| Pasteurella multocida is a pathogenic Gram-negative bacterium that has been classified into five serogroups(A, B, D, E and F) and 16 serotypes based on capsular antigens and somatic antigens, respectivly. It can cause variety diseases of animals, such as haemorrhagic septicaemia in cattle, avian cholera, atrophic rhinitis in pig, and snuffles in rabbit. Those diseases brought serious economic losses to livestock husbandry all over the world.During the pathogen infection, adhesins of the bacterial cells have an important role in growth and colonization of pathogenic bacteria. Recently, there has found a new non-pili adhesin, a trimeric autotransporter adhesin(TAAs) which is consisted of the head, stalk and anchor domains,was considered to be an important virulence factor involved in bacterial adhesion, invasion, biofilm formation, and it can be selfaggregation. Further more, it can be used as a new subunit vaccine candidate. At present, TAAs are still poorly understood, which had been found in only a few bacteria strains such as H. parasuis and Pleural Actinobacillus.According to bioinformatics analysis, we also found one of TAAs in bovine P.mulotocida capsular serogroup A CQ2 isolated from lung of calf with pnuemenia, gene was nominated as pm1g0001.In this study, we employed the molecular biological techniques proved pm1g0001 was a Yad A trimeric autotransporter adhesin from theoretical and experimental aspects. In addition, we had done some experiments to indendify a few biological functions of pm1g0001, such as immunogenicity, adhesion activity and biofilm formation. Main contents are as follows: 1. Bioinformatics analysis of trimeric autotransporter adhesin pm1g0001Based on the genome data of bovine Pasteurella multocida serotype A strain PmCQ2,using online bioinformatics tools such as SignalP-3.0 server, TMHMM Server v. 2.0, SMART, analysised and predicted the signal peptide, transmembrane domain of Pm1g0001 protein, also employed autotransporter adhesin protein analysis software to analysis the structure and adhesion motifs of trimeric autotransporter adhesin. The results showed that the 1-22 aa of Pm1g0001 was signal peptide region, 1-84 aa was transmembrane domain, and 211-411 aa was TAAs. Where at the N-terminus 211-284 aa was the Ylhead motif of TAAs, 295-341 aa was stems, and 323-411 aa was C end anchor portion motifs, which indicated that this protein contains typical head- stem- anchor structure of trimeric autotransporter adhesin, theoretically proved Pm1g0001 hypothetical protein was belong to trimeric autotransporter protein family. Therefor, we used the Bcepred Server tool to predict the linear of B cell antigenic epitopes, and the software of BepiPred 1.0 Server and D.Astar packages can predicte the secondary structure antigen index, hydrophilicity and polarity. Analysis results inidcated that the main epitopes of Pm1g0001 located at 21-315 aa. 2. Cloning and expression of pm1g0001 and recombinant proteins analysis by Western-blotFrom the analysis of pm1g0001 above, 211-411 aa was the TAAs, so, here we designed 7 segments of gene pm1g0001: the entire gene was named Pm1g0001, the head and neck of TAAs was named Pm1g0001-1, the anchoring portion of TAAs was named Pm1g0001-2, the TAAs part was named Pm1g0001-3, the non-TAAs part was named Pm1g0001-4, the head of TAAs was named Pm1g0001-5, the neck of TAAs was named Pm1g0001-6. The primers of all segments were designed and synthesised by Shenggong company, the DNA fragments of those segments were purified after PCR and then cloned into recombinant expression plasmid pET30a(+) or pET32a(+), the recombinant plasmids were transformed into the competent cell of BL21(DE3) and induced by IPTG to get recombinant proteins. Used the His affinity chromatography to purify the recombinant proteins and found that those recombinant proteins were good immunoactive with antiserum of P.multocida detected by Western-blot. In addition, SDS-PAGE and Western blot showed that Pm1g0001-2 had a special band at 78 KDa, the size was three times of the monomeric band at 26 KDa, which was also the exactly proof that the anchoring portion of Pm1g0001 has a trimerization function and the pm1g0001 protein is a real trimeric autotransporter protein. 3. Functions of trimeric autotransporter protein pm1g0001In this study, we prepared immune antigens by mixing each purified recombinant protein of Pm1g0001 and Pm1g0001-1~Pm1g0001-6 with Freund’s Adjuvant Complete and immunized mices. After the third immunization, collected the blood of mices to separate the serum and detected the antibody titer by ELISA. Then immunized mice were challenged with the lethal dosage of 6.8×105CFU of PmCQ2 by intramuscular injection to detect the protection of those recombinant proteins against P.multocida. The results showed that all recombinant proteins can stimulate the mices to produce high levels antibodies, the titers range of 1:51200 to 1: 102400. The antibody valence of recombinant protein Pm1g0001-3 is higher than the other six proteins, which indicated that the trimeric autotransporter protein had a good immunogenicity can stimulate mice to produce high titers of antibodies. The results of immune protective test showed the protection rate of Pm1g0001-3 was 70%, which was better than other proteins, that meaned the trimeric autotransporter protein had a good protective effect against Pasteurella multocida, so it can be used as a vaccine candidate for preventing pasteurellosis. In addition, the protection rate of recombinant proteins Pm1g0001-5 and Pm1g0001 reached 60%, the protection rate of recombinant protein Pm1g0001-1 and Pm1g0001-6 was 40%, the protection rate of Pm1g0001-2 was only 10%, all results above illustrated the functional area of entire protein Pm1g0001 was the trimeric autotransporter portion, and the role of heads is better than necks.Fist using the negative serum and polyclonal antibodies against recombinant proteins Pm1g0001-1, Pm1g0001-3, Pm1g0001-5 and Pm1g0001-6 dealt with the macrophage cells of C57 BL / 6 mice, and then added PmCQ2 in the wells, after 4h, washed away the non-adherent bacterial cells with cell culture medium, then counted the adhesions of bacteria after cell eluted. Through the experiment, we know that if those antibodies against recombinant proteins binding with P. multocida cells can inhibite the adhesion of P. multocida cells with mouse macrophage cells. The results of adhesion experiment showed that four polyclonal antibodies of proteins had very significantly effects in inhibition of adherence of PmCQ2 to macrophage cells. Among them, with the anti-Pm1g0001-3 had a better inhibition effects than anti-Pm1g0001-1 、 anti-Pm1g0001-5and anti-Pm1g0001-6. The results indicated that trimeric autotransporter proteins of P.multocida can inhance the adhesion of pathogen cells with host cells and the functional areas of TAAs played a major role.In order to investigate whether recombinant proteins have founction of bacterial biofilm formation, we added the PmCQ2(1×108 CFU) into the cell culture plates to culture 24 h, 48 h and 72 h, then detected the biofilm formation. On the basis of this, we used anti-Pm1g0001-1, anti-Pm1g0001-3, anti-Pm1g0001-5, anti-Pm1g0001-6 and the negative serum to deal with the cell culture plates containing bacteria, and then measured the A630 value of each group. The results showed that Pasteurella multocida can form bacterial biofilm and the best was at 48 h. We found that the A630 in the groups pretreated by antibodies were significantly lower than the control group, results showed that four antibodies inhibited the biofilms formation, and the best inhibitory was also at 48 h after treatment. At the same time, Anti-Pm1g0001-1 had the best inhibitory effect, the inhibiting effect of anti-Pm1g0001-3 was better than Pm1g0001-5 and Pm1g0001-6, Anti-Pm1g0001-5 was better than anti-Pm1g0001-6. The results indicated that the functional areas of TAAs had main effects on biofilms formation and the neck played a major role. |
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