Study On The Function Of The Eag Gene In Bacillus Anthracis

Bacillus anthracis is a spore-forming, rod-shaped, gram-positive bacterium. It is the causative agent of the disease anthrax of animals and human, including cutaneous, gastrointestinal and inhalational anthrax. B.anthracis is used as a biological warfare agent and a terrorist weapon causing a serious threat. Relative research shows that the S-layer of bacteria can protect cells, adhere to other cells, work as a molecular sieve, and provide the adhesion sides for the cell-associated exoenzymes, in pathogenic bacteria, the S-layer can even resist the aggression of host’s immune system, and is considered to a potential virulence factor. B.anthracis indicates that there are considerable genes contributed to virulence. These genes locate among plasmids and chromosome. For example the S-layer proteins are encoded by the genes of B.anthracis chromosome.The studies indicate that S-layer protein of B.anthracis might be important targets for vaccines and drugs. The S-layer protein of B.anthracis consists of Sap (surface array protein) and EA1 (extracellular antigen 1).In this study, the B.anthracis vaccine strain A16R was used as experiment strain, the eag deletion mutant was constructed by the homologous recombination and checked by the PCR and proteomics. In order to get the function of gene eag, this study researched the growth curve and carbohydrate metabolism, comparative proteomic, cell and animal model experiments of mutant strain and wild strain.Results are as follow.1. Construction of mutantIn this study a new integrational vector pKSV7 was used to construct a recombination vector pKSV7usd. It was introduced into the B.anthracis vaccine strain A16R by electroporation and screened the mutant A16R△eag: :spc using the principle of homologous recombination.2. The growth curve and carbohydrate metabolism experiment of the mutant strain and wild strainThe growth curve was no differences between mutant strain and wild strain in exponential phase, stationary phase and decline phase. The result of utilizing carbohydrate showed that both the mutant strain and wild strain used ribose, fructose, glucose, N-acetyl-glucosamine, aesculin, cellubiose, maltose, sucrose, trehalose, glycogen ten kinds of carbohydrate. There were no differences about utilizing carbohydrate between mutant strain and wild strain after cultivated 48h. So eag gene was uncorrelated with carbohydrate metabolism.3. The virulence analysis of the mutant strain and wild strainThe study of damage effects the spores of the mutant and wild strain on CHO cell showed that the mean OD490 was 0.457, 0.407 respectively.The t-test revealed that the virulence of the mutant strain was weaker than the virulence of the wild strain. The competitive interaction experiment of spores and J774A. 1 cell showed that the competition index was 0.52, in another word the virulence of the mutant strain was about half of wild strain. Animal attack tests showed that the mean survival time of the mice after attacked by the spores of the mutant and wild strain was 31.37h, 21.03h respectively and the mean survival time of the mice after attacked by the vegetative of the mutant and wild strain was 27.12h, 20.68h respectively. The result revealed that the virulence of the mutant strain was weaker than the virulence of the wild strain. Animal’s lung infected test showed the competition index was 0.55, that is to say the virulence of the mutant strain was half of wild strain.Both the cell and animal model experiments showed that the virulence of the mutant strain was about half of the wild strain. The cell model analysis showed the EA1 protein function as protective coats.4. The comparative proteomic research of the mutant strain and wild strainThe whole cell proteins extract of 8h, 23h, 35h of the mutant strain and wild strain were prepared and 2-DE reference maps and database of the the mutant strain and wild strain in different pH were constructed. In total, 95 difference spots were processed and 54 spots representing 25protein entries were identified. There were 8 proteins not found in the whole 2-DE maps of Bacillus anthracis which had been reported. 30 spots were identified EA1 protein. The location of the proteins in the cell showed majority of these proteins located on Cytoplasmic except EA1. The function of the proteins analysis showed majority of these proteins focus on energy production and conversion, amino acid transport and metabolism, inorganic ion transport and metabolism. These proteins in the mutant strain about all of them were down-regulation proteins, it revealed that after deleted the EA1 protein affected their metabolism.The protein-to-spots analysis revealed that EA1 protein has post-translational modifications character (Phosphorylation). The result of analysis not found any Sap protein expression has changed, so deleted the eag gene could not affect the Sap protein expression.There were protein spots identified as two kinds of putative S-layer proteins: BA3338 (two protein spots) and BA1127 which were up-regulated in the mutant strain. These proteins function is still unknown. Both the BA3338 and BA1127 proteins were up-regulated in the mutant strain. It was supposed that the BA3338 and BA1127 possible to compensate the function of EA1 protein. This research analysis revealed: 1) BA3338 protein correspond to two spots also cause of Phosphorylation2) the structure analysis showed BA3338, BA1127 and EA1 three proteins all had three typical SLH-motifs at the N-terminal part which is responsible for binding of the S-layer subunits to the underlying cell envelope layer and there were difference structure of C-terminal part which is involved in the self assembly process.Comparative proteomic showed there was not identified any protein correlated with the virulence. It was revealed that the virulence of the mutant strain was lower than the wild strain because of EA1 protein was deleted. So eag gene acts as a virulence factor. The results of this study might be helpful to the research of B. anthracis vaccine.