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Christine Citti - One of the best experts on this subject based on the ideXlab platform.
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impacts of Mycoplasma agalactiae restriction modification systems on pan epigenome dynamics and genome plasticity
bioRxiv, 2021Co-Authors: Emilie Dordetfrisoni, Eric Baranowski, Eveline Sagne, C Vandecasteele, R Contarin, Christophe Klopp, X Nouvel, Christine CittiAbstract:DNA methylation plays an important role in the biology of bacteria. Often associated with restriction-modification (RM) systems, they also provide a defence against foreign DNA. Little is known regarding the methylome of the Mycoplasma genus, which encompasses several pathogenic species with small genomes. Here, single molecule real-time (SMRT) and bisulphite sequencing combined with whole-genome analysis identified 19 methylated motifs associated with three orphan methyltransferases (MTases) and eight RM systems in Mycoplasma agalactiae, a ruminant pathogen and a model organism. All systems had a homolog in at least one phylogenetically distinct Mycoplasma spp. Our study also revealed that several superimposed genetic events may participate in the M. agalactiae dynamic epigenome landscape. These included (i) DNA shuffling and frameshift mutations that affect the MTase and restriction endonuclease content of a clonal population and (ii) gene duplication, erosion, and horizontal transfer that modulate MTase and RM repertoires of the species. Some of these systems were experimentally shown to play a major role in Mycoplasma conjugative, horizontal DNA transfer. While the versatility of DNA methylation may contribute to regulating essential biological functions at cell and population levels, RM systems may be key in Mycoplasma genome evolution and adaptation by controlling horizontal gene transfers.
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the integrative conjugative element ice of Mycoplasma agalactiae key elements involved in horizontal dissemination and influence of coresident ices
Mbio, 2018Co-Authors: Eric Baranowski, Marie Claude Hygonenq, Eveline Sagne, Emilie Dordetfrisoni, Laurentxavier Nouvel, Gabriela Pretre, Stéphane Claverol, Laura Fernandez, Christine CittiAbstract:ABSTRACT The discovery of integrative conjugative elements (ICEs) in wall-less Mycoplasmas and the demonstration of their role in massive gene flows within and across species have shed new light on the evolution of these minimal bacteria. Of these, the ICE of the ruminant pathogen Mycoplasma agalactiae (ICEA) represents a prototype and belongs to a new clade of the Mutator-like superfamily that has no preferential insertion site and often occurs as multiple chromosomal copies. Here, functional genomics and mating experiments were combined to address ICEA functions and define the minimal ICEA chassis conferring conjugative properties to M. agalactiae. Data further indicated a complex interaction among coresident ICEAs, since the minimal ICEA structure was influenced by the occurrence of additional ICEA copies that can trans -complement conjugation-deficient ICEAs. However, this cooperative behavior was limited to the CDS14 surface lipoprotein, which is constitutively expressed by coresident ICEAs, and did not extend to other ICEA proteins, including the cis -acting DDE recombinase and components of the mating channel whose expression was detected only sporadically. Remarkably, conjugation-deficient mutants containing a single ICEA copy knocked out in cds14 can be complemented by neighboring cells expressing CDS14. This result, together with those revealing the conservation of CDS14 functions in closely related species, may suggest a way for Mycoplasma ICEs to extend their interaction outside their chromosomal environment. Overall, this report provides a first model of conjugative transfer in Mycoplasmas and offers valuable insights into understanding horizontal gene transfer in this highly adaptive and diverse group of minimal bacteria. IMPORTANCE Integrative conjugative elements (ICEs) are self-transmissible mobile genetic elements that are key mediators of horizontal gene flow in bacteria. Recently, a new category of ICEs was identified that confer conjugative properties to Mycoplasmas, a highly adaptive and diverse group of wall-less bacteria with reduced genomes. Unlike classical ICEs, these mobile elements have no preferential insertion specificity, and multiple Mycoplasma ICE copies can be found randomly integrated into the host chromosome. Here, the prototype ICE of Mycoplasma agalactiae was used to define the minimal conjugative machinery and to propose the first model of ICE transfer in Mycoplasmas. This model unveils the complex interactions taking place among coresident ICEs and suggests a way for these elements to extend their influence outside their chromosomal environment. These data pave the way for future studies aiming at deciphering chromosomal transfer, an unconventional mechanism of DNA swapping that has been recently associated with Mycoplasma ICEs.
