mSphere最新文献

{"title":"Antimicrobial peptide LL37 is potent against non-growing <i>Escherichia coli</i> cells despite a slower action rate.","authors":"Salimeh Mohammadi, Derek Saucedo, Sattar Taheri-Araghi","doi":"10.1128/msphere.00211-24","DOIUrl":"10.1128/msphere.00211-24","url":null,"abstract":"<p><p>Antimicrobial peptides (AMPs) have long been considered as potential agents against non-growing, dormant cells due to their membrane-targeted action, which is largely independent of the cell's growth state. However, the relationship between the action of AMPs and the physiological state of their target cells has been unclear, with recent reports offering conflicting views on the efficacy of AMPs against bacteria in a stationary phase. In this study, we employ single-cell approaches combined with population-level experiments to examine the action of human LL37 peptides against <i>Escherichia coli</i> cells in different growth phases. Time-lapse, single-cell data from our experiments reveal that LL37 peptides act faster on large, dividing cells than on small, newborn cells. We extend this investigation to non-growing <i>E. coli</i> cells in a stationary phase, where we observe that the action of LL37 peptides is slower on non-growing cells compared to exponentially growing cells. This slower action rate is, however, not mirrored in the minimum bactericidal concentration (MBC) measurements. Notably, we find that the MBC for non-growing cells is lower than for exponentially growing cells, indicating that, given sufficient time, LL37 peptides exhibit strong potency against non-growing cells. We propose that the enhanced potency of LL37 peptides against non-growing cells, despite their slower action, can be attributed to continuous absorption of AMPs on the cell membrane over time.</p><p><strong>Importance: </strong>Antibiotic treatments can fail because of the regrowth of a bacterial subpopulation that resumes proliferation once the treatment ceases. This resurgence is primarily driven by non-growing, dormant bacterial cells that withstand the action of antibiotics without developing resistance. In this study, we explore the potency of the human antimicrobial peptide LL37 against non-growing <i>Escherichia coli</i> cells. Our findings reveal that despite a slower initial action, LL37 peptides, given sufficient time, demonstrate strong efficacy against non-growing cells. These insights suggest a potential role of antimicrobial peptides in combating persistent bacterial infections by targeting the non-growing cells.</p>","PeriodicalId":19052,"journal":{"name":"mSphere","volume":" ","pages":"e0021124"},"PeriodicalIF":3.7,"publicationDate":"2025-01-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11774018/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142877467","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Role of Vigilin and RACK1 in dengue virus-<i>Aedes aegypti-Wolbachia</i> interactions.","authors":"Guijie Wang, Mazhar Hussain, Zhi Qi, Sassan Asgari","doi":"10.1128/msphere.00482-24","DOIUrl":"10.1128/msphere.00482-24","url":null,"abstract":"<p><p>Vigilin is a large and evolutionary conserved RNA-binding protein (RBP), which can interact with RNA through its KH domain. Vigilin is, therefore, a multifunctional protein reported to be associated with RNA transport and metabolism, sterol metabolism, chromosome segregation, carcinogenesis, and heterochromatin-mediated gene silencing. The receptor for activated C kinase 1 (RACK1) is another highly conserved protein involved in many cellular pathways. Functional studies in human cells indicated that RACK1 interacts with Vigilin to promote dengue virus (DENV) replication. Both proteins are associated with the endoplasmic reticulum. Here, we investigated the significance of Vigilin and RACK1 homologs in <i>Aedes aegypti</i> mosquitoes concerning DENV replication and <i>Wolbachia</i> infection. We identified the homologs of the two genes in <i>Ae. aegypti</i> (<i>AeVigilin</i> and <i>AeRACK1</i>), which were upregulated in DENV-infected Aag2 cells and mosquitoes, and silencing them in Aag2 cells resulted in reduced DENV replication. Co-immunoprecipitation showed that AeRACK1 and AeVigilin interact in mosquito cells. Interestingly, we also found upregulation of both genes in a <i>Wolbachia</i>-infected cell line (Aag2.<i>w</i>AlbB). Furthermore, silencing <i>AeVigilin</i> and <i>AeRACK1</i> in Aag2.