The Mechanisms Of TolC Homologs Involved In The Virulence Of Actinobacillus Pleuropneumoniae

Porcine contagious pleuropneumonia,caused by the Actinobacillus pleuropneumoniae,is a significant respiratory disease in swine and causes serious economic losses to the swine industry worldwide.The infection process of A.pleuropneumoniae is complicated and potential virulence factors with their pathogenic mechanisms remain to be studied.TolC,an outer membrane channel,is widespread in Gram-negative bacteria and has been identified as an important virulence factor in numerous pathogens.The identification of the functions of TolC in the virulence of A.pleuropneumoniae would play crucial roles in efforts to prevent and control this disease.In this study,tolC1 and tolC2 mutants were generated via recombinant techniques using a wild-type A.pleuropneumoniae strain,SC1516.The biological characteristics of SC1516 and its mutants were investigated to reveal the correlations of TolC1 and TolC2 with the virulence of A.pleuropneumoniae.Ultimately,our findings allowed determination of the functions of TolC1 and TolC2 in the secrection of virulence-associated proteins and in the biofilm formation.A brief summary of our work is presented below.1.Construction of tolC1 and tolC2 mutants in A.pleuropneumoniaeUsing homologous alignment,we characterized two tolC genes in the A.pleuropneumoniae genome,named tolC1 and tolC2.To uncover the roles of TolC1 and TolC2 in the virulence of A.pleuropneumoniae,we constructed tolC1 and tolC2 mutants via recombinant techniques.For creation of the tolC1 mutant,an internal fragment of tolC1was joined with the chloramphenicol acetyl transferase gene and cloned into a pMD19-T vector to give pLCT.pLCT was integrated into the genome of SC1516 by electroporation.The tolC1 mutant was selected using resistant plates and named tolC1::cat.To delete the full length of tolC2 gene,the upstream and downstream regions flanking the tolC2 gene was fused and cloned into the suicide vector pEMOC2 to generate plasmid pEMOC2-?tolC2.The pEMOC2-?tolC2 was integrated into the genome of SC1516 by conjugation.After two homologous recombination steps,the tolC2 mutant was identified by PCR and named?tolC2.For genetic complementation,the entire coding regions of tolC1 and tolC2 together with their promoters were cloned into the shuttle vector pLS88 to generate plasmids pLStolC1 and pLStolC2,respectively.The resulting plasmids were electroporated into the corresponding mutant strains to give the complemented strains,tolC1::cat tolC1~+and?tolC2/tolC2.To confirm the mutations,western blotting was performed with TolC1 and TolC2 rabbit antisera respectively,and the results confirmed that both tolC1::cat and tolC2 have lost their ability to express corresponding proteins.In contrast,the expression of TolC1 and TolC2 were restored in both genetically complemented strains.In this study,the tolC1 and tolC2 mutants and their complemented strains were constructed,which are of great help for the study on virulence correlations and functions of TolC1 and TolC2.2.Characterization of tolC1 and tolC2 mutants of A.pleuropneumoniae.To determine the biological functions of TolC1 and TolC2,we investigated the biological characteristics of SC1516 and its derivatives.The growth curves showed that the tolC1::cat strain showed a slight growth delay in the log phage,but reached similar optical density in the stationary phase.The transmission electron microscopy examination revealed a significant morphological variation between the cells of SC1516 and tolC1::cat,which showed mutiple vacuole-shape inside the cell and vesicle-like structures in the cell envelope.In the hemolytic test,the wild type showed alpha hemolytic,while the tolC1mutant lost its hemolysis activity.The biofilm formation assays showed that the amount of biofilm in the tolC1 mutant showed was significantly reduced compared to that in the wild type.Besides,the antibiotic susceptibility of the tolC1::cat was significantly increased both in planktonic culture as well as in biofilms.However,the tolC2 mutant did not show significant difference with wild type in any of these aforementioned experiments.The stress resistance assays showed that the survival rates of both the tolC1 and tolC2mutants were significantly reduced compared with that of SC1516 under oxidative,acid and heat shock circumstance.The tolC1 mutant also showed significantly increased susceptibility to the hyperosmotic challenge,while the?tolC2 did not.In the serum killing assays,the tolC2 mutant showed significantly lower percent survival compared to the SC1516,but the tolC1::cat showed no difference.Under the same dose conditions,the mortality of mice intraperitoneally infected by the tolC1 and tolC2 mutants were 20%and40%,respectively,both were significantly lower compared to that of SC1516 with a 90%mortality.After intranasal infections,the number of CFU from bronchoalveolar lavage fluid of mice infected with?tolC2 was significantly higher than that infected with SC1516.The bacterial loads of the lungs of mice infected with tolC1::cat