Study On The Function Of The S-layer Genes Sap And Eag In Bacillus Anthracis

Bacillus anthracis is a spore-forming, rod-shaped, gram-positive bacterium. It is the etiological agent of the disease anthrax of animals and human, including cutaneous, gastrointestinal and inhalational anthrax. Anthrax is harmful for animal husbandry and human health. There are maybe some virulence factors on the chromosome of B. anthracis beside of the known genes on the plasmids pXO1 and pXO2, which are including the virulence gene and the capsule synthesis gene separately. It has been reported that the S-layer protein maybe also is a virulence factor. The S-layer of B. anthracis is major composed of Sap and EA1 proteins which are encoded by sap and eag gene separately.In order to investigate the function of Sap and EA1 proteins, we constructed the sap and double mutants based on B. anthracis vaccine strain A16R using Cre/loxp system by homologous recombination principle. We verified the mutants by PCR and western blot. Then we researched the growth curve, carbohydrate metabolism and the surface of A16R and its mutants strain by electron microscope. Different animal models and comparative proteomics were also used to get the function of sap and eag gene. Results are as follow.1 construction of the A16RAsap and double deletion mutants A16R△sapAeag with markerlessIt is a basic method to research gene function through knockout the target gene. The vaccine without any antibiotics is of great benefit to medical. In this study, we used the shuttle vector pKSV7 to construct the targeting vector, and introduced it into A16R, then screening the mutant A16R△sap::spc based on homologous recombination principle. Finally, we obtained the A16RAsap by removal of the antibiotic gene using Cre/loxp system. Double deletion mutant A16RAsap△eag was constructed by the same method.2 Analysis of the physiology, biochemistry and the surface of A16R and its mutantsThe growth curve was no differences between mutants strain and A16R in exponential phase, stationary phase and decline phase. The result of utilizing carbohydrate showed that the mutants and A16R used the same kinds of carbohydrate. We found that the sap and eag double deletion mutant could not synthesize S-layer structure, while the single deletion mutants could synthesize a 7nm or 19nm thiner S-layer structure than A16R separately.3 Animal models for A16R and its mutants to evaluate the virulenceWhen the experiments of the spore or the vegetative form infect the different animals shows the virulence of A16R is reduced after eag was deleted, while sap gene has no effect onA16R. 4 The comparative proteomic research of A16R and the mutants strainThe whole cell proteins of A16R and the mutants were prepared and two-dimensional electrophoresis reference maps and database of A16R and the mutants in pH 4-7 were constructed. In total,198 difference spots were obtained and the major different function protein was energy production and conversion protein when sap or eag gene was deleted. There was no other virulence factor when identificated of the defferent proteins between A16R and its mutants.We found the B. anthracis putative S-layer protein BA3338. When sap gene was deleted, BA3338 would up-regulate at exponential phase. When eag gene was deleted, one BA3338 spot was up-regulated and the other BA3338 spot was down-regulated at late-exponential phase, but the two BA3338 spots were both up-regulated from stationary phase to decline phase. As we know the Sap and EA1 expressed at the different phase, we presumed that BA3338 possible to compensate the function of Sap or EA1 protein. The expression of BA3338 in the double deletion mutant was the same as in A16R.Two protein spots on the spore formation (Ad07, BA1530; Ad18, BA5641) were lost after the Sap and EA1 proteins were both deleted by comparing of the two-dimensional electrophoresis maps of A16R and A16RAsap△eag. While the double deficient mutant in the culture could form spores which indicated that the two proteins BA1530 and BA5641 have no influence on spore formation.The results of virulence evaluation using different animal models and the comparative proteomics analysis showed that eag gene was a virulence factor, but sap gene was merely the structure gene consisted of S-layer structure of B. anthracis.This is not only providing theory for the gene function research about S-layer of B. anthracis, but also promoting to manufacture more safer and effective B. anthracis vaccine and consummate therapeutics.