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The Integrative Conjugative Element (ICE) of Mycoplasma agalactiae: Key Elements Involved in Horizontal Dissemination and Influence of Coresident ICEs
American Society for Microbiology, 2018Co-Authors: Eric Baranowski, Marie Claude Hygonenq, Eveline Sagne, Laurentxavier Nouvel, Emilie Dordet-frisoni, Gabriela Pretre, Stéphane Claverol, Laura Fernandez, Christine CittiAbstract:The discovery of integrative conjugative elements (ICEs) in wall-less Mycoplasmas and the demonstration of their role in massive gene flows within and across species have shed new light on the evolution of these minimal bacteria. Of these, the ICE of the ruminant pathogen Mycoplasma agalactiae (ICEA) represents a prototype and belongs to a new clade of the Mutator-like superfamily that has no preferential insertion site and often occurs as multiple chromosomal copies. Here, functional genomics and mating experiments were combined to address ICEA functions and define the minimal ICEA chassis conferring conjugative properties to M. agalactiae. Data further indicated a complex interaction among coresident ICEAs, since the minimal ICEA structure was influenced by the occurrence of additional ICEA copies that can trans-complement conjugation-deficient ICEAs. However, this cooperative behavior was limited to the CDS14 surface lipoprotein, which is constitutively expressed by coresident ICEAs, and did not extend to other ICEA proteins, including the cis-acting DDE recombinase and components of the mating channel whose expression was detected only sporadically. Remarkably, conjugation-deficient mutants containing a single ICEA copy knocked out in cds14 can be complemented by neighboring cells expressing CDS14. This result, together with those revealing the conservation of CDS14 functions in closely related species, may suggest a way for Mycoplasma ICEs to extend their interaction outside their chromosomal environment. Overall, this report provides a first model of conjugative transfer in Mycoplasmas and offers valuable insights into understanding horizontal gene transfer in this highly adaptive and diverse group of minimal bacteria.Integrative conjugative elements (ICEs) are self-transmissible mobile genetic elements that are key mediators of horizontal gene flow in bacteria. Recently, a new category of ICEs was identified that confer conjugative properties to Mycoplasmas, a highly adaptive and diverse group of wall-less bacteria with reduced genomes. Unlike classical ICEs, these mobile elements have no preferential insertion specificity, and multiple Mycoplasma ICE copies can be found randomly integrated into the host chromosome. Here, the prototype ICE of Mycoplasma agalactiae was used to define the minimal conjugative machinery and to propose the first model of ICE transfer in Mycoplasmas. This model unveils the complex interactions taking place among coresident ICEs and suggests a way for these elements to extend their influence outside their chromosomal environment. These data pave the way for future studies aiming at deciphering chromosomal transfer, an unconventional mechanism of DNA swapping that has been recently associated with Mycoplasma ICEs
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Experimental infections with Mycoplasma agalactiae identify key factors involved in host-colonization
PLoS ONE, 2014Co-Authors: Eric Baranowski, Dominique Bergonier, Xavier Berthelot, Marie Claude Hygonenq, Patricia Ronsin, Christine CittiAbstract:Mechanisms underlying pathogenic processes in Mycoplasma infections are poorly understood, mainly because of limited sequence similarities with classical, bacterial virulence factors. Recently, large-scale transposon mutagenesis in the ruminant pathogen Mycoplasma agalactiae identified the NIF locus, including nifS and nifU, as essential for Mycoplasma growth in cell culture, while dispensable in axenic media. To evaluate the importance of this locus in vivo, the infectivity of two knock-out mutants was tested upon experimental infection in the natural host. In this model, the parental PG2 strain was able to establish a systemic infection in lactating ewes, colonizing various body sites such as lymph nodes and the mammary gland, even when inoculated at low doses. In these PG2-infected ewes, we observed over the course of infection (i) the development of a specific antibody response and (ii) dynamic changes in expression of M. agalactiae surface variable proteins (Vpma), with multiple Vpma profiles co-existing in the same animal. In contrast and despite a sensitive model, none of the knock-out mutants were able to survive and colonize the host. The extreme avirulent phenotype of the two mutants was further supported by the absence of an IgG response in inoculated animals. The exact role of the NIF locus remains to be elucidated but these data demonstrate that it plays a key role in the infectious process of M. agalactiae and most likely of other pathogenic Mycoplasma species as many carry closely related
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ICEA of Mycoplasma agalactiae: a new family of self-transmissible integrative elements that confers conjugative properties to the recipient strain
Molecular Microbiology, 2013Co-Authors: Emilie Dordet-frisoni, Marc S. Marenda, Eric Baranowski, Florence Tardy, Xavier Nouvel, Romain Guérillot, Philippe Glaser, Alain Blanchard, Pascal Sirand-pugnet, Christine CittiAbstract:Horizontal gene transfer (HGT) is a major force of microbial evolution but was long thought to be marginal in Mycoplasmas. In silico detection of exchanged regions and of loci encoding putative Integrative Conjugative Elements (ICE) in several Mycoplasma genomes challenged this view, raising the prospect of these simple bacteria being able to conjugate. Using the model pathogen Mycoplasma agalactiae, we demonstrated for the first time that one of these elements, ICEA, is indeed self-transmissible. As a hallmark of conjugative processes, ICEA transfers were DNase resistant and required viable cells. ICEA acquisition conferred ICE-negative strains with the new ability to conjugate, allowing the spread of ICEA. Analysis of transfer-deficient mutants indicated that this process requires an ICEA-encoded lipoprotein of unknown function, CDS14. Formation of a circular extrachromosomal intermediate and the subsequent chromosomal integration of ICEA involved CDS22, an ICEA-encoded product distantly related to the ISLre2 transposase family. Remarkably, ICEA has no specific or no preferential integration site, often resulting in gene disruptions. Occurrence of functional Mycoplasma ICE offers these bacteria with a means for HGT, a phenomenon with far-reaching implications given their minute-size genome and the number of species that are pathogenic for a broad host-range.
