Caractérisation moléculaire des mécanismes de résistance aux macrolides chez Campylobacter

Campylobacter, is one of the leading cause of human gastroenteritis, is reported worldwide to be increasingly resistant to antibiotics. Apart from target mutations, an efflux mechanism (CmeABC efflux system) has been described. My thesis work is about the molecular characterization of macrolide resistance in Campylobacter. This work of thesis allowed us to put in evidence the important capacity of adaptation of Campylobacter to its environment, whether it is by a deregulation of the expression of the cmeABC operon or an important genetic variability of the CmeB transporter. Furthermore, for the first time to this bacterium, we identified modifications in L4 and L22 ribosomal proteins who can be directly responsible for an increase of the resistance in macrolides.Campylobacter est responsable de la majorité des gastro-entérites bactériennes humaines dans les pays développés. A ce problème de prévalence, s ajoute celui de la résistance aux antibiotiques de ce genre bactérien. Lorsqu une antibiothérapie est nécessaire, les molécules de choix, sont l érythromycine (macrolide) ou la ciprofloxacine (fluoroquinolone). L objectif de ce travail de thèse était de mieux comprendre les mécanismes de résistance aux macrolides. Ce travail de thèse nous a permis de mettre en évidence la formidable capacité d'adaptation de Campylobacter à son environnement, que ce soit par une dérégulation de l'expression de l'opéron cmeABC ou une importante variabilité génétique du transporteur CmeB. De plus, pour la première fois chez ce genre bactérien, nous avons identifié des modifications dans les protéines ribosomales L4 et L22 pouvant être directement responsables d'une augmentation de la résistance aux macrolides.TOURS-BU Sciences Pharmacie (372612104) / SudocSudocFranceF

Diversité, dynamique et mobilité des éléments intégratifs conjugatifs (ICE) de Streptococcus agalactiae intégrés dans l'extrémité 3' du gène codant un ARNt Lysine

Les éléments intégratifs conjugatifs (ICE) et les éléments en dérivant jouent un rôle important dans le transfert horizontal de gènes chez les bactéries. Les ICE s'excisent par recombinaison site-spécifique sous forme circulaire, se transfèrent par conjugaison et s'intègrent dans un réplicon de la cellule réceptrice. Streptococcus agalactiae est une bactérie pathogène opportuniste responsable d'infections néonatales sévères chez l'Homme et d'infections chez les animaux (bovins, poissons, ...). Une analyse in silico antérieure de 8 génomes séquencés de Streptococcus agalactiae avait permis d'identifier plusieurs éléments intégrés dans l'extrémité 3' d'un gène codant un ARNtLys CTT dont 4 ICE putatifs. Cette étude élargie à 246 génomes a confirmé la prévalence et la diversité des éléments intégrés dans ce locus (présence d'ICE, éléments mobilisables en trans ou en cis, éléments composites, ... chez 98 % des souches). Une nouvelle famille d'éléments mobilisables putatifs s'intégrant dans l'oriT d'ICE a été caractérisée. L'étude fonctionnelle de 5 ICE a montré que 4 s'excisent du chromosome mais que seuls ICE_FSL S3-026_tRNALys et ICE_515_tRNALys se transfèrent par conjugaison au sein de l'espèce et vers S. pyogenes pour l'un des 2. Des éléments composites ont été obtenus par transfert d'ICE_515_tRNALys vers une souche possédant déjà un élément intégré dans ce locus. Un de ces éléments composites est capable de s'exciser et de se transférer par conjugaison conduisant à une mobilisation en cis de l'élément résident. En conclusion, les ICE et les éléments mobilisables (en cis ou en trans) sont très répandus chez S. agalactiae et contribuent à la plasticité génomique chez cette espèceIntegrative and Conjugative Elements (ICEs) and related elements are widespread in bacteria and play a key role in horizontal gene transfer. ICEs excise by site-specific recombination as a circular intermediate, promote their own transfer by conjugation and then integrate into a replicon of the recipient cell. Streptococcus agalactiae is an opportunistic pathogen that causes severe human invasive neonatal infections as well as infections in animals (bovine, fish...). Previous in silico analysis of eight sequenced genomes of S. agalactiae identified in each genome a different element integrated in the tRNALys CTT gene with four putative ICEs. This study, carried on 246 other genomes of S. agalactiae, confirmed the prevalence and diversity of elements integrated in this locus with 98% of the strains carrying an element (ICE, trans or cis mobilizable elements composite elements...). A novel family of putative mobilisable elements which can integrate in the oriT of ICE has been characterized. Functional analysis of 5 ICEs demonstrated that four can excise of the chromosome but that only ICE_FSLS3-026_tRNALys and ICE_515_tRNALys can transfer by conjugation inside the species or to S. pyogenes for one of them. Composite elements have been obtained after transfer of ICE_515_tRNALys to a recipient strain already carrying an element integrated in the same locus. One of this composite element is able to excise and transfer by conjugation to a new strain leading to cis-mobilization of the resident element.In conclusion, ICEs and cis and trans mobilizable elements are widespread in S. agalactiae and contribute to the genomic plasticity in this bacterial speciesMETZ-SCD (574632105) / SudocNANCY1-Bib. numérique (543959902) / SudocNANCY2-Bibliotheque electronique (543959901) / SudocNANCY-INPL-Bib. électronique (545479901) / SudocSudocFranceF

