Bacteria of the genus Xenorhabdus are symbionts of soil entomopathogenic nematodes of the genus Steinernema. This symbiotic association constitutes an insecticidal complex active against a wide range ...of insect pests. Within Xenorhabdus bovienii species, the X. bovienii CS03 strain (Xb CS03) is nonvirulent when directly injected into lepidopteran insects, and displays a low virulence when associated with its Steinernema symbiont. The genome of Xb CS03 was sequenced and compared with the genome of a virulent strain, X. bovienii SS-2004 (Xb SS-2004). The genome size and content widely differed between the two strains. Indeed, Xb CS03 had a large genome containing several specific loci involved in the inhibition of competitors, including a few NRPS-PKS loci (nonribosomal peptide synthetases and polyketide synthases) producing antimicrobial molecules. Consistently, Xb CS03 had a greater antimicrobial activity than Xb SS-2004. The Xb CS03 strain contained more pseudogenes than Xb SS-2004. Decay of genes involved in the host invasion and exploitation (toxins, invasins, or extracellular enzymes) was particularly important in Xb CS03. This may provide an explanation for the nonvirulence of the strain when injected into an insect host. We suggest that Xb CS03 and Xb SS-2004 followed divergent evolutionary scenarios to cope with their peculiar life cycle. The fitness strategy of Xb CS03 would involve competitor inhibition, whereas Xb SS-2004 would quickly and efficiently kill the insect host. Hence, Xenorhabdus strains would have widely divergent host exploitation strategies, which impact their genome structure.
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•The Steinernema/Xb pairs are entomopathogenic nematode-bacteria associations.•The S. weiseri/Xb CS03 pair has attenuated virulence and fitness.•Xb CS03 alone is non-virulent when ...injected into G. mellonella and S. littoralis.•Xb CS03 is sensitive to antimicrobial peptides.•Xb CS03 is a new model for the study of Xenorhabdus virulence.
Xenorhabdus bacteria (γ-proteobacteria: Enterobacteriaceae) have dual lifestyles. They have a mutualistic relationship with Steinernema nematodes (Nematoda: Steinernematidae) and are pathogenic to a wide range of insects. Each Steinernema nematode associates with a specific Xenorhabdus species. However, a Xenorhabdus species can have multiple nematode hosts. For example, Xenorhabdus bovienii (Xb) colonizes at least nine Steinernema species from two different phylogenetic clades. The Steinernema–Xb partnership has been found in association with different insect hosts. Biological and molecular data on the Steinernema jollieti–Xb strain SS-2004 pair have recently been described. In particular, the Xb SS-2004 bacteria are virulent alone after direct injection into insect, making this strain a model for studying Xb virulence. In this study, we searched for Xb strains attenuated in virulence. For this purpose, we underwent infection assays with five Steinernema spp.–Xb pairs with two insects, Galleria mellonella (Lepidoptera: Pyralidae) and Spodoptera littoralis (Lepidoptera: Noctuidae). The S. weiseri–Xb CS03 pair showed attenuated virulence and lower fitness in S. littoralis in comparison to the other nematode-bacteria pairs. Furthermore, when injected alone into the hemolymph of G. mellonella or S. littoralis, the Xb CS03 bacterial strain was the only non-virulent strain. By comparison with the virulent Xb SS-2004 strain, Xb CS03 showed an increased sensitivity to the insect antimicrobial peptides, suggesting an attenuated response to the insect humoral immunity. To our current knowledge, Xb CS03 is the first non-virulent Xb strain identified. We propose this strain as a new model for studying the Xenorhabdus virulence.
Expansion of antimicrobial resistance monitoring and epidemiological surveillance are key components of the WHO strategy towards zero leprosy. The inability to grow Mycobacterium leprae in vitro ...precludes routine phenotypic drug susceptibility testing, and only limited molecular tests are available. We evaluated a culture-free targeted deep sequencing assay, for mycobacterial identification, genotyping based on 18 canonical SNPs and 11 core variable-number tandem-repeat (VNTR) markers, and detection of rifampicin, dapsone and fluoroquinolone resistance-associated mutations in rpoB/ctpC/ctpI, folP1, gyrA/gyrB, respectively, and hypermutation-associated mutations in nth.
