Nicotinamide Adenine Dinucleotide-Dependent Flavin Oxidoreductase of Mycoplasma hyopneumoniae Functions as a Potential Novel Virulence Factor and Not Only as a Metabolic Enzyme

Abstract

Mycoplasma hyopneumoniae (Mhp) is the main pathogen that causes enzootic pneumonia, a disease that has a significant impact on the pig industry worldwide. The pathogenesis of enzootic pneumonia, especially possible virulence factors of Mhp, has still not been fully elucidated. The transcriptomic and proteomic analyses of different Mhp strains reported in the literature have revealed differences in virulence, and differences in RNA transcription levels between high- and low-virulence strains initially indicated that nicotinamide adenine dinucleotide (NADH)-dependent flavin oxidoreductase (NFOR) was related to Mhp pathogenicity. Prokaryotic expression and purification of the NFOR protein from Mhp were performed, a rabbit-derived polyclonal antibody against NFOR was prepared, and multiple sequence alignment and evolutionary analyses of Mhp NFOR were performed. For the first time, it was found that the NFOR protein was conserved among all Mhp strains, and NFOR was localized to the cell surface and could adhere to immortalized porcine bronchial epithelial cells (hTERT-PBECs). Adhesion to hTERT-PBECs could be specifically inhibited by an anti-NFOR polyclonal antibody, and the rates of adhesion to both high- and low-virulence strains, 168 and 168L, significantly decreased by more than 40%. Moreover, Mhp NFOR not only recognized and interacted with host fibronectin and plasminogen but also induced cellular oxidative stress and apoptosis in hTERT-PBECs. The release of lactate dehydrogenase by hTERT-PBECs incubated with Mhp NFOR was significantly positively correlated with the virulence of Mhp. Overall, in addition to being a metabolic enzyme related to oxidative stress, NFOR may also function as a potential novel virulence factor of Mhp, thus contributing to the pathogenesis of Mhp; these findings provide new ideas and theoretical support for studying the pathogenic mechanisms of other mycoplasmas.

NADH oxidase of Mycoplasma hyopneumoniae functions as a potential mediator of virulence

“…that bind ISMPs remain unknown. However, recent studies have shown that extracellular enolase is bound to a rhamnose residue in the cell membrane of mycoplasmas [ 21 ], while enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) can covalently bind to lipoteichoic acid on L. crispatus [ 20 ], and NADH-dependent flavin oxidoreductase (NFOR) of M. hyopneumoniae has been shown to interact with host fibronectin and plasminogen [ 22 ]. Although it is still unclear how NOX acts as an enzyme in the cell matrix, stimulating adhesion on cell surfaces, the investigations described here provide findings that can help to guide further study.…”

“…Strain JS is a virulent strain that can induce typical characteristics of EP with a lung lesion score of approximately 15, as described previously [ 35 ]. Strain LH was also a virulent clinical strain isolated in our lab and capable of inducing typical characteristics of EP (GenBank accession number: CP079799) [ 22 ]. These five M. hyopneumoniae strains were cultured in modified Friis’ medium designated KM2 cell-free medium containing 20% (v/v) swine serum (produced in our lab from a clean snatch-farrowed, porcine-colostrum-deprived (sF-pCD) piglet after irradiation sterilization) at 37 °C in a humidified incubator.…”

“…To investigate whether NOX is present on the surface of M. hyopneumoniae strains and to probe NOX surface distribution differences between highly virulent strain 168 and virulence-attenuated strain 168L, fluorescence intensity was measured using a flow cytometer as previously described with modifications [ 22 ]. Briefly, M. hyopneumoniae strains 168 and 168L (each 1 × 10 8 CCU/mL) were incubated with anti-rNOX serum at a 1:100 dilution (1:100 diluted pre-immune serum was used as a negative control).…”

A genetic and virulence characterization of Brazilian strains of Mycoplasma hyopneumoniae

“…The M. hyopneumoniae strain J was obtained by Friis in the United Kingdom in 1957 as first isolated (Kobisch and Friis, 1996) and its genome was sequenced in 2005 (Vasconcelos et al, 2005). The other isolates sequenced originate 232 United States (Minion et al, 2004);7,422 and 7,448 Brazil (Vasconcelos et al, 2005;Siqueira et al, 2013); 168/168-L China (Liu et al, 2011); 11 Netherlands (Kamminga et al, 2017); 98 Netherlands; F7.2C Switzerland (Trueeb et al, 2019); KM014 South Korea (Han et al, 2017); LE China (Xie et al, 2021); TB1 China (Qiu et al, 2019); ES2-L China (Zong et al, 2022) ES2 China; MHP699, MHP650, MHP653, MHP679, MHP682, MHP691, MHP694, MHP709, MHP696 France; NCTC10127 UK and UFV01/UFV02 Brazil.…”

Unveiling the stealthy tactics: mycoplasma’s immune evasion strategies

“…Certain inherent constituents of the cell membrane of mycoplasma, including lipids and membrane lipoproteins, can elicit inflammatory reactions and induce tissue injury through diverse mechanisms (Yiwen et al, 2021). Other pathogenic materials, such as metabolic enzymes, phosphatase, cytotoxic nucleases, etc., are also considered essential mycoplasma pathogens (Xie et al, 2021). At the same time, mycoplasma utilizes nutrients from host cells and secrete substantial quantities of metabolites, such as hydrogen peroxide (H 2 O 2 ) and hydrogen sulfide (H 2 S) (Großhennig et al, 2016;Blötz and Stülke, 2017).…”