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

<i>Bacillus thuringiensis</i> Spores and Vegetative Bacteria: Infection Capacity and Role of the Virulence Regulon PlcR Following Intrahaemocoel Injection of <i>Galleria mellonella</i>

Bacillus thuringiensis is an invertebrate pathogen that produces insecticidal crystal toxins acting on the intestinal barrier. In the Galleria mellonella larvae infection model, toxins from the PlcR virulence regulon contribute to pathogenicity by the oral route. While B. thuringiensis is principally an oral pathogen, bacteria may also reach the insect haemocoel following injury of the cuticle. Here, we address the question of spore virulence as compared to vegetative cells when the wild-type Bt407cry- strain and its isogenic ∆plcR mutant are inoculated directly into G. mellonella haemocoel. Mortality dose-response curves were constructed at 25 and 37 &#176;C using spores or vegetative cell inocula, and the 50% lethal dose (LD50) in all infection conditions was determined after 48 h of infection. Our findings show that (i) the LD50 is lower for spores than for vegetative cells for both strains, while the temperature has no significant influence, and (ii) the ∆plcR mutant is four to six times less virulent than the wild-type strain in all infection conditions. Our results suggest that the environmental resistant spores are the most infecting form in haemocoel and that the PlcR virulence regulon plays an important role in toxicity when reaching the haemocoel from the cuticle and not only following ingestion

Quorum Sensing in Bacillus thuringiensis Is Required for Completion of a Full Infectious Cycle in the Insect

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

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

International audienceThe 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

Faecalibacterium : a bacterial genus with promising human health applications

In humans, many diseases are associated with alterations in gut microbiota, namely increases or decreases in the abundance of specific bacterial groups. One example is the genus Faecalibacterium. Numerous studies have underscored that low levels of Faecalibacterium are correlated with inflammatory conditions, with inflammatory bowel disease (IBD) in the forefront. Its representation is also diminished in the case of several diseases, including colorectal cancer (CRC), dermatitis, and depression. Additionally, the relative presence of this genus is considered to reflect, at least in part, intestinal health status because Faecalibacterium is frequently present at reduced levels in individuals with gastrointestinal diseases or disorders. In this review, we first thoroughly describe updates to the taxonomy of Faecalibacterium, which has transformed a single-species taxon to a multispecies taxon over the last decade. We then explore the links discovered between Faecalibacterium abundance and various diseases since the first IBD-focused studies were published. Next, we examine current available strategies for modulating Faecalibacterium levels in the gut. Finally, we summarize the mechanisms underlying the beneficial effects that have been attributed to this genus. Together, epidemiological and experimental data strongly support the use of Faecalibacterium as a next-generation probiotic (NGP) or live biotherapeutic product (LBP)

Integration of bacterial RNA-Seq & human gut microbiota metaproteomic datasets highlights Faecalibacterium duncaniae A2-165’s import systems

Abstract text (incl. references):Faecalibacterium are among the main bacteria responsible for the consumption of acetate and the production of butyrate, which has anti-inflammatory properties beneficial to intestinal health (1). Yet, little is known about how acetate availability affects this bacterium’s gene expression strategies either in a pure culture or in a complex environnement. Here, we show that, in the early stationary phase, F. duncaniae strain A2-165 can strongly regulate the expression of metabolic, transport and stress-response genes based on acetate availability in a pure culture model. Using RNA-seq, we compared gene expression patterns between two growth phases (late exponential vs. early stationary) and two acetate levels (low: 3 mM vs. high: 23 mM). At low-acetate levels, a general stress response was activated, and protein synthesis expression was down-regulated. At high- acetate levels, there was greater expression of genes related to butyrate synthesis and to the importation of B vitamins and iron. We analyzed a metaproteomic dataset obtained from eight healthy individuals (2), dedicated to the envelope fraction of the gut microbiota. Among the 42 transporter genes upregulated in high-acetate conditions, we found 236 peptides that matched with 10 transporter genes. The second highest degree of protein coverage (43.6%, 51 peptides) was found for the FeoB transporter. Of those 51 peptides, 9 were specific to a single protein in the metaproteomic dataset. Through multiomics and targeted approaches, this work highlights the crucial role that the feoAABC operon might play in iron homeostasis in F. duncaniae strain A2-165’s physiology (3). 1. R. Martin, D. Rios-Covian, E.Huillet, et al.. Faecalibacterium: a genus with expanding potential as a live biotherapeutic product in humans revue soumise à FEMS Microbiol Rev. 2. Henry C, Bassignani A, Berland M, Langella O, Sokol H, Juste C. (2022) Modern Metaproteomics: A Unique Tool to Characterize the Active Microbiome in Health and Diseases, and Pave the Road towards New Biomarkers—Example of Crohn’s Disease and Ulcerative Colitis Flare-Ups. Cells, 8 (11) 1340 3. S. Verstraeten, S. Layec, S. Auger, C. Juste, C.Henry, S. Charif, Y. Jaszczyszyn, H. Sokol, L.Beney, P. Langella, M. Thomas, E. Huillet. Faecalibacterium duncaniae A2-165 regulates the expression of butyrate synthesis, ferrous irouptake, and stress-response genes based on acetate level in early-stationary cultures. https://doi.org/10.21203/rs.3.rs-2481125/v1 preprint en révisio

