Emerging Microbes & Infections最新文献

{"title":"An attenuated African swine fever virus expressing the E2 glycoprotein of classical swine fever virus protects pigs against challenge of both viruses.","authors":"Jiwen Zhang, Fang Li, Weiye Chen, Yongfeng Li, Zhenjiang Zhang, Ronghong Hua, Renqiang Liu, Yuanmao Zhu, Encheng Sun, Huaji Qiu, Zhigao Bu, Dongming Zhao","doi":"10.1080/22221751.2025.2469636","DOIUrl":"10.1080/22221751.2025.2469636","url":null,"abstract":"<p><p>African swine fever (ASF) and classical swine fever (CSF) are highly contagious diseases with high morbidity and mortality rates resulting in an enormous impact on the global pig industry. A bivalent vaccine that simultaneously protects against both ASF and CSF is highly desirable. We previously developed a seven-gene-deleted African swine fever virus (ASFV) attenuated vaccine candidate (HLJ/18-7GD) that provides complete protection against homologous strains. Herein, we constructed a recombinant virus HLJ/18-7GD-E2 by inserting the classical swine fever virus (CSFV) E2 gene into the HLJ/18-7GD via homologous recombination. After continuous <i>in vitro</i> passaging, Western blotting analysis showed that the E2 gene was expressed and stably maintained within the ASFV genome. Next, the immunogenicity and protective efficacy of the recombinant HLJ/18-7GD-E2 virus was evaluated in pigs. The results revealed that a single dose of 10⁶ TCID₅₀ of HLJ/18-7GD-E2 induced an efficient immune response and provided complete protection against lethal challenges with ASFV or CSFV. These results demonstrate that recombinant ASFV expressing the CSFV E2 protein has potential as a bivalent live attenuated vaccine, providing solid protection against ASFV and CSFV infection in pigs.</p>","PeriodicalId":11602,"journal":{"name":"Emerging Microbes & Infections","volume":" ","pages":"2469636"},"PeriodicalIF":8.4,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11881660/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143440101","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Lessons for medical countermeasure development from unforeseen outbreaks.","authors":"Marie Mura, Aurélie Trignol, Erwan Le Dault, Jean-Nicolas Tournier","doi":"10.1080/22221751.2025.2471035","DOIUrl":"10.1080/22221751.2025.2471035","url":null,"abstract":"<p><p>The unanticipated emergence of the COVID-19 pandemic and the rapid spread of the mpox epidemic in 2022 and 2024 brought unforeseen challenges to public health. While distinct in nature, these outbreaks share some similarities and offer valuable insights into responding to novel virus dissemination in vulnerable populations. In light of these two experiences, we aim to discern the prioritization of medical countermeasures (MCM) among antivirals, antibodies, and vaccines. Comparative analysis of MCMs reveals that while antivirals serve essential roles as therapeutic tools, monoclonal antibodies can be used for both prevention and treatment, and vaccines remain of paramount importance for controlling epidemics as mass or targeted prophylaxis. Variability in production processes, administration methods, logistics, and costs distinguish these countermeasures. Vaccines, by inducing long-lasting immunity and ideally promoting herd effects, exhibit substantial advantages over other options. To enhance future pandemic readiness, proactive measures must include ready-to-use vaccine platforms with regulatory approval and manufacturing capacities, as well as prototype vaccines for representative pathogens and preexisting protocols to evaluate their efficacies and side effects. The comparison underscores the challenges of social acceptance and equity, particularly in vaccine production and distribution. As the world faces unknown agents, the three major types of MCMs do not have equal and symmetrical effects in terms of epidemic control. Thus, a vaccine-oriented strategy with a community-centered approach, proves essential for effective pandemic preparedness, encouraging continued innovation in vaccinology.