| Haemophilus parasuis(H.parasuis)is a kind of parasitic conditional pathogenic bacteria of the upper respiratory tract in swine,when the defense mechanism of upper respiratory tract is destroyed by environmental stress factors and pathogenic condition,H.parasuis could invade the host,resulting in Glasser's disease characterized by polyserositis,arthritis and meningitis,and seriously affect the health of swine especially piglets and weaned pigs.Our previous research has showed emodin,the main ingredient of Polygonum cuspidatum was active substance against H.parasuis.Emodin as a broad-spectrum antimicrobial agents,but its systematic and in-depth researches about antibacterial mechanism are rarely reported.Therefore,the study is aimed to evaluate the antibacterial properties of emodin to H.parasuis firstly,then on the basis of antibacterial properties,we adopted iTRAQ-LC-MS/MS high-throughput proteomics technology and bioinformatics analysis to analyze the global protein alteration of H.parasuis in response to the emodin treatment,and combined with verification experiments of adhering to and invading in cells,resistance to phagocytosis,and resistance to serum complement of H.parasuis to elucidate the potential antibacterial mechanism of emodin.The study is helpful to excavate potential targets.The main research methods and results are as follows:1.Antibacterial properties of emodin to H.parasuis in vitroThe MIC and MBC values of emodin against H.parasuis were determined by standard broth microdilution method and agar plate counting method respectively.Time-kill curves were constructed based on the time course of CFU/mL measurements.And the protein expression profile of H.parasuis in response to emodin treatment was analyzed by SDS-PAGE.Results are as follows:? The MIC and MBC values of emodin was 32 ?g/mL and 64 ?g/mL,respectively.? Time-kill curves shown that when treated with 1/2 MIC and lower concentraion of emodin,H.parasuis had the same growth trendwith the bacteria in the control group,but reproduce slowed down.When treated with the concentration of MIC,the amount of bacteria decreased and keeped in a certain range.However,when the concentrations of emodin were 2MIC and 4MIC,H.parasuis directly entered into decline phase and all bacterial cells were killed within 6 h and 4 h.?SDS-PAGE showed protein bands of untreated H.parasuis were more than that of treated cells,some protein bands of treated bacteria became shallow and even disappeared at concentration of 2MIC.These results suggested that emodin has a good antibacterial activity against H.parasuis,and the antibacterial activity was in a concentration-dependent manner.Moreover,emodin could inhibit the protein synthesis of H.parasuis,2.Proteomic analysis of H.parasuis in response to emodin based on the iTRAQ technologyiTRAQ-LC-MS/MS was used to analyze the protein expression profile of H.parasuis treated with or without emodin,the protein identification was using a Mascot library.Then combined with bioinformatics analysis methods to analysis the function of the differentially expressed proteins.Results are as follows:? A total of 2219 proteins wereidentified.Among these proteins,the expression levels of 338 proteins were shown to be significantly different(>1.2-fold change and<0.83-fold change,p<0.05)between treated and untreated bacteria including 95 up-regulations and 243 down-regulations in three independent experiments.? The GO annotation of differentially expressed proteins showed,these altered proteins were mainly annotated into eight cellular components,including cell,macromolecular complex,membrane etc,and annotated into twelve biological processes,i.e.,metabolic process and cellular process,also annotated into ten molecular function,i.e.,catalytic activity and binding.The down-regulated structural components of ribosomes,i.e.,30s ribosomal protein S8,50S ribosomal protein L2,L4,and some core proteins associated with cell division,i.e.,FtsB,FtsL,FtsQ were commented in three GO systems.? KEGG pathway analysis showed that these differential proteins were mainly enriched into ABC transporter system,carbon metabolism,bacterial chemotaxis,citrate cycle and virulence factors etc.These results suggested that the growth inhibition of H.parasuis treated with emodin was likely to attribute to the ribosomal protein synthesis was inhibited,result in the repression of some key protein expressions of ABC transport system,carbohydrate metabolism pathway and bacterial cell division proteins.Moreover,emodin also could regulate the protein expression of some virulence factors,such as OmpP2,OmpP5,neuraminidase etc.3.Research on gene transcription of differential virulence factors treated with emodinTaking ppiB as the internal reference,the gene transcriptions of ompP2,ompP5,nanH,gnd,vtaA2,vtaA4,etc under the treatment with emodin were studied by establishing reverse transcription fluorescent quantitative PCR method and using 2-??ct.Results showed the method has good specificity and sensitivity,and after treatment with emodin,the relative quantity of gnd mRNA expression was>1,and the relative quantities of mRNA expression of nanH,ompP2,ompP5,vtaA2,vtaA4,vtaA6,vtaA11,vtaA16 were<1.It illustrated that emodin could promote the gene transcription of gnd,at the same time could inbibit the gene transcription of ompP2,ompP5,nanH,vtaA2,vtaA4,vtaA6,vtaAl 1,vtaA16.It showed that transcription level agree with protein expression level of these virulence factors treated with emodin.4.Influence of emodin on the related functions of differential virulence factorsIn order to verify whether the changs of virulence factors protein expression treated with emodin result in corresponding changes of their related functions,CCK-8 method was used to screen security action concentrations of emodin,and colony count method was adopted to evaluate the abilities of adhering to and invading in cells,resistance to phagocytosis,and resistance to serum complement of H.parasuis under the treatment of emodin.Besides,the released amounts of IL-6,IL-8,IL-1? and TNF-a under the treatment of emodin were tested based on the establishment of H.parasuis induced RAW264.7 cells producing inflammation factors model.Results are as follows:? The security concentrations of emodin on the tested cells were 16 ?g/mL and lower concentrations.? After treatment with different concentrations of emodin,the adherence percentages and invasive percentages of H.parasuis were gradually decreased.when the concentrations of emodin were 16 ?g/mL and 8 ?g/mL,the adherence percentage and invasive percentage of bacteria were significantly decreased(P<0.05).? Compared with normal control group,when theconcentrations of emodin were 16 and 8?g/mL,it significantly increased the bacteria uptake by PAM,with the phagocytosis percentage were 12.43%and 9.10%respectively(P<0.05).? When treated with 16 ?g/mL emodin,the survival rates of H.parasuis in different concentrations of swine serum were lower,and the survival rate of untreated bacteria was 1.24%in 90%serum,neverthless after treated with emodin,bacteria were completely killed.? About the H.parasuis induced RAW264.7 cells producing inflammation factors model,the optimal amount of bacterial infection was 109 CFU/mL,and 6 h was optimal infection time.The releases of IL-6,IL-8,IL-1? and TNF-a were inhibited by emodin in a concentration dependent manner.These results suggested that emodin could reduce the adherence ability of H.parasuis to PK-15 cells,weaken the abilities of phagocytosis of PAM cells and complement-mediated serum killing,and inhibit the release of inflammatory cytokines.These functional changes were associated with differently expressed virulence factors,and the down-regulation of OmpP2 protein likely is the most critical factor.In conclusion,emodin exhibited a good antibacterial activity in vitro against H.parasuis,and its action mechanism is mainly reflected in the following two aspects:firstly,emodin achieves the effect of inhibiting bacterial growth might owing to the inhibition of ribosomal protein synthesis,it led to the inhibition of some key protein expressions of ABC transport system and carbohydrate metabolism pathway,result in the attenuation of bacterium material energy activities,and the cell division protein expressions were down-regulated,thus the binary fission was inhibited.Secondly,emodin could weaken the H.parasuis infection attributed to inhibition of transcription and expression of some virulence factors. |
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