Integration of bacterial RNA-Seq & human gut microbiota metaproteomic datasets highlights Faecalibacterium duncaniae A2-165’s import systems

Abstract text (incl. references):Faecalibacterium are among the main bacteria responsible for the consumption of acetate and the production of butyrate, which has anti-inflammatory properties beneficial to intestinal health (1). Yet, little is known about how acetate availability affects this bacterium’s gene expression strategies either in a pure culture or in a complex environnement. Here, we show that, in the early stationary phase, F. duncaniae strain A2-165 can strongly regulate the expression of metabolic, transport and stress-response genes based on acetate availability in a pure culture model. Using RNA-seq, we compared gene expression patterns between two growth phases (late exponential vs. early stationary) and two acetate levels (low: 3 mM vs. high: 23 mM). At low-acetate levels, a general stress response was activated, and protein synthesis expression was down-regulated. At high- acetate levels, there was greater expression of genes related to butyrate synthesis and to the importation of B vitamins and iron. We analyzed a metaproteomic dataset obtained from eight healthy individuals (2), dedicated to the envelope fraction of the gut microbiota. Among the 42 transporter genes upregulated in high-acetate conditions, we found 236 peptides that matched with 10 transporter genes. The second highest degree of protein coverage (43.6%, 51 peptides) was found for the FeoB transporter. Of those 51 peptides, 9 were specific to a single protein in the metaproteomic dataset. Through multiomics and targeted approaches, this work highlights the crucial role that the feoAABC operon might play in iron homeostasis in F. duncaniae strain A2-165’s physiology (3). 1. R. Martin, D. Rios-Covian, E.Huillet, et al.. Faecalibacterium: a genus with expanding potential as a live biotherapeutic product in humans revue soumise à FEMS Microbiol Rev. 2. Henry C, Bassignani A, Berland M, Langella O, Sokol H, Juste C. (2022) Modern Metaproteomics: A Unique Tool to Characterize the Active Microbiome in Health and Diseases, and Pave the Road towards New Biomarkers—Example of Crohn’s Disease and Ulcerative Colitis Flare-Ups. Cells, 8 (11) 1340 3. S. Verstraeten, S. Layec, S. Auger, C. Juste, C.Henry, S. Charif, Y. Jaszczyszyn, H. Sokol, L.Beney, P. Langella, M. Thomas, E. Huillet. Faecalibacterium duncaniae A2-165 regulates the expression of butyrate synthesis, ferrous irouptake, and stress-response genes based on acetate level in early-stationary cultures. https://doi.org/10.21203/rs.3.rs-2481125/v1 preprint en révisio

The CodY-dependent <i>clhAB2</i> operon is involved in cell shape, chaining and autolysis in <i>Bacillus cereus</i> ATCC 14579

