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

Abstract

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.

Role of the GapA and CrmA Cytadhesins of Mycoplasma gallisepticum in Promoting Virulence and Host Colonization

“…While R high is known to contain 29 additional mutations that could contribute to its avirulence, data obtained with RCL2 raised the question of the in vivo translation of gapA via ribosome slippage and/or of crmA, whose transcription could be initiated by a cryptic promoter located downstream of the gapA point mutation. Such a situation was recently reported for Mycoplasma agalactiae, in which a cryptic promoter was shown to be active in a mutant with a mutation affecting a group of genes required for survival (31). In this scenario, RCL2 would produce CrmA at a level that might differ slightly from that in R low , thus generating a pattern of infection that is intermediate between those of the wild-type, parent strain and its sibling, mHAD3, in which the disruptive mutation cannot be compensated for by the activity of the cryptic promoter.…”

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

“…Despite causing significant economic losses, its pathogenic mechanisms and attributes are largely unknown. Earlier studies in this regard were based on in vitro analysis of genes involved in host-cell interaction (5), and a recent report has focused on a single factor in an experimental infection study (6). In general, the previously reported in vivo studies dealt with either characterizing of field strains or deciphering events occurring in mammary epithelial cells during M. agalactiae infection (7)(8)(9)(10).…”

“…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).…”

The Integrative Conjugative Element (ICE) ofMycoplasma agalactiae: key elements involved in horizontal dissemination and influence of co-resident ICEs

“…Protein expression in M. agalactiae was performed as previously described by using the plasmid p20-1miniO/T (designated in the present study as pO/T) (19, 25). Briefly, mycoplasma coding sequences were cloned downstream of the lipoprotein P40 gene (MAG2410) promoter region.…”

“…Peptides were identified as previously described by using a database consisting of M. agalactiae strain 5632 entries (26). ICEA products were detected by specific anti-sera on Western and colony blots (25, 27). Triton-X114 soluble proteins were extracted from M. agalactiae as previously described (28).…”