Une population minoritaire résistante aux peptides antimicrobiens chez la bactérie Photorhabdus est responsable de la virulence

Une population minoritaire résistante aux peptides antimicrobiens chez la bactérie [i]Photorhabdus[/i] est responsable de la virulence. Immuninv 201

Antibiose et antibiorésistance: tuer et ne pas être tué

Antibiose et antibiorésistance: tuer et ne pas être tu

Bistability and antimicrobial peptide resistance in the entomopathogenic bacteria Photorhabdus

Bistability and antimicrobial peptide resistance in the entomopathogenic bacteria [i]Photorhabdus[/i]. 5. Journée du Réseau thématique transversal « INTERACTION MICRO-ORGANISMES HOTES (IMH)

- PM1072 - Bistability and antimicrobial peptide resista nce in the entomopathogenic bacteria Photorhabdus

- PM1072 - Bistability and antimicrobial peptide resista nce in the entomopathogenic bacteria Photorhabdus. International Union of Microbiological Societies Congresses - IUMS201

The complete methylome of an entomopathogenic bacterium reveals the existence of loci with unmethylated Adenines

DNA methylation can serve to control diverse phenomena in eukaryotes and prokaryotes, including gene regulation leading to cell differentiation. In bacteria, DNA methylomes (i.e., methylation state of each base of the whole genome) have been described for several species, but methylome profile variation during the lifecycle has rarely been studied, and only in a few model organisms. Moreover, major phenotypic changes have been reported in several bacterial strains with a deregulated methyltransferase, but the corresponding methylome has rarely been described. Here we report the first methylome description of an entomopathogenic bacterium, Photorhabdus luminescens. Eight motifs displaying a high rate of methylation (> 94%) were identified. The methylome was strikingly stable over course of growth, but also in a subpopulation responsible for a critical step in the bacterium's lifecycle: successful survival and proliferation in insects. The rare unmethylated GATC motifs were preferentially located in putative promoter regions, and most of them were methylated after Dam methyltransferase overexpression, suggesting that DNA methylation is involved in gene regulation. Our findings bring key insight into bacterial methylomes and encourage further research to decipher the role of loci protected from DNA methylation in gene regulation

Ail and PagC-Related Proteins in the Entomopathogenic Bacteria of Photorhabdus Genus

Among pathogenic Enterobacteriaceae, the proteins of the Ail/OmpX/PagC family form a steadily growing family of outer membrane proteins with diverse biological properties, potentially involved in virulence such as human serum resistance, adhesion and entry into eukaryotic culture cells. We studied the proteins Ail/OmpX/PagC in the bacterial Photorhabdus genus. The Photorhabdus bacteria form symbiotic complexes with nematodes of Heterorhabditis species, associations which are pathogenic to insect larvae. Our phylogenetic analysis indicated that in Photorhabdus asymbiotica and Photorhabdus luminescens only Ail and PagC proteins are encoded. The genomic analysis revealed that the Photorhabdus ail and pagC genes were present in a unique copy, except two ail paralogs from P. luminescens. These genes, referred to as ail1Pl and ail2Pl, probably resulted from a recent tandem duplication. Surprisingly, only ail1Pl expression was directly controlled by PhoPQ and low external Mg2+ conditions. In P. luminescens, the magnesium-sensing two-component regulatory system PhoPQ regulates the outer membrane barrier and is required for pathogenicity against insects. In order to characterize Ail functions in Photorhabdus, we showed that only ail2Pl and pagCPl had the ability, when expressed into Escherichia coli, to confer resistance to complement in human serum. However no effect in resistance to antimicrobial peptides was found. Thus, the role of Ail and PagC proteins in Photorhabdus life cycle is discussed

An antimicrobial peptide-resistant minor subpopulation of Photorhabdus luminescens is responsible for virulence

Some of the bacterial cells in isogenic populations behave differently from others. We describe here how a new type of phenotypic heterogeneity relating to resistance to cationic antimicrobial peptides (CAMPs) is determinant for the pathogenic infection process of the entomopathogenic bacterium Photorhabdus luminescens. We demonstrate that the resistant subpopulation, which accounts for only 0.5% of the wild-type population, causes septicemia in insects. Bacterial heterogeneity is driven by the PhoPQ two-component regulatory system and expression of pbgPE, an operon encoding proteins involved in lipopolysaccharide (LPS) modifications. We also report the characterization of a core regulon controlled by the DNA-binding PhoP protein, which governs virulence in P. luminescens. Comparative RNAseq analysis revealed an upregulation of marker genes for resistance, virulence and bacterial antagonism in the pre-existing resistant subpopulation, suggesting a greater ability to infect insect prey and to survive in cadavers. Finally, we suggest that the infection process of P. luminescens is based on a bet-hedging strategy to cope with the diverse environmental conditions experienced during the lifecycle

Only <i>ail1</i>_Pl is directly regulated by PhoP.

<p><b>A</b>. RT-qPCR: <i>ail1</i>_Pl expression is PhoP-dependent. Total RNA from <i>phoP</i> mutant or TT01 wild-type strain of <i>Photorhabdus luminescens</i> was used for RT-qPCR analysis with internal primers specific for the indicated genes. mRNA levels were normalized against those of a reference gene (<i>gyrB</i>). Data are presented as a ratio of values for <i>phoP</i> mutant and TT01 wild-type strain. A ratio of 1 indicates no difference in expression level between both strains. The bars indicate standard errors calculated using Taylor's series. Significant differences (<i>p</i>-value <0.05) are indicated by asterisks (*). The relative quantification results were obtained from three independent experiments with the REST 2009 program. <b>B</b>. Gene transcription monitored by GFP quantification: <i>ail1</i>_Pl promoter region is positively regulated by PhoP. The dynamic expression of <i>ail1</i>_Pl and <i>ail2</i>_Pl promoter in TT01 and <i>phoP</i> backgrounds was monitored over time after growth in LB medium. Each histogram represents the specific fluorescence at the peak of expression for each condition. One experiment representative of more than three independent experiments is shown. Standard deviations represent technical replicates. <b>C</b>. PhoP-His binds the promoter region of <i>ail1</i>_Pl. Electrophoretic mobility shift assay was carried out to test the binding of PhoP-His protein activated <i>in vitro</i> with ACP 10 mM (P-PhoP-His) or non activated PhoP-His (PhoP-His) on <i>ail1</i>_Pl promoter. The PhoP-His concentrations indicated are in micromolar. To ensure that the fixation is specific, we used BSA proteins and poly(dI-dC) in the binding buffer. <b>D</b>. <i>ail1</i>_Pl expression is higher at low MgSO₄ concentrations. We evaluated the impact of low and high MgSO₄ concentrations on <i>ail1</i>_Pl and <i>ail2</i>_Pl expression. Cultures diluted at 1/200 were grown in M9 minimal medium supplemented with 10 µM or 10 mM MgSO₄. Each histogram represents specific fluorescence at the peak of expression for each condition. Experiments were realized at least three times.</p

Human serum resistance of <i>Escherichia coli</i> XL1Blue strains carrying the plasmid pUC19 and its derivatives harboring <i>ail</i> or <i>pagC</i> genes.

<p>Overnight grown bacteria were tested for viability at 37°C in 50% serum (black histograms) or heat-inactivated serum (hatched histograms). The resistance was expressed as the difference in log kill between XL1-Blue harboring pUC19 incubated in 50% human serum and XL1Blue harboring the recombinant plasmid incubated either in 50% human serum or heat-inactivated serum. Means and standard errors of results from triplicate experiments are shown.</p