| Actinobacillus pleuropneumoniae(App)is a Gram-negative capsulated rod with typical coccobacillary morphology that can cause contagious pleuropneumonia in pigs which is one of the common infectious diseases in intensive pig farms.Actinobacillus pleuropneumoniae App6 strain is a virulent strain isolated from a typical case of porcine contagious pleuropneumonia,and it belongs to serotype 5b.From the consequence of drug susceptibility test,the strain shows multiple drug resistance,including florfenicol,chloramphenicol,tetracycline,amikacin,kanamycin,streptomycin,tilmicosin,erythromycin,Clindamycin,lincomycin,and sulfamethoxazole.In order to analyze the multi-drug resistance of the virulent strain and provide information for the following study of this virulent isolate,a genome-wide sequencing analysis of this strain was conducted.The App6 genome has a total length of 2,490,072 bp with an average GC content of 41.49%,which is similar to other reported App strains;a total of 2265 open reading frames,61 tRNAs,and 19 rRNA operons were predicted;Plasmid DNA was not found.In addition,ICEAplChn1,belong to SXT/R391 of integrated conjugative element,was discovered in the App6 genome.ICEAplChn1 contains eight variable insertion regions,including VRII,VRIII,HS1,HS2,HS3,HS4,HS5 and a new Insertion zone,named VRVI;ICEAplChn1 carries 7 antibiotic resistance genes,including tet(A),erm(42),floR,aphA6,strB(two copies),strA and sul2,which can explain the multidrug resistance of this strain.The discovery of ICEAplChn1 showed that,in addition to the plasmids,the SXT/R391 ICE is another kind of transferable element that can mediate the drug resistance of App.The outcome of the bacteria infections is largely determined by the genomes of the pathogen and host and the appropriate expression of their genes.In vivo RNA-seq can truly and comprehensively reflect the gene expression and regulation in the process of bacterial invasion into the body.In order to further study the virulence-related genes of Actinobacillus pleuropneumoniae and its expression regulation mechanism,the virulent strain of App6 was used as the object.A mouse model of porcine contagious pleuropneumonia was first established;then the genome of the strain was completed.For the reference genome,the transcriptome analysis of bacterial cultures in vitro and in vivo was performed using the strand-specific RNA-seq technique,and 471 differentially expressed genes were screened.These significantly differentially expressed genes reflect the bacterial infection in vivo.Of these differential genes,316 genes were up-regulated,accounting for 13.95% of the entire open reading frame of the App6 genome(including hypothetical proteins),and they reflected many genes recently studied by the bacteria in recent years.In addition,with the help of a variety of biological software,172 differentially expressed genes were predicted to yield 82 secreted proteins.It is believed that the prediction of these differentially expressed genes and their secreted proteins will provide a certain reference for further investigation of the pathogenic mechanism of App and for potential vaccine targets.Previous experiments confirmed that the Sec secretion pathway-related genes were generally upregulated when App6 strains were infected in mice.Of them,the secD,secF,and yajC genes were all located on the same operon.Firstly,a yajC gene-deleted strain of the bacterium was constructed.By this mutant,the characteristics of the mutant was studied to reveal the biological function of the yajC gene of Actinobacillus pleuropneumoniae.The study investigated the biofilm ability,mouse virulence and mouse lung clearance ability of the yajC gene-deleted mutant.It was found that the deletion of this gene did not affect the growth curve of the bacteria,but resulted in a decrease in the biofilm formation capacity;Deletions do not affect the virulence of the bacteria,but they can accelerate the rate of clearance of the bacteria including lethal doses and a recoverable healthy dose;the absence of yajC does not affect the adherence of bacteria to cells,but reduces the ability of bacteria to invade cells.The specific role of yajC gene in the establishment of bacterial infections in vivo,in the expression and regulation of virulence factors,and in the contribution to the secretory system of bacteria,etc.,need more further studies. |