The limit of detection (LOD) was determined using DNA of M. leprae reference strains and from 246 skin biopsies and 74 slit skin smears of leprosy patients, with genome copies quantified by RLEP qPCR. Sequencing results were evaluated versus whole genome sequencing (WGS) data of 14 strains, and versus VNTR-fragment length analysis (FLA) results of 89 clinical specimens.
The LOD for sequencing success ranged between 80 and 3000 genome copies, depending on the sample type. The LOD for minority variants was 10%. All SNPs detected in targets by WGS were identified except in a clinical sample where WGS revealed two dapsone resistance-conferring mutations instead of one by Deeplex Myc-Lep, due to partial duplication of the sulfamide-binding domain in folP1. SNPs detected uniquely by Deeplex Myc-Lep were missed by WGS due to insufficient coverage. Concordance with VNTR-FLA results was 99.4% (926/932 alleles).
Deeplex Myc-Lep may help improve the diagnosis and surveillance of leprosy. Gene domain duplication is an original putative drug resistance-related genetic adaptation in M. leprae.
EDCTP2 programme supported by the European Union (grant number RIA2017NIM-1847 -PEOPLE). EDCTP, R2Stop: Effect:Hope, The Mission To End Leprosy, the Flemish Fonds Wetenschappelijk Onderzoek.
Bacteria of the genus Xenorhabdus are symbionts of soil entomopathogenic nematodes of the genus Steinernema. This symbiotic association constitutes an insecticidal complex active against a wide range ...of insect pests. Unlike other Xenorhabdus species, Xenorhabdus poinarii is avirulent when injected into insects in the absence of its nematode host. We sequenced the genome of the X. poinarii strain G6 and the closely related but virulent X. doucetiae strain FRM16. G6 had a smaller genome (500–700 kb smaller) than virulent Xenorhabdus strains and lacked genes encoding potential virulence factors (hemolysins, type 5 secretion systems, enzymes involved in the synthesis of secondary metabolites, and toxin–antitoxin systems). The genomes of all the X. poinarii strains analyzed here had a similar small size. We did not observe the accumulation of pseudogenes, insertion sequences or decrease in coding density usually seen as a sign of genomic erosion driven by genetic drift in host-adapted bacteria. Instead, genome reduction of X. poinarii seems to have been mediated by the excision of genomic blocks from the flexible genome, as reported for the genomes of attenuated free pathogenic bacteria and some facultative mutualistic bacteria growing exclusively within hosts. This evolutionary pathway probably reflects the adaptation of X. poinarii to specific host.
Background: Expansion of antimicrobial resistance monitoring and epidemiological surveillance are key components of the WHO strategy towards zero leprosy. The inability to grow Mycobacterium leprae ...in vitro precludes routine phenotypic drug susceptibility testing, and only limited molecular tests are available. We evaluated a culture-free targeted deep sequencing assay, for mycobacterial identification, genotyping based on 18 canonical SNPs and 11 core variable-number tandem-repeat (VNTR) markers, and detection of rifampicin, dapsone and fluoroquinolone resistance-associated mutations in rpoB/ctpC/ctpI, folP1, gyrA/gyrB, respectively, and hypermutation-associated mutations in nth. Methods: The limit of detection (LOD) was determined using DNA of M. leprae reference strains and from 246 skin biopsies and 74 slit skin smears of leprosy patients, with genome copies quantified by RLEP qPCR. Sequencing results were evaluated versus whole genome sequencing (WGS) data of 14 strains, and versus VNTR-fragment length analysis (FLA) results of 89 clinical specimens. Findings: The LOD for sequencing success ranged between 80 and 3000 genome copies, depending on the sample type. The LOD for minority variants was 10%. All SNPs detected in targets by WGS were identified except in a clinical sample where WGS revealed two dapsone resistance-conferring mutations instead of one by Deeplex Myc-Lep, due to partial duplication of the sulfamide-binding domain in folP1. SNPs detected uniquely by Deeplex Myc-Lep were missed by WGS due to insufficient coverage. Concordance with VNTR-FLA results was 99.4% (926/932 alleles). Interpretation: Deeplex Myc-Lep may help improve the diagnosis and surveillance of leprosy. Gene domain duplication is an original putative drug resistance-related genetic adaptation in M. leprae. Funding: EDCTP2 programme supported by the European Union (grant number RIA2017NIM-1847 -PEOPLE). EDCTP, R2Stop: Effect:Hope, The Mission To End Leprosy, the Flemish Fonds Wetenschappelijk Onderzoek.
