Photorhabdus luminescens is an enterobacterium establishing a mutualistic symbiosis with nematodes, that also kills insects after septicaemia and connective tissue colonization. The role of the ...bacterial mdtABC genes encoding a putative multidrug efflux system from the resistance/nodulation/cell division family was investigated. We showed that a mdtA mutant and the wild type had similar levels of resistance to antibiotics, antimicrobial peptides, metals, detergents and bile salts. The mdtA mutant was also as pathogenic as the wild-type following intrahaemocoel injection in Locusta migratoria, but had a slightly attenuated phenotype in Spodoptera littoralis. A transcriptional fusion of the mdtA promoter (PmdtA) and the green fluorescent protein (gfp) encoding gene was induced by copper in bacteria cultured in vitro. The PmdtA-gfp fusion was strongly induced within bacterial aggregates in the haematopoietic organ during late stages of infection in L. migratoria, whereas it was only weakly expressed in insect plasma throughout infection. A medium supplemented with haematopoietic organ extracts induced the PmdtA-gfp fusion ex vivo, suggesting that site-specific mdtABC expression resulted from insect signals from the haematopoietic organ. Finally, we showed that protease inhibitors abolished ex vivo activity of the PmdtA-gfp fusion in the presence of haematopoietic organ extracts, suggesting that proteolysis by-products play a key role in upregulating the putative MdtABC efflux pump during insect infection with P. luminescens.
Xenorhabdus is a bacterial symbiont of entomopathogenic Steinernema nematodes and is pathogenic for insects. Its life cycle involves a stage inside the insect cadaver, in which it competes for ...environmental resources with microorganisms from soil and the insect gut. Xenorhabdus is, thus, a useful model for identifying new interbacterial competition systems. For the first time, in an entomopathogenic bacterium, Xenorhabdus doucetiae strain FRM16, we identified a cdi-like locus. The cdi loci encode contact-dependent inhibition (CDI) systems composed of proteins from the two-partner secretion (TPS) family. CdiB is the outer membrane protein and CdiA is the toxic exoprotein. An immunity protein, CdiI, protects bacteria against inhibition. We describe here the growth inhibition effect of the toxic C-terminus of CdiA from X. doucetiae FRM16, CdiA-CTFRM16, following its production in closely and distantly related enterobacterial species. CdiA-CTFRM16 displayed Mg2+-dependent DNase activity, in vitro. CdiA-CTFRM16-mediated growth inhibition was specifically neutralized by CdiIFRM16. Moreover, the cdi FRM16 locus encodes an ortholog of toxin-activating proteins C that we named CdiCFRM16. In addition to E. coli, the cdiBCAI-type locus was found to be widespread in environmental bacteria interacting with insects, plants, rhizospheres and soils. Phylogenetic tree comparisons for CdiB, CdiA and CdiC suggested that the genes encoding these proteins had co-evolved. By contrast, the considerable variability of CdiI protein sequences suggests that the cdiI gene is an independent evolutionary unit. These findings further characterize the sparsely described cdiBCAI-type locus.
Antibiotic resistance is an increasing threat to human health. A direct link has been established between antimicrobial self-resistance determinants of antibiotic producers, environmental bacteria, ...and clinical pathogens. Natural odilorhabdins (ODLs) constitute a new family of 10-mer linear cationic peptide antibiotics inhibiting bacterial translation by binding to the 30S subunit of the ribosome. These bioactive secondary metabolites are produced by entomopathogenic bacterial symbiont
(
), vectored by the soil-dwelling nematodes. ODL-producing Xenorhabdus nematophila symbionts have mechanisms of self-protection. In this study, we cloned the 44.5-kb
biosynthetic gene cluster (
-BGC) of the symbiont by recombineering and showed that the
-acetyltransferase-encoding gene,
, is responsible for ODL resistance.
acetylation and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses showed that OatA targeted the side chain amino group of ODL rare amino acids, leading to a loss of translation inhibition and antibacterial properties. Functional, genomic, and phylogenetic analyses of
revealed an exclusive
-link to the odilorhabdin BGC, found only in
and a specific phylogenetic clade of
. This work highlights the coevolution of antibiotic production and self-resistance as ancient features of this unique tripartite complex of host-vector-symbiont interactions without
-BGC dissemination by lateral gene transfer.
