Mbov_0503 Encodes a Novel Cytoadhesin that Facilitates Mycoplasma bovis Interaction with Tight Junctions

Abstract

Molecules contributing to microbial cytoadhesion are important virulence factors. In Mycoplasma bovis, a minimal bacterium but an important cattle pathogen, binding to host cells is emerging as a complex process involving a broad range of surface-exposed structures. Here, a new cytoadhesin of M. bovis was identified by producing a collection of individual knock-out mutants and evaluating their binding to embryonic bovine lung cells. The cytoadhesive-properties of this surface-exposed protein, which is encoded by Mbov_0503 in strain HB0801, were demonstrated at both the mycoplasma cell and protein levels using confocal microscopy and ELISA. Although Mbov_0503 disruption was only associated in M. bovis with a partial reduction of its binding capacity, this moderate effect was sufficient to affect M. bovis interaction with the host-cell tight junctions, and to reduce the translocation of this mycoplasma across epithelial cell monolayers. Besides demonstrating the capacity of M. bovis to disrupt tight junctions, these results identified novel properties associated with cytoadhesin that might contribute to virulence and host colonization. These findings provide new insights into the complex interplay taking place between wall-less mycoplasmas and the host-cell surface.

An emerging role for cyclic dinucleotide phosphodiesterase and nanoRNase activities in Mycoplasma bovis: Securing survival in cell culture

“…These laboratory conditions have largely contributed to the understanding of the biology of these minimal bacteria but provided only limited information on the strategies these organisms have evolved to secure their survival in the animal host and get access to essential nutritional resources. In the absence of small animal models, eukaryotic cell cultures as re-emerged as a valuable environment to revisit the minimal cell concept at the light of host-pathogen interactions [30][31][32][33]44]. Our functional genomic study with the genome-reduced pathogen M. bovis revealed that phosphodiesterases involved in cyclic dinucleotides and nanoRNAs degradation are critical for survival under cell culture conditions, while dispensable in axenic medium.…”

“…DNA constructions and oligonucleotide primers used in the present study are listed in S2 Table. Plasmid pOH/P was used as a backbone for complementation studies [16]. This plasmid was derived from p20-1miniO/T by replacing (i) the tetM region by the pac gene encoding a puromycin N-acetyltransferase [12], and (ii) the replication origin of M. agalactiae by its counterpart in HB0801 [33,51]. The plasmid pCP-T9.386 contains a pOH/P backbone in which the Mbov_0328/0327 coding region was inserted under the control of the M. agalactiae P40 lipoprotein promoter [30].…”

“…Recombinant proteins used in the present study are listed in S2 Table. DNA constructions were cloned into pET28b (+) and transferred into E. coli BL21 cells for protein expression as previously described [33]. Briefly, protein expression was induced with 0.8 mM isopropyl-β-D-thiogalactose (IPTG) for 20 h at 16˚C or 4 h at 37˚C.…”

“…This technology has been successfully applied to several phylogenetically related species but is facing some limitations with other species, including the ruminant pathogens Mycoplasma agalactiae and Mycoplasma bovis [29]. For these species, random transposon mutagenesis, when combined with relevant screening systems, remains an efficient strategy for functional genomics studies [30][31][32][33] M. bovis is a re-emerging cause of pneumonia and mastitis in cattle worldwide, whose management is challenging because of the ineffectiveness of current vaccines and the high level of resistance to antibiotic treatments. In the present study, we used this pathogenic species as a model system to identify key factors regulating the proliferation of mycoplasmas under cocultivation with host cells.…”

“…This technology has been successfully applied to several phylogenetically related species but is facing some limitations with other species, including the ruminant pathogens Mycoplasma agalactiae and Mycoplasma bovis [ 29 ]. For these species, random transposon mutagenesis, when combined with relevant screening systems, remains an efficient strategy for functional genomics studies [ 30 – 33 ]…”