Caractérisation des fonctions codées par les éléments intégratifs conjugatifs (ICE) intégrés dans un gène codant un ARNt lysine chez Streptococcus agalactiae (rôle dans le maintien des ICE, l'adaptation et la virulence de l'hôte)

Le transfert horizontal participe à l'évolution rapide des génomes bactériens. Les éléments intégratifs et conjugatifs (ICE) sont des îlots génomiques capables de se transférer par conjugaison vers une bactérie receveuse. Streptococcus agalactiae est une bactérie pathogène opportuniste qui est à l'origine de problèmes sanitaires et économiques majeurs. Des études ont révélé la présence de nombreux ICE chez cette espèce, notamment à l'extrémité 3' d?un gène codant un ARNtLys. La fonctionnalité de l'ICE intégré à ce locus chez la souche 515 de S. agalactiae a été démontrée. Les fonctions véhiculées par ICE_515_tRNALys et pouvant conférer un avantage adaptatif ont été caractérisées et leur transfert vers d'autres espèces a été évalué. Les résultats ont montré que l'ICE confère à S. agalactiae des propriétés d'adhésion à l'hôte et de formation de biofilm et pourrait être impliqué dans l'agrégation cellulaire. Un antigène I/II codé par l'ICE est impliqué dans des phénotypes d'adhésion. De plus, un nouveau facteur co-hémolytique de type CAMP, codé par l'ICE et qui pourrait être impliqué dans la virulence et la survie des souches, a été caractérisé. La fonctionnalité de ces facteurs de virulence chez des espèces bactériennes pathogènes et non pathogènes a été établie. Les travaux ont également révélé la prévalence et la dynamique évolutive des ICE appartenant à la famille d'ICE_515_tRNALys et des fonctions adaptatives codées par ces éléments chez plusieurs espèces de streptocoques. En conclusion, les ICE de la famille d'ICE_515_tRNALys représentent des vecteurs de traits phénotypiques importants pour la virulence et la survie chez les streptocoquesHorizontal gene transfer is a rapid mechanism of evolution. Integrative and conjugative elements (ICEs) are genomic islands which can transfer by conjugation to recipient bacteria. Streptococcus agalactiae is a human and animal opportunistic pathogen that is responsible for major health and economic problems. Studies revealed the presence of numerous ICEs in S. agalactiae, in particular at the 3' end of a tRNALys encoding gene. The functionality of the element present in strain S. agalactiae 515 was demonstrated and was thus chosen as a model for this study. This work focused on the characterization of adaptive and virulence functions encoded by ICE_515_tRNALys and their transfer to other species. Results indicated that this ICE confers adhesion properties to host, increases biofilm formation and may be involved in cell aggregation. A new protein belonging to the antigens I/II family is involved in fibronectin binding and contributes to the biofilm phenotype. In addition, a new co-hemolytic CAMP factor encoded by ICE_515_tRNALys, which could be involved in virulence and bacterial survival, was identified and characterized. These virulence factors are functional in other bacterial species. This work also revealed the prevalence and evolutionary dynamics of ICE belonging to the family of ICE_515_tRNALys and adaptive functions encoded by these elements in several species of streptococci. In conclusion, ICEs of the ICE_515_tRNALys family represent vectors of phenotypic features important for virulence and survival in streptococciMETZ-SCD (574632105) / SudocNANCY1-Bib. numérique (543959902) / SudocNANCY2-Bibliotheque electronique (543959901) / SudocNANCY-INPL-Bib. électronique (545479901) / SudocSudocFranceF

Diversity of Integrative and Conjugative Elements of Streptococcus salivarius and Their Intra- and Interspecies Transfer