</p>","PeriodicalId":11602,"journal":{"name":"Emerging Microbes & Infections","volume":" ","pages":"2471035"},"PeriodicalIF":8.4,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11894751/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143457290","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"As pertussis returns to pre-COVID19 endemicity, vaccination remains our best ally against an evolving <i>Bordetella pertussis</i>.","authors":"Zeng Mei, Macina Denis","doi":"10.1080/22221751.2025.2466691","DOIUrl":"10.1080/22221751.2025.2466691","url":null,"abstract":"","PeriodicalId":11602,"journal":{"name":"Emerging Microbes & Infections","volume":" ","pages":"2466691"},"PeriodicalIF":8.4,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11869333/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143406475","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Discovery of a pan anti-SARS-CoV-2 monoclonal antibody with highly efficient infected cell killing capacity for novel immunotherapeutic approaches.","authors":"Daouda Abba Moussa, Mario Vazquez, Christine Chable-Bessia, Vincent Roux-Portalez, Elia Tamagnini, Mattia Pedotti, Luca Simonelli, Giang Ngo, Manon Souchard, Sebastien Lyonnais, Myriam Chentouf, Nathalie Gros, Soledad Marsile-Medun, Heiko Dinter, Martine Pugnière, Pierre Martineau, Luca Varani, Manel Juan, Hugo Calderon, Mar Naranjo-Gomez, Mireia Pelegrin","doi":"10.1080/22221751.2024.2432345","DOIUrl":"10.1080/22221751.2024.2432345","url":null,"abstract":"<p><p>Unlocking the potential of broadly reactive coronavirus monoclonal antibodies (mAbs) and their derivatives offers a transformative therapeutic avenue against severe COVID-19, especially crucial for safeguarding high-risk populations. Novel mAb-based immunotherapies may help address the reduced efficacy of current vaccines and neutralizing mAbs caused by the emergence of variants of concern (VOCs). Using phage display technology, we discovered a pan-SARS-CoV-2 mAb (C10) that targets a conserved region within the receptor-binding domain (RBD) of the virus. Noteworthy, C10 demonstrates exceptional efficacy in recognizing all assessed VOCs, including recent Omicron variants. While C10 lacks direct neutralization capacity, it efficiently binds to infected lung epithelial cells and induces their lysis via natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC). Building upon this pan-SARS-CoV-2 mAb, we engineered C10-based, Chimeric Antigen Receptor (CAR)-T cells endowed with efficient killing capacity against SARS-CoV-2-infected lung epithelial cells. Notably, NK and CAR-T-cell mediated killing of lung infected cells effectively reduces viral titers. These findings highlight the potential of non-neutralizing mAbs in providing immune protection against emerging infectious diseases. Our work reveals a pan-SARS-CoV-2 mAb effective in targeting infected cells and demonstrates the proof-of-concept for the potential application of CAR-T cell therapy in combating SARS-CoV-2 infections. Furthermore, it holds promise for the development of innovative antibody-based and cell-based therapeutic strategies against severe COVID-19 by expanding the array of therapeutic options available for high-risk populations.<b>Trial registration:</b> ClinicalTrials.gov identifier: NCT04093596.</p>","PeriodicalId":11602,"journal":{"name":"Emerging Microbes & Infections","volume":" ","pages":"2432345"},"PeriodicalIF":8.4,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11632933/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142709662","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The E3 ligase RAD18-mediated ubiquitination of henipavirus matrix protein promotes its nuclear-cytoplasmic trafficking and viral egress.","authors":"Dongning Jin, Linliang Zhang, Cheng Peng, Mingbin He, Weiwei Wang, Zhifei Li, Cong Liu, Jinhong Du, Jin Zhou, Lei Yin, Chao Shan, Yali Qin, Mingzhou Chen","doi":"10.1080/22221751.2024.2432344","DOIUrl":"10.1080/22221751.2024.2432344","url":null,"abstract":"<p><p>The nuclear-cytoplasmic trafficking of matrix proteins (M) is essential for henipavirus budding, with M protein ubiquitination playing a pivotal role in this dynamic process. Despite its importance, the intricacies of the M ubiquitination cascade have remained elusive. In this study, we elucidate a novel mechanism by which Nipah virus (NiV), a highly pathogenic henipavirus, utilizes a ubiquitination complex involving the E2 ubiquitin-conjugating enzyme RAD6A and the E3 ubiquitin ligase RAD18 to ubiquitinate the virus's M protein, thereby facilitating its nuclear-cytoplasmic trafficking. We demonstrate that RAD18 interacts with RAD6A, enabling the latter to supply ubiquitins for the RAD18-mediated transfer of ubiquitin to M through RAD18-M interactions. Specifically, M is ubiquitinated by the RAD6A-RAD18 complex at lysine (K) 258 through a K63-linked ubiquitination, a modification crucial for M's function. This ubiquitination drives M's relocation to the cytoplasm, directing it to plasma membranes for effective viral