Screenning Atoxic Mutants Of Anthrax Lethal Toxin And Studying The Immunogenicity And Anti-Toxin Mechanism

Anthrax is an acute, febrile, septicemic, zoonotic infectious disease caused by Bacillus anthracis. The bacteria are highly dangerous, easily to be produced on a large scale, can be stored for a long time, and simply to be handled. The humans and animals are infected when they inhale the floating aerosol with Bacillus anthracis. And pulmonary anthrax is highly fatal; this bacterium is one of the earliest bacteria used as biological weapon. The anthrax letters affair after "9.11" event in 2001 caused anthrax terror in the world and let this ancient disease becomes a great concern for human again.The anthrax spores will germinate in vivo to produce vegetative forms which replicate in large number and secret highly pathogenic anthrax lethal toxins when they enter the blood. At this stage, all antibiotics treatments are ineffective. Only drugs able to block the activity of anthrax lethal toxins can protect the hosts from damage or even death. Thus anthrax lethal toxins are major determinants of virulence for Bacillus anthracis. The toxins are composed of protective antigen （PA） and lethal factor （LF）. The study of inhibitors and vaccines to block the activity of these two toxins plays an important role on anthrax prevention and treatment.In this study, we screened dominant inhibitory mutants of PA and detected the immunogenicity and inhibitory activity in vitro and in vivo of dominant inhibitory mutants of PA, evaluated the cellular and humoral immunity and protection against lethal bacterial challenge induced by foreign antigen Streptococcus suis surface antigen Sao carried by LF N termimal, screening atoxic LF mutants, and explored the mechanism responsible for loss of toxicity. The mainly findings were summarized as follows:1. Expression and purification of PA and LF and used in cytotoxicity assaysThe two components of anthrax letahal toxin （LT）, PA and LF were expressed and purified. The activities of these two proteins were detected on LT sensitive cell line RAW264.7 macrophage cells. The cells were cultivated in 96-well plates and the viabilities of cells were appraised and calculated based on the fluorescense of alamarBlue dye taken by cells. When LT was fixed at 1μg/mL, the EC₅₀ of PA is 0.078μg /mL. On other hand the EC₅₀ of LF is 0.003μg/mL when PA was fixed at 1μg/ml, showing this cytotoxicity assay was sensitive to test cytotoxicity of the toxins. 2. Scanning dominant negative inhibitors of anthrax PA427 mutants for anthrax therapy and vaccineResidue phenylalanine-427 （F427） is a crucial aminao acid for PA function. To obtain potential novel therapeutic agents and vaccines for anthrax, the site saturated mutation library at F427 was constructed and screened for dominant negative inhibitory （DNI） phenotype which was non-functional and inhibited toxin intoxication. The F427 was firstly mutated to 19 naturally occurring amino acids. The cytotoxicity and DNI phenotype of recombinant proteins were tested and proved to depend on individual amino acid replacements in RAW264.7 cells. A total of 16 non-toxic mutants with various DNI activities were identified in vitro. Among them, PA F427D and F427N mutants were determined to have the highest DNI activities in RAW264.7 cells. The further mouse experiments demonstrated that these two mutants possessed therapeutic effect on LT intoxication in a dose-dependent way. Furthermore, they induced Th2 predominant immune response and protected mice against 5 LD50 LT challenge. The protection was mainly correlated to low level of IL-1βand high level of IgG1, IL-6 and TNF-α. Therefore the PA DNI mutants such as F427D and F427N PA DNI may have great potential to be novel therapeutic agents and vaccines to fight B. anthracis infection.3. Recombinant Streptococcus suis surface protein Sao with LFn boosted Th1 response in micePreviously studies have shown that the atoxic N terminal of anthrax lethal factor （1-254aa） can transport forgein antigen fused to its C-terminal when LF enters the cytosol of antigen presenting cells by the same mechanism as LF protein transformation. Once in the cytosol, the heterologous antigen fused to LFn can lead to peptide fragmentation and then is loaded onto both MHC-I and MHC-II molecules to stimulate both CD8⁺ and CD4⁺ T cellular immune response.Hereby, LFn was appraised as a gene delivery vehicle to deliver Streptococcus suis （S. suis） surface antigen Sao. We demonstrated that higher Sao specific IgG titers can be induced in BALB/c mice immunized with PA plus a fusion protein LFn-Sao than in mice immunized with Sao alone. Furthermore, the ratio of IgG2a to IgG1 was enhanced showing a Th1 biased atmosphere. The expression of IFN-γdetected by flowcytometry assay and quantitive ELISA and the results indicated that higher IFN-γlevels were expressed in LFn-Sao immunized mouse splenocytes than Sao immunized mouse splenocytes. When challenged intraperitoneally with log-phase S. suis strain, the LFn-Sao gave significantly higher protection in the immunized mice than Sao immunized mice. However, there was no difference in IL-4 level between these two groups. Therefore, the fusion expression of foreign antigen with LFn is a way to promote the immunogenicity of the antigens and enhance Th 1 immune response.4. Scanning LF mutation libray for atoxic LF mutations and the atoxic mechanism of the LF mutationsThe LF gene was amplified and a mutation （TAT-TTT） at the residue 236 resulting in an amino acid replacement （Y236F） was obtained. Compared to the WLF, an increase by 3700 folds of the purified Y236F was needed to achieve a similar level of cytotoxicity of the WLF. Y236F was highly immunogenicity, one week after the third vaccination, the antibody titers reached 1:819200, showing that the Y236F kept a high immunogenicity. In vitro neutralization assay was further performed to detect the anti-LF antibodies titers. It was observed that 1:20 dilution of the antiserum conferred 100% protection of cells against the cytotoxic effects of LT.There are 12 histidine residues in the LF domain 2, 3 and 4. To locate the important residue（s）, site-directed mutagenesis of these histidines was performed. H686-H690 is a reported Zn²⁺ motif （HEXXH）. Residues 745 to 749 （HSTDH）, which are similar to the inverted zinc metalloprotease motif HXXEH, could potentially act as another zinc site, either structural or enzymatic. H745-H749 was mutated by saturated site mutagenesis separately and 150 clones with mutation were scanned for the toxicities. For H745 mutation, 35 of 150 clones lost cytotoxicity, while all clones mutated at other residues kept remained cytotoxicity of LF on the cells.The remaining 8 histidine residues were mutated to alanine individually. H669A was totally nontoxic to cells even when H669A as high as 20μg/mL was added. H669A can inhibit the activities of LT in a competitive way with LF for the binding site on the heptermer of PA. H669A could be transported to the cell cytosol as the same mechanism of LF. The zinc content of H669A mutant is as the same as WLF, indicating that H669 is not correlated to the zinc binding inside the LF structure.