or?tolC2 were both significantly lower than that infected with SC1516.Besides,the histopathological analysis showed that the tissue infected with the tolC1 or tolC2 mutant both showed milder pulmonary lesion compared to that of the wild type.These results showed that TolC1 and TolC2,as important virulence determinants,are both required for the full virulence of A.pleuropneumoniae.3.Roles of TolC homologous in the secretion of ApxIIA and ApxIVA-S toxins of A.pleuropneumoniae.The relatively simple type I secretion systems(TISS)in Gram-negative bacteria are highly efficient in transporting substrate to the exterior and are required for the secretion of several important virulence-associated proteins.TolC is the outer membrane channel component used by the type I secretion system.To explore the potential role of TolC1 and TolC2 of A.pleuropneumoniae in the secretion of extracellular virulence-associated proteins,comparative analysis were performed on the secretome between the wild type and tolC1 or tolC2 mutant in this study.The two-dimensional gel electrophoresis identified 7protein spots whose expression were significantly reduced in the?tolC2 compared to SC1516.Mass spectrometry and bioinformatics analysis revealed that the differentially expressed proteins are located in intracellular,and the 7 spots are identified as false positive results.SDS-PAGE analysis found that the protein bands of the secreted protein by the tolC1::cat showed noticeable difference with that of SC1516.The differential protein bands were analysed using LC-MS and the results identified 6 protein spots that were absence in the tolC1::cat.Further bioinformatics analysis suggested that only two proteins,ApxIIA and ApxIVA-S,were likely to be the substrates of TolC1 dependent type I secretion systems.Western blotting assays using ApxIIA and ApxIVA-S rabbit antisera showed that ApxIIA and ApxIVA-S could not be detected in the secreted protein of the tolC1 mutant,confirming that TolC1 is responsible for the secretion of ApxIIA and ApxIVA-S.Bioinformatics analysis showed that 50%of the amino acids of ApxIVA-S were identical to the classical ApxIVA.And the identical region is centered on the carboxyl terminus.Considering that the secretion signals recognized by T1SS are located at the carboxyl terminus of the secreted proteins,we speculate that TolC1 is also involved in the secretion of ApxIVA.The apxIVA-S gene is proposed to be a partial copy of classical apxIVA on the genome and is able to expression in vitro.This is the first study to identify the ApxIVA-S protein in vitro culture of A.pleuropneumoniae.Together,our study reveals that TolC1 is required for the secretion of ApxIIA and ApxIVA-S,while TolC2 is not involved in the transportation of secretory protein of A.pleuropneumoniae.4.Roles of TolC homologous in the biofilm formation of A.pleuropneumoniae.Biofilm formation plays an important role in conferring resistance to antimicrobials and protection against host defenses.The previous biofilm formation assays showed the tolC1mutant formed significantly less biofilm compared to the wild type.In this study,we tried to uncover the role of TolC1 in the biofilm formation of A.pleuropneumoniae.The time course of biofilm formation for SC1516,tolC1::cat and tolC1::cat tolC1~+showed that the biofilm biomass peaked at 6,and decreased gradually over time in all groups after 12 h.The consistency of the curves suggested that the TolC1 was not involved in the biofilm maturation process.Results of the surface attachment assays and observations by confocal laser scanning microscopy both showed that the number of attached cells of the tolC1mutant was significantly reduced in the early stage of biofilm formation.To solve this,we found that the tolC1::cat cells showed a reduced autoaggregation phenotype and decreased cell hydrophobicity values in the aggregative and hydrophobicity assays.Digestions with DspB showed that loss of TolC1 reduced PGA proportion in early-stage biofilms.The qRT-PCR assays further suggested that the reduced PGA production in the tolC1 mutant was due to the decreased transcription of pgaA.Besides,digestions with proteinase K and DNase I showed reduced extracellular proteins and DNA proportion in the biofilms of tolC1::cat strain.Tanswell assays suggested that inactivation of TolC1 caused the secretion deficit of certain biofilm promoting factor in A.pleuropneumoniae.All the results above showed that TolC1 contributes to maintaining the natural surface hydrophobicity and autoaggregation of the bacterial cells,regulating the transcription of pgaA and ultimately the production of PGA.In these ways,TolC1 is required for the surface adherence of biofilm cells and thus plays an important role in the initial attachment step during biofilm formation.Besides,it is proposed that TolC1 is involved in the secretion of certain biofilm promoting factor during biofilm formation.Our findings demonstrated the role of TolC in the biofilm formation,which is helpful for the studies that targeting TolC as the antibiofilm strategies of A.pleuropneumoniae.