Pretre, G. | Search bioRxiv

bioRxiv2026

BoviScan: a Phage ImmunoPrecipitation Sequencing library to characterize the antibody response against Mycoplasma bovis.

Mycoplasma bovis is a major cattle pathogen and a global concern due to the limited efficient prophylactic tools and the emergence of antibiotic-resistant strains. Current control strategies rely primarily on surveillance, diagnostic and segregation or culling of infected animals. Despite substantial efforts to develop vaccines against M. bovis, no broadly effective solution is currently available. This situation is partly attributable to important gaps in our understanding of the host humoral immune response following infection, vaccination, or challenge. ELISA and Western blot assays have been widely used to assess M. bovis-specific antibody responses. While robust and easy to implement, these approaches are difficult to scale for hundreds of antigens in large animal cohorts and provide limited information on the specific antigens and epitopes targeted by the antibody response. To address these limitations, we developed BoviScan, a Phage Immunoprecipitation Sequencing (PhIP-Seq) library covering the pan-proteome of 295 M. bovis strains. We describe the design, synthesis, and validation of BoviScan using a panel of known-positive sera. We then applied the library to characterize antibody responses in a small cohort of experimentally immunized cattle and to monitor the longitudinal evolution of the humoral response in a single animal. Our results show that BoviScan enables high-throughput, high-resolution mapping of the antibody responses against M. bovis. This approach could be highly valuable for the study of host-pathogen interactions in mycoplasmas and the development of improved vaccines and serological assays.

microbiology↗

bioRxiv2018

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

The discovery of integrative conjugative elements (ICEs) in wall-less mycoplasmas and the demonstration of their role in massive gene flows within and across species has shed new light on the evolution of these minimal bacteria. Of these, ICEA of the ruminant pathogen Mycoplasma agalactiae represents a prototype and belongs to a new clade of the Mutatorlike superfamily that has no preferential insertion site and often occurs as multiple chromosomal copies. Here, functional genomics and mating experiments were combined to address ICEA functions and define the minimal ICEA chassis conferring conjugative properties to M. agalactiae. Data further indicated a complex interaction among co-resident ICEAs, since the minimal ICEA structure was influenced by the occurrence of additional ICEA copies that can trans-complement conjugative-deficient ICEAs. However, this cooperative behavior was limited to the CDS14 surface lipoprotein, which is constitutively expressed by co-resident ICEAs, and did not extend to other ICEA proteins including the cis-acting DDE recombinase and components of the mating channel whose expression was only sporadically detected. Remarkably, conjugative-deficient mutants containing a single ICEA copy knocked-out in cdsl4 can be complemented by neighboring cells expressing CDS14. This result, together with the conservation of CDS14 functions in closely related species, may suggest a way for mycoplasma ICEs to extend their interaction outside of their chromosomal environment. Overall, this study provides a first model of conjugative transfer in mycoplasmas and offers valuable insights towards the understanding of horizontal gene transfer in this highly adaptive and diverse group of minimal bacteria.\n\nIMPRTANCEIntegrative conjugative elements (ICEs) are self-transmissible mobile genetic elements that are key mediators of horizontal gene flow in bacteria. Recently, a new category of ICEs has been identified that confer conjugative properties to mycoplasmas, a highly adaptive and diverse group of wall-less bacteria with reduced genomes. Unlike classical ICEs, these mobile elements have no preferential insertion specificity and multiple mycoplasma ICE copies can be found randomly integrated into the host chromosome. Here, the prototype ICE of Mycoplasma agalactiae was used to define the minimal conjugative machinery and propose the first model of ICE transfer in mycoplasmas. This model unveils the complex interactions taking place among co-resident ICEs and suggests a way for these elements to extend their influence outside of their chromosomal environment. These data pave the way for future studies aiming at deciphering chromosomal transfer, an unconventional mechanism of DNA swapping that has been recently associated with mycoplasma ICEs.

microbiology↗