoculated separately. In order to evaluate whether G. mellonella can function as an oral infection model for human and entomo-bacterial pathogens. we tested strains of Bacillus cereus, Bacillus anthracis, Enterococcus faecalis, Listeria monocytogenes, Pseudomonas aeruginosa and a Drosophila targeting Pseudomonas entomophila strain. Six B. cereus strains (5 diarrheal, I environmental isolate) were first screened in 2nd instar G. mellonella larvae by free ingestion and four of them were analyzed by force-feeding 5th instar larvae. The virulence of these B. cereus strains did not differ from the B. thuringiensis virulent reference strain 407Cry(-) with the exception of strain D19 (NVH391/98) that showed a lower virulence. Following force-feeding, 5th instar G. mellonella larvae survived infection with B. anthracis, L. monocytogenes, E. faecalis and P. aeruginosa strains in contrast to the P. entomophila strain which led to high mortality even without Cry1C toxin co-ingestion. Thus, specific virulence factors adapted to the insect intestine might exist in B. thuringiensis/B. cereus and P. entomophila. This suggests a co-evolution between host and pathogens and supports the close links between B. thuringiensis and B. cereus and more distant links to their relative B. anthracis