egress. Conversely, disrupting the RAD6A-RAD18-M axis, mutating RAD18's E3 ligase activity, or inhibiting RAD6A activity with TZ9 (a RAD6-ubiquitin thioester formation inhibitor) impairs M ubiquitination, resulting in defective nuclear export and budding of NiV. Significantly, live NiV and Hendra virus infection is attenuated in RAD18 knockout cells or in cells treated with TZ9, highlighting the critical physiological role of RAD6A-RAD18-mediated M ubiquitination in the henipavirus life cycle. Our findings not only reveal how NiV manipulates a nucleus-localized ubiquitination complex to promote virus's M protein ubiquitination and nuclear export, but also suggest that the small molecule inhibitor TZ9 could serve as a potential therapeutic against henipavirus infection.</p>","PeriodicalId":11602,"journal":{"name":"Emerging Microbes & Infections","volume":" ","pages":"2432344"},"PeriodicalIF":8.4,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11632932/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142767274","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Clinical manifestations and pathogenicity of Clade IIb monkeypox virus in rabbits.","authors":"Chao Shang, Shaowen Shi, Qiwei Jiang, Xiaohan Wang, Xiaohong Yao, Wanzi Li, Gaojie Song, Yiquan Li, Yongyang Sun, Jinglei Hu, Cuiling Zhang, Yilong Zhu, Zirui Liu, Chaode Gu, Yan Liu, Wanyu Shi, Zongzheng Zhao, Xiao Li","doi":"10.1080/22221751.2025.2465309","DOIUrl":"10.1080/22221751.2025.2465309","url":null,"abstract":"<p><p>The 2022 monkeypox outbreak involved rapid global dissemination, prompting research into animal models for the monkeypox virus (MPXV), including non-human primates and mice. However, studies utilizing rabbits as models remain limited. In this study, we established three rabbit models using the current epidemic MPXV strain. Following intravenous MPXV injection, adult rabbits exhibited characteristic clinical manifestations, including widespread rash and fever, with viral replication in the skin, lungs, and testes, resulting in severe pathological damage by 6 days post-infection (dpi). Intradermal injection of MPXV into the dorsal skin of adult rabbits produced red lesions with central necrosis and hemorrhage accompanied by dense inflammatory infiltrates. Abundant viral particles were observed in epidermal cells at 6 dpi. Additionally, a fatal MPXV model was developed in 10-day-old rabbits using intranasal virus administration. These young rabbits exhibited lethargy and diarrhea beginning at 2 dpi, significant weight loss, and a 50% mortality rate by 15 dpi. Viral dissemination was detected in multiple organs, leading to extensive multi-organ damage. This study highlights the utility of rabbit models for MPXV, displaying typical clinical features and pathogenic mechanisms.</p>","PeriodicalId":11602,"journal":{"name":"Emerging Microbes & Infections","volume":" ","pages":"2465309"},"PeriodicalIF":8.4,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11849023/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143406478","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"<i>In vivo</i> determination of protective antibody thresholds for SARS-CoV-2 variants using mouse models.","authors":"Peilan Wei, Ruoxi Cai, Lu Zhang, Jingjun Zhang, Zhaoyong Zhang, Airu Zhu, Hai Li, Zhen Zhuang, Lan Chen, Jiantao Chen, Yuting Zhang, Xinyi Xiong, Bin Qu, Jianfen Zhuo, Tian Tang, Yuanyuan Zhang, Lei Chen, Qier Zhong, Zhiwei Lin, Xindan Xing, Fang Li, Qingtao Hu, Jun Dai, Yongxia Shi, Jingxian Zhao, Jincun Zhao, Yanqun Wang","doi":"10.1080/22221751.2025.2459140","DOIUrl":"10.1080/22221751.2025.2459140","url":null,"abstract":"<p><p>Neutralizing antibody titres have been shown to correlate with immune protection against COVID-19 and can be used to estimate vaccine effectiveness. Numerous studies have explored the relationship between neutralizing antibodies and protection. However, there remains a lack of quantitative data directly assessing the minimum effective protective neutralizing antibody titre in <i>in vivo</i>. In this study, we utilized eight cohorts of participants with diverse immune backgrounds for evaluation of protective antibody response. To precisely assess the lower threshold of neutralizing antibody titres required for effective protection against SARS-CoV-2 infections, we employed plasma adoptive transfer from different cohorts into mice. This study demonstrated that neutralizing titres in the plasma of recipient mice correlated well with those in human donors, and a positive linear correlation was observed between the human and mouse recipients of transferred plasma neutralizing titre. A pseudotyped virus neutralizing titres greater than 7 was identified as the minimum threshold necessary to reduce viral titres in infected mice, establishing a crucial baseline for effective protection. Furthermore, despite the variability in immune backgrounds, these diverse cohorts' plasma exhibited a similar neutralizing antibody threshold necessary for protection. This finding has significant implications for vaccine design and the assessment of immune competence.