Sebastiana Tola - One of the best experts on this subject based on the ideXlab platform.
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development of a specific immunomagnetic capture pcr for rapid detection of viable Mycoplasma agalactiae in sheep milk samples
Journal of Applied Microbiology, 2014Co-Authors: Giovanna Sanna, Valentina Lecca, Antonio Foddai, Sebastiana TolaAbstract:Aims To develop an immunomagnetic capture (IMC) to detect viable Mycoplasma agalactiae in routine ovine milk samples. Methods and Results Polyclonal antibodies against two M. agalactiae membrane surface proteins (P80 and P55) were covalently conjugated to magnetic beads (MBs) to form MB-Ab80 and MB-Ab55. Mycoplasma agalactiae cells were captured by a specific antigen–antibody reaction and magnetic separation. Immunomagnetic capture (IMC) was used to isolate and concentrate M. agalactiae in serial decimal dilutions and in artificially contaminated milk to facilitate subsequent detection by PCR. A 375-bp fragment of M. agalactiae was amplified using a pair of M. agalactiae-specific primers in PCR. The limit of detection of IMC-PCR method ranged from 10 to 102 CCU ml−1 when Mycoplasmas were resuspended in PBS and from 102 to 103 CCU ml−1 when Mycoplasmas were resuspended in uncontaminated ovine milk. This study also describes the application of IMC-PCR method to test for M. agalactiae in 516 milk samples collected from sheep with suspected contagious agalactia. Its performance was evaluated relative to culture. Conclusions This report has demonstrated for the first time, the effective use of rapid and reliable IMC combined with PCR assay for the detection of viable M. agalactiae. Significance and Impact of the Study The method IMC-PCR provides an alternative to conventional microbiological detection, method and it could be applied to quick detection of M. agalactiae in routine sheep milk samples.
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field trial of two dual vaccines against Mycoplasma agalactiae and Mycoplasma mycoides subsp mycoides large colony type in goats
Vaccine, 2007Co-Authors: Patricia Assunção, Sebastiana Tola, Carlos Poveda, P Saavedra, José B. PovedaAbstract:Two vaccines against Mycoplasma agalactiae and Mycoplasma mycoides subsp. mycoides (LC type) were developed using inactivated strains selected in previous characterization studies. The vaccines differed in terms of the adjuvants used: aluminium hydroxide (vaccine A) or aluminium hydroxide plus purified saponin (vaccine B). These vaccines were tested on 60 pregnant goats and 60 seronegative kids that were challenged by placing in a herd with a history of caprine contagious agalactia (CCA). Our findings indicate the effectiveness of the vaccines in preventing the appearance of new clinical signs such as mastitis, abortion, pneumonia and polyarthritis in CCA affected herds.
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Sequence, cloning, expression and characterisation of the 81-kDa surface membrane protein (P80) of Mycoplasma agalactiae
Fems Microbiology Letters, 2001Co-Authors: Sebastiana Tola, G. Idini, S. Crobeddu, Gavino Chessa, B Ibba, Sergio Uzzau, S RoccaAbstract:Mycoplasma agalactiae, the causative agent of contagious agalactia in small ruminants, produces a protein, named P80, that is detectable in all wild-type isolates examined to date and that appears expressed during the early phase of infection. We describe here the identification, cloning and expression of the gene encoding P80 (ma-mp81). The deduced amino acid sequence is consistent with a hydrophobic and basic protein that possesses a lipoprotein signal peptide. Sequence analysis of gene ma-mp81 suggests that P80 is a membrane lipoprotein that shows significant homology with other putative lipoproteins of M. pneumoniae. An internal 1-kb fragment of ma-mp81 was expressed in Escherichia coli as a 6×His-tagged protein. The purified recombinant protein greatly reacted with polyclonal anti-P80 sera raised in lamb.
