Genetic loci of Mycoplasma agalactiae involved in systemic spreading during experimental intramammary infection of sheep

Abstract

Mycoplasmas are amongst the most successful pathogens of both humans and animals yet the molecular basis of mycoplasma pathogenesis is poorly understood. This is partly due to the lack of classical virulence factors and little similarity to common bacterial pathogenic determinants. Using Mycoplasma agalactiae as a model we initiated research in this direction by screening a transposon mutant library in the natural sheep host using a negative selection method. Having successfully identified putative factors involved in the colonization of local infection and lymphogenic sites, the current study assessed mutants unable to spread systemically in sheep after experimental intramammary infection. Analysis of distant body sites for complete absence of mutants via SSM PCR revealed that additional set of genes, such as pdhB, oppC, oppB, gtsB, MAG1890, MAG5520 and MAG3650 are required for systemic spreading apart from those that were necessary for initial colonization. Additional in vitro studies with the mutants absent at these systemic sites confirmed the potential role of some of the respective gene products concerning their interaction with host cells. Mutants of pdhB, oppC and MAG4460 exhibited significantly slower growth in the presence of HeLa cells in MEM medium. This first attempt to identify genes exclusively required for systemic spreading provides a basis for further in-depth research to understand the exact mechanism of chronicity and persistence of M. agalactiae.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-016-0387-0) contains supplementary material, which is available to authorized users.

Characterization of Mycoplasma gallisepticum pyruvate dehydrogenase alpha and beta subunits and their roles in cytoadherence

“…agalactiae PDHB influences initial colonization and systemic spreading of M . agalactiae during experimental infection of sheep [47], suggesting that PDHB may help microorganisms colonize and invade host cells and cross host tissue barriers. M .…”

Large-Scale Analysis of the Mycoplasma bovis Genome Identified Non-essential, Adhesion- and Virulence-Related Genes

“…This approach has already led to the identification of the function of some genes in M. bovis (Sharma et al, 2015; Rasheed et al, 2017; Zhao et al, 2017). In the closely related species M. agalactiae , this technique was used to investigate growth phenotypes of individual or pools of mutants (Baranowski et al, 2010; Hegde et al, 2016).…”

“…Thus, mutagenesis of single genes does not allow us to predict genes that could be simultaneously dispensable or mutants that are mutually exclusive (Hutchison et al, 2016). Moreover, the definition of essentiality versus non-essentiality is dependent on several factors including the selective environment, bacterial growth conditions, and makes use of an artificial environment potentially neglecting differences present in in vivo situations (Acevedo-Rocha et al, 2013; Tseng et al, 2013; Hegde et al, 2016; Hutchison et al, 2016).…”

Comprehensive RNA-Seq Profiling to Evaluate the Sheep Mammary Gland Transcriptome in Response to Experimental Mycoplasma agalactiae Infection

“…agalactiae enabling survival in the mammary gland are not completely understood. We had recently identified potential pathogenicity factors by negative selection of transposon mutants that were unable to survive in the experimentally infected udders [11, 12]. Although the exact role of most of the identified factors remains to be elucidated, pyruvate dehydrogenase was shown to be involved in cell invasiveness [13], a strategy that M .…”