</p>","PeriodicalId":11602,"journal":{"name":"Emerging Microbes & Infections","volume":" ","pages":"2459140"},"PeriodicalIF":8.4,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11809195/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143032629","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Rational mpox vaccine design: immunogenicity and protective effect of individual and multicomponent proteins in mice.","authors":"Xueting Cheng, Yawei Wang, Baoying Huang, Jialuo Bing, Tangqi Wang, Ruiwen Han, Shuting Huo, Shucai Sun, Li Zhao, Chang Shu, Yao Deng, Wenjie Tan","doi":"10.1080/22221751.2025.2482702","DOIUrl":"https://doi.org/10.1080/22221751.2025.2482702","url":null,"abstract":"<p><p>The 2022 global mpox virus (MPXV) outbreak highlights the urgent need for safer, next-generation vaccines. We compared the immunogenicity and protective efficacy of individual and multicomponent membrane proteins of MPXV virions in mice to inform the development of a recombinant subunit vaccine against mpox. BALB/c mice were immunized with eukaryotically expressed A35R, A29L, B6R, and M1R proteins, administered individually or in multicomponent combinations with an Al(OH)₃ + CpG oligodeoxynucleotide adjuvant. Three multicomponent protein vaccines (A29/B6, A29/B6/M1, and A29/B6/M1/A35) provided complete protection, but others (individual protein and A35/M1 combinations) provided partial protection against challenge with high-lethal doses of vaccinia virus Western Reserve (VACV-WR). Additionally, A29/B6 conferred partial protection, whereas A29/B6/M1 and A29/B6/M1/A35 provided complete protection against ectromelia virus (ECTV), with A29/B6/M1 being most effective. All vaccines induced strong antigen-specific immunoglobulin G (IgG) and cellular immunity, whereas only four (M1, A35/M1, A29/B6/M1, A29/B6/M1/A35) exhibited significant neutralizing activity against MPXV, VACV-Tiantan, and ECTV. Correlation analysis suggested that neutralizing antibodies and A35-/A29-/B6-specific cellular immunity act as complementary defense mechanisms, potentially providing first- and second-line protection against MPXV and related orthopoxviruses. Collectively, A29/B6/M1 demonstrated the best protective efficacy. This study provides novel insights into immunogen optimization and potential mechanisms for the development of vaccines against MPXV and other orthopoxviruses.</p>","PeriodicalId":11602,"journal":{"name":"Emerging Microbes & Infections","volume":" ","pages":"2482702"},"PeriodicalIF":8.4,"publicationDate":"2025-03-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143656340","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Genomic characterisation of invasive <i>Neisseria meningitidis</i> in Spain (2011/12-2022/23): Expansion of clonal complex 213 and the potential threat to 4CMenB vaccine strain coverage.","authors":"Josep Roca-Grande, Alba Mir-Cros, Carmen Muñoz-Almagro, Mayli Lung, Alba Bellés-Bellés, Jordi Càmara, Emilia Cercenado, M A Galán-Ladero, Andrea Martín-Nalda, Albert Moreno-Mingorance, Daniel Navarro de la Cruz, M Ángeles Orellana, Begoña Palop, Amaresh Pérez-Argüello, Guillem Puigsech-Boixeda, M Dolores Quesada, Alba Rivera, Ana Rodriguez-Fernandez, Enrique Ruiz de Gopegui, Carolina Sarvisé, Aleix Soler-Garcia, Belén Viñado, Nieves Larrosa, Juan José González-López","doi":"10.1080/22221751.2025.2482696","DOIUrl":"https://doi.org/10.1080/22221751.2025.2482696","url":null,"abstract":"<p><p>Invasive meningococcal disease (IMD) is associated with significant global morbidity and mortality and is addressed by conjugated polysaccharide and subcapsular vaccines. In Spain, data on 4CMenB vaccine strain coverage and antimicrobial susceptibility are limited. This study aimed to describe the genomic epidemiology, predict 4CMenB vaccine strain coverage, and assess antimicrobial susceptibility of 323 <i>Neisseria meningitidis</i> isolates causing IMD, collected from 57 Clinical Microbiology Laboratories in Spain over 12 years (2011/12-2022/23). Whole genome sequencing was performed to identify serogroup, clonal complex (cc), and antimicrobial resistance determinants. Vaccine strain coverage for serogroup B (MenB) isolates was predicted using the genetic Meningococcal Antigen Typing System approach. The most prevalent serogroups were B (57.9%), W (21.4%), C (10.4%), and Y (8.4%). MenB predominated throughout most seasons, except during the 2019/20 season when serogroup W peaked. Post-COVID-19 pandemic, MenB remained the most frequent (70.2%). Thirteen cc were identified among MenB isolates, with cc213 being the most prevalent (40.1%). Only 28.9% of MenB isolates were predicted to be covered by 4CMenB, with cc213 showing an exceptionally low coverage rate (5.3%) due to antigenic variants poorly targeted by the vaccine. Notably, cc213 was responsible for twice the proportion of MenB cases in 4CMenB-vaccinated versus unvaccinated. All isolates were susceptible to third generation cephalosporins, and 13.5% showed penicillin resistance. This study highlights the alarming prevalence of cc213 among MenB IMD cases in Spain and the limited 4CMenB coverage against this cc. The disproportionate representation of cc213 in vaccinated individuals underscores its potential to compromise vaccine effectiveness.</p>","PeriodicalId":11602,"journal":{"name":"Emerging Microbes & Infections","volume":" ","pages":"2482696"},"PeriodicalIF":8.4,"publicationDate":"2025-03-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143656409","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Hypermutability of <i>Mycolicibacterium smegmatis</i> due to ribonucleotide reductase-mediated oxidative homeostasis and imbalanced dNTP pools.","authors":"Xiao Zhang, Yuchang Di, Yu Zhang, Youwei Hu, Mingzhe Chi, Jian Kang, Yuqing Zheng, Hengyu Wang, Yu Wang, Jiazhen Chen, Xuelian Zhang","doi":"10.1080/22221751.2025.2480698","DOIUrl":"https://doi.org/10.1080/22221751.2025.2480698","url":null,"abstract":"<p><p><b>ABSTRACT</b>Ribonucleotide reductase (RNR) is a crucial enzyme that catalyzes the synthesis and regulation of the four deoxyribonucleoside triphosphates (dNTPs) required for DNA synthesis. Perturbations in dNTP concentrations are known to reduce DNA replication fidelity, increasing mutation rates. However, despite genetic mutation of <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) is a critical factor in its ability to develop drug resistance, no studies have investigated that impaired RNR activity and mutations has any impact on <i>Mtb</i> physiology or drug resistance development. Here, we constructed inducible knockdown strains for the RNR R1 subunit NrdE in <i>Mtb</i> and <i>Mycolicibacterium smegmatis</i> (<i>Msm</i>). NrdE knockdown significantly impaired growth and metabolic imbalances in Mycobacteria, indirectly disrupting oxidative homeostasis and mycolic acid synthesis, while increasing levels of intracellular reactive oxygen species (ROS) accumulation and enhancing cell wall permeability. Additionally, we developed two genomic mutant strains, <i>Msm-Y252A</i> and <i>Msm-Q255A</i>, featuring targeted point mutations in the substrate-specific site (S-site) of the RNR loop domain, which determines NDP reduction specificity. The <i>Msm-Y252A</i> mutant displayed a 1.9-fold decrease in dATP and increases in dGTP (1.6-fold), dTTP (9.0-fold), and dCTP (1.3-fold). In contrast, <i>Msm-Q255A</i> exhibited elevated intracellular levels of dGTP (1.6-fold), dTTP (6.1-fold), and dATP (1.5-fold), while dCTP levels remained unchanged. Neither the NrdE knockdown strain nor the S-site mutants exhibited direct resistance development; however, they both showed genomic instability, enhancing the emergence of rifampicin-resistant mutants, even with a 70-fold and a 25-fold increase in mutation frequency for <i>Msm-Y252A</i> and <i>Msm-Q255A</i>, respectively, compared to the <i>WT</i>. This study demonstrates that NrdE is integral to <i>Mycobacterium</i> survival and genomic stability, and that its RNR dysfunction creates a mutagenic environment under selective pressure, indirectly contributes to the development of drug resistance, positioning NrdE as an effective target for therapeutic strategies and a valuable molecular marker for early detection of drug-resistant <i>Mtb</i>.</p>","PeriodicalId":11602,"journal":{"name":"Emerging Microbes & Infections","volume":" ","pages":"2480698"},"PeriodicalIF":8.4,"publicationDate":"2025-03-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143647678","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}