Les entérobactéries du genre Xenorhabdus sont pathogènes de larves d'insectes et symbiotiques de nématodes du genre Steinernema. En lutte biologique, les couples Steinernema-Xenorhabdus sont utilisés ...contre un large spectre d'insectes ravageurs de culture. Les deux partenaires du couple modèle Steinernema carpocapsae-Xenorhabdus nematophila peuvent être expérimentalement dissociés tout en restant pathogènes pour les insectes. En revanche, certaines souches de Xenorhabdus sont non-virulentes lorsqu'elles sont injectées directement dans une larve d'insecte. L'objectif de cette thèse est de caractériser deux souches non-virulentes de Xenorhabdus, X. poinarii G6 (Xp G6) et X. bovienii CS03 (Xb CS03). Les souches appartenant à l'espèce non-virulente X. poinarii possèdent des génomes de petite taille. Nous avons mis en évidence un phénomène de réduction génomique due à la délétion de larges régions génomiques chez la souche Xp G6. Cette évolution pourrait avoir eu lieu suite à un transfert des fonctions bactériennes de virulence à son nématode hôte et/ou à sa spécialisation envers certains coléoptères. Au sein de l'espèce X. bovienii, Xb CS03 est non-virulente par injection dans les lépidoptères Spodoptera littoralis et Galleria mellonella. Par rapport à d'autres couples némato-bactériens Steinernema sp.-X. bovienii, le couple formé par Xb CS03 et son nématode symbiotique S. weiseri 583 présente également une virulence atténuée sur ces lépidoptères. Le génome de Xb CS03 est de très grande taille et contient un grand nombre de gènes dégradés (pseudogènes). Une comparaison génomique entre Xb CS03 et une souche virulente appartenant à la même espèce, X. bovienii SS-2004 (Xb SS-2004), montre que Xb CS03 est plus riche que Xb SS-2004 en gènes codant des chaînes d'assemblage enzymatiques NRPS/PKS (non-ribosomal peptide synthase/polyketide synthethase) produisant des métabolites antimicrobiens potentiels. A l'inverse, Xb SS-2004 contient davantage de gènes codant des facteurs de virulence de type hémolysine, adhésine ou systèmes de sécrétion. Ceci suggère deux scénarios évolutifs différents, privilégiant une forte virulence pour Xb SS-2004 et l'élimination des compétiteurs au sein du cadavre de l'insecte pour Xb CS03. Enfin, une recherche de facteurs de virulence potentiels a été effectuée par une approche de génomique comparative entre les souches non-virulentes Xp G6 et Xb CS03, d'une part et trois souches de Xenorhabdus virulentes, d'autre part. L'analyse fonctionnelle de gènes candidats a été entamée. En conclusion, la caractérisation de nouveaux modèles bactériens dans le genre Xenorhabdus ouvre le champ à l'identification de nouvelles stratégies de virulence et de nouveaux facteurs de virulence chez les bactéries entomopathogènes.
