| Mycoplasma hyopneumoniae(M.hyopneumoniae,Mhp)is the causative agent of mycoplasma pneumonia of swine,a chronic respiratory infectious disease worldwide.Despite its low direct mortality,M.hyopneumoniae causes highly prevalent infections and is extremely difficult to eradicate.What's more,this pathogen increases host susceptibility to secondary respiratory infections by damaging cilia and epithelial cells,intensifying the difficulty of disease prevention and control,and seriously threatening the development of pig industry.The studies on pathogenesis can provide scientific instruction for drug design and vaccine production against M.hyopneumoniae,and are of great benefit to controlling infections.To date,the progress in research for the molecular pathogenic mechanisms of M.hyopneumoniae has been hindered by its fastidious growth requirements and the lack of tools for its genetic manipulation.Identifying novel virulence-associated factors,and unveiling those biological processes potentially related to virulence attenuation,help to elucidate the pathogenesis of M.hyopneumoniae and effectively prevent and control its infection.In this study,we undertook a large-scale comparative proteomic study between the virulent M.hyopneumoniae strain 168 and its highly passaged attenuated strain 168 L,and identified differentially displayed proteins using the non-gel based isobaric tags for relative and absolute quantification(i TRAQ)approach.Based on the proteomic results,a further study was performed on a potential virulence-associated factor,lipoprotein mhp390 which was overrepresented in strain 168.The main research works were as follows: 1.Comparative proteomic analyses of a pathogenic M.hyopneumoniae strain and its highly passaged attenuated strainThe growth curves of M.hyopneumoniae strains 168 and 168 L in the modified Friis medium were measured at first to collect bacterial cells as they grew to late-log phase.Whole proteins were extracted from the two M.hyopneumoniae strains,digested and labeled with i TRAQ regents,followed by downstream LC-MS/MS analysis.A total of 489 proteins were detected,?70% of the predicted proteome of M.hyopneumoniae,including 125 proteins assigned no function and annotated as “hypothetical protein” or “uncharacterized protein”.70 proteins showed significant differences in expression level between the two strains,of which 35 were overrepresented and 35 were underrepresented in strain 168.Gene Ontology(GO)enrichment analysis was performed for the differentially expressed proteins.For the GO category “molecular function”,“catalytic activity”,“N-methyltransferase activity” and “kinase activity” were the top three enriched items.The highest enrichment for “biological process” was associated with “small molecule catabolic process”,following by “inositol catabolic process”,“organic hydroxy compound catabolic process”,“cellular carbohydrate catabolic process”,“alcohol catabolic process” and “polyol catabolic process”.KEGG enrichment analysis showed that “microbial metabolism in diverse environments” and “inositol phosphate metabolism” were pathways significantly enriched.In our study,proteins covered all the enzymes comprising the classical myo-inositol bacterial catabolic pathway in M.hyopneumoniae were significantly down-regulated in the virulent strain.It may be assumed that M.hyopneumoniae retains its ability to degrade myo-inositol in vitro,and the capability is increased with passage.Proteins involved in nucleoside metabolism(Ula A,MHP168?564,Prs A,Deo D,Tdk,Hpt and Pyr G)were up-regulated in M.hyopneumoniae strain 168,possibly increasing the synthesis of phosphoribosyl pyrophosphate(PRPP),nicotinamide adenine dinucleotide(NAD)and nucleotide.These increments could be treated as enhanced defense to reactive oxygen species(ROS)damage.The up-regulated proteins in M.hyopneumoniae strain 168 were screened through Virulent Pred and manually searched for virulent proteins,and 25 of them were assigned as virulence-associated factors.All the overrepresented proteins with unknown function in strain 168 were predicted to be associated with virulence.In addition,a series of proteins which confirmed to be virulence-associated in other pathogens,such as moonlighting adhesins(elongation factor-Tu,enolase and endo-1,4-beta-glucanase),transporters(Mgt E and Pac L),lipoate-protein ligase(Lpl A-1 and Lpl A)and ribonuclease,might be virulence determinants in M.hyopneumoniae.Lipoprotein mhp390(Mhp168?418,P68)was found to be significantly up-regulated in strain 168 and predicted as a virulent protein.In view of the important roles that lipoproteins play in the pathogenesis of mycoplasma,and the function of mhp390 is still unclear in M.hyopneumoniae,thus,mhp390 was selected for further investigation.2.Functional research of differentially expressed protein mhp390Bioinformatic analysis of lipoprotein mhp390 in M.hyopneumoniae strain 168 showed that it was a membrane protein with a typical lipoprotein signal peptide.Recombinant protein mhp390 was obtained through E.coli prokaryotic expression system.Antiserum against mhp390 was prepared and used to detect whole cell extracts,membrane proteins and cytosolic proteins of M.hyopneumoniae.The protein was detected in both whole cell extracts and membrane proteins,but couldn't be detected in cytosolic fraction,suggesting that mhp390 is a membrane lipoprotein.The ability of recombinant protein mhp390 binding to swine tracheal cilia was determined via a microtiter plate assay,and the result showed that mhp390 bound specifically to cilia in a does-dependent manner,suggesting that mhp390 is a novel cilia adhesin and may play an important role during the adherence of M.hyopneumoniae to respiratory epithelium.The percentages of apoptotic cells,including early apoptotic cells and late apoptotic or necrotic cells,in both lymphocytes and monocytes of PBMCs treated with recombinant protein mhp390 were significantly increased.However,the pro-apoptotic effects of mhp390 on lymphocytes and monocytes could be completely inhibited by anti-mhp390 antibody.Protein mhp390 could induce apoptosis of porcine alveolar macrophages(PAMs)via significantly increasing the caspase-3 activity.The expression levels of TNF-? and IL-1? were both significantly up-regulated in PAMs treated with mhp390,while no inhibition effect of anti-mhp390 antibody on the inflammatory response was observed.In conclusion,the virulence and physiological differences between virulent and attenuated strains of M.hyopneumoniae were analyzed preliminarily,and 25 virulence-associated factors were screened.The results verified the function of lipoprotein mhp390 in M.hyopneumoniae infection,indicating that mhp390 is a new virulence factor of M.hyopneumoniae.These findings will provide scientific bases for further clariying the pathogenic mechanism of M.hyopneumoniae. |
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