α-Enolase, an Adhesion-Related Factor of Mycoplasma bovis

Mycoplasma bovis is the causative agent of Mycoplasma bovis-associated disease (MbAD). Although the mechanisms underlying M. bovis adherence to host cells is not clear, recent studies have shown that the cell surface protein α-enolase facilitates bacterial invasion and dissemination in the infected host. In this study, we cloned, expressed and purified recombinant M. bovis α-enolase and induced polyclonal anti-α-enolase antibodies in rabbits. M. bovis α-enolase was detected in the cytoplasmic and membrane protein fractions by these antibodies. Triple immunofluorescence labeling combined with confocal laser scanning microscopy (CLSM) revealed that the plasminogen (Plg) enhanced the adherence of M. bovis to embryonic bovine lung (EBL) cells; the values obtained for adherence and inhibition are consistent with this finding. Interestingly, we found that trace amounts of trypsin acted as a more effective enhancer of cell adherence than Plg. Hence, our data indicate that surface-associated M. bovis α-enolase is an adhesion-related factor of M. bovis that contributes to adherence by binding Plg

The Complete Genome Sequence of Mycoplasma bovis Strain Hubei-1

Infection by Mycoplasma bovis (M. bovis) can induce diseases, such as pneumonia and otitis media in young calves and mastitis and arthritis in older animals. Here, we report the finished and annotated genome sequence of M. bovis strain Hubei-1, a strain isolated in 2008 that caused calf pneumonia on a Chinese farm. The genome of M. bovis strain Hubei-1 contains a single circular chromosome of 953,114 bp with a 29.37% GC content. We identified 803 open reading frames (ORFs) that occupy 89.5% of the genome. While 34 ORFs were Hubei-1 specific, 662 ORFs had orthologs in the M. bovis type strain PG45 genome. Genome analysis validated lateral gene transfer between M. bovis and the Mycoplasma mycoides subspecies mycoides, while phylogenetic analysis found that the closest M. bovis neighbor is Mycoplasma agalactiae. Glycerol may be the main carbon and energy source of M. bovis, and most of the biosynthesis pathways were incomplete. We report that 47 lipoproteins, 12 extracellular proteins and 18 transmembrane proteins are phase-variable and may help M. bovis escape the immune response. Besides lipoproteins and phase-variable proteins, genomic analysis found two possible pathogenicity islands, which consist of four genes and 11 genes each, and several other virulence factors including hemolysin, lipoate protein ligase, dihydrolipoamide dehydrogenase, extracellular cysteine protease and 5′-nucleotidase

Molecular cloning and characterization of a surface-localized adhesion protein in Mycoplasma bovis Hubei-1 strain.

Mycoplasma bovis (M. bovis) is an important pathogen that causes various bovine diseases, such as mastitis in cows and pneumonia in calves. The surface proteins are generally thought to play a central role in the pathogenesis of this organism. We screened the entire genome of M. bovis Hubei-1 and discovered a gene named vpmaX that encodes the 25 kDa variable surface lipoprotein A (VpmaX). Sequence analysis revealed that VpmaX contains several repetitive units and a typical bacterial lipoprotein signal sequence. The vpmaX gene was cloned and expressed in E. coli to obtain recombinant VpmaX (rVpmaX). Western blot analysis using a rabbit antibody against rVpmaX demonstrated that VpmaX is a membrane protein. Immunostaining visualized via confocal laser scanning microscopy showed that rVpmaX was able to adhere to embryonic bovine lung cells (EBL), and this was also confirmed by a sandwich ELISA. In summary, a surface-localized adhesion protein was identified in M. bovis Hubei-1

Localization of <i>Mycoplasma bovis</i> α-enolase.

<p>Western blot analysis of bovine serum albumin (BSA; lane 1), cell soluble cytosolic fraction proteins (lane 2), cell membrane fraction proteins (lane 3), whole cell protein (lane 4), and purified recombinant <i>Mycoplasma bovis</i> α-enolase blotted onto a nylon membrane and detected with rabbit anti-recombinant enolase antibodies (lane 5) blotted onto a nylon membrane and detected with rabbit anti-recombinant enolase antibodies. M: protein marker.</p

Ligand blotting assay.

<p>Membrane fraction proteins(lane 1), soluble cytosolic fraction proteins(lane 2), commercial α-enolase(lane 3), recombinant <i>Mycoplasma bovis</i> α-enolase (lane 4), and BSA(lane 5) were blotted onto nitrocellulose membranes following SDS-PAGE, and then incubated with plasminogen post-blocking. Bound plasminogen was detected with sheep anti-plasminogen polyclonal antibody. M: protein marker.</p

Adherence and inhibition assays.

<p>Experiments were performed in triplicate. Hank's Balanced Salt Solution (HBSS). *P<0.05, compared with the corresponding group using non-immune rabbit antibodies.</p

Localization of VpmaX in <i>M. bovis</i> Hubei-1.

<p>Western blot analysis of rVpmaX (lane 1), <i>M. bovis</i> total proteins (lane 2), cell membrane fraction proteins (lane 3), cell soluble cytosolic fraction proteins (lane 4), and bovine serum albumin (lane 5) using rabbit anti-rVpmaX serum and a peroxidase-conjugated secondary antibody.</p

Assay of rVpmaX adhesion and adhesion inhibition to EBL cells visualized by confocal laser scanning microscopy.

<p>Active rVpmaX interacted with fixed EBL cells, and the surplus protein was rinsed away by washing with PBST. The attached protein was immunostained with rabbit anti-rVpmaX antibody and mouse anti-rabbit IgG-FITC. The EBL cell membranes were labeled with 1,19-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI), and the cell nuclei were counter-labeled with 49,6-diamidino-2-phenylindole (DAPI). (A1–A2) 10 µg rVpmaX adhering to EBL cells. (B) Adhesion inhibition of 10 µg rVpmaX to EBL cells by 10 µl rabbit anti-rVpmaX serum. (C) Adhesion of 20 µg rVpmaX to EBL cells. (D) The adhesion of 20 µg rVpmaX to EBL cells was inhibited by 20 µl rabbit anti-rVpmaX serum. (E) EBL cells without protein added.</p

Overlapping PCR primers using for amplification of α-Enolase.

<p>Overlapping PCR primers using for amplification of α-Enolase.</p