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a physical map of the Mycoplasma agalactiae strain pg2 genome
Veterinary Microbiology, 2001Co-Authors: Sebastiana Tola, A.m. Rocchigiani, G. Idini, D. Manunta, Stefano Rocca, Guido LeoriAbstract:We have constructed a physical map of the Mycoplasma agalactiae strain PG2 chromosome analyzing it by pulsed field gel electrophoresis in a contour-clamped homogeneous electric-field system. We mapped 33 cleavage sites generated with SmaI, XhoI, SalI, EclXI and BsiWI restriction endonucleases using double digestions, one- and two-dimensional pulsed electrophoresis, cross-hybridization and linking clones. We have also mapped the loci of some genes by Southern hybridization.
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experimental vaccination against Mycoplasma agalactiae using different inactivated vaccines
Vaccine, 1999Co-Authors: Sebastiana Tola, A.m. Rocchigiani, G. Idini, D. Manunta, P. P. Angioi, Stefano Rocca, Guido LeoriAbstract:Abstract Five sets of vaccines were prepared using Mycoplasma agalactiae washed cultures inactivated with phenol (1), formalin (2), heat-treatment (3), sodium hypochlorite (4) and saponin (5). All sets, except for saponin-vaccine, were adjuvated with aluminium hydroxide. Five groups of six sarda ewes were inoculated twice before pregnancy, once during pregnancy and challenged during the lactation period. Monthly blood samples were taken from the vaccinated sheep and from 12 controls: sera were assayed by immunoblotting, ELISA and growth inhibition tests. Four control sheep were infected by intracanalicular route with pooled Mycoplasmas at concentrations of 10 4 , 10 5 , 10 6 and 10 7 CCU. The challenge involved using infected milk to contaminate the remaining sheep. All the controls and some ewes from groups 2, 3 and 4 developed contagious agalactia. Ewes vaccinated with phenol- and saponin-inactivated Mycoplasmas resisted experimental challenge. These results suggest that these two vaccines are effective and that their use could limit the diffusion of M. agalactiae infection.
Eric Baranowski - One of the best experts on this subject based on the ideXlab platform.
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impacts of Mycoplasma agalactiae restriction modification systems on pan epigenome dynamics and genome plasticity
bioRxiv, 2021Co-Authors: Emilie Dordetfrisoni, Eric Baranowski, Eveline Sagne, C Vandecasteele, R Contarin, Christophe Klopp, X Nouvel, Christine CittiAbstract:DNA methylation plays an important role in the biology of bacteria. Often associated with restriction-modification (RM) systems, they also provide a defence against foreign DNA. Little is known regarding the methylome of the Mycoplasma genus, which encompasses several pathogenic species with small genomes. Here, single molecule real-time (SMRT) and bisulphite sequencing combined with whole-genome analysis identified 19 methylated motifs associated with three orphan methyltransferases (MTases) and eight RM systems in Mycoplasma agalactiae, a ruminant pathogen and a model organism. All systems had a homolog in at least one phylogenetically distinct Mycoplasma spp. Our study also revealed that several superimposed genetic events may participate in the M. agalactiae dynamic epigenome landscape. These included (i) DNA shuffling and frameshift mutations that affect the MTase and restriction endonuclease content of a clonal population and (ii) gene duplication, erosion, and horizontal transfer that modulate MTase and RM repertoires of the species. Some of these systems were experimentally shown to play a major role in Mycoplasma conjugative, horizontal DNA transfer. While the versatility of DNA methylation may contribute to regulating essential biological functions at cell and population levels, RM systems may be key in Mycoplasma genome evolution and adaptation by controlling horizontal gene transfers.
