The CpxAR Two-Component System Is Required For Actinobacillus Pleuropneumoniae Resistence To Fever-Like Temperature

Actinobacillus pleuropneumoniae(APP)is an important endemic pathogen,the causative agent of porcine pleuropneumonia(PCP)responsible for huge economic losses to the pig industry worldwide.Porcine pleuropneumonia is a very contagious respiratory disease that affects pigs of all ages,characterized by fibrino-haemorrhagic and necrotizing pleuropneumonia.Based on differences in surface polysaccharides,mainly capsule,18 serotypes(types 1 to 18)have been described for APP.With the rapid development of large-scale farm in the 1990 s,the disease severely outbroke and rapidly spread.Since PCP was first reported by Pattison et al in 1957,it has widely spread in the world,such as Switzerland,Denmark,and the United States.PCP brings a huge threat to the modern pig industry,and is recognized as one of the major diseases that cause substantial economic damages in the pork industry globally by the international community.While infecting the host,APP encounters various adverse conditions and requires a regulatory system to sense and respond to environmental signals.After the pig is infected with acute pleuropneumonia,the body temperature rises rapidly to about 42°C.APP still grows at fever-like temperature and causes disease and death to a large number of pigs.However,there is still no report on the mechanism of APP survival at feverlike temperature in vivo.CpxAR is an important two-component regulation system ubiquitous in Gram-negative bacteria,which senses and responds to envelope alterations that are mostly associated with protein misfolding in the periplasm.Our previous study has showed that CpxAR is necessary for APP survival at feverlike temperature.In this study,ten CpxR-regulated genes(apfA,apfB,apfC,apfD,pilM,pilN,pilO,pilP,pilQ and csgG)were selected,and their roles in APP survival at feverlike temperature were studied.The results indicated that CpxAR contributes to APP resistance to fever-like temperature by directly suppressing the expression of the major pilin protein ApfA to prevent its aggregation.The main contents are as following:1.Predicting pili-related genes involved in CpxAR regulation of APP resistance to fever-like temperatureOur previous study has showed that CpxAR plays an important role in APP survival at fever-like temperature(42°C).We explored the mechanism of CpxAR in regulating APP resistance to fever-like temperature by strand-specific RNA-seq analysis on APP strain S4074 strain and its isogenic cpxAR mutants in 37°C and 42°C condition.The expression of 9 genes randomly selected which regulated by CpxAR was confirmed by real-time PCR in 37°C and 42°C condition.The real-time PCR result was in accordance to the transcriptional data.The results showed there were 73 genes regulated by CpxAR in 37°C condition,among which 52 genes were upregulated,and 21 genes were downregulated.And there were 54 genes regulated by CpxAR in 42°C condition,among which 41 genes were upregulated,and 13 genes were downregulated.While analyzing the CpxAR-regulated genes which may involve in the mechanism of APP resistance to fever-like temperature,we found that 10 genes susceptible to misfolded proteins were significantly upregulated.These 10 genes are apfA,apfB,apfC and apfD encoding type IV pilus proteins,pilM,pilN,pilO,pilP and pilQ predictive encoding type IV pilus proteins,and csgG predictive encoding the curli fibre protein.2.Biological role of Actinobacillus pleuropneumoniae type IV pilus proteins encoded by the apf and pil operonsIn this study,we predicted that pilM,pilN,pilO,pilP and pilQ may be required for IV pilus biogenesis and function in Actinobacillus pleuropneumoniae by bioinformatics comparison analysis.By RT-PCR,we found that the pilMNOPQ(pil)gene cluster is operon.Sequencing analysis revealed that the apf and pil operons are highly conserved among different serotypes of A.pleuropneumoniae.Our data demonstrate that the transcription of the pil operon was greatly upregulated upon bacterial contact with host cells.Mutants with single deletions of each gene in the pil and apf operons had defective adherence when they were in contact with host cells and,additionally,these mutants were also deficient in biofilm formation and IV pilus biogenesis.In conclusion,these results indicate that the products of each gene within the apf and pil operons are necessary for the normal biogenesis and function of IV pilus in A.pleuropneumoniae.3.CpxAR contributes to APP resistance to fever-like temperature by directly suppressing the expression of the major pilin protein ApfA to prevent its aggregationBased on the RNA-seq analysis,ten CpxAR-regulated genes may play an important role in resisting fever-like temperature of A.pleuropneumoniae,but need further verification.Ten double mutant strains were constructed on the basis of the ?cpxAR mutant strain through homologous recombination.To analysis the role of the ten genes in resisting fever-like temperature,growth curves of the ten double mutants under 37°C and 42°C conditions were compared with those of the ?cpxAR mutant.Confocal microscopy and flow cytometry were used to assess the ability of the mutant and WT strains to resist fever-like temperature.The results showed that ApfA is involved in resisting fever-like temperature of A.pleuropneumoniae.The results of Western blot and MALDI-TOF showed that ApfA is the major pilin subunit in A.pleuropneumoniae.The ApfA protein leves in both cell surface and whole cell lysates from each strain were analyzed by Western blot.Transmission electron microscopy was used to observe the type IV pilus of each strain.These results suggested that the ability of the ?cpxAR mutant to resist fever-like temperature was descreased because the over-expressed ApfA protein aggregated in periplasm and could not be secreted extracellularly to participate in the assembly of type IV pilus,resulting in bacteria death.By analyzing the promoter regions of apfA,pilM and csgG genes,we found that they all have the predictive CpxR binding box.Moreover,we also showed that the CpxR protein can bind to the promoter of the genes by EMSA.DNase I footprinting assay identified the site of CpxR binding to the promoter region of the apfA gene.Capping RACE assay identified the transcription start site of apfA.In conclution,our results revealed that CpxAR plays an improtent role in APP resistance to fever-like temperature by directly suppressing the expression of type IV pilus major protein ApfA to prevent its aggregation.