<i>w</i>AlbB cells reduced DENV replication but increased <i>Wolbachia</i> density. However, we did not find a significant effect on DENV replication after silencing both genes in <i>Ae. aegypti</i> mosquitoes. Overall, our results support the involvement and significance of AeVigilin and AeRACK1 in DENV replication in <i>Ae. aegypti</i>.IMPORTANCEDengue virus (DENV), transmitted mainly by <i>Aedes aegypti</i> mosquitoes, poses significant health risks. Identifying factors involved in the virus replication in mosquitoes and human hosts is essential for devising control measures. In this study, we show that Vigilin and the receptor for activated C kinase 1 (RACK1), two proteins shown to play a role in the replication of DENV in human cells, are induced in mosquitoes and cell lines following DENV replication. Both proteins reside in the cytoplasm, where they interact similarly to human cells. Silencing the genes in mosquito cells significantly reduced virus replication. Furthermore, we found that both genes are induced in mosquito cells transinfected with <i>Wolbachia</i>, a bacterium that blocks DENV replication. The results help better understand the role of the common factors supporting DENV replication in mosquitoes and human cells.</p>","PeriodicalId":19052,"journal":{"name":"mSphere","volume":" ","pages":"e0048224"},"PeriodicalIF":3.7,"publicationDate":"2025-01-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11774047/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142877471","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Backtranslation of human RNA biosignatures of tuberculosis disease risk into the preclinical pipeline is condition dependent.","authors":"Hannah Painter, Sasha E Larsen, Brittany D Williams, Hazem F M Abdelaal, Susan L Baldwin, Helen A Fletcher, Andrew Fiore-Gartland, Rhea N Coler","doi":"10.1128/msphere.00864-24","DOIUrl":"10.1128/msphere.00864-24","url":null,"abstract":"<p><p>It is unclear whether human progression to active tuberculosis disease (TB) risk signatures are viable endpoint criteria for evaluations of treatments in development. TB is the deadliest infectious disease globally and more efficacious vaccines are needed to reduce this mortality. However, the immune correlates of protection for either preventing infection with <i>Mycobacterium tuberculosis</i> or preventing TB disease have yet to be completely defined, making the advancement of candidate vaccines through the pipeline slow, costly, and fraught with risk. Human-derived correlate of risk (COR) gene signatures, which identify an individual's risk of progressing to active TB disease, provide an opportunity for evaluating new therapies for TB with clear and defined endpoints. Though prospective clinical trials with longitudinal sampling are prohibitively expensive, the characterization of COR gene signatures is practical with preclinical models. Using a 3Rs (replacement, reduction, and refinement) approach we reanalyzed heterogeneous publicly available transcriptional data sets to determine whether a specific set of COR signatures are viable endpoints in the preclinical pipeline. We selected RISK6, Sweeney3, and BATF2 human-derived blood-based RNA biosignatures because they require relatively few genes and have been carefully evaluated across several clinical cohorts. These data suggest that in certain experimental designs and in several tissue types, human COR signatures correlate with disease progression as measured by the bacterial burden in the preclinical TB model pipeline. We observed the best performance when the model most closely reflected human infection or disease conditions. Human-derived COR signatures offer an opportunity for high-throughput preclinical endpoint criteria of vaccine and drug therapy evaluations.</p><p><strong>Importance: </strong>Understanding the strengths or limitations of back-translating human-derived correlate of risk (COR) RNA signatures into the preclinical pipeline may help streamline down-selection of therapeutic vaccine and drug candidates and better align preclinical models with proposed clinical trial efficacy endpoints.</p>","PeriodicalId":19052,"journal":{"name":"mSphere","volume":" ","pages":"e0086424"},"PeriodicalIF":3.7,"publicationDate":"2025-01-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11774039/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142801307","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"2024 Acknowledgment of <i>mSphere Ad Hoc</i> Reviewers.","authors":"Michael J Imperiale","doi":"10.1128/msphere.01041-24","DOIUrl":"10.1128/msphere.01041-24","url":null,"abstract":"","PeriodicalId":19052,"journal":{"name":"mSphere","volume":"10 1","pages":"e0104124"},"PeriodicalIF":3.7,"publicationDate":"2025-01-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11774017/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143053024","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"MecA in <i>Streptococcus mutans</i> is a multi-functional protein.","authors":"Kassapa Ellepola, Robert C Shields, Jessica K Kajfasz, Hua Zhang, Jose