Xenorhabdus are enterobacteria pathogenic of insect larvae and symbiotic of nematodes from the Steinernema genus. The Steinernema-Xenorhabdus associations are used against a wide range of insect pests. The two partners of the model Steinernema carpocapsae-Xenorhabdus nematophila association can be experimentally dissociated. Each partner is pathogenic for insect larvae. Contrarily, some other Xenorhabdus strains are non-virulent when injected directly into insect larvae. In this thesis, we characterized two non-virulent Xenorhabdus strains, X. poinarii G6 (Xp G6) and X. bovienii CS03 (Xb CS03). Strains from the X. poinarii species had small-sized genomes. We showed that the Xp G6 strain had undergone a genome reduction due to the deletion of large genomic regions. Transfer of virulence functions from the bacteria to the nematode and/or the specialization of the association towards coleopteran insects are likely the cause of this evolution. Within the X. bovienii species, Xb CS03 was non-virulent strain when injected into the Spodoptera littoralis and Galleria mellonella lepidopteran insects. When compared to other Steinernema-X. bovienii pairs, the association between Xb CS03 and its symbiotic nematode S. weiseri 583 had also a lower virulence on those insects. Xb CS03 had a large-sized genome and harbored numerous degraded genes (pseudogenes). Genome comparison between Xb CS03 and a virulent strain from the same species, X. bovienii SS-2004 (Xb SS-2004), showed that Xb CS03 contained more loci encoding NRPS/PKS enzymes (non-ribosomal peptide synthase/polyketide synthethase), producing potential antimicrobial metabolites, than Xb SS-2004. On the other hand, Xb SS-2004 contained more genes encoding virulence factors such as hemolysins, adhesins or secretion systems. This suggests that the two strains followed different evolutionary scenarios, favoring strong virulence in Xb SS-2204 and elimination of competitors for Xb CS03.Finally, we searched for potential virulence factors by comparing the genomes of the non-virulent strains Xp G6 and Xb CS03 with three virulent strains. Functional analyses of the candidates are in progress. In conclusion, characterizing new bacterial models in the Xenorhabdus genus paves the way for the identification of new virulence strategies and new virulence genes in entomopathogenic bacteria.
Many insects rely on bacterial symbionts to supply essential amino acids and vitamins that are deficient in their diets, but metabolic comparisons of closely related gut bacteria in insects with ...different dietary preferences have not been performed. Here, we demonstrate that herbivorous ants of the genus Dolichoderus from the Peruvian Amazon host bacteria of the family Bartonellaceae, known for establishing chronic or pathogenic infections in mammals. We detected these bacteria in all studied Dolichoderus species, and found that they reside in the midgut wall, that is, the same location as many previously described nutritional endosymbionts of insects. The genomic analysis of four divergent strains infecting different Dolichoderus species revealed genes encoding pathways for nitrogen recycling and biosynthesis of several vitamins and all essential amino acids. In contrast, several biosynthetic pathways have been lost, whereas genes for the import and conversion of histidine and arginine to glutamine have been retained in the genome of a closely related gut bacterium of the carnivorous ant Harpegnathos saltator. The broad biosynthetic repertoire in Bartonellaceae of herbivorous ants resembled that of gut bacteria of honeybees that likewise feed on carbohydrate-rich diets. Taken together, the broad distribution of Bartonellaceae across Dolichoderus ants, their small genome sizes, the specific location within hosts, and the broad biosynthetic capability suggest that these bacteria are nutritional symbionts in herbivorous ants. The results highlight the important role of the host nutritional biology for the genomic evolution of the gut microbiota-and conversely, the importance of the microbiota for the nutrition of hosts.
