The Pathogenicity Of Haemophilus Parasuis Cytolethal Didtending Toxin And The Putative Serine Proteases Espps

Haemophilus parasuis(HPS) is a kind of opportunistic pathogen. It exists on the upper respiratory tract of swine and causes swine with suppressed immunity to Glasser’s disease which is characterized by serofibrinous pleurisy, pericarditis, peritonitis, arthritis, and meningitis. In recent years, infections caused by this bacterial pathogen have led to great economic losses in the pig industry worldwide and it is urgent to analyze and demonstrate its molecular pathogenesis in order to generate a highly effective and comprehensive strategy for the prevention and control of this disease.Two objects were included in this research:cytolethal distending toxin(CDT) which has two copys on SH0165genome and putative serine protease EspPs(EspPl and EspP2included). Both of the two objects are potential causative agents of HPS. The previous studys of CDT were restricted to the recombinated CDT protein expressed in vitro and there was few report about the founction of CDT in vivo. Moreover, there was no report represented that CDT of HPS led cells to apoptosis. Recently, H.parasuis was found with IgA protease activity but there was no gene found with homology to the H. influenzae IgA protease genes iga and igaB, and only some of the HPS strains were found with IgA protease activity. However, the blast search showed the IgA protease homolog in H. parasuis SH0165was a complete ORF annotated as a putative extracellular serine protease. The main research contents were as follows:1. Constructed cdt mutants and researched the roles of CDT on cells and guinea pigs1.1Construction of cdt mutants by natural transformationConstructed3cdt mutants in HPS serotype4strain JS0135by natural transformation, including JS0135Δcdt1, JS0135Δcdt2and JS0135Δcdt1Δcdt2. The first step of natural transformation was to clone the upstream, downstream and a resistance cassette to the suicide plasmid pK18mobsacB to construct the plasmids pK18-C1G and pK18-C2K. Then the plasmids were transformed into the standard strains and wild strains stored in our laboratory by natural transformation. The wild strain JS0135(cdt also have two copys on the genome of this strain, and the sequence was same to the cdt sequence on SH0165genome by sequencing) was selected for its ability to transform both the plasmids pK18-C1G and pK18-C2K. Afer selected by solid medium with resistance and detected by PCR, Western blot and Southern blot, the cdt1and cdt2were replaced by the resistance cassettes gm and kan, respectively. It means the3cdt mutants of JS0135has been constructed successfully.1.2HPS CDT led to mophological changes but not apoptosis of PK15Three different methods of Flow cytometry, DNA ladder and nuclear staining technology were used separately to detect the apoptosis of PK15which had been incubated with secreted proteins of JS0135and the cdt mutants, respectively. May the different apoptosis rate reflects the relationship between CDT and apoptosis of PK15. Among the three method, results of Flow cytometry suggested CDT lead PK15to rapid cell death in48-72h while the rate of early apoptosis was low; method of DNA ladder resulted no obvious gene fragmentation, which implied no apoptosis of PK15. Nucleus of PK15incubited with JS0135were expansive compared with that incubited with cdt mutants but there was no nuclear fragmentation which was the typical feature of cells apoptosis. So the three methods above gave evidence to that HPS CDT led to the distending and death but not apoptosis of PK15.Cell morphology of PK15after incubated with secreted proteins of JS0135compared with the cdt mutants:expansion of the nucleus to3-4times, uneven of the cell size, decrease in the cell numbers, emptybubble around the nucleus, dense of some nucleus.1.3Roles of HPS CDT to guinea pigs2teams of guinea pigs were given intraperitoneal injection of the wild strain JS0135and its cdt deletion mutant strain JS0135Acdt1Acdt2in MLD dose, after that they were observated continuously for7days. The roles of CDT to guinea pigs mainly contains: First, CDT contributes to the virulence of HPS for JS0135Δcdt1Acdt2led lower mortality. Second, led guinea pigs’organ lesions including pulmonary congestion, brain hyperemia, pleural effusion and ascites. Third, HPS was isolated from the pleural effusion of the guinea pigs dead from JS0135but not JS0135Δcdt1Δcdt2implied that CDT was related to the invasion and metastasis of HPS or helped HPS to defence host immunity.2. HPS putative serine proteases EspPland EspP2have IgA protease activityWe incubated swine IgA with HPS serotype5wild strain CF7066and its espPs mutants CF7066ΔespP1、CF7066ΔespP2pd and CF7066ΔespP1ΔespP2pd. CF7066and CF7066ΔespP1、CF7066ΔespP2pd were found to cleave the heavy chain of swine IgA. To further prove the IgA protease activity of these two proteins, recombinated proteins EspPland EspP2were cloned, expressed in E. coli BL21(DE3) and incubated with swine IgA, the result was the same. The in vivo and in vitro results proved the IgA protease activity of HPS putative serine proteases EspPland EspP2.