Integrative and conjugative elements (ICEs) are widespread chromosomal mobile genetic elements which can transfer autonomously by conjugation in bacteria. Thirteen ICEs with a conjugation module closely related to that of ICESt3 of Streptococcus thermophilus were characterized in Streptococcus salivarius by wholegenomesequencing. Sequence comparison highlighted ICE evolution by shuffling of 3 different integration/excision modules (for integration in the 3= end of the fda, rpsI, or rpmG gene) with the conjugation module of the ICESt3 subfamily. Sequence analyses also pointed out a recombination occurring at oriT (likely mediated by the relaxase) as a mechanism of ICE evolution. Despite a similar organization in two operons including three conserved genes, the regulation modules show a high diversity (about 50% amino acid sequence divergence for the encoded regulators and presence of unrelated additional genes) with a probable impact on the regulation of ICE activity. Concerning the accessory genes, ICEs of the ICESt3 subfamily appearparticularly rich in restriction-modification systems and orphan methyltransferase genes. Other cargo genes that could confer a selective advantage to the cell hosting the ICE were identified, in particular, genes for bacteriocin synthesis and cadmium resistance. The functionality of 2 ICEs of S. salivarius was investigated. Autonomous conjugative transfer to other S. salivarius strains, to S. thermophilus, and to Enterococcusfaecalis was observed. The analysis of the ICE-fda border sequence in these transconjugants allowed the localization of the DNA cutting site of the ICE integrase. IMPORTANCE The ICESt3 subfamily of ICEs appears to be widespread in streptococci and targets diverse chromosomal integration sites. These ICEs carry diverse cargo genes that can confer a selective advantage to the host strain. The maintenance of these mobile genetic elements likely relies in part on self-encoded restrictionmodification systems. In this study, intra- and interspecies transfer was demonstrated for 2 ICEs of S. salivarius. Closely related ICEs were also detected in silico in other Streptococcus species (S. pneumoniae and S. parasanguinis), thus indicating thatdiffusion of ICESt3-related elements probably plays a significant role in horizontal gene transfer (HGT) occurring in the oral cavity but also in the digestive tract, where S. salivarius is present

Diversity of Integrative and Conjugative Elements of Streptococcus salivarius and Their Intra- and Interspecies Transfer

Integrative and conjugative elements (ICEs) are widespread chromosomal mobile genetic elements which can transfer autonomously by conjugation in bacteria. Thirteen ICEs with a conjugation module closely related to that of ICESt3 of Streptococcus thermophilus were characterized in Streptococcus salivarius by wholegenomesequencing. Sequence comparison highlighted ICE evolution by shuffling of 3 different integration/excision modules (for integration in the 3= end of the fda, rpsI, or rpmG gene) with the conjugation module of the ICESt3 subfamily. Sequence analyses also pointed out a recombination occurring at oriT (likely mediated by the relaxase) as a mechanism of ICE evolution. Despite a similar organization in two operons including three conserved genes, the regulation modules show a high diversity (about 50% amino acid sequence divergence for the encoded regulators and presence of unrelated additional genes) with a probable impact on the regulation of ICE activity. Concerning the accessory genes, ICEs of the ICESt3 subfamily appearparticularly rich in restriction-modification systems and orphan methyltransferase genes. Other cargo genes that could confer a selective advantage to the cell hosting the ICE were identified, in particular, genes for bacteriocin synthesis and cadmium resistance. The functionality of 2 ICEs of S. salivarius was investigated. Autonomous conjugative transfer to other S. salivarius strains, to S. thermophilus, and to Enterococcusfaecalis was observed. The analysis of the ICE-fda border sequence in these transconjugants allowed the localization of the DNA cutting site of the ICE integrase. IMPORTANCE The ICESt3 subfamily of ICEs appears to be widespread in streptococci and targets diverse chromosomal integration sites. These ICEs carry diverse cargo genes that can confer a selective advantage to the host strain. The maintenance of these mobile genetic elements likely relies in part on self-encoded restrictionmodification systems. In this study, intra- and interspecies transfer was demonstrated for 2 ICEs of S. salivarius. Closely related ICEs were also detected in silico in other Streptococcus species (S. pneumoniae and S. parasanguinis), thus indicating thatdiffusion of ICESt3-related elements probably plays a significant role in horizontal gene transfer (HGT) occurring in the oral cavity but also in the digestive tract, where S. salivarius is present

New Insights into the Classification and Integration Specificity of Streptococcus Integrative Conjugative Elements through Extensive Genome Exploration

