A miniature CRISPR-Cas12f has been demonstrated to serve as an effective genome editing tool in gram negative bacteria as well as human cells. Here, we developed an alternative method to edit the genome of Bacillus anthracis based on the AsCas12f1 nuclease from Acidibacillus sulfuroxidans. When the htrA gene on the chromosome and the lef gene on the plasmid pXO1 were selected as targets, the CRISPR-AsCas12f1 system showed very high efficiency (100%). At the same time, a high efficiency was observed for large-fragment deletion. Our results also indicated that the length of the homologous arms of the donor DNA had a close relationship with the editing efficiency. Furthermore, a two-plasmid CRISPR-AsCas12f1 system was also constructed and combined with the endonuclease I-SceI for potential multi-gene modification. This represents a novel tool for mutant strain construction and gene function analyses in B. anthracis and other Bacillus cereus group bacteria.
Helicobacter pylori (H. pylori) is a gram-negative pathogen classified as a class I carcinogen. The H. pylori urease B subunit (UreB) and heat shock protein A (HspA) are two important vaccine candidate antigens. In this study, we evaluated the immunogenicity and immunoprotective effect of the attenuated Shigella vector vaccine SH02 expressing the UreB-HspA fusion protein of H. pylori in a mouse model. Oral SH02 with or without subcutaneous injection of rUreB-HspA induced antigen-specific serum IgG, mucosal sIgA, and T cells immune response. Subcutaneous injection of the candidate antigen rUreB-HspA enhanced the level of serum antigen-specific IgG antibodies (p < 0.0001) and the levels of IgG1/IgG2a/IgG2b subtypes. In addition, injection boost also increased the proportion of spleen antigen-specific CD4+CD154+ T cells (p < 0.001), and the proportion of CD4+CD154+ T cells that secrete IFN-γ and IL-17A. Following the H. pylori challenge, the levels of H. pylori colonization in the two experimental groups (Groups A and B) significantly reduced compared with the control group (p < 0.001), indicating that the candidate vaccine yielded a preventive effect of anti-H.pylori infection. Compared with the non-subcutaneous booster injection group (Group A), the subcutaneous booster injection group (Group B) exhibited less gastric inflammation, but there was no significant difference in the level of colonization (p > 0.05). These results lay a foundation for the development of a vaccine against H. pylori and the optimization of immunization methods and procedures to prevent H. pylori infection.
Helicobacter pylori (H. pylori), heat-shock protein A (HspA), is a bacterial heat-shock chaperone that serves as a nickel ion scavenging protein. Ni2+ is an important co-factor required for the maturation and enzymatic activity of H. pylori urease and [NiFe] hydrogenase, both of which are key virulence factors for pathogen survival and colonization. HspA is an important target molecule for the diagnosis, treatment, and immune prevention of H. pylori. In this work, HspA was truncated into five fragments to determine the location of an antigen immunodominant peptide. A series of overlapping, truncated 11-amino-acid peptides in immunodominant peptide fragments were synthesized chemically and screened by ELISA. The immunogenicity and antigenicity of the screened epitope peptides were verified by ELISA, Western blot, and lymphocyte proliferation tests. Two novel B-cell epitopes were identified, covering amino acids 2–31 of HspA, which are HP11 (2–12; KFQPLGERVLV) and HP19 (18–28; ENKTSSGIIIP). The antiserum obtained from HP11-KLH and HP19-KLH immunized mice can bind to naive HspA in H. pylori SS2000, rHspA expressed in E. coli, and the corresponding GST fusion peptide. Among HspA seropositive persons, the seropositive rates of HP11 and HP19 were 21.4% and 33.3%, respectively. Both of the B-cell epitopes of HspA are highly conserved epitopes with good antigenicity and immunogenicity.
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