<div><p>The Gram-positive pathogen <i>Bacillus cereus</i> 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 <i>B</i>. <i>cereus</i> 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 <i>clhAB</i>_<i>2</i> operon is required for this chain lengthening phenotype. The <i>clhAB</i>_<i>2</i> operon is specific to the <i>B</i>. <i>cereus</i> group (i.e., <i>B</i>. <i>thuringiensis</i>, <i>B</i>. <i>anthracis</i> and <i>B</i>. <i>cereus</i>) and encodes two membrane proteins of unknown function, which are homologous to the <i>Staphylococcus aureus</i> CidA and CidB proteins involved in cell death control within glucose-grown cells. A deletion mutant (Δ<i>clhAB</i>_<i>2</i>) was constructed and our quantitative image analyses show that Δ<i>clhAB</i>_<i>2</i> cells formed abnormal short chains regardless of the presence of glucose. We also found that glucose-grown cells of Δ<i>clhAB</i>_<i>2</i> were significantly wider than wild-type cells (1.47 μm ±CI_95% 0.04 vs 1.19 μm ±CI_95% 0.03, respectively), suggesting an alteration of the bacterial cell wall. Remarkably, Δ<i>clhAB</i>_<i>2</i> cells showed accelerated autolysis under autolysis-inducing conditions, compared to wild-type cells. Overall, our data suggest that the <i>B</i>. <i>cereus clhAB</i>_<i>2</i> 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 <i>clhAB</i>_<i>2</i> using a <i>lacZ</i> transcriptional reporter in wild-type, <i>ccpA</i> and <i>codY</i> deletion-mutant strains. We found that the global transcriptional regulatory protein CodY is required for the basal level of <i>clhAB</i>_<i>2</i> 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 <i>clhAB</i>_<i>2</i> in glucose-grown cells.</p></div

Two models for <i>B</i>. <i>cereus clhAB</i>_<i>2</i> regulation.

<p>Left: regulation of <i>clhAB</i>_<i>2</i> expression in an amino acid- rich medium. In a nutrient-rich medium (such as LB medium [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184975#pone.0184975.ref013" target="_blank">13</a>]), ILV uptake is sufficient to maintain the endogenous pool of ILV. The CodY global regulatory protein displays enhanced affinity for its DNA target when bound to ILV[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184975#pone.0184975.ref027" target="_blank">27</a>]. ILV-bound CodY binds to the CodY binding sequence upstream of <i>clhAB</i>_<i>2</i> and assists RNA polymerase with transcribing the <i>clhAB</i>_<i>2</i> operon (Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184975#pone.0184975.g003" target="_blank">3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184975#pone.0184975.g004" target="_blank">4</a>). The expression level of <i>clhAB</i>_<i>2</i> is constant and moderate (i.e. basal level) during the late exponential and transition growth phases (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184975#pone.0184975.g001" target="_blank">Fig 1</a>). Right: regulation of <i>clhAB</i>_<i>2</i> expression in an amino acid- glucose- rich medium. In LB medium with 0.35% glucose, ILV-CodY binds to the CodY motif and activates <i>clhAB</i>_<i>2</i> expression, but the expression profile is different (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184975#pone.0184975.g001" target="_blank">Fig 1</a>). CcpA plays a positive role by indirectly regulating the transcription of <i>clhAB</i>_<i>2</i> (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184975#pone.0184975.g002" target="_blank">Fig 2</a>) and this regulatory pathway remains to be characterized. The role of known or hypothetical effector molecules is depicted with a dashed arrow. In the left part, the ILV effector [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184975#pone.0184975.ref027" target="_blank">27</a>] is depicted with a green dashed arrow; in right part, the Fru-6-P [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184975#pone.0184975.ref025" target="_blank">25</a>] and unknown glycolytic intermediate effectors are depicted with blue dashed arrows. Moderate and constant <i>clhAB</i>_<i>2</i> expression is depicted by three identical gray arrows. High and gradual <i>clhAB</i>_<i>2</i> expression is depicted by three non-identical gray arrows. The intracellular ILV pool is depicted as a green box. The CodY motif sequence is depicted as a red box. The unknown DNA-binding motif is depicted as a blue box.?, unknown CcpA-dependent signaling pathway; ?, unknown transcriptional regulator. Fru-6-P, fructose-6-phosphate, Glc-6-P, glucose-6-phosphate. ILV, isoleucine, leucine, valine. CodY, CodY transcriptional regulator. CcpA, CcpA global carbon regulator; PTS, phosphotransferase system. T, unknown transporter of ILV.</p