New Biotechnology
·
2021
·
IF 5.079
Ke Liu,Yang Gao,Zhen-Hai Li,Min Liu,Feng-Qing Wang,Dong-Zhi Wei
Efficient and convenient genetic manipulation of mycobacteria, important microorganisms in human healthcare and the pharmaceutical industry, is limited. In this study, using a model strain Mycolicibacterium neoaurum ATCC 25795, the classical bacterium for the production of valuable steroidal pharmaceuticals, a genome editing system employing CRISPR-Cas12a to achieve efficient and precise genetic manipulation has been developed. Targeted genome mutations could be easily achieved by the CRISPR-Cas12a system without exogenous donor templates, assisted by innate non-homologous end-joining (NHEJ). CRISPR-Cas12a enabled rapid one-step genomic DNA fragment deletions of 1 kb, 5 kb, 10 kb, 15 kb, 20 kb and 24 kb with efficiencies of 70 %, 30 %, 30 %, 20 %, 20 % and 10 %, respectively. Combined with the pNIL/pGOAL system, CRISPR-Cas12a successfully integrated the gene of interest into the targeted genomic site by single crossover and double crossovers with efficiencies of 100 % and 9 %, respectively, using a two-plasmid system. The robust CRISPR systems developed demonstrated strong potential for precise genome editing in M. neoaurum , including targeted deletion of DNA sequences of various lengths and integration of targeted genes into desired sites in the genome.
研究领域:
微生物学
·
基因工程
·
制药工业
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DOI
PubMed
高影响因子
Microbiology Spectrum
·
2025
·Vol. 13
·期 10
·
IF 3.8
Daoyan Tang,Minggui Wang,Dan Wang,Danni Yang,Yi Cai,Tao Luo,Jianqing He,Qinglan Wang
Efficient DNA delivery is essential for genetic manipulation of mycobacteria and for dissecting their physiology, pathogenesis, and drug resistance. Although electroporation enables transformation efficiencies exceeding 10⁵ CFU per µg DNA in Mycobacterium smegmatis and Mycobacterium tuberculosis, it remains highly inefficient in many nontuberculous mycobacteria (NTM), including Mycobacterium abscessus. Here, we discovered that NTM such as M. abscessus exhibit exceptional tolerance to ultra-high electric field strengths and that hypertonic preconditioning partially protects cells from electroporation-induced damage. Using ultra-high electric field strength (3 kV/mm) electroporation, we achieved dramatic improvements in plasmid transformation efficiency—up to 106-fold in M. abscessus, 83-fold in Mycobacterium marinum, and 37-fold in Mycobacterium kansasii—compared to standard conditions (1.25 kV/mm). Transformation efficiency was further influenced by the choice of selectable marker. Ultra-high field strength electroporation also markedly enhanced allelic exchange in M. abscessus expressing Che9c RecET recombinases, increasing the recovery of gene deletion mutants by over 1,000-fold relative to conventional electroporation. In parallel, oligonucleotide-mediated recombineering for targeted point mutations produced nearly 10,000-fold more mutants under ultra-high field conditions. Together, these findings establish ultra-high field electroporation as a robust, broadly applicable platform for genetic engineering of NTMs. This method substantially enhances transformation efficiency and enables construction of advanced genetic tools—including expression libraries and CRISPRi knockdown libraries—in species that have historically resisted genetic manipulation.
研究领域:
微生物学
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分子生物学
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遗传学
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生物技术
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感染性疾病
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转化医学
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产品关联
DOI
PubMed
高影响因子
INFECTION GENETICS AND EVOLUTION
·
2019
·
IF 2.611
Cao Ruan,Jiang Li,Jingjing Niu,Ping Li,Yu Huang,Xue Li,Wei Duan,Shuangquan Yan,Junfeng Zhen,Jianping Xie
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb) is still a leading cause of death worldwide. M. tuberculosis has evolved multipronged strategies to subvert host immune defenses and establish an immunologically privileged niche in macrophages. Rv0426c has been predicted to be an effector involved in the Mtb-host interactions. To investigate the potential role played by Rv0426c, we constructed recombinant M. smegmatis strains with heterologous expression of Rv0426c. We observed that Rv0426c recombinants became more susceptible to various stresses by increasing cell wall permeability, however with elevated early survival rate within macrophages. This was accompanied by decreased levels of pro-inflammatory cytokines and host cell apoptosis . The data suggested that Rv0426c was a new player involved in the interactions between Mtb and macrophages.