Odilorhabdins (ODLs) constitute a novel antibiotic family with promising properties for treating problematic multidrug-resistant Gram-negative bacterial infections. ODLs are 10-mer linear cationic peptides inhibiting bacterial translation by binding to the small subunit of the ribosome. These natural peptides are produced by Xenorhabdus nematophila, a bacterial symbiont of entomopathogenic nematodes well known to produce large amounts of specialized secondary metabolites. Like other antimicrobial producers, ODL-producing Xenorhabdus nematophila has mechanisms of self-protection. In this study, we cloned the ODL-biosynthetic gene cluster of the symbiont by recombineering and showed that the
-acetyltransferase-encoding gene,
, is responsible for ODL resistance.
acetylation and LC-MS/MS analyses showed that OatA targeted the side chain amino group of ODL rare amino acids, leading to a loss of translation inhibition and antibacterial properties. Functional, genomic, and phylogenetic analyses of
revealed the coevolution of antibiotic production and self-resistance as ancient feature of this particular niche in soil invertebrates without resistance dissemination.
Bacterial infections are often composed of cells with distinct phenotypes that can be produced by genetic or epigenetic mechanisms. This phenotypic heterogeneity has proved to be important in many ...pathogens, because it can alter both pathogenicity and transmission. We studied how and why it can emerge during infection in the bacterium
, a pathogen that kills insects and multiplies in the cadaver before being transmitted by the soil nematode vector
We found that phenotypic variants cluster in three groups, one of which is composed of
defective mutants. These mutants, together with variants of another group, have in common that they maintain high survival during late stationary phase. This probably explains why they increase in frequency: variants of
with a growth advantage in stationary phase (GASP) are under strong positive selection both in prolonged culture and in late infections. We also found that the within-host advantage of these variants seems to trade off against transmission by nematode vectors: the variants that reach the highest load in insects are those that are the least transmitted.
Pathogens can evolve inside their host, and the importance of this mutation-fueled process is increasingly recognized. A disease outcome may indeed depend in part on pathogen adaptations that emerge during infection. It is therefore important to document these adaptations and the conditions that drive them. In our study, we took advantage of the possibility to monitor within-host evolution in the insect pathogen
We demonstrated that selection occurring in aged infection favors
defective mutants, because these metabolic mutants benefit from a growth advantage in stationary phase (GASP). We also demonstrated that these mutants have reduced virulence and impaired transmission, modifying the infection outcome. Beyond the specific case of
, we propose that metabolic mutants are to be found in other bacterial pathogens that stay for many generations inside their host.
Photorhabdus luminescens is an entomopathogenic bacterium found in symbiosis with the nematode Heterorhabditis. Dam DNA methylation is involved in the pathogenicity of many bacteria, including P. ...luminescens, whereas studies about the role of bacterial DNA methylation during symbiosis are scarce. The aim of this study was to determine the role of Dam DNA methylation in P. luminescens during the whole bacterial life cycle including during symbiosis with H. bacteriophora. We constructed a strain overexpressing dam by inserting an additional copy of the dam gene under the control of a constitutive promoter in the chromosome of P. luminescens and then achieved association between this recombinant strain and nematodes. The dam overexpressing strain was able to feed the nematode in vitro and in vivo similarly as a control strain, and to re-associate with Infective Juvenile (IJ) stages in the insect. No difference in the amount of emerging IJs from the cadaver was observed between the two strains. Compared to the nematode in symbiosis with the control strain, a significant increase in LT50 was observed during insect infestation with the nematode associated with the dam overexpressing strain. These results suggest that during the life cycle of P. luminescens, Dam is not involved the bacterial symbiosis with the nematode H. bacteriophora, but it contributes to the pathogenicity of the nemato-bacterial complex.
The Tetraconata (Pancrustacea) concept proposes that insects are more closely related to aquatic crustaceans than to terrestrial centipedes or millipedes. The question therefore arises whether ...insects have kept crustacean-specific genetic traits that could be targeted by specific toxins. Here we show that a toxin (nigritoxin), originally identified in a bacterial pathogen of shrimp, is lethal for organisms within the Tetraconata and non-toxic to other animals. X-ray crystallography reveals that nigritoxin possesses a new protein fold of the α/β type. The nigritoxin N-terminal domain is essential for cellular translocation and likely encodes specificity for Tetraconata. Once internalized by eukaryotic cells, nigritoxin induces apoptotic cell death through structural features that are localized in the C-terminal domain of the protein. We propose that nigritoxin will be an effective means to identify a Tetraconata evolutionarily conserved pathway and speculate that nigritoxin holds promise as an insecticidal protein.