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the integrative conjugative element ice of Mycoplasma agalactiae key elements involved in horizontal dissemination and influence of coresident ices
Mbio, 2018Co-Authors: Eric Baranowski, Marie Claude Hygonenq, Eveline Sagne, Emilie Dordetfrisoni, Laurentxavier Nouvel, Gabriela Pretre, Stéphane Claverol, Laura Fernandez, Christine CittiAbstract:ABSTRACT The discovery of integrative conjugative elements (ICEs) in wall-less Mycoplasmas and the demonstration of their role in massive gene flows within and across species have shed new light on the evolution of these minimal bacteria. Of these, the ICE of the ruminant pathogen Mycoplasma agalactiae (ICEA) represents a prototype and belongs to a new clade of the Mutator-like superfamily that has no preferential insertion site and often occurs as multiple chromosomal copies. Here, functional genomics and mating experiments were combined to address ICEA functions and define the minimal ICEA chassis conferring conjugative properties to M. agalactiae. Data further indicated a complex interaction among coresident ICEAs, since the minimal ICEA structure was influenced by the occurrence of additional ICEA copies that can trans -complement conjugation-deficient ICEAs. However, this cooperative behavior was limited to the CDS14 surface lipoprotein, which is constitutively expressed by coresident ICEAs, and did not extend to other ICEA proteins, including the cis -acting DDE recombinase and components of the mating channel whose expression was detected only sporadically. Remarkably, conjugation-deficient mutants containing a single ICEA copy knocked out in cds14 can be complemented by neighboring cells expressing CDS14. This result, together with those revealing the conservation of CDS14 functions in closely related species, may suggest a way for Mycoplasma ICEs to extend their interaction outside their chromosomal environment. Overall, this report provides a first model of conjugative transfer in Mycoplasmas and offers valuable insights into understanding horizontal gene transfer in this highly adaptive and diverse group of minimal bacteria. IMPORTANCE Integrative conjugative elements (ICEs) are self-transmissible mobile genetic elements that are key mediators of horizontal gene flow in bacteria. Recently, a new category of ICEs was identified that confer conjugative properties to Mycoplasmas, a highly adaptive and diverse group of wall-less bacteria with reduced genomes. Unlike classical ICEs, these mobile elements have no preferential insertion specificity, and multiple Mycoplasma ICE copies can be found randomly integrated into the host chromosome. Here, the prototype ICE of Mycoplasma agalactiae was used to define the minimal conjugative machinery and to propose the first model of ICE transfer in Mycoplasmas. This model unveils the complex interactions taking place among coresident ICEs and suggests a way for these elements to extend their influence outside their chromosomal environment. These data pave the way for future studies aiming at deciphering chromosomal transfer, an unconventional mechanism of DNA swapping that has been recently associated with Mycoplasma ICEs.
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The Integrative Conjugative Element (ICE) of Mycoplasma agalactiae: Key Elements Involved in Horizontal Dissemination and Influence of Coresident ICEs
American Society for Microbiology, 2018Co-Authors: Eric Baranowski, Marie Claude Hygonenq, Eveline Sagne, Laurentxavier Nouvel, Emilie Dordet-frisoni, Gabriela Pretre, Stéphane Claverol, Laura Fernandez, Christine CittiAbstract:The discovery of integrative conjugative elements (ICEs) in wall-less Mycoplasmas and the demonstration of their role in massive gene flows within and across species have shed new light on the evolution of these minimal bacteria. Of these, the ICE of the ruminant pathogen Mycoplasma agalactiae (ICEA) represents a prototype and belongs to a new clade of the Mutator-like superfamily that has no preferential insertion site and often occurs as multiple chromosomal copies. Here, functional genomics and mating experiments were combined to address ICEA functions and define the minimal ICEA chassis conferring conjugative properties to M. agalactiae. Data further indicated a complex interaction among coresident ICEAs, since the minimal ICEA structure was influenced by the occurrence of additional ICEA copies that can trans-complement conjugation-deficient ICEAs. However, this cooperative behavior was limited to the CDS14 surface lipoprotein, which is constitutively expressed by coresident ICEAs, and did not extend to other ICEA proteins, including the cis-acting DDE recombinase and components of the mating channel whose expression was detected only sporadically. Remarkably, conjugation-deficient mutants containing a single ICEA copy knocked out in cds14 can be complemented by neighboring cells expressing CDS14. This result, together with those revealing the conservation of CDS14 functions in closely related species, may suggest a way for Mycoplasma ICEs to extend their interaction outside their chromosomal environment. Overall, this report provides a first model of conjugative transfer in Mycoplasmas and offers valuable insights into understanding horizontal gene transfer in this highly adaptive and diverse group of minimal bacteria.Integrative conjugative elements (ICEs) are self-transmissible mobile genetic elements that are key mediators of horizontal gene flow in bacteria. Recently, a new category of ICEs was identified that confer conjugative properties to Mycoplasmas, a highly adaptive and diverse group of wall-less bacteria with reduced genomes. Unlike classical ICEs, these mobile elements have no preferential insertion specificity, and multiple Mycoplasma ICE copies can be found randomly integrated into the host chromosome. Here, the prototype ICE of Mycoplasma agalactiae was used to define the minimal conjugative machinery and to propose the first model of ICE transfer in Mycoplasmas. This model unveils the complex interactions taking place among coresident ICEs and suggests a way for these elements to extend their influence outside their chromosomal environment. These data pave the way for future studies aiming at deciphering chromosomal transfer, an unconventional mechanism of DNA swapping that has been recently associated with Mycoplasma ICEs
