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

Abstract

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.

Gut-associated bacteria invade the midgut epithelium of Aedes aegypti and stimulate innate immunity and suppress Zika virus infection in cells

“…Taken together, these data suggested that the Toll and IMD pathways were induced by invasion of Cedecea into mosquito cells. This is consistent with previous observations which demonstrated interplay between native microbiota and mosquito innate immune pathways [61, 66–68]. We observed dramatic modulation of effector molecules with Defensin , Cecropin and Gambicin, all significantly enhanced by Cedecea .…”

“…Midgut-associated bacteria can affect pathogens transmitted by mosquitoes by direct or indirect interactions [59–61]. Therefore, we examined how intracellular C. neteri affected viral infection.…”

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 .…”

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

“…These studies have demonstrated the role of the vpma locus in M. agalactiae pathogenesis [ 8 ]. Also, recently we attempted to identify potential pathogenicity factors of M. agalactiae in the natural sheep host by negative selection of transposon mutants [ 9 ]. Other independent studies using transposon mutants were able to show the role of different genomic loci in interaction with host cells [ 10 ] and the results obtained were further evaluated in vivo to conclude that the NIF locus was important for the pathogenicity of M. agalactiae [ 11 ].…”

“…Systemic spread of M. agalactiae after intramammary infection is well established [ 12 ]. In a recent experimental infection trial using pools of transposon mutants for intramammary infection of lactating ewes, we identified transposon mutants absent at the local site of infection and lymph nodes (LNs) [ 9 ]. The present study is an extension of the latter work, where we have further assessed the presence or absence of specific mutants in several inner organs distant from the site of inoculation.…”