A genome‐wide transposon mutagenesis screening identifies LppB as a key factor associated with Mycoplasma bovis colonization and invasion into host cells

Abstract

Mycoplasma spp., the smallest self‐replicating and genome‐reduced organisms, have raised a great concern in both the medical and veterinary fields due to their pathogenicity. The molecular determinants of these wall‐less bacterium efficiently use their limited genes to ensure successful infection of the host remain unclear. In the present study, we used the ruminant pathogen Mycoplasma bovis as a model to identify the key factors for colonization and invasion into host cells. We constructed a nonredundant fluorescent transposon mutant library of M. bovis using a modified transposon plasmid, and identified 34 novel adhesion‐related genes based on a high‐throughput screening approach. Among them, the ΔLppB mutant exhibited the most apparent decrease in adhesion to embryonic bovine lung (EBL) cells. The surface‐localized lipoprotein LppB, which is highly conserved in Mycoplasma species, was then confirmed as a key factor for M. bovis adhesion with great immunogenicity. LppB interacted with various components (fibronectin, vitronectin, collagen IV, and laminin) of host extracellular matrix (ECM) and promoted plasminogen activation through tPA to degrade ECM. The 439–502 amino acid region of LppB is a critical domain, and F465 and Y493 are important residues for the plasminogen activation activity. We further revealed LppB as a key factor facilitating internalization through clathrin‐ and lipid raft‐mediated endocytosis, which helps the Mycoplasma invade the host cells. Our study indicates that LppB plays a key role in Mycoplasma infection and is a potential new therapeutic and vaccine target for Mycoplasma species.

Mycoplasma synoviae elongation factor thermo stable is an adhesion-associated protein that enters cells by endocytosis and stimulates DF-1 cell proliferation.

“…Activated by a plasminogen activator, plasminogen produces a highly e cient serine protease that can hydrolyze brin and various ECM components. Numerous invasive pathogens can capture plasminogen and bind to plasminogen receptors (PlgRs) on their surfaces, activating the brinolytic system, degrading the ECM system, and facilitating its adhesion and colonization, leading to tissue dissemination [18,30,46]. Examples include LppA and LppB in Mycoplasma bovis and GAPDH and enolase in Mycoplasma hyorhinis, which act as PlgRs to bind and activate plasminogen, enabling the pathogen to degrade the host ECM [28, 30,47,48].…”

“…Numerous invasive pathogens can capture plasminogen and bind to plasminogen receptors (PlgRs) on their surfaces, activating the brinolytic system, degrading the ECM system, and facilitating its adhesion and colonization, leading to tissue dissemination [18,30,46]. Examples include LppA and LppB in Mycoplasma bovis and GAPDH and enolase in Mycoplasma hyorhinis, which act as PlgRs to bind and activate plasminogen, enabling the pathogen to degrade the host ECM [28, 30,47,48]. This mechanism potentially represents how Mycoplasma breaks through the tissue barrier in the host and causes persistent infection in the animal organism.…”

“…For instance, Edwardsiella tarda invades cells through clathrin-and caveolin-mediated pathways [49], Ureaplasma parvum and Mycoplasma bovis are internalized through clathrin-mediated pathways [50,51], Mycoplasma gallisepticum is internalized through cholesterol [52], and Mycoplasma hyopneumoniae colonizes porcine epithelial cells through clathrin-and caveolin-mediated pathways [53]. In addition, pathogenic virulence factors, such as the CARDS toxin of Mycoplasma pneumoniae [29] and LppB of Mycoplasma bovis [30], can be internalized through the aforementioned pathways. In this study, in addition to degrading the ECM, EF-Ts was internalized by DF-1 cells.…”

“…The colocalization of rEF-Ts with caveolin and clathrin was examined using laser confocal microscopy. The endocytosis inhibition assay was modi ed as previously described to detect the endocytosis pathway of rEF-Ts using two speci c inhibitors[29,30]. Pitstop-2 (HY-115604, MCE) is an inhibitor of clathrin protein-mediated endocytosis, and simvastatin (HY-17502, MCE) is an inhibitor of caveolin protein pathway-mediated endocytosis.…”