International audienceRecent genome analyses suggest that integrative and conjugative elements (ICEs) are widespread in bacterial genomes and therefore play an essential role in horizontal transfer. However, only a few of these elements are precisely characterized and correctly delineated within sequenced bacterial genomes. Even though previous analysis showed the presence of ICEs in some species of Streptococci, the global prevalence and diversity of ICEs was not analyzed in this genus. In this study, we searched for ICEs in the completely sequenced genomes of 124 strains belonging to 27 streptococcal species. These exhaustive analyses revealed 105 putative ICEs and 26 slightly decayed elements whose limits were assessed and whose insertion site was identified. These ICEs were grouped in seven distinct unrelated or distantly related families, according to their conjugation modules. Integration of these streptococcal ICEs is catalyzed either by a site-specific tyrosine integrase, a low-specificity tyrosine integrase, a site-specific single serine integrase, a triplet of site-specific serine integrases or a DDE transposase. Analysis of their integration site led to the detection of 18 target-genes for streptococcal ICE insertion including eight that had not been identified previously (ftsK, guaA, lysS, mutT, rpmG, rpsI, traG, and ebfC). It also suggests that all specificities have evolved to minimize the impact of the insertion on the host. This overall analysis of streptococcal ICEs emphasizes their prevalence and diversity and demonstrates that exchanges or acquisitions of conjugation and recombination modules are frequent

A Glimpse into the World of Integrative and Mobilizable Elements in Streptococci Reveals an Unexpected Diversity and Novel Families of Mobilization Proteins

International audienceRecent analyses of bacterial genomes have shown that integrated elements that transfer by conjugation play an essential role in horizontal gene transfer. Among these elements, the integrative and mobilizable elements (IMEs) are known to encode their own excision and integration machinery, and to carry all the sequences or genes necessary to hijack the mating pore of a conjugative element for their own transfer. However, knowledge of their prevalence and diversity is still severely lacking. In this work, an extensive analysis of 124 genomes from 27 species of Streptococcus reveals 144 IMEs. These IMEs encode either tyrosine or serine integrases. The identification of IME boundaries shows that 141 are specifically integrated in 17 target sites. The IME-encoded relaxases belong to nine superfamilies, among which four are previously unknown in any mobilizable or conjugative element. A total of 118 IMEs are found to encode a non-canonical relaxase related to rolling circle replication initiators (belonging to the four novel families or to MobT). Surprisingly, among these, 83 encode a TcpA protein (i.e., a non-canonical coupling protein (CP) that is more closely related to FtsK than VirD4) that was not previously known to be encoded by mobilizable elements. Phylogenetic analyses reveal not only many integration/excision module replacements but also losses, acquisitions or replacements of TcpA genes between IMEs. This glimpse into the still poorly known world of IMEs reveals that mobilizable elements have a very high prevalence. Their diversity is even greater than expected, with most encoding a CP and/or a non-canonical relaxase

Impact of cell surface molecules on conjugative transfer of the integrative and conjugative element ICESt3 of Streptococcus thermophilus

International audienceIntegrative Conjugative Elements (ICEs) are chromosomal elements that are widely distributed in bacterial genomes, hence contributing to genome plasticity, adaptation and evolution of bacteria. Conjugation requires a contact between both the donor and the recipient cells and thus likely depends on the composition of the cell surface envelope. In this work, we investigated the impact of different cell surface molecules including cell surface proteins, wall teichoic acids, lipoteichoic acids and exopolysaccharides on the transfer and acquisition of ICESt3 from Streptococcus thermophilus The transfer of ICESt3 from wild type donor towards mutated recipient cells increased 5- to 400-fold compared to WT when recipients cells were affected in lipoproteins, teichoic acids or exopolysaccharides. These mutants displayed an increased biofilm-forming ability compared to WT suggesting better cell interactions that could contribute to the increase of ICESt3 acquisition. Microscopic observations of S. thermophilus cell surface mutants showed different phenotypes (aggregation in particular) that can also have an impact on conjugation.By contrast, the same mutations did not have the same impact when the donor cells, instead of recipient cells, were mutated. In that case, the transfer frequency of ICESt3 decreased compared to WT. The same observation was made when both donor and recipient cells were mutated. The dominant effect of mutations in donor suggests that modifications of the cell envelope could impair the establishment or activity of the conjugation machinery required for DNA transport. IMPORTANCE: ICEs contribute to horizontal gene transfer of adaptive traits (for example virulence, antibiotic resistance or biofilm formation) and play a considerable role in bacterial genome evolution thus underlying the need of a better understanding of their conjugative mechanism of transfer. While most studies are focusing on the different functions encoded by ICEs, little is known about the effect of host factors on their conjugative transfer. Using ICESt3 of S. thermophilus as a model of study, we demonstrated the impact of lipoproteins, teichoic acids and exopolysaccharides on ICE transfer and acquisition. This opens up new avenues to control gene transfers mediated by ICEs

The ICE/IME Finder approach and its application to ICE/IME annotation in Streptococcus.

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Mobile Genetic Elements transferring by conjugation in the human commensal bacterium Streptococcus salivarius: abundance, diversity and role in the adaptation to environment.

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