Studies On Expression Of Adhesion-related Genes In Vivo And Functions Of FimA Protein Of Bovine Pasteurella Multocida

P. multocida subspecies multocida, the most important pathogenic member of the genus, is a Gram-negative, non-motile and facultative coccobacillus bacteria, which has a broad disease spectrum and can infect many wild and domestic animal species. Diseases caused by P. multocida include fowl cholera in birds, atrophic rhinitis in pigs, haemorrhagic septicaemia in cattle and buffalo, enzootic pneumonia in cattle, sheep and goats, snuffles in rabbits and, more rarely, wound abscesses and meningitis in humans, predominantly following cat - or dog-inflicted injuries. The capsule and lipopolysaccharide (LPS) form the basis for classification into serogroups (A, B, D, E, or F) and serotypes 1～16, respectively. Pasteurella multocida is responsible for significant losses on large farms worldwide. Capsular serotype B, which mainly cause hemorrhagic septicemia, is more prevalent in our country previously. However, recent studies found that capsular serotype A mainly results in pulmonary infection is inceasing. This bacterial has caused diseases happened in several provinces and cities in our country.P. multocida possesses various virulence factors, including capsule, lipopolysacch-aride, fimbriae and adhesins, toxins, iron-regulated and iron acquisition proteins, sialic acid metabolism proteins, hyaluronidase and outer membrane proteins. These virulence factors play important roles in the pathogenesis of P. multocida. Serotype B strains of P. multocida have been reported to adhere and entry into the host cells, but serotype A strains of P. multocida was found loosely adhering to the cell surface of host cells and were not detected intracellularly. At present, the adherence mechanism between serotype A strains and serotype B strains is not clear. The fimA gene as an important virulence may be code fimbriae subunit, but there is no report for its functions. In this research, we studied on expression of adhesion-related genes among different serotype strains in vivo in order to explain the adherence mechanism at the level of transcription at first. Next, the fimA gene of PmCQ2 was cloned into vector pET-30a (+) for prokaryotic expression, we prepared polyclonal antibody of FimA and analysised the functions of PmCQ2 FimA protein by several methods, which base on antigen-antibody reaction. Finally, we tried to construct PmCQ2 AfimA mutant for our lab. Main research contents and results are as follows:1. The study on expression of adhesion-related genes of three strains bovine P.multocida capsular in vivoIn the research, we take three strains bovine P. multocida serotype A CQ2, CQ6 and serotype B(PmCQ2, CQ6 and PmB) as objects, studied 5 adhesion-related gene (ptfA, fimA, pfhA, hsf-2 and hsf-1) in lung of mice at transcription level. The results show that: expression of ptfA in PmCQ6 is almostly same as in PmB, suggested that ptfA gene is not the impotant adhesion factor between P. multocida serotype A and B, but can be used as one of the important signs of P. multocida proliferation in vivo. At the same time, expression of adhesion related genes in PmCQ6 which is an attenuated strain were lower in each time period except ptfA gene, while the transcription level of adhesion related genes in PmCQ2 is significantly increased on 24h, we hypothesized that fimA, hsf-1, hsf-2 and pfhA may be an important factor in the pathogenic mechanism of virulent and avirulent strains. This study will provide a molecular basis for further study of the pathogenesis of bovine capsular serotype A P. multocida.2. Prokaryotic expression and the analysis of functions of PmCQ2 FimA Protein.The fimA gene of PmCQ2 was cloned into prokaryotic expression vector pET-30a (+), then the recombinant protein FimA(rFimA) was induced by IPTG, and the antigenicity of rFimA was analysised by western blot. The results showed that the homology between fimA gene and znuA gene in other P. multocida strains from NCBI was more than 97%, and 68% to 74% with other species of bacteria; The results of SDS-PAGE and western blot showed that rFimA was a molecular weight of 38kDa as expect, and with good antigenicity; PmCQ2 whole cell protein and purified rFimA proteins were prepared to immunize mice prior to challenge with the lethal dosage of 4.4x105 CFU of PmCQ2, the results showed that the protective rates were 100% and 20%, respectively; the results of adhesion inhibition and antibody blocking test showed that rFimA can partly inhibit the adherence of PmCQ2 to vero cells, but there is no significant difference in rFimA group and the control. Meanwhile, indirect immunofluorescence assay(IFA) were employed to determine the distribution of FimA in PmCQ2 and the results showed that FimA was mainly located in cytoderm.3. The construction of suicide recombinant plasmid of PmCQ2 fimADifferent homologous arms of fimA gene was amplified by PCR and cloned into pBC-SK and pWSK-29 plasmid, respectively. Kanamycin or Tetracycline resistance gene cassette was inserted into the corresponding homologous arm of fimA, and the recombinant plasmids include pBC-fimA-Km, pBC-fimA-Tet, pWSK- fimA-Tet①, pWSK-fimA-Tet①, pWSK- fimA-Tet③ and pWSK- fimA-Tet④; Then the recombiantion plasmid was transferred into PmCQ2 competent cells by electroporation, transferring and protoplast three methods, and we also tried different shock conditions. However, the results showed that:the colony on Kanamycin resistance plate is more than on tetracycline resistance plate after transformation, further study found that, Kanamycin resistance gene cassette may be in the genome of PmCQ2, but we have not screened the mutant by tetracycline resistance. The study on the construction of PmCQ2 mutant will provide a molecular basis in order to further construction of bovine P. multocida gene knockout mutant.