Functional Study Of AtxA Gene In Bacillus Anthracis

Bacillus anthracis is the etiologic agent of anthrax-a common disease of livestock and humans,which is one of the five major anthropozoonosis.Anthrax occurs mainly in herbivorous mammals such as cattle and sheep,but it can be transmitted to humans as well when they are exposed to infected animals and anthrax spores present in the air.Spores,the main transmission and infection form of Bacillus anthracis,may infect human beings via three routes,i.e.,skin,intestine,and lung,leading to cutaneous anthrax,intestinal anthrax,and pulmonary anthrax,respectively.Pulmonary anthrax among others has the highest mortality rate,which is explained by the fact that spores are inhaled in alveolus and engulfed by macrophages,followed by spore germination,proliferation,lysis of macrophages,and release of large amount of bacteria.In addition,Bacillus anthracis capsule inhibits the fusion of phagosome and lysosome to prevent from being digested by phagocytes,and these bacteria may hide in deep tissues instead,finally resulting in the invasion of Bacillus anthracis propagules in blood and the release of toxins as well as the consequent death of patients.Bacillus anthracis is a Gram-positive,spore-forming capsule bacterium without flagella.It can grow under aerobic or anaerobic conditions in two life forms,namely vegetative form and spore.Spores of Bacillus anthracis have extremely strong stress resistance and cannot be inactivated by commonly used disinfection approaches,including high temperature,chemical reagent,ionizing radiation,and ultraviolet.Due to the persistent communicability（about 20-30 years）,convenient storage and transportation,Bacillus anthracis is often used as a material of biological weapons,which seriously threatens national security and social stability.Therefore,it is very important to understand the virulence regulatory network of Bacillus anthracis and elucidate its pathogenic mechanism so as to prevent and combat the occurrence of anthrax.Toxins and capsule are considered the major virulence factors of Bacillus anthracis.Toxins consist of three factors,including lethal factor（LF）,edema factor（EF）and protective antigen（PA）,from which two complexes are formed,respectively,to exhibit its function.The three toxin components are encoded by lef,cy A and pagA genes,respectively,and capsule is encoded by capBCADE gene.All these genes encoding toxins and capsule are located on the two virulence plasmid pXO1（181,654bp）and pXO2（96,231bp）in Bacillus anthracis,respectively,and loss of any of the two plasmids can reduce the toxicity of Bacillus anthracis.Meanwhile,some virulence factors,such as S-layer gene,are located in the chromosome of Bacillus anthracis（5.3Mb）.atxA（anthracis toxin activator）,a key regulatory factor of the virulence regulatory network of Bacillus anthracis,is located in the pXO1 plasmid and positively regulates the transcription of the genes encoding the three toxin factors and the capsule as well as other virulence genes the on the chromosome of Bacillus anthracis.A distinct transcription start site is found 99bp upstream of the translation initiation site of atxA gene.Atx A,the toxin activator protein,is synthesized when Bacillus anthracis infects macrophages and geminates.This protein is composed of476 amino acids with a molecular weight of 5.6kDa.Genes encoding three toxin components（lef,cya,and pagA）and the capsule on plasmids pXO1 and pXO2,and that encoding the S-layer protein（sap and eag）on the chromosome,are regulated by AtxA protein.Additionally,atxA gene interacts with abrB,spoOA,codY genes to play a role in mutual regulation.Silent mutation of plcR gene is found in Bacillus anthracis.When plcR and atxA genes are co-expressed in Bacillus anthracis,the formation of spores is inhibited.atxA gene is involved in the regulation of toxin genes and is the core of the regulatory network in Bacillus anthracis.However,the whole regulatory network of Bacillus anthracis is not thoroughly studied and further exploration is necessary.Therefore,the present study is designed to focus on atxA gene to further investigate the function of atxA gene and improve the virulence regulatory network in Bacillus anthracis,laying the foundation for the prevention of anthrax.Through constructing atx A gene-deleted mutant strain B17D2△atxA::spc from B.anthracis strain B17D2,this study is designed to investigate the proteomic differences between the constructed mutant strain and the original strain,analyze atxA-regulated proteins and obtain all differentially expressed genes via gene chip hybridization experiment.Considering the coordinating function of CO₂ for atxA gene regulation,this experiment was conducted in the atmospheres of 5%CO₂ induction and non-CO₂induction respectively.The regulatory effect and function of atxA gene were comprehensively analyzed according to the overall gene expression differences and those differences under different conditions between the original strain and the constructed mutant strain.Providing the low gene-knockout efficiency in B.anthracis,the gene knockout method was improved at first in our study through developing a B.anthracis gene knockout system universally applicable to the knockout of any genes.The Golden Gate method and rational design of Type IIs restriction endonuclease cleavage site allowed the simultaneous cleavage and ligation in the same aforesaid system subject to the principle that type IIs restriction endonuclease cleavage site and recognition site were located in different regions;the framework plasmid pKSV7 was used to further construct atxA gene knockout vector,to which cleavage site I-Sce I was then added;by introducing I-SceI endonuclease in the homologous recombination to improve recombination efficiency,the atxA gene-deleted mutant strain B17D2△atxA::spc from B.anthracis strain B17D2 was successfully constructed and subsequently verified using sequencing,RT-PCR and Western Blot.Based on above efforts,two-dimensional electrophoresis on whole-cell proteins and proteins in supernatant,HRMS/MS and gene chip assay were performed in these two strains to transcriptionally and proteomically analyze atxA gene-regulated genes.The results showed that the proteomic analysis identified 50 distinct proteins between B17D2 and B17D2△atxA::spc strains,including 30 upregulated genes and 20downregulated genes;the obviously changed genes revealed by mass spectrometry findings included upregulated genes pfl,BA0541,rpsB and sodA-1 as well as downregulated genes eag,tkt-2,BA3760,yqiQ,BA4202 and BA2500.According to the gene chip hybridization,the atxA gene-deleted strain exhibited 2487 differentially expressed genes compared to the original strain,including 1111 upregulated ones and1376 downregulated ones;2042 differentially expressed genes,including 1065upregulated ones and 977 downregulated ones,were determined in the original strain cultured in 5%CO₂ relative to that cultured in non-5%CO₂;under the condition of 5%CO₂,1049 differentially expressed genes were found between the atxA gene-deleted strain and the original strain,including 445 upregulated ones and 604 downregulated ones;the atxA gene-deleted strain cultured in 5%CO₂ showed 1833 differentially expressed genes,including 1037 upregulated ones and 796 downregulated ones,relative to the results under non-5%CO₂ condition.The most significant differences were reported in genes sipW,acoL,acoB,acoA,acoC,ywdH and argF,which are warranted to perform further exploration.In summary,we can conclude that atxA gene regulates the expression of multiple virulence factor-related genes,as its regulation on pagA,pagR,cya,eag and sap genes has been verified by the statistically significant differences in gene chip hybridization.Furthermore,atxA gene also exerts a regulatory role in some spore-forming and metabolism-related proteins,such as BA1290,sipW and acoL.In addition,CO₂ may have a certain compensating effect on atxA gene regulation,which,however,requires further experiments for verification.In terms of the intra-strain comparison,the outcomes under 5%CO₂ culture of both original strain and atxA gene-deleted strain exhibit differences compared to those under non-5%CO₂ culture,where the upregulated genes hold higher proportion.