Screening And Characterization Of Biofilm-forming Mutants Of Actinobacillus Pleuropneumoniae

Actinobacillus pleuropneumoniae(APP) is the causative agent of porcine contagious pleuropneumonia(PCP),a severe infectious respiratory disease of swine causing great economic losses to the pig industry worldwide.APP is obligatory parasitic bacteria of porcine respiratory,spreading through air and direct contact.Due to many distinct serotypes and lack of cross-protection among them,it is difficult to prevent and control the disease using traditional attenuated bacterins and/or subunit vaccines.So novel safe and effective vaccines are urgently needed to prevent and control this disease.The overall identification of virulence factors is the basis to understand the pathogenesis and develop new control products.Signature-tagged mutagenesis(STM) is an improved transposon mutagenesis technique.Using mini-transposon carrying different sequence tags,a series of transposal mutant pools could be constructed.Then attenuated mutant strains could be negatively selected from the mutant pools in host and/or experimental animals.Since the first application in 1995,STM is widely used to pick up the infecteion-associated genes in many kinds of bacteria.In order to adapt to the changed environments,many bacteria could form biofilms in vitro and in vivo.A plenty of evidence demonstrated that biofilm formation is involved in the immune escape and antibiotics resistance in many species of pathogenic bacteria.It plays an important role in the full infection and pathogenesis.In the present study,an STM mutant bank of APP serotype 1(a non-biofilm forming strain) was constructed.Two biofilm-forming mutant strains were identified from the mutant bank.The mutated gene was mapped,cloned and sequenced.Further characterization of the two biofilm-forming mutants provided useful experimental data to understand the mechanism of biofilm formation in this important pathogen.1.Constrcution of an STM mutant bank of APP serotype 1A nalidixic acid-resistant strain 4074-N of APP serotypes 1 was used as recipient strain to mate with E.coli CC 118λpir or Sm17-1λpir containing mini-Tn 10 tag plasmids pLOF/TAG1-48,with or without the help of E.coli DH5α(pRK2073).Mutant strains were screened by kanamycin resistance selection,ampicillin negative selection,PCR and Southern blot identification.In the mating experiments,the bi-parental mating was more effective and easy than tri-parental mating.Using the bi-parental mating method eatablished for APP,thirty two STM mutant pools have been constructed.Each pool contained at least 50 mutant strains. 2.Screening of biofilm-producing mutants and identification of inactivated genesUsing 96-well microtiter plate and glass tube assays,two strong biofilm-producing strains STM1-21 and STM6-42 were screened from the STM mutant pools described above.The genomic DNA of the mutants was extracted,digested with SspⅠlocated in the transposon,and self ligated.Using the ligated mixture as template,the both flank fragments of the transposon were amplified by reverse PCR with the transposon-specific primers STM10/11 and STM10/12 respectively.Sequence determination and analysis revealed that the hns gene encoding the histone-like nucleoid structuring protein(H-NS) was inactivated by the insertion of the mini-Tn10 transposon.Further PCR and Southern blots analysis confirmed the insertion.3.Structural and functional characterization of the hns gene in APPThe hns gene of APP encodes a histone-like nucleoid structuring protein(H-NS) consisting of 135 amino acids.The H-NS protein consists of an N-terminal dimerization domain and a C-terminal nucleic acid-binding domain that are separated by a linker region.It can form homodimer,tetramer or oligomer via its N-terminal dimerization domain,and the C-terminal nucleic acid-binding domain is important to bind DNA in regulation of gene expression.In order to understand the function of the hns gene in APP,the hns expression cassette,containing the complete coding sequence of hns and its native promoter and terminator,was amplified from the genomic DNA of APP strain 4074-N,and then cloned into the shuttle vector pJN105.To obtain a complementary strain and an over-expressing strain,the resultant expression plasmid pJN-hns was transformed into the transposal mutant strain STM1-21(M) and parental strain 4074-N(P) respectively,resulting in strain C-3 and 0-2.The in vitro growth property,biofilm formation and virulence were compared among the 4 strains P,M,C-3 and O-2.The results showed that no obvious difference in the in vitro growth was observed among the four strains.Except strain M forming obvious biofilms in vitro,no biofilm formation was visible in the other three strains.The virulence was attenuated in the strains M,C-3 and O-2 when their haemolytic activity and 50%lethal doses in mice were compared with the parental strain P.In order to explain the tmexpected phenotypes of strains C-3 and O-2,Real-time RT-PCR analysis was performed to compare the expression levels of hns gene and the two major virulence genes apxⅠA and apxⅡA.The results showed that the hns gene was in deed expressed in strains C-3 and O-2,but their levels were lower than that in the parental strain P.This indicated that the hns deletion was not completely complemented in strain C-3,and hns was not overexpressed in strain O-2 as well.Both strains C-3 and O-2 were actually hns knockdown strains.This may be due to the auto-regulation mechanism of hns expression which was reported in E.coli previously.H-NS may regulate the expression of target genes in a dose dependent manner,and the down-regulation of the exotoxin genes(apxⅠA and apxⅡA) in the hns deletion and knockdown strains may partly contribute to the virulence attenuation.To further investigate the effect of H-NS on APP biofilm formation,the 408 bp complete coding sequence of hns gene was amplified by PCR from the genomic DNA of APP serotype 1 strain 4074 and cloned into the prokaryotic expression vector pET-28c.The resultant recombinant plasmid pET28c-hns was transformed into Escherichia coli BL21(DE3).A 19 kD of recombinant protein(rH-NS) was obtained by IPTG induction,and purified using the Ni-NTA agarose columns.Different concentrations of rH-NS were added into the culture medium of the hns transposal mutant strain 1-21 and its parental strain 4074, and biofilms were quantified using the microtiter plate biofilm assay.Without rH-NS in the medium,strain 1-21 formed obvious biofims,but strain 4074 did not.Supplemented with 0.1～0.3μmol/L rH-NS in the culture medium,the biomass of biofilms formed by strain 1-21 continuously decreased,whereas that formed by strain 4074 was increased. No obvious changes could be observed when more than 0.4μmol/L of rH-NS was supplemented in both strains.Results indicated that H-NS negatively regulates biofilm formation of A.pleuropneumoniae in a dose-dependent manner.Our data indicate that H-NS plays important roles in regulating biofilm formation and virulence in A. pleuropneumoniae.The H-NS regulated genes and regulatory mechanism need futher investigation.