NprR is a protein of Bacillus anthracis that exhibits moonlighting functions as either a phosphatase or a neutral protease regulator that belongs to the RNPP family. We previously observed that the extracellular protease activity of an nprR deletion mutant significantly decreased within in vitro cultures. To identify the genes within the regulatory network of nprR that contribute to its protease activity, integrated transcriptomic and proteomic analyses were conducted here by comparing the nprR deletion mutant and parent strains. A total of 366 differentially expressed genes (DEGs) between the strains were observed via RNA-seq analysis. In addition, label-free LC-MS/MS analysis revealed 503 differentially expressed proteins (DEPs) within the intracellular protein fraction and 213 extracellular DEPs with significant expressional differences between the strains. The majority of DEGs and DEPs were involved in environmental information processing and metabolism. Integrated transcriptomic and proteomic analyses indicated that oxidation-reduction-related GO terms for intracellular DEPs and endopeptidase-related GO terms for extracellular DEPs were significantly enriched in the mutant strain. Notably, many genes involved in protease activity were largely downregulated in the nprR deletion mutant cultures. Moreover, western blot analysis revealed that the major extracellular neutral protease Npr599 was barely expressed in the nprR deletion mutant strain. The mutant also exhibited impaired degradation of protective antigen, which is a major B. anthracis toxin component, thereby resulting in higher protein yields. Concomitantly, another global transcriptional regulator, SpxA1, was also dramatically downregulated in the nprR deletion mutant, resulting in higher sensitivity to oxidative and disulfide stress. These data consequently indicate that NprR is a transcriptional regulator that controls genes whose products function as extracellular proteases and also is involved in oxidative stress responses. This study thus contributes to a more comprehensive understanding of the biological function of NprR, and especially in the middle growth stages of B. anthracis .

Extracellular antigen 1 (EA1), a major component of the Bacillus anthracis surface layer (S-layer), was used as a fusion partner for the expression of heterologous antigen. A recombinant B. anthracis strain was constructed by integrating a translational fusion harboring the DNA fragments encoding the cell wall–targeting domain of the S-layer protein EA1 and the 20-kDa N-terminal fragment of anthrax protective antigen (PA20) into the chromosome. A thermosensitive plasmid expressing Cre recombinase was introduced at a permissive temperature to remove the antibiotic marker. Cre recombinase action at the loxP sites excised the spectinomycin resistance cassette. The final derivative strains were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, Western blot analysis, and immunofluorescence analysis. PA20 was successfully expressed on the S-layer of the recombinant antibiotic marker-free strain. Guinea pigs were immunized with the attenuated recombinant B. anthracis strain, and the bacilli elicited a humoral response to PA20. This antibiotic marker-free strain and the correlative experiment method may have potential applications for the generation of a live attenuated anthrax vaccine.

目的:应用生物信息学分析方法筛选幽门螺杆菌新的疫苗候选抗原。方法:从TIGRCMR下载幽门螺杆菌26695和J99株全基因组序列,应用生物信息学SignalP、PredTMBB、LipoP、TMHMM、Phobius、PSORT-B和SubLoc等分析软件,筛选幽门螺杆菌新的外膜蛋白和分泌蛋白疫苗候选抗原。结果:从幽门螺杆菌26695株筛选得到54个编码β-桶型跨膜蛋白、脂蛋白或分泌表达蛋白的疫苗候选蛋白抗原,从幽门螺杆菌J99株得到61个呈现上述表达方式的疫苗候选蛋白抗原;且这2株细菌的疫苗候选蛋白呈现良好的交集状况,即有43个候选疫苗蛋白是相同的。结论:用生物信息学分析方法可以从全基因组范围内快速筛选到保守的分泌或表面暴露的疫苗候选抗原,为疫苗抗原的快速筛选与鉴定奠定了基础。

Objective:To construct rfaH gene deletion mutant of Salmonella typhi Ty2(S.ty2) with λRed recombination system.Methods:Homologous regions and kanamycin cassette with two FRT sites were amplified from the corresponding templates and were inserted into plasmid pET22b-kana to construct a recombinant vector.The resultant fragments were then amplified from the recombinant vector and transformed into S.ty2.Under the pressure of antibiotic and with the work of λRed system, homologous recombination occurred between the fragments and genome of host strain, and the recombinants were selected on kanamycin agar plate.Plasmid pCP20 was introduced into the recombinant to remove the kanamycin resistant relevant DNA fragment, resulting in a single FRT site within the targeted genomic segment.The markerless mutant strains were detected by genome PCR.Phenotype analysis was performed by transmission electron micrographs.Results:rfaH gene deletion mutant was obtained and identified by PCR method and sequencing.Phenotype analysis indicated that the flagella synthesis of mutant decreased dramatically.Conclusions:The rfaH deletion mutant of Salmonella typhi Ty2 was obtained, and it will be modified further to be the live attenuated bacterial vector.

It was to obtain the receptor-binding domain of Bacillus anthracis protective antigen from prokaryotic cells and prepare its antibody used for further studies of PA-D4. The Bacillus anthracis protective antigen receptor-binding domain gene (the domain 4 of PA ,PA-D4) was obtained by PCR from Bacillus anthracis A16R genome and cloned into the prokaryotic expression vector pET-2b(+) containing 6×His coding sequence .The recombinant plasmid was transformed into host strain E.coli BL21(DE3) and induced to express recombinant PA-D4 by IPTG. The recombinant protein was purified through HiTrapTM Chelating HP affinity columns and then identified by Western blot analysis. Purified protein was used as the antigen to immunize New Zealand rabbits for three times to raise polyclonal antibody. Antiserum was analyzed by ELISA and Western blot. Results indicated that reconstructed PA-D4 gene was successfully expressed in E.coli BL21(DE3) in soluble form,and when purified with Ni column,PA-D4 protein covered more than 90% total proteins. The polyclonal antibody against the protein with high titer was obtained,and ELISA assay titer of antiserum from vaccinated rabbits reached 1∶102 400. The results of Western blot showed PA-D4 protein exhibited preferable immunogenicity and its polyclonal antibody could specifically recognize endogenous PA protein. Thus,PA-D4 protein and its specific antibodies were obtained successfully,which would lay foundation for further research of protein function and immune mechanism of the anthrax vaccine.

Cre-LoxP homologous recombinant system was used to disrupt eag gene in B. anthracis AP422. To construct the recombinant vector, homologous regions and Spc(r) cassette with two loxP sites were amplified from the corresponding templates. The produced shuttle vector was then transformed into B. anthracis AP422. Under the pressure of temperature and antibiotic, homologous recombination occurred between the vector and genome of the host bacterium and the recombinants were selected by agar media with spectinomycin (Spc) and X-gal. To remove the SpC(r) cassette, a plasmid with Cre recombinase was introduced into the recombinant to delete the relevant DNA fragment, resulting in a single loxP site within the targeted genomic segment. The markerless mutant strains were detected by genome PCR, RT-PCR, total proteins SDS-PAGE analysis and Western blot. The results showed that the eag gene was successfully deleted.