| The Gram-negative bacterium Gallibacterium anatis(G.anatis)is a typical species of Gallibacterium within the Pasteurellaceae.The pathogenic bacterium is a major cause of salpingitis and peritonitis in egg-laying chickens,leading to decreased egg-production and higher mortality worldwide.Widespread multidrug resistance largely prevents treatment of this organism using traditional antimicrobial agents.Currently,vaccine candidates based on several konown virulence factors of G.anatis have been evaluated,no ideal protective effect was got.It is imperative to develop novel effective vaccine.The identification of novel virulence factors and pathogesis is the key issues need to be resolved.Outer membrane protein W plays an important role in biofilm formation,adhesion and stress tolerance of gram-negative bacteria.For many bacterial pathogens,adhesive fimbriea play a key role in the colonization of host tissues.However,the specific role of these two factors in G.anatis has not been elucidated.To better understand the role of pili and OmpW in biological functions of G.anatis and further elucidate its pathogenesis.experiments in the following aspects were acrried out.1.The construction and characterization of a OmpW deletion mutant of G.anatisGiven the outer membrane proteins of gram-negative bacteria play an important role in adhesion,stress adaption and a variety of biological functions,ompW was selected as the research object in this study.Natural competence was induced in G.anatis by transfer from rich medium to the starvation medium M-?.Then competence cells of G.anatis were transformated with linearized plasmid DNA carrying homologous arm sequences of ompW and chloromycetin resistance cassette.The transformants were identified by using PCR,SDS-PAGE and Western-Blotting analysis.The growth and hemolysis activity of G.anatis RZ and AompW were quite similar,showing that the deletion of OmpW has no negative effect on growth,colony morphology and hemolysis activity of G.anatis.The results indicated OmpW don't involve these biological characteristics in G.anatis.The MIC-determinations of 12 kinds of antibacterial agents were performed for both wild type and mutant AompW using Mueller Hinton-II.Comparing with G.anatis wild type,the MICs of terramycin and tetracycline for AompW were decreased to 1/8-fold(16/128)and 1/32-fold(4/128)respectively.suggesting OmpW plays an important role in the formation of tetracyclines resistance phenotype of G.anatis strains.2.Analysis of stress resistance of G.anatis RZ and AompWIn view of OmpW playing an important role in stress adaption in gram-negative bacteria,the growth of G.anatis RZ and AompW in medium of different salt content were determined,then the corresponding expression and mRNA transcriotion of OmpW were analyzed by Western Blotting and RT-qPCR respectively.The results demonstrated the growth of both G.anatis strains were affected,and their logarithmic phase were delayed.However,the growth rate of G.anatis strain RZ was higher than that of AompW in medium with 3%salt content.With the increase of incubation time,the impact on growth rate of AompW was more serious than G.antis RZ.These results showed AompW adapt high osmolality stress worse than RZ,and that AompW growth affected more by high osmolality.The expression of OmpW in RZ was up-regulated with an increase of NaCl concentration.Meanwhile the transcription of mRNA of OmpW was also up-regulated,suggestting OmpW might paly a role in the adaption of the organism under osmotic stress conditions.In addition,the survival rates of AompW in heat stress,osmotic stress and oxidative stress environment were lower than that of G.anatis RZ,indicating that ompW may related to the ability to stress resistance.3.Study of serum-resistance in G.anatis RZ and ?ompWIn this study,the survival ability of RZ and AompW in different serum content were analyzed,then the expression and mRNA transcription of OmpW were derermined by Western Blotting and RT-qPCR respectively.The Results demonstrated that G.anatis RZ and AompW show similar survival rate in low concentration of serum(2%).Along with the increase of serum content,the survival ability of AompW is lower than that of G.anatis RZ,suggesting OmpW may play a critical role in antiserum bactericidal activity in G.anatis.Further,in order to further illuminate the regulation of OmpW in respone to complement-mediated killing,G.anatis RZ was incubated with normal chicken serum or heat inactivated serum for one hour,then OmpW was analyzed by Western Blotting.Compared with the control group(incubated with heat inactivated serum),the expression of OmpW and its mRNA transcription in RZ increased significantly after incubation in normal chicken serum.These results indicated that OmpW contributed to bacterial resistance to complement-mediated killing in G.anatis.To further understand the contribution of the different pathways of complement activation.G.anatis RZ and AompW suspension were incubated in normal chicken serum or heat inactivated serum supplemented with EDTA(inhibition of all complement pathways)or EGTA and MgCl2(with only the effect of an alternative complement pathway allowed).The results indicated that there were no significant changes among the two strains in normal chicken serum in the presence of EDTA.However,when EGTA-MgCI2 was added in the normal chicken serum,the survival of the two strains all decreased.These results suggested that OmpW is required for bacterial resistance to alternative complement pathway-mediated attack.4.Study of pathogenicity of G.anatis RZ and ?ompWThe median lethal dose(LDso)of mice and the viability of the mice after infection of G.anatis RZ or ?ompW.The results showed that the LD50 of G.anatis RZ and ? ompW in mice were 2.93×108 CFU and 4.08×108 CFU.Obviously,the LD50 of RZ was slightly higher than that of AompW.There no obvious differences in mortality and clinical symptoms of mice in two groups infected with RZ and AompW respectively.The pathological changes of the mice mainly included the intestinal wall thinning,and hemorrhage in testine,lung,liver and spleen.However,there no visible difference in pathological lesions of mice in the two groups,showing OmpW deletion lead to pathogenicity of G.anatis for mice was slightly reduced.The determination biofilm formation and adhesion performance of RZ and ompW were carried out,the results showed that both RZ and AompW have similar biofilm formation ability.The amount of G.anatis RZ and AompW adhered to primary chicken oviduct epithelial cells(COECs)increased with the incubation time increased.the amount of?ompW adhered to COECs was lower than that of RZ,but the difference was not significant.These results indicated that OmpW contribute to the adhesion of G.anstis to COECs and had little effect on biofilm formation.5.Construction,identification and biological characteristics of fimbrial protein FlfA deletion mutant ?flfA of G.anatisTo explore the role of fimbrial protein FlfA in the pathogenicity of G.anatis.The flfA gene knock-out mutant of G.anatis was constructed by natural transformation.The competent cells of G.anatis were induced following transfer of cells to M-IV medium from colonies on a blood agar plate.Then,transformation was performed by incubating the M-IV compentent cells with linear DNA fragment consisting of homologous arms and Cmpr gene.The transformants were identified by using PCR,SDS-PAGE and Western-blot analysis.Results showed fimbrial protein FlfA.deletion mutant ?flfA was constructed sucessfully.The growth and colonial morphology of G.anatis RZ and ? ompW were determined.The results demostrated both strains have similar growth performance,showing that the deletion of FlfA has no obvious negative effect on growth,colony morphology of G.anatis.The amount of G.anatis RZ and ?flfA.adhered to primary chicken oviduct epithelial cells(COEC)increased with the incubation time increased.the amount of ?flfA adhered to COEC was lower than that of RZ significantly.These results indicated that FlfA play a critical role in the adhesion of G.anstis to COECs. |
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