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Experimental infections with Mycoplasma agalactiae identify key factors involved in host-colonization
PLoS ONE, 2014Co-Authors: Eric Baranowski, Dominique Bergonier, Xavier Berthelot, Marie Claude Hygonenq, Patricia Ronsin, Christine CittiAbstract:Mechanisms underlying pathogenic processes in Mycoplasma infections are poorly understood, mainly because of limited sequence similarities with classical, bacterial virulence factors. Recently, large-scale transposon mutagenesis in the ruminant pathogen Mycoplasma agalactiae identified the NIF locus, including nifS and nifU, as essential for Mycoplasma growth in cell culture, while dispensable in axenic media. To evaluate the importance of this locus in vivo, the infectivity of two knock-out mutants was tested upon experimental infection in the natural host. In this model, the parental PG2 strain was able to establish a systemic infection in lactating ewes, colonizing various body sites such as lymph nodes and the mammary gland, even when inoculated at low doses. In these PG2-infected ewes, we observed over the course of infection (i) the development of a specific antibody response and (ii) dynamic changes in expression of M. agalactiae surface variable proteins (Vpma), with multiple Vpma profiles co-existing in the same animal. In contrast and despite a sensitive model, none of the knock-out mutants were able to survive and colonize the host. The extreme avirulent phenotype of the two mutants was further supported by the absence of an IgG response in inoculated animals. The exact role of the NIF locus remains to be elucidated but these data demonstrate that it plays a key role in the infectious process of M. agalactiae and most likely of other pathogenic Mycoplasma species as many carry closely related
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ICEA of Mycoplasma agalactiae: a new family of self-transmissible integrative elements that confers conjugative properties to the recipient strain
Molecular Microbiology, 2013Co-Authors: Emilie Dordet-frisoni, Marc S. Marenda, Eric Baranowski, Florence Tardy, Xavier Nouvel, Romain Guérillot, Philippe Glaser, Alain Blanchard, Pascal Sirand-pugnet, Christine CittiAbstract:Horizontal gene transfer (HGT) is a major force of microbial evolution but was long thought to be marginal in Mycoplasmas. In silico detection of exchanged regions and of loci encoding putative Integrative Conjugative Elements (ICE) in several Mycoplasma genomes challenged this view, raising the prospect of these simple bacteria being able to conjugate. Using the model pathogen Mycoplasma agalactiae, we demonstrated for the first time that one of these elements, ICEA, is indeed self-transmissible. As a hallmark of conjugative processes, ICEA transfers were DNase resistant and required viable cells. ICEA acquisition conferred ICE-negative strains with the new ability to conjugate, allowing the spread of ICEA. Analysis of transfer-deficient mutants indicated that this process requires an ICEA-encoded lipoprotein of unknown function, CDS14. Formation of a circular extrachromosomal intermediate and the subsequent chromosomal integration of ICEA involved CDS22, an ICEA-encoded product distantly related to the ISLre2 transposase family. Remarkably, ICEA has no specific or no preferential integration site, often resulting in gene disruptions. Occurrence of functional Mycoplasma ICE offers these bacteria with a means for HGT, a phenomenon with far-reaching implications given their minute-size genome and the number of species that are pathogenic for a broad host-range.
Guido Leori - One of the best experts on this subject based on the ideXlab platform.
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a physical map of the Mycoplasma agalactiae strain pg2 genome
Veterinary Microbiology, 2001Co-Authors: Sebastiana Tola, A.m. Rocchigiani, G. Idini, D. Manunta, Stefano Rocca, Guido LeoriAbstract:We have constructed a physical map of the Mycoplasma agalactiae strain PG2 chromosome analyzing it by pulsed field gel electrophoresis in a contour-clamped homogeneous electric-field system. We mapped 33 cleavage sites generated with SmaI, XhoI, SalI, EclXI and BsiWI restriction endonucleases using double digestions, one- and two-dimensional pulsed electrophoresis, cross-hybridization and linking clones. We have also mapped the loci of some genes by Southern hybridization.
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experimental vaccination against Mycoplasma agalactiae using different inactivated vaccines
Vaccine, 1999Co-Authors: Sebastiana Tola, A.m. Rocchigiani, G. Idini, D. Manunta, P. P. Angioi, Stefano Rocca, Guido LeoriAbstract:Abstract Five sets of vaccines were prepared using Mycoplasma agalactiae washed cultures inactivated with phenol (1), formalin (2), heat-treatment (3), sodium hypochlorite (4) and saponin (5). All sets, except for saponin-vaccine, were adjuvated with aluminium hydroxide. Five groups of six sarda ewes were inoculated twice before pregnancy, once during pregnancy and challenged during the lactation period. Monthly blood samples were taken from the vaccinated sheep and from 12 controls: sera were assayed by immunoblotting, ELISA and growth inhibition tests. Four control sheep were infected by intracanalicular route with pooled Mycoplasmas at concentrations of 10 4 , 10 5 , 10 6 and 10 7 CCU. The challenge involved using infected milk to contaminate the remaining sheep. All the controls and some ewes from groups 2, 3 and 4 developed contagious agalactia. Ewes vaccinated with phenol- and saponin-inactivated Mycoplasmas resisted experimental challenge. These results suggest that these two vaccines are effective and that their use could limit the diffusion of M. agalactiae infection.