A Lemos, Hui Wu, Zezhang T Wen","doi":"10.1128/msphere.00308-24","DOIUrl":"10.1128/msphere.00308-24","url":null,"abstract":"<p><p>Our recent studies have shown that deficiency of MecA in <i>Streptococcus mutans</i> significantly affects cell division, growth, and biofilm formation. In this study, an <i>in vitro</i> mixed-species model, proteomics, and affinity pull-down assays were used to further characterize the MecA-mediated regulation in <i>S. mutans</i>. The results showed that compared with the wild type, UA159, the <i>mecA</i> mutant significantly reduced its production of glucans and weakened its ability to facilitate mixed-species biofilm formation. Relative to the wild type, the <i>mecA</i> mutant also displayed unique characteristics, including colony morphology, growth rate, and biofilm formation that did not fully resemble any of the <i>clpP, clpX, clpE, clpCE,</i> and <i>clpC</i> individual or combinational mutants. Deletion of <i>mecA</i> was shown to result in alteration of >337 proteins, including down expression of GtfBC&D and adhesin P1. More than 277 proteins were differentially expressed in response to <i>clpP</i> deletion, including increased expression of GtfB. By cross-referencing the two proteomes, a distinctive set of proteins was found to be altered in the <i>mecA</i> mutant, indicating a ClpP-independent role of MecA in the regulation of <i>S. mutans</i>. When analyzed using affinity pull-down, ClpC, ClpX, ClpE, and CcpA were among the members identified in the MecA-associated complex. Further analysis using a bacterial two-hybrid system confirmed CcpA, ClpX, and ClpE as members of the MecA interactome. These results further suggest that MecA in <i>S. mutans</i> is more than an adapter of the Clp-proteolytic machinery, although the mechanism that underlies the Clp-independent regulation and its impact on <i>S. mutans</i> pathophysiology await further investigation.</p><p><strong>Importance: </strong>MecA is known as an adaptor protein that works in concerto with ATPase ClpC and protease ClpP in the regulated proteolysis machinery. The results presented here provide further evidence that MecA in <i>S. mutans</i>, a keystone cariogenic bacterium, plays a significant role in its ability to facilitate mixed-species biofilm formation, a trait critical to its cariogenicity. Proteomics analysis, along with affinity pull-down and bacterial two-hybrid system, further confirm that MecA can also regulate <i>S. mutans</i> physiology and biofilm formation through pathways independent of the Clp proteolytic machinery, although how it functions independently of Clp awaits further investigation.</p>","PeriodicalId":19052,"journal":{"name":"mSphere","volume":" ","pages":"e0030824"},"PeriodicalIF":3.7,"publicationDate":"2024-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11656736/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142624693","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Erratum for Longley et al., \"Signatures of Mollicutes-related endobacteria in publicly available Mucoromycota genomes\".","authors":"Reid Longley, Aaron J Robinson, Olivia A Asher, Earl Middlebrook, Gregory Bonito, Patrick S G Chain","doi":"10.1128/msphere.00881-24","DOIUrl":"10.1128/msphere.00881-24","url":null,"abstract":"","PeriodicalId":19052,"journal":{"name":"mSphere","volume":" ","pages":"e0088124"},"PeriodicalIF":3.7,"publicationDate":"2024-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11656730/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142648473","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Erratum for Hassall et al., \"Dissecting Individual Interactions between Pathogenic and Commensal Bacteria within a Multispecies Gut Microbial Community\".","authors":"Jack Hassall, Jeffery K J Cheng, Meera Unnikrishnan","doi":"10.1128/msphere.00893-24","DOIUrl":"10.1128/msphere.00893-24","url":null,"abstract":"","PeriodicalId":19052,"journal":{"name":"mSphere","volume":" ","pages":"e0089324"},"PeriodicalIF":3.7,"publicationDate":"2024-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11656729/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142682453","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"<i>Chlamydomonas</i> IC97, an intermediate chain of the flagellar dynein f/I1, is required for normal flagellar and cellular motility.","authors":"Ryosuke Yamamoto, Yui Tanaka, Shunsuke Orii, Kogiku Shiba, Kazuo Inaba, Takahide Kon","doi":"10.1128/msphere.00558-24","DOIUrl":"10.1128/msphere.00558-24","url":null,"abstract":"<p><p>Motile flagella (also called \"motile cilia\") play a variety of important roles in lower and higher eukaryotes, including cellular motility and fertility. Flagellar motility is driven by several species of the