| Actinobacillus pleuropneumoniae (APP) is the pathogen of porcine contagiouspleuropneumonia causing great economic losses worldwide. However, the15currently knownserotypes of APP show significant differences in pathogenicity and immunogenicity, the maininactivated vaccines lack of cross immunity protection to other serotypes. Up to now, there is nocommercialization of the vaccine in the world which is broad-spectrum and can offer crossprotection for various serotypes. Trimeric autotransporter adhesins (TAAs) are novelnon-fimbrial adhesins with high-affinity multivalent adhesive and invasion interactions with host,Some can inhibit phagocytosis or complement-mediated killing, helping the bacteria to overcomethe innate immune response. Proteins associated with virulence and can inducea good antibodybactericidal response, suggesting them to be potential candidates in protein based vaccines likeVtaA, DrsA, NadA.We have identified a TAAs (GenBank: ABN73547.1, named Apa1) from APP5bL20andcharacterized its influence to bacteria. In this paper, Sequence analysis of the genomic sequenceof APP5bL20(GenBank: CP000569.1), another autotransporter adhesins gene（GenBank：ABN73212.1）was founded and named Apa2. Complete sequencing of Apa2was speculated byusing SignalP3.0sofeware, daTAA software, Predicting Antigenic Peptides software, the resultshowed that Apa2was a potential TAAs gene, its YadA-like head binding motifs were distributedin the N terminal162-485aa and656-1070aa, named Apa2H1and Apa2H2. Also, the C terminalof Apaa2in3045-3154aa was a putative translocator domain, named Apa2C. To investigate thefunction of Apa2, the Apa2H1domain, Apa2H2domain and Apa2C domain was cloned andexpressed to obtain His-Apa2H1fusion protein, His-Apa2H2fusion protein, HAT-Apa2C fusionprotein. Both His-Apa2H1fusion protein and His-Apa2H2fusion protein show adhesive activesor antigenicity, The result of protective immunity test indicates that His-Apa2H1fusion protein,protein plays a certain role in immune protection against APP5b infection, with survival rate of70%,and His-Apa2H2fusion protein,with survival rate of80%, indicating that they could begood candidates to be used as immunogens for vaccine development. Western blot analysisrevealed that the Apa2C domain could form trimeric molecules. To further inspect the function of TAAs in APP5bL20, Apa1and Apa2gene deleted mutantwas constructed by homologous replacement utilizing high performance suicide vector system,single and double crossover strains were obtained by antibiotic selection and PCR identification,the deleted mutant was named△Apa1/△Apa2. And the effect of Apa1and Apa2deletion on thefunction of APP5bL20was comparatively studied with the wild type.In vitro the△Apa1/△Apa2showed rough and irregular on bacteria surfaces, bacterial aggregation andbiofilm formation were decreased, For the adhesion and invasion,△Apa1/△Apa2reduced bondof RAW264.7cells and CRL2845cells. In animal experiments, a mouse infection model byintranasal inoculation was established and the virulence of mutant was evaluated. Firstly, thedegree of LD50about△Apa1/△Apa2mutant was higher than wild type with2.93×108CFU byintraperitoneal infection and8.36×107CFU by intranasal inoculation, secondly, although it isinteresting that no excessive reduction in clinical symptoms and pathology about lung lesionscan be show in two strains, the mutant strain has the decreasing bacterial colonization of lungand spleen than wild type by intraperitoneal infection or intranasal inoculation, it is inferred thatTAAs play an essential role in adhesion and colonization of APP5b.Here, all the results highlight TAAs-mediated interactions of APP5bL20infection, the rolesof Apa1and Apa2have shown promise in the control of APP. |