研究领域:
微生物学
·
免疫学
·
传染病学
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产品关联
DOI
PubMed
高影响因子
Microbiology Spectrum
·
2023
·Vol. 11
·期 3
·
IF 3.7
Kun Wang,Yimin Deng,Xujie Cui,Mengli Chen,Yanzhe Ou,Danting Li,Minhao Guo,Weihui Li
Lipids are prominent components of the mycobacterial cell wall, and they play critical roles not only in maintaining biofilm formation but also in resisting environmental stress, including drug resistance. However, information regarding the mechanism mediating mycobacterial lipid synthesis remains limited. PatA is a membrane-associated acyltransferase and synthesizes phosphatidyl-myo-inositol mannosides (PIMs) in mycobacteria. Here, we found that PatA could regulate the synthesis of lipids (except mycolic acids) to maintain biofilm formation and environmental stress resistance in Mycolicibacterium smegmatis. Interestingly, the deletion of patA significantly enhanced isoniazid (INH) resistance in M. smegmatis, although it reduced bacterial biofilm formation. This might be due to the fact that the patA deletion promoted the synthesis of mycolic acids through an unknown synthesis pathway other than the reported fatty acid synthase (FAS) pathway, which could effectively counteract the inhibition by INH of mycolic acid synthesis in mycobacteria. Furthermore, the amino acid sequences and physiological functions of PatA were highly conserved in mycobacteria. Therefore, we found a mycolic acid synthesis pathway regulated by PatA in mycobacteria. In addition, PatA also affected biofilm formation and environmental stress resistance by regulating the synthesis of lipids (except mycolic acids) in mycobacteria.IMPORTANCE Tuberculosis, caused by Mycobacterium tuberculosis, leads to a large number of human deaths every year. This is so serious, which is due mainly to the drug resistance of mycobacteria. INH kills M. tuberculosis by inhibiting the synthesis of mycolic acids, which are synthesized by the FAS pathway. However, whether there is another mycolic acid synthesis pathway is unknown. In this study, we found a PatA-mediated mycolic acid synthesis pathway that led to INH resistance of in patA-deleted mutant. In addition, we first report the regulatory effect of PatA on mycobacterial biofilm formation, which could affect the bacterial response to environmental stress. Our findings represent a new model for regulating biofilm formation by mycobacteria. More importantly, the discovery of the PatA-mediated mycolic acid synthesis pathway indicates that the study of mycobacterial lipids has entered a new stage, and the enzymes might be new targets of antituberculosis drugs.
研究领域:
微生物学
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分子生物学
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传染病学
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细菌生理学
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抗生素耐药性
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DOI
高影响因子
JOURNAL OF MICROBIOLOGY
·
2024
·Vol. 62
·期 1
·49-62
·
IF 3.3
Xu Tao,Wang Chutong,Li Minying,Wei Jing,He Zixuan,Qian Zhongqing,Wang Xiaojing,Wang Hongtao
Tuberculosis (TB), a bacterial infectious disease caused by Mycobacterium tuberculosis (M. tuberculosis ), is a significant global public health problem. Mycobacterium tuberculosis expresses a unique family of PE_PGRS proteins that have been implicated in pathogenesis. Despite numerous studies, the functions of most PE_PGRS proteins in the pathogenesis of mycobacterium infections remain unclear. PE_PGRS45 (Rv2615c) is only found in pathogenic mycobacteria. In this study, we successfully constructed a recombinant Mycobacterium smegmatis ( M. smegmatis ) strain which heterologously expresses the PE_PGRS45 protein. We found that overexpression of this cell wall-associated protein enhanced bacterial viability under stress in vitro and cell survival in macrophages. MS_PE_PGRS45 decreased the secretion of pro-inflammatory cytokines such as IL-1β, IL-6, IL-12p40, and TNF-α. We also found that MS_PE_PGRS45 increased the expression of the anti-inflammatory cytokine IL-10 and altered macrophage-mediated immune responses. Furthermore, PE_PGRS45 enhanced the survival rate of M. smegmatis in macrophages by inhibiting cell apoptosis. Collectively, our findings show that PE_PGRS45 is a virulent factor actively involved in the interaction with the host macrophage.
研究领域:
微生物学
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免疫学
·
传染病学
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DOI
高影响因子
MOLECULAR PHARMACEUTICS
·
2024
·
IF 4.5
Xueyu Pu,Yuanyuan Wang,Xi Wang,Xiaoqing Sang,Miaomiao Jiang,DaWei Qi,Xin Zhao,Rong Chen,Jianwei Li,Xiang Liu,Zhidong Liu,Jian Yang
Tuberculosis (TB) is a chronic disease caused byMycobacterium tuberculosis (Mtb), which shows a long treatment cycle often leads to drug resistance, making treatment more difficult. Immunogens present in the pathogen’s cell membrane can stimulate endogenous immune responses. Therefore, an effective lipid-based vaccine or drug delivery vehicle formulated from the pathogen’s cell membrane can improve treatment outcomes. Herein, we extracted and characterized lipids fromMycobacterium smegmatis, and the extracts contained lipids belonging to numerous lipid classes and compounds typically found associated with mycobacteria. The extracted lipids were used to formulate biomimetic lipid reconstituted nanoparticles (LrNs) and LrNs-coated poly(lactic-co-glycolic acid) nanoparticles (PLGA-LrNs). Physiochemical characterization and results of morphology suggested that PLGA-LrNs exhibited enhanced stability compared with LrNs. And both of these two types of nanoparticles inhibited the growth of M. smegmatis. After loading different drugs, PLGA-LrNs containing berberine or coptisine strongly and synergistically prevented the growth of M. smegmatis. Altogether, the bacterial membrane lipids we extracted with antibacterial activity can be used as nanocarrier coating for synergistic antibacterial treatment of M. smegmatis─an alternative model of Mtb, which is expected as a novel therapeutic system for TB treatment.
研究领域:
药学
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纳米技术
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微生物学
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药物递送系统
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抗菌治疗
✓ 已验证
产品关联
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）