Vpma phase variation is important for survival and persistence of Mycoplasma agalactiae in the immunocompetent host

Abstract

Despite very small genomes, mycoplasmas retain large multigene families encoding variable antigens whose exact role in pathogenesis needs to be proven. To understand their in vivo significance, we used Mycoplasma agalactiae as a model exhibiting high-frequency variations of a family of immunodominant Vpma lipoproteins via Xer1-mediated site-specific recombinations. Phase-Locked Mutants (PLMs) expressing single stable Vpma products served as first breakthrough tools in mycoplasmology to study the role of such sophisticated antigenic variation systems. Comparing the general clinical features of sheep infected with a mixture of phase-invariable PLMs (PLMU and PLMY) and the wild type strain, it was earlier concluded that Vpma phase variation is not necessary for infection. Conversely, the current study demonstrates the in vivo indispensability of Vpma switching as inferred from the Vpma phenotypic and genotypic analyses of reisolates obtained during sheep infection and necropsy. PLMY and PLMU stably expressing VpmaY and VpmaU, respectively, for numerous in vitro generations, switched to new Vpma phenotypes inside the sheep. Molecular genetic analysis of selected ‘switchover’ clones confirmed xer1 disruption and revealed complex new rearrangements like chimeras, deletions and duplications in the vpma loci that were previously unknown in type strain PG2. Another novel finding is the differential infection potential of Vpma variants, as local infection sites demonstrated an almost complete dominance of PLMY over PLMU especially during early stages of both conjunctival and intramammary co-challenge infections, indicating a comparatively better in vivo fitness of VpmaY expressors. The data suggest that Vpma antigenic variation is imperative for survival and persistence inside the immunocompetent host, and although Xer1 is necessary for causing Vpma variation in vitro, it is not a virulence factor because alternative Xer1-independent mechanisms operate in vivo, likely under the selection pressure of the host-induced immune response. This singular study highlights exciting new aspects of mycoplasma antigenic variation systems, including the regulation of expression by host factors.

Genome-wide detection of conservative site-specific recombination in bacteria

“…Recent evidence of lipoprotein phase variation in Mycoplasma gallisepticum and M . agalactiae also points towards a nonstochastic model during infection [ 12 – 14 ].…”

Genomic Islands in Mycoplasmas

“…Genes 2020, 11, x FOR PEER REVIEW 3 of 16 addition, the largest cluster, locus I, is closed to a tRNALys gene and harbors two unrelated genes, abiGI and abiGII, whose products present similarities to the abortive infection system AbiG found in more classical bacteria. Discovered in the 90s, the M. agalactiae vpma loci were shown to encode phase-variable surface proteins that are key in allowing host-colonization and immune-escape [21,22]. These GIs could match the definition of a pathogenicity island (PAI), a GI involved in virulence.…”

Removal of a subset of non-essential genes fully attenuates a highly virulentMycoplasmastrain

“…For many years, the lack of genetic tools has limited our basic understanding of Mycoplasma pathogenicity. Due to their regressive evolution by gene loss, mycoplasmas appear to lack many of the common bacterial effectors and toxins used to interact with their hosts or to escape the hosts’ immune systems (6, 7). Lipoproteins have been proposed to be involved in both aspects by using their cytoadherent properties and allowing antigenic variability through phase or sequence variation (8).…”