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A genome-wide transposon mutagenesis screening identifies LppB as a key factor associated with Mycoplasma bovis
Shimei Lan1,2,3, Zhangcheng Li1,2,3, Huafang Hao1,2,3
1State Key Laboratory for Animal Disease Control and Prevention, College of Veterinary Medicine, Lanzhou University, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China.
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.
Insights
Mycoplasma bovis infection relies on the surface lipoprotein LppB for host cell adhesion and invasion. This study identifies LppB as a crucial factor for Mycoplasma pathogenesis and a potential therapeutic target.
Area of Science:
- Veterinary Microbiology
- Molecular Pathogenesis
- Bacterial Adhesion
Background:
- Mycoplasma species are significant pathogens in medical and veterinary fields.
- The molecular mechanisms underlying Mycoplasma host colonization and invasion are not fully understood.
- Mycoplasma bovis is a major ruminant pathogen necessitating research into its virulence factors.
Purpose of the Study:
- To identify key factors involved in Mycoplasma bovis colonization and host cell invasion.
- To elucidate the role of identified factors in the infection process.
- To explore potential therapeutic and vaccine targets for Mycoplasma infections.
Main Methods:
- Construction of a nonredundant fluorescent transposon mutant library of Mycoplasma bovis.
- High-throughput screening to identify adhesion-related genes.
- Biochemical assays to confirm LppB function, including interaction with extracellular matrix components and plasminogen activation.
- Analysis of host cell internalization mechanisms.
Main Results:
- Identification of 34 novel adhesion-related genes in Mycoplasma bovis.
- The surface lipoprotein LppB was identified as a critical factor for M. bovis adhesion to embryonic bovine lung cells.
- LppB interacts with host extracellular matrix components and promotes ECM degradation via plasminogen activation.
- LppB facilitates host cell internalization through clathrin- and lipid raft-mediated endocytosis.
Conclusions:
- LppB is a key virulence factor in Mycoplasma bovis infection, mediating adhesion, ECM degradation, and host cell invasion.
- The identified LppB functional domain and residues are crucial for its pathogenic activity.
- LppB represents a promising new target for therapeutic and vaccine development against Mycoplasma infections.
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