gigantic motor-protein complexes, flagellar dyneins, that reside within these organelles. Among the flagellar-dynein species, a hetero-dimeric dynein called \"IDA f/I1\" has been shown to be particularly important in controlling the flagellar waveform, and defects in this dynein species in humans cause ciliopathies such as multiple morphological abnormalities of the flagella and asthenoteratozoospermia. IDA f/I1 is composed of many subunits, including two HCs (HCα and HCβ) and three ICs (IC140, IC138, and IC97), and among the three ICs of IDA f/I1, the exact molecular function(s) of IC97 in flagellar motility is not well understood. In this study, we isolated a <i>Chlamydomonas</i> mutant lacking IC97 and analyzed the phenotypes. The <i>ic97</i> mutant phenocopied several aspects of the previously isolated IDA-f/I1-related mutants in <i>Chlamydomonas</i> and showed slow swimming compared to the wild type but retained the ability to phototaxis. Further analysis revealed that the mutant had low flagellar beat frequency and miscoordination between the two (<i>cis</i>- and <i>trans</i>-) flagella. In addition, the mutant cells swam in a comparatively straight path compared to the wild-type cells. Taken together, our results highlight the importance of proper assembly of IC97 in the IDA-f/I1 complex for the regulation of flagellar and cellular motility in <i>Chlamydomonas</i> and provide valuable insights into both the molecular functions of IC97 orthologs in higher eukaryotes and the pathogenetic mechanisms of human ciliopathies caused by IDA-f/I1 defects.</p><p><strong>Importance: </strong>IDA f/I1 is a hetero-dimeric flagellar dynein that is particularly important for the regulation of flagellar waveform and whose defects are associated with human ciliopathies. IC97 is an evolutionarily conserved intermediate chain of IDA f/I1, but the detailed molecular functions of IC97 in flagellar motility have not been elucidated. In this study, mutational and biochemical analyses of the previously uncharacterized <i>Chlamydomonas ic97</i> mutant revealed that IC97 is required for both the normal flagellar and cellular motility. In particular, IC97 appears to play an important role in both the control of flagellar beat frequency and the coordination between the two (<i>cis</i>- and <i>trans</i>-) flagella in <i>Chlamydomonas</i>. Our results provide important insights into the regulation of IDA-f/I1 activity by IC97 and the pathogenetic mechanisms of human ciliopathies caused by IDA-f/I1 defects.</p>","PeriodicalId":19052,"journal":{"name":"mSphere","volume":" ","pages":"e0055824"},"PeriodicalIF":3.7,"publicationDate":"2024-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11656769/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142730962","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Recombinant chimeric horsepox virus (TNX-801) is attenuated relative to vaccinia virus strains in both <i>in vitro</i> and <i>in vivo</i> models.","authors":"Stephanie V Trefry, Mayanka Awasthi, Christy N Raney, Amy L Cregger, Chase A Gonzales, Brittney L Layton, Robert N Enamorado, Nelson A Martinez, Deborah S Gohegan, Masoudeh Masoud-Bahnamiri, Jennifer Y Cho, Dawn M Myscofski, Tinoush Moulaei, Natasza E Ziółkowska, Scott J Goebel, Seth Lederman, Sina Bavari, Farooq Nasar","doi":"10.1128/msphere.00265-24","DOIUrl":"10.1128/msphere.00265-24","url":null,"abstract":"<p><p>Recombinant chimeric horsepox virus (TNX-801) is a preclinical vaccine in development against mpox and smallpox. In this report, we investigated the potential phenotypic differences in <i>in vitro</i> and <i>in vivo</i> models between TNX-801 and older vaccinia virus (VACV)-based vaccine strains (VACV-Lis and VACV-NYCBH) used in the eradication of smallpox as well as VACV-WR, VACV-IHD, and MVA. TNX-801 displayed a small plaque phenotype (~1-2 mm) in BSC-40 and Vero-E6 cells. Multi-step replication kinetics in immortalized nonhuman primate cell lines, and human primary cells from dermal and respiratory tracts yielded >10- to 100-fold lower infectious titers than the VACV strains. In addition, the infectious particle-to-genome copy ratio data suggests that TNX-801 genome packaging is ~10- to 100-fold less efficient than the VACV strains and the potential mechanism of TNX-801 attenuation is at the packaging/egress stage. Lastly, the susceptibility to VACV and TNX-801 infection of three new immunocompromised murine models (C56BL/6 <i>Ifnar</i>^-/-, C56BL/6 <i>Ifngr</i>^-/-, and C56BL/6 <i>Ifnar</i>^-/-/<i>Ifngr</i>^-/-) was investigated. VACV strains were able to produce severe disease including decrease in body weight and temperature, as well as lethality in murine models via the intraperitoneal or intranasal routes. In contrast to VACV strains, TNX-801 was unable to produce any disease in murine models. These data demonstrate that TNX-801 is >10- to 1,000-fold more attenuated compared to older VACV-based smallpox vaccine strains in human primary cell lines and immunocompromised mice.