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Comparison of restriction pattern polymorphism of Mycoplasma agalactiae and Mycoplasma bovis by pulsed field gel electrophoresis.
Zentralblatt fur Veterinarmedizin. Reihe B. Journal of veterinary medicine. Series B, 1999Co-Authors: Sebastiana Tola, A.m. Rocchigiani, G. Idini, D. Manunta, P. P. Angioi, Stefano Rocca, M. Cocco, Guido LeoriAbstract:Mycoplasma agalactiae and Mycoplasma bovis are closely related species in phylogenetic terms. Pulsed Field Gel Electrophoresis (PFGE) of SmaI, EclXI, Bsi WI, MluI, BssHII, SalI, XhoI, NruI and ApaI digested DNAs were used to analyse and to compare restriction fragment length polymorphism between M. agalactiae and M. bovis and to estimate their genome sizes. SmaI, EclXI and Bsi WI enzymes cleaved DNAs of both microrganisms. MluI, BssHII, SalI, XhoI and NruI digested only M. agalactiae DNA whereas ApaI cut only M. bovis DNA. The total DNA length was established to be 945 +/- 8.4 Kb for M. agalactiae and 961 +/- 18.9 Kb for M. bovis.
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Detection of Mycoplasma agalactiae in sheep milk samples by polymerase chain reaction
Veterinary Microbiology, 1998Co-Authors: Sebastiana Tola, Antonio Angioi, A.m. Rocchigiani, G. Idini, D. Manunta, Grazia Galleri, Guido LeoriAbstract:We developed a simple and rapid method for DNA extraction from sheep milk to use for polymerase chain reaction (PCR) diagnosis of Mycoplasma agalactiae. We tested 357 samples from 21 newly infected flocks (group 1) and 87 samples from 8 flocks infected in the past (group 2). PCR results were compared with those of conventional culture. By PCR we detected 175 positives in group 1, while by culture we detected only 153. Milk samples from group 2 were negative, both by PCR assay and by culture. Our PCR is much faster than culture and reduces the time required for diagnosis from several days to 5 h. The method could be used for the routine diagnosis of contagious agalactia caused by Mycoplasma agalactiae.
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comparison of Mycoplasma agalactiae isolates by pulsed field gel electrophoresis sds page and immunoblotting
Fems Microbiology Letters, 1996Co-Authors: Sebastiana Tola, Antonio Angioi, A.m. Rocchigiani, G. Idini, D. Manunta, Ida Casciano, Guido LeoriAbstract:We have analyzed 81 isolates of Mycoplasma agalactiae from four different regions of Italy between 1990 and 1995 in order to identify antigenic differences through SDS-PAGE and Western blotting and chromosomal DNA restriction endonuclease cleavage pattern differences. Antigenic variability in M. agalactiae isolates was investigated analyzing hydrophobic membrane protein fractions by immunoblotting using pooled sheep antiserum from naturally infected sheep. Large restriction fragments obtained cleaving genomic DNAs with SmaI, NruI, SalI, XhoI, BssHII and KpnI were analyzed by pulsed field gel electrophoresis. Genetic analysis indicates that isolates are all similar without intraspecific differences. This homogeneity was confirmed by immunoblotting: 80 and 50 kDa antigens are present in all strains analyzed.
Marc S. Marenda - One of the best experts on this subject based on the ideXlab platform.
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ICEA of Mycoplasma agalactiae: a new family of self-transmissible integrative elements that confers conjugative properties to the recipient strain
Molecular Microbiology, 2013Co-Authors: Emilie Dordet-frisoni, Marc S. Marenda, Eric Baranowski, Florence Tardy, Xavier Nouvel, Romain Guérillot, Philippe Glaser, Alain Blanchard, Pascal Sirand-pugnet, Christine CittiAbstract:Horizontal gene transfer (HGT) is a major force of microbial evolution but was long thought to be marginal in Mycoplasmas. In silico detection of exchanged regions and of loci encoding putative Integrative Conjugative Elements (ICE) in several Mycoplasma genomes challenged this view, raising the prospect of these simple bacteria being able to conjugate. Using the model pathogen Mycoplasma agalactiae, we demonstrated for the first time that one of these elements, ICEA, is indeed self-transmissible. As a hallmark of conjugative processes, ICEA transfers were DNase resistant and required viable cells. ICEA acquisition conferred ICE-negative strains with the new ability to conjugate, allowing the spread of ICEA. Analysis of transfer-deficient mutants indicated that this process requires an ICEA-encoded lipoprotein of unknown function, CDS14. Formation of a circular extrachromosomal intermediate and the subsequent chromosomal integration of ICEA involved CDS22, an ICEA-encoded product distantly related to the ISLre2 transposase family. Remarkably, ICEA has no specific or no preferential integration site, often resulting in gene disruptions. Occurrence of functional Mycoplasma ICE offers these bacteria with a means for HGT, a phenomenon with far-reaching implications given their minute-size genome and the number of species that are pathogenic for a broad host-range.
