Critical Role of Dispensable Genes in<i>Mycoplasma agalactiae</i>Interaction with Mammalian Cells

Baranowski, Éric; Guiral, Sébastien; Sagné, Eveline; Skapski, Agnès; Citti, Christine

doi:10.1128/iai.01195-09

Cited by 32 publications

(94 citation statements)

References 48 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Some of the loci identified in mutants selected on caprine cell lines (Tables 3 and 4), were also identified as being required for growth on HeLa cells, either in this study (Tables 3 and 4) or in our previous study [20]. This suggests that these loci may be involved in general processes mediating interactions between M. agalactiae and mammalian cells.…”

Section: Resultssupporting

confidence: 66%

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

et al. 2011

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: Resultssupporting

confidence: 66%

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

et al. 2011

Self Cite

View full text Add to dashboard Cite

show abstract

Section: Methodsmentioning

confidence: 99%

“…Plasmid p20-1miniO/T (here termed "pO/T"), which contains parts of the M. agalactiae origin of replication (oriC), was used as a stable vector for protein expression in this species (26). The gsmA gene from strain 14628 (MAGb_1260) was cloned downstream of the P40 gene promoter region, as previously described (26). Briefly, the promoter region was amplified first by using oligonucleotide primers p40RF-CC (5=-ACG GGGCTAAAGAAGCTGAT-3=) and p40/MAGb1260 (5=-ACACATTT TGTTTCTTTTTCATAATTATTTATATCCTTTTC-3=) to generate a 200-bp DNA fragment overlapping MAGb_1260 at the ATG codon.…”

Section: Methodsmentioning

confidence: 99%

Mycoplasma agalactiae Secretion of β-(1→6)-Glucan, a Rare Polysaccharide in Prokaryotes, Is Governed by High-Frequency Phase Variation

Gaurivaud

Baranowski

Pau-Roblot

et al. 2016

Appl Environ Microbiol

View full text Add to dashboard Cite

Mycoplasmas are minimal, wall-less bacteria but have retained the ability to secrete complex carbohydrate polymers that constitute a glycocalyx. In members of the Mycoplasma mycoides cluster, which are important ruminant pathogens, the glycocalyx includes both cell-attached and cell-free polysaccharides. This report explores the potential secretion of polysaccharides by M. agalactiae, another ruminant pathogen that belongs to a distant phylogenetic group. Comparative genomic analyses showed that M. agalactiae possesses all the genes required for polysaccharide secretion. Notably, a putative synthase gene (gsmA) was identified, by in silico reconstruction of the biosynthetic pathway, that could be involved in both polymerization and export of the carbohydrate polymers. M. agalactiae polysaccharides were then purified in vitro and found to be mainly cell attached, with a linear ␤-(1¡6)-glucopyranose structure [␤-(1¡6)-glucan]. Secretion of ␤-(1¡6)-glucan was further shown to rely on the presence of a functional gsmA gene, whose expression is subjected to high-frequency phase variation. This event is governed by the spontaneous intraclonal variation in length of a poly(G) tract located in the gsmA coding sequence and was shown to occur in most of the M. agalactiae clinical isolates tested in this study. M. agalactiae susceptibility to serum-killing activity appeared to be dictated by ON/OFF switching of ␤-(1¡6)-glucan secretion, suggesting a role of this phenomenon in survival of the pathogen when it invades the host bloodstream. Finally, ␤-(1¡6)-glucan secretion was not restricted to M. agalactiae but was detected also in M. mycoides subsp. capri PG3 T , another pathogen of small ruminants. IMPORTANCEMany if not all bacteria are able to secrete polysaccharides, either attached to the cell surface or exported unbound into the extracellular environment. Both types of polysaccharides can play a role in bacterium-host interactions. Mycoplasmas are no exception despite their poor overall metabolic capacity. We showed here that M. agalactiae secretes a capsular ␤-(1¡6)-glucopyranose thanks to a specific glycosyltransferase with synthase activity. This secretion is governed by high-frequency ON/OFF phase variation that might be crucial in mycoplasma host dissemination, as cell-attached ␤-(1¡6)-glucopyranose increases serum-killing susceptibility. Our results provide functional genetic data about mycoplasmal glycosyltransferases with dual functions, i.e., assembly and export of the sugar polymers across the cell membrane. Furthermore, we demonstrated that nonprotein epitopes can be subjected to surface antigenic variation in mycoplasmas. Finally, the present report contributes to unravel the role of secreted polysaccharides in the virulence and pathogenicity of these peculiar bacteria.

show abstract

“…However, there is no report of any such study in ruminant mycoplasmas, which is imperative in understanding and preventing infections caused by this economically important group of mycoplasmas. Even though transposition in M. agalactiae was attained much earlier (13), it was only recently that this approach was used to identify genes involved in host-cell interactions during in vitro cell culture studies (5,24).…”

mentioning

confidence: 99%

Simultaneous Identification of Potential Pathogenicity Factors of Mycoplasma agalactiae in the Natural Ovine Host by Negative Selection

Hegde

Zimmermann

et al. 2015

Infect Immun

View full text Add to dashboard Cite

Mycoplasmas possess complex pathogenicity determinants that are largely unknown at the molecular level. Mycoplasma agalactiae serves as a useful model to study the molecular basis of mycoplasma pathogenicity. The generation and in vivo screening of a transposon mutant library of M. agalactiae were employed to unravel its host colonization factors. Tn4001mod mutants were sequenced using a novel sequencing method, and functionally heterogeneous pools containing 15 to 19 selected mutants were screened simultaneously through two successive cycles of sheep intramammary infections. A PCR-based negative selection method was employed to identify mutants that failed to colonize the udders and draining lymph nodes in the animals. A total of 14 different mutants found to be absent from >95% of samples were identified and subsequently verified via a second round of stringent confirmatory screening where 100% absence was considered attenuation. Using this criterion, seven mutants with insertions in genes MAG1050, MAG2540, MAG3390, uhpT, eutD, adhT, and MAG4460 were not recovered from any of the infected animals. Among the attenuated mutants, many contain disruptions in hypothetical genes, implying their previously unknown role in M. agalactiae pathogenicity. These data indicate the putative role of functionally different genes, including hypothetical ones, in the pathogenesis of M. agalactiae. Defining the precise functions of the identified genes is anticipated to increase our understanding of M. agalactiae infections and to develop successful intervention strategies against it.

show abstract

Critical Role of Dispensable Genes inMycoplasma agalactiaeInteraction with Mammalian Cells

Cited by 32 publications

References 48 publications

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

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

Mycoplasma agalactiae Secretion of β-(1→6)-Glucan, a Rare Polysaccharide in Prokaryotes, Is Governed by High-Frequency Phase Variation

Simultaneous Identification of Potential Pathogenicity Factors of Mycoplasma agalactiae in the Natural Ovine Host by Negative Selection

Contact Info

Product

Resources

About