Mycoplasma bovis NADH oxidase functions as both a NADH oxidizing and O2 reducing enzyme and an adhesin

Abstract

Mycoplasma bovis causes considerable economic losses in the cattle industry worldwide. In mycoplasmal infections, adhesion to the host cell is of the utmost importance. In this study, the amino acid sequence of NOX was predicted to have enzymatic domains. The nox gene was then cloned and expressed in Escherichia coli. The enzymatic activity of recombinant NOX (rNOX) was confirmed based on its capacity to oxidize NADH to NAD+ and reduce O2 to H2O2. The adherence of rNOX to embryonic bovine lung (EBL) cells was confirmed with confocal laser scanning microscopy, enzyme-linked immunosorbent assay, and flow cytometry. Both preblocking EBL cells with purified rNOX and preneutralizing M. bovis with polyclonal antiserum to rNOX significantly reduced the adherence of M. bovis to EBL cells. Mycoplasma bovis NOX–expressed a truncated NOX protein at a level 10-fold less than that of the wild type. The capacities of M. bovis NOX– for cell adhesion and H2O2 production were also significantly reduced. The rNOX was further used to pan phage displaying lung cDNA library and fibronectin was determined to be potential ligand. In conclusion, M. bovis NOX functions as both an active NADH oxidase and adhesin, and is therefore a potential virulence factor.

Gap analysis ofMycoplasma bovisdisease, diagnosis and control: An aid to identify future development requirements

“…A recent metabolomics study has greatly increased both our knowledge base of key metabolites and our understanding of potential pathways important for M. bovis growth in vitro (Masukagami et al., ). One product of metabolism is hydrogen peroxide, which may be a pathogenicity factor in mycoplasma infections (Khan, Miles, & Nicholas, ; Zhao et al., ).…”

“…Intriguingly, TrmFO exhibits reduced expression in an attenuated, high‐passage M. bovis isolate. Recently, NADH oxidase (Nox) was shown to have both hydrogen peroxide generating activity (a possible virulence attribute) and an adherence function (Zhao et al., ). Based on a high degree of sequence similarity (44% amino acid sequence identity; 63% similarity) and conserved genomic context, M. bovis MBOVPG45_0378 is an unambiguous ortholog of the recently described P19 adhesin of the bovine pathogen M. mycoides subsp mycoides (Small Colony); (Zhou et al., ).…”

Mbov_0503 Encodes a Novel Cytoadhesin that Facilitates Mycoplasma bovis Interaction with Tight Junctions

“…A number of nonclassical secretory proteins have been identified in mycoplasmas, such as EF-Tu and DnaK in M. ovipneumoniae [52]. Several of these proteins can have moonlighting activities, such as the EF-Tu in M. hypneumoniae [53] and the TrmFO and NADH oxidase in M. bovis that were identified to be cytoadherence factors, [16,17]. Additional studies are needed to characterize these proteins and their potential role in M. bovis pathogenesis.…”

“…Colony immunoblotting analysis was performed by transferring mycoplasma cells onto PVDF membranes by close contact with colonies grown on agar plates. After blocking with 5% skim milk, membranes were incubated with rMbovP0503 mouse antiserum (1:250) or rVpsX rabbit antiserum [17] (1:250) at RT for 2 h, then incubated with HRP-conjugated goat anti-mouse or -rabbit IgG antibodies (1:5000, SouthernBiotech, Birmingham, AL, USA). Western and colony blots were developed by using the enhanced chemiluminescence (ECL) substrate kit (Thermo Fisher Scientific, Waltham, MA, USA).…”

TrmFO, a Fibronectin-Binding Adhesin of Mycoplasma bovis

“…Adhesins serve as vital virulence attributes in mycoplasmas, and decline of adhesion ability generally attenuates the bacteria [ 8 ]. Several adhesion proteins have been identified in M. bovis , including a 32 kDa membrane-localized protein P26 [ 9 ], a plasminogen binding enzyme α-enolase [ 10 ], NADH oxidase [ 11 ] and several variable surface lipoproteins (VspA, VspB, VspE, VspF and VpmaX) [ 12 , 13 ]. Identification and characterization of other adhesins will facilitate a better understanding of interactions between M. bovis and host cells.…”