Construction Of Mutant Strains (ΔnarQ/narP) And Function Of Two-Component Regulatory System NarQ/narP On Actinobacillus Pleuropneumoniae

Porcine contagious pleuropneumonia (PCP) is an infectious porcine respiratory tract disease causing severe economic losses in the swine industry, caused by Actinobacillus pleuropneumoniae (APP).There are many virulence factors exist in APP. The role of virulence factors and their interactions formed a complex relationships, which is very important for the pathogenesis of APP. In bacteria, two-component regulatory systems (TCS) control the expression of virulence factors in a wide range of bacterial species in response to external stimuli. For bacterial pathogens, they have evolved virulence factors and regulation systems that allow adaptation to different environments within a host ensuring their survival.It has been demonstrated that there are five TCSs exists in APP, namely arcA/arcB, cpxA/cpxR, narQ/narP, phoB/phoR, and qseB/qseC. In many pathogens, narQ/narP interact with narX/narL. It has been reported that there is no narX/narL in APP. Which kind of regulatory mechanizm of narQ/narP is still unclear in pathogenesis of APP.To study the regulatory function of the narQ/narP of APP, the deleted narQ/narP mutant strain of APP serotype 1 (SLWO1) not carrying antibiotic resistance determinant was constructed based on whole-genome sequencing of serum type 3 strain(JL03)of APP. The following experiments were conducted in this dissertation:1. Bioinformation analysis of narP, narQBased on the information of APP JL03 strain genome sequencing in GeneBank, the analysis of location of the narP, narQ on the genome, base composition, G+C ratio, concervative region of protein, and phylogenic trees were conducted. This provided constructive strategies for the following steps.2. Construction and biological characterization of AnarP, AnarQ and AnarPQTo construct the narP deletion reconbinant suicidal plasmid PEMAnarP and PEMAnarQ, the narP and narQ upstream and downstream fragments from SLWOl genomic DNA were amplified, respectively and linked to the suicidal plasmid PEMOC2 which carring sucose-sensitive gene(SacB). Thus the narQ-deletion reconbinant suicidal plasmid PEMAnarP and PEMAnarQ was constructed.AnarP, AnarQ single gene deleted mutant strain were constructed by conjugating E.coliβ2155 contain suisidal plasmid PEM-AnarP and PEM-AnarQ with SLWOl strain as receptor, respectively. The deleted mutant strain was constructed based on counter-selection and two-step homologous recombination. The AnarPQ double gene deleted mutant strain was conducted by conjugating E.coliβ2155 contain suisidal plasmid PEM-ΔnarP as doner with AnarQ strain as receptor. Finally, three deletion mutants were identified by PCR, RT-PCR and sequencing. Construction and characterization of AnarP, AnarQ and AnarPQ mutant strain was successful.3. Construction and characterization of AnarP, AnarQ complementary strains (CnarP, CnarQ)The narP, narQ genes from APP SLW01 genomic were amplified, respectively, and linked to shuttle vector pJff-XN. The recombination expressive plasmids PJff-narP, PJff-narQ were constructed, respectively. The plasmid was electrotransformed into AnarP and AnarQ mutant strains. Therefore, the complement strains CnarP, CnarQ were constructed and identified by RT-PCR.4. Expression of response regulator narPTo construct expressive plasmid PET-narP, narP gene from SLW01 genomic DNA was amplified and linked to expression vector PET-28a(+). Then the PET-narP plasmid was transformed into E.coli BL21 strain. Its expression was induced by IPTG. Expression activity was testified by SDS-PAGE.5. Bio-characteristics of deletion strainsThe AnarP, AnarQ and AnarPQ strains were cultured in TSA plate for 20 generations. Identification of genetic stability was conducted by PCR. The results showed that the heredity of three deletion mutant strains was stable. The hemolytic activity of AnarP, AnarQ and AnarPQ were tested in 9%(v/v) sheep blood plate. The result showed that there is no significant difference between mutant strains and parental strain. To detect growth of mutants, parental strain and mutant strains were cultured overnight at 37℃in TSB, respectively. The next day, strains were transfered into freshly prepared TSB at a 1:999.9 dilution. The OD600 was recorded every 1h. Growth of mutant strains were slightly slower than parental strain during log phase. By testing LD50 of mutants and parental strain in Balb/C mice, virulence of AnarP was slightly lower than parentalal strain, while virulence of CnarP was slightly higher than parental strain.6. Gene Differential expression of deletion mutants and parental strain.Parental strain and mutant strains were cultured at 37℃overnight. The next day strains were inoculated into freshly prepared supplemented TSB at a dilution of 1:100. Then, parental strain, AnarP, AnarQ and AnarPQ were cultured at the same time respectively under aerobic and anaerobic conditions in vitro. Expression differencnce of deletion mutant and parental strains were analyzed by using DNA Microarray, qRT-PCR and clustering. The result shed light on regulatory network of narQ/narP two-component regulatory system.Under arobic condition,75 genes of AnarP were differencially expressed compared to SLW01, following by 31 genes of AnarQ and 21 genes of AnarPQ.Under anarobic condition,94 genes of AnarPQ were differencially expressed, following by 61 genes of AnarQ and 22 genes of AnarP.Under anarobic condition,151 genes of SLW01 were differencially expressed compared to arobic conditions, of which 41 were up-regulated, and 112 were down-regulated.