Xenorhabdus bacteria (γ-proteobacteria: Enterobacteriaceae) have dual lifestyles. They have a mutualistic relationship with Steinernema nematodes (Nematoda: Steinernematidae) and are pathogenic to a ...wide range of insects. Each Steinernema nematode associates with a specific Xenorhabdus species. However, a Xenorhabdus species can have multiple nematode hosts. For example, Xenorhabdus bovienii (Xb) colonizes at least nine Steinernema species from two different phylogenetic clades. The Steinernema-Xb partnership has been found in association with different insect hosts. Biological and molecular data on the Steinernema jollieti-Xb strain SS-2004 pair have recently been described. In particular, the Xb SS-2004 bacteria are virulent alone after direct injection into insect, making this strain a model for studying Xb virulence. In this study, we searched for Xb strains attenuated in virulence. For this purpose, we underwent infection assays with five Steinernema spp.- Xb pairs with two insects, Galleria mellonella (Lepidoptera: Pyralidae) and Spodoptera littoralis (Lepidoptera: Noctuidae). The S. weiseri-Xb CS03 pair showed attenuated virulence and lower fitness in S. littoralis in comparison to the other nematode-bacteria pairs. Furthermore, when injected alone into the hemolymph of G. mellonella or S. littoralis, the Xb CS03 bacterial strain was the only non-virulent strain. By comparison with the virulent Xb SS-2004 strain, Xb CS03 showed an increased sensitivity to the insect antimicrobial peptides, suggesting an attenuated response to the insect humoral immunity. To our current knowledge, Xb CS03 is the first non-virulent Xb strain identified. We propose this strain as a new model for studying the Xenorhabdus virulence.
Bacteria of the genus Xenorhabdus are symbionts of soil entomopathogenic nematodes of the genus Steinernema. This symbiotic association constitutes an insecticidal complex active against a wide range ...of insect pests. Unlike other Xenorhabdus species, Xenorhabdus poinarii is avirulent when injected into insects in the absence of its nematode host. We sequenced the genome of the X. poinarii strain G6 and the closely related but virulent Xenorhabdus doucetiae strain FRM16. G6 had a smaller genome (500-700 kb smaller) than virulent Xenorhabdus strains and lacked genes encoding potential virulence factors (haemolysins, type 5 secretion systems, enzymes involved in the synthesis of secondary metabolites, toxin-antitoxin systems). The genomes of all the X. poinarii strains analysed here had a similar small size. We did not observe the accumulation of pseudogenes, insertion sequences or decrease in coding density usually seen as a sign of genomic erosion driven by genetic drift in host-adapted bacteria. Instead, genome reduction of X. poinarii seems to have been mediated by the excision of genomic blocks from the flexible genome, as reported for the genomes of attenuated free pathogenic bacteria and some facultative mutualistic bacteria growing exclusively within hosts. This evolutionary pathway probably reflects the adaptation of X. poinarii to specific host.
Bacteria of the genus Xenorhabdus are symbionts of soil entomopathogenic nematodes of the genus Steinernema. This symbiotic association constitutes an insecticidal complex active against a wide range ...of insect pests. Unlike other Xenorhabdus species, Xenorhabdus poinarii is avirulent when injected into insects in the absence of its nematode host. We sequenced the genome of the X. poinarii strain G6 and the closely related but virulent Xenorhabdus doucetiae strain FRM16. G6 had a smaller genome (500-700 kb smaller) than virulent Xenorhabdus strains and lacked genes encoding potential virulence factors (haemolysins, type 5 secretion systems, enzymes involved in the synthesis of secondary metabolites, toxin-antitoxin systems). The genomes of all the X. poinarii strains analysed here had a similar small size. We did not observe the accumulation of pseudogenes, insertion sequences or decrease in coding density usually seen as a sign of genomic erosion driven by genetic drift in host-adapted bacteria. Instead, genome reduction of X. poinarii seems to have been mediated by the excision of genomic blocks from the flexible genome, as reported for the genomes of attenuated free pathogenic bacteria and some facultative mutualistic bacteria growing exclusively within hosts. This evolutionary pathway probably reflects the adaptation of X. poinarii to specific host.
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