</p><p><strong>Importance: </strong>Variola and monkeypox viruses are medically important pathogens that can cause fatal human disease. The two FDA-approved vaccines, ACAM-2000 and JYNNEOS, have important advantages and disadvantages. ACAM-2000 offers durable immunity; however, it has high adverse event rates. In contrast, JYNNEOS has a safer profile but requires two doses 4-weeks apart to achieve comparable immunity. Consequently, there is a need for vaccines offering durable immunity via single immunization with minimal adverse events. TNX-801 is a preclinical stage vaccine that can stimulate potent immunity via a single dose and provides protection against lethal mpox disease in the nonhuman primate model. Here, we show that TNX-801 is >10- to 1,000-fold attenuated in <i>in vitro</i> and <i>in vivo</i> models including human primary cells and immunocompromised murine models than vaccine strains utilized in smallpox eradication. The natural attenuation of TNX-801 and its ability to induce protective immunity via a single vaccination are promising and warrants further development.</p>","PeriodicalId":19052,"journal":{"name":"mSphere","volume":" ","pages":"e0026524"},"PeriodicalIF":3.7,"publicationDate":"2024-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11656774/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142624746","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"CRISPR-Cas9-based approaches for genetic analysis and epistatic interaction studies in <i>Coxiella burnetii</i>.","authors":"Samuel Steiner, Craig R Roy","doi":"10.1128/msphere.00523-24","DOIUrl":"10.1128/msphere.00523-24","url":null,"abstract":"<p><p><i>Coxiella burnetii</i> is an obligate intracellular bacterial pathogen that replicates to high numbers in an acidified lysosome-derived vacuole. Intracellular replication requires the Dot/Icm type IVB secretion system, which translocates over 100 different effector proteins into the host cell. Screens employing random transposon mutagenesis have identified several <i>C. burnetii</i> effectors that play an important role in intracellular replication; however, the difficulty in conducting directed mutagenesis has been a barrier to the systematic analysis of effector mutants and to the construction of double mutants to assess epistatic interactions between effectors. Here, two CRISPR-Cas9 technology-based approaches were developed to study <i>C. burnetii</i> phenotypes resulting from targeted gene disruptions. CRISPRi was used to silence gene expression and demonstrated that silencing of effectors or Dot/Icm system components resulted in phenotypes similar to those of transposon insertion mutants. A CRISPR-Cas9-mediated cytosine base editing protocol was developed to generate targeted loss-of-function mutants through the introduction of premature stop codons into <i>C. burnetii</i> genes. Cytosine base editing successfully generated double mutants in a single step. A double mutant deficient in both <i>cig57</i> and <i>cig2</i> had a robust and additive intracellular replication defect when compared to either single mutant, which is consistent with Cig57 and Cig2 functioning in independent pathways that both contribute to a vacuole that supports <i>C. burnetii</i> replication. Thus, CRISPR-Cas9-based technologies expand the genetic toolbox for <i>C. burnetii</i> and will facilitate genetic studies aimed at investigating the mechanisms this pathogen uses to replicate inside host cells.</p><p><strong>Importance: </strong>Understanding the genetic mechanisms that enable <i>C. burnetii</i> to replicate in mammalian host cells has been hampered by the difficulty in making directed mutations. Here, a reliable and efficient system for generating targeted loss-of-function mutations in <i>C. burnetii</i> using a CRISPR-Cas9-assisted base editing approach is described. This technology was applied to make double mutants in <i>C. burnetii</i> that enabled the genetic analysis of two genes that play independent roles in promoting the formation of vacuoles that support intracellular replication. This advance will accelerate the discovery of mechanisms important for <i>C. burnetii</i> host infection and disease.</p>","PeriodicalId":19052,"journal":{"name":"mSphere","volume":" ","pages":"e0052324"},"PeriodicalIF":3.7,"publicationDate":"2024-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11656778/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142668534","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}