Dam, the most described bacterial DNA-methyltransferase, is widespread in gamma-proteobacteria. Dam DNA methylation can play a role in various genes expression and is involved in pathogenicity of ...several bacterial species. The purpose of this study was to determine the role played by the
ortholog identified in the entomopathogenic bacterium
. Complementation assays of an
mutant showed the restoration of the DNA methylation state of the parental strain. Overexpression of
in
did not impair growth ability
. In contrast, compared to a control strain harboring an empty plasmid, a significant decrease in motility was observed in the
-overexpressing strain. A transcriptome analysis revealed the differential expression of 208 genes between the two strains. In particular, the downregulation of flagellar genes was observed in the
-overexpressing strain. In the closely related bacterium
overexpression also impaired motility. In addition, the
-overexpressing
strain showed a delayed virulence compared to that of the control strain after injection in larvae of the lepidopteran
. These results reveal that Dam plays a major role during
insect infection.
Heterogeneity in the expression of various bacterial genes has been shown to result in the presence of individuals with different phenotypes within clonal bacterial populations. The genes specifying ...motility and flagellar functions are coordinately regulated and form a complex regulon, the flagellar regulon. Complex interplay has recently been demonstrated in the regulation of flagellar and virulence gene expression in many bacterial pathogens. We show here that FliZ, a DNA-binding protein, plays a key role in the insect pathogen, Xenorhabdus nematophila, affecting not only hemolysin production and virulence in insects, but efficient swimming motility. RNA-Seq analysis identified FliZ as a global regulatory protein controlling the expression of 278 Xenorhabdus genes either directly or indirectly. FliZ is required for the efficient expression of all flagellar genes, probably through its positive feedback loop, which controls expression of the flhDC operon, the master regulator of the flagellar circuit. FliZ also up- or downregulates the expression of numerous genes encoding non-flagellar proteins potentially involved in key steps of the Xenorhabdus lifecycle. Single-cell analysis revealed the bimodal expression of six identified markers of the FliZ regulon during exponential growth of the bacterial population. In addition, a combination of fluorescence-activated cell sorting and RT-qPCR quantification showed that this bimodality generated a mixed population of cells either expressing ("ON state") or not expressing ("OFF state") FliZ-dependent genes. Moreover, studies of a bacterial population exposed to a graded series of FliZ concentrations showed that FliZ functioned as a rheostat, controlling the rate of transition between the "OFF" and "ON" states in individuals. FliZ thus plays a key role in cell fate decisions, by transiently creating individuals with different potentials for motility and host interactions.
Xenorhabdus nematophila, a member of the Enterobacteriaceae, kills many species of insects by strongly depressing the immune system and colonizing the entire body. A peptide cytotoxin has been ...purified from X. nematophila broth growth, and the cytolytic effect on insect immunocytes and hemolytic effect on mammalian red blood cells of this toxin have been described (Ribeiro, C., Vignes, M., and Brehélin, M. (2003) J. Biol. Chem. 278, 3030–3039). We show here that this toxin, Xenorhabdus α-xenorhabdolysin (Xax), triggers apoptosis in both insect and mammalian cells. We also report the cloning and sequencing of two genes, xaxAB, encoding this toxin in X. nematophila. The expression of both genes in recombinant Escherichia coli led to the production of active cytotoxin/hemolysin. However, hemolytic activity was observed only if the two peptides were added in the appropriate order. Furthermore, we report here that inactivation of xaxAB genes in X. nematophila abolished the major cytotoxic activity present in broth growth, called C1. We also show that these genes are present in various entomopathogenic bacteria of the genera Xenorhabdus and Photorhabdus, in Pseudomonas entomophila, in the human pathogens Yersinia enterocolitica and Proteus mirabilis, and in the plant pathogen Pseudomonas syringae. This toxin cannot be classified in any known family of cytotoxins on the basis of amino acid sequences, locus organization, and activity features. It is, therefore, probably the prototype of a new family of binary toxins.
There is a complex interplay between the regulation of flagellar motility and the expression of virulence factors in many bacterial pathogens. We investigated the role of FliZ in the regulation of ...flagellar and virulence genes in Xenorhabdus nematophila, an insect pathogen. The fliZ gene is the second gene in the fliAZ operon in X. nematophila. In vivo transcription analysis revealed a positive feedback loop of fliAZ transcription in which FliZ activates flhDC, the master operon of flagellar regulon in X. nematophila, leading to an increased transcription of the FlhDC-dependent promoter of fliAZ. We also showed that fliAZ and flhDC mutants lacked motility, had no haemolysin or Tween lipase activity and displayed an attenuated virulence phenotype in insects. Lipase activity is controlled by FliA, whereas haemolysin production and full virulence phenotype have been reported to be FliZ-dependent. Transcriptional analysis revealed that FliZ directly controlled expression of the xhlBA and xaxAB operons, which encode haemolysins from the two-partner secretion system and the binary XaxAB toxin family respectively. We suggest that this regulatory pathway may also occur in other pathogenic enterobacteria with genes encoding members of these two growing families of haemolysins.
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