Exploration fonctionnelle du génome de mycoplasma agalactiae : recherche des facteurs de virulence en culture cellulaire

Mycoplasma agalactiae est l'agent étiologique de l'agalactie contagieuse des petits ruminants, maladie inscrite sur la liste de l'OIE. Le développement d'un système de criblage en culture cellulaire a permis de mener une étude à haut-débit des interactions entre M. agalactiae et les cellules de l'hôte. Au cours de cette étude, 62 loci potentiellement impliqués dans les interactions hôte-mycoplasme ont été identifiés, une majorité codant pour des protéines membranaires. Par ailleurs, le locus NIF, un facteur de virulence impliqué dans la formation des groupes fer-soufre chez certaines bactéries pathogènes, s'est révélé essentiel pour la croissance de M. agalactiae en culture cellulaire. Cette étude montre également le rôle important que pourraient jouer certaines régions intergéniques dans les interactions hôte-mycoplasme. Ces résultats offrent de nouvelles pistes pour la compréhension des mécanismes de virulence chez les mycoplasmes et des outils pour le développement de stratégies vaccinales adaptées à ces pathogènes atypiques.Mycoplasma agalactiae is the etiological agent of contagious agalactia, a disease of small ruminants that is listed by the OIE. A cell culture assay was developed for the high-throughput analysis of the interactions between M. agalactiae and host cells. This approach identified 62 loci potentially involved in host-mycoplasma interactions, most of them encoding membrane proteins. Moreover, the NIF locus, a virulence factor involved in the synthesis of iron-sulfur cluster in several pathogenic bacteria, was found as essential for M. agalactiae proliferation in cell culture, while dispensable for axenic growth. Remarkably, this study also revealed that intergenic regions may play unexpected roles in mycoplasma-host interactions. These results provide a new approach to study pathogenic processes in mycoplasma infections and new means for the development of efficient vaccine strategies adapted to these unconventional pathogens

Genome-Scale Analysis of Mycoplasma agalactiae Loci Involved in Interaction with Host Cells

Mycoplasma agalactiae is an important pathogen of small ruminants, in which it causes contagious agalactia. It belongs to a large group of “minimal bacteria” with a small genome and reduced metabolic capacities that are dependent on their host for nutrients. Mycoplasma survival thus relies on intimate contact with host cells, but little is known about the factors involved in these interactions or in the more general infectious process. To address this issue, an assay based on goat epithelial and fibroblastic cells was used to screen a M. agalactiae knockout mutant library. Mutants with reduced growth capacities in cell culture were selected and 62 genomic loci were identified as contributing to this phenotype. As expected for minimal bacteria, “transport and metabolism” was the functional category most commonly implicated in this phenotype, but 50% of the selected mutants were disrupted in coding sequences (CDSs) with unknown functions, with surface lipoproteins being most commonly represented in this category. Since mycoplasmas lack a cell wall, lipoproteins are likely to be important in interactions with the host. A few intergenic regions were also identified that may act as regulatory sequences under co-culture conditions. Interestingly, some mutants mapped to gene clusters that are highly conserved across mycoplasma species but located in different positions. One of these clusters was found in a transcriptionally active region of the M. agalactiae chromosome, downstream of a cryptic promoter. A possible scenario for the evolution of these loci is discussed. Finally, several CDSs identified here are conserved in other important pathogenic mycoplasmas, and some were involved in horizontal gene transfer with phylogenetically distant species. These results provide a basis for further deciphering functions mediating mycoplasma-host interactions

Exploration fonctionnelle du génome de mycoplasma agalactiae (recherche des facteurs de virulence en culture cellulaire)

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

The Response to Nitric Oxide of the Nitrogen-Fixing Symbiont <i>Sinorhizobium meliloti</i>

Nitric oxide (NO) is crucial in animal– and plant–pathogen interactions, during which it participates in host defense response and resistance. Indications for the presence of NO during the symbiotic interaction between the model legume Medicago truncatula and its symbiont Sinorhizobium meliloti have been reported but the role of NO in symbiosis is far from being elucidated. Our objective was to understand the role or roles played by NO in symbiosis. As a first step toward this goal, we analyzed the bacterial response to NO in culture, using a transcriptomic approach. We identified approximately 100 bacterial genes whose expression is upregulated in the presence of NO. Surprisingly, most of these genes are regulated by the two-component system FixLJ, known to control the majority of rhizobial genes expressed in planta in mature nodules, or the NO-dedicated regulator NnrR. Among the genes responding to NO is hmp, encoding a putative flavohemoglobin. We report that an hmp mutant displays a higher sensitivity toward NO in culture and leads to a reduced nitrogen fixation efficiency in planta. Because flavohemoglobins are known to detoxify NO in numerous bacterial species, this result is the first indication of the importance of the bacterial NO response in symbiosis. </jats:p

Critical Role of Dispensable Genes in Mycoplasma agalactiae Interaction with Mammalian Cells▿

Mycoplasmas are minimal bacteria whose genomes barely exceed the smallest amount of information required to sustain autonomous life. Despite this apparent simplicity, several mycoplasmas are successful pathogens of humans and animals, in which they establish intimate interactions with epithelial cells at mucosal surfaces. To identify biological functions mediating mycoplasma interactions with mammalian cells, we produced a library of transposon knockout mutants in the ruminant pathogen Mycoplasma agalactiae and used this library to identify mutants displaying a growth-deficient pheonotype in cell culture. M. agalactiae mutants displaying a 3-fold reduction in CFU titers to nearly complete extinction in coculture with HeLa cells were identified. Mapping of transposon insertion sites revealed 18 genomic regions putatively involved in the interaction of M. agalactiae with HeLa cells. Several of these regions encode proteins with features of membrane lipoproteins and/or were involved in horizontal gene transfer with phylogenetically distant pathogenic mycoplasmas of ruminants. Two mutants with the most extreme phenotype carry a transposon in a genomic region designated the NIF locus which encodes homologues of SufS and SufU, two proteins presumably involved in [Fe-S] cluster biosynthesis in Gram-positive bacteria. Complementation studies confirmed the conditional essentiality of the NIF locus, which was found to be critical for proliferation in the presence of HeLa cells and several other mammalian cell lines but dispensable for axenic growth. While our results raised questions regarding essential functions in mycoplasmas, they also provide a means for studying the role of mycoplasmas as minimal pathogens

Recherche de gènes essentiels pour la multiplication de Mycoplasma agalactiae chez l'höte mais accessoires en milieu axénique : Identification du locus conditionnel NIF

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