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molecular typing of Mycoplasma agalactiae tracing european wide genetic diversity and an endemic clonal population
Comparative Immunology Microbiology and Infectious Diseases, 2012Co-Authors: Laurentxavier Nouvel, Marc S. Marenda, Eveline Sagne, Michelle D Glew, Philippe GiammarinaroAbstract:Mycoplasma agalactiae causes chronic infections in small ruminants and remains endemic in many regions of the world, despite intensive and costly eradication programs. In this study, the innate genomic plasticity of M. agalactiae was exploited to design and assess a combination of molecular epidemiological tools to trace the pathogen in different geographic locations and to understand its emergence or re-emergence after eradication campaigns. For this purpose, two collections of M. agalactiae isolates, representing European outbreaks or localized endemic disease in a single region of France, were subjected to RFLP (Restriction Fragment Length Polymorphism) analyses using two sets of DNA probes (distributed across the genome and specific for the vpma gene locus), and a previously described VNTR (Variable Number Tandem Repeats) analysis. A combination of four genome-specific DNA probes and two VNTRs gave the highest discriminative power. Molecular typing revealed that, while isolates from diverse geographical origins fell into clearly different groups, the endemic disease repeatedly observed in the Western Pyrenees region over the past 30 years has been caused by a unique subtype of M. agalactiae. This indicates that the re-emergence of the pathogen after seemingly successful eradication programs is not due to the importation of exotic strains, but to the persistence of local reservoirs of infection.
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a new integrative conjugative element occurs in Mycoplasma agalactiae as chromosomal and free circular forms
Journal of Bacteriology, 2006Co-Authors: Marc S. Marenda, Valerie Barbe, Geraldine Gourgues, Sophie Mangenot, Evelyne Sagne, Christine CittiAbstract:An integrative conjugative element, ICEA, was characterized in Mycoplasma agalactiae strain 5632, in which it occurs as multiple chromosomal copies and as a free circular form. The distribution of ICEA sequences in M. agalactiae strains and their occurrence in Mycoplasma bovis suggest the spreading of the element within or between species.
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Construction of the first shuttle vectors for gene cloning and homologous recombination in Mycoplasma agalactiae
Fems Microbiology Letters, 2005Co-Authors: Rohini Chopra-dewasthaly, Renate Rosengarten, Wolfgang Jechlinger, Marc S. Marenda, Christine CittiAbstract:Mycoplasma agalactiae is a worldwide ruminant pathogen that causes significant economic losses by inflicting contagious agalactia in sheep and goats. The development of efficient control strategies requires a better understanding of the Mycoplasma factors that promote successful infection. However, lack of genetic tools has been a major impediment in studying the pathogenic mechanisms of M. agalactiae. This study describes the identification and cloning of the M. agalactiae origin of replication (oriC) in order to construct the first shuttle vectors for targeted gene disruption, gene complementation and expression studies. Additionally, this report provides the first evidence of the occurrence of homologous recombination and the functionality of heterologous tetM determinant in this pathogen.
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validation of the suppressive subtractive hybridization method in Mycoplasma agalactiae species by the comparison of a field strain with the type strain pg2
Veterinary Research, 2004Co-Authors: Marc S. Marenda, Joachim Frey, Edy M. Vilei, Francois Poumarat, X BerthelotAbstract:The subtractive suppressive hybridization (SSH), a method that allows the identification of sequences that are present in one genome (tester) but not in the other (driver), is a promising technique for the comparison of Mycoplasma agalactiae pathogenic strains. The optimal conditions for SSH were established by subtracting the M. agalactiae type strain PG2 DNA from the M. agalactiae strain 5632 DNA. Because these two strains possess different vpma gene repertoires, 5632-specific vpma sequences (and possibly other 5632-specific sequences) were predicted to be retrieved by SSH. The subtracted tester DNA was PCR-amplified and cloned into the pGEM-T easy E. coli vector. Two independent libraries were generated and used to prepare individual probes that were tested by Southern blot with genomic DNA from various field isolates and Mycoplasma reference strains. Sequence analysis of two overlapping clones showed that they potentially code for a large carboxyterminal portion of a new vpma ORF. Several DNA fragments homologous to insertion sequences were also found in 5632 and related strains. These preliminary data suggest that SSH is a powerful method to investigate differences between Mycoplasma strains, and may be applied to molecular epidemiology, diagnostic, and host specificity or pathogenicity determinant discovery. Mycoplasma agalactiae / subtractive hybridization / vpma / insertion sequence
