mBio最新文献

{"title":"Activation-independent capture of free fatty acids at the bacterial cell envelope by BrtB.","authors":"Amaranta Kahn, Cristina I F Sousa, João P A Reis, David A Russo, Adriana Rego, Ricardo J C V Queirós, Marine Cuau, Julie A Z Zedler, Sandra A C Figueiredo, Paulo Oliveira, Pedro N Leão","doi":"10.1128/mbio.01132-26","DOIUrl":"10.1128/mbio.01132-26","url":null,"abstract":"<p><p>Fatty acids (FAs) are key metabolites in living organisms, shaping membrane architecture, and helping cellular acclimatization to changing conditions. Bacteria synthesize FAs <i>de novo</i> but can also reclaim them from membrane lipids or uptake them from the environment, usually converting free fatty acids (FFAs) into activated FAs that can then be further metabolized. In cyanobacteria, it is the acyl-acyl carrier protein synthetase (Aas) that activates exogenous FFAs. However, the cyanobacterial enzyme BrtB was recently shown to esterify, <i>in vitro</i>, FFAs directly onto abundant chlorinated glycolipids (bartolosides), generating bartoloside fatty acid esters (B-FAs). Whether this chemistry operates <i>in vivo</i>, where it occurs, and what its implications are for cell physiology have remained unclear. Here, we show that in the cyanobacterium <i>Synechocystis salina</i> LEGE 06099, BrtB likely esterifies exogenous FFAs at or near the cell envelope without prior activation to generate B-FAs. We found that supplemented FFAs were converted into B-FAs within minutes. Unexpectedly, this response occurred with minimal changes in gene expression and little alteration of the extracellular proteome, consistent with a pathway already in place. Additionally, we observed that B-FAs can further be hydrolyzed into hydroxybartolosides, the levels of which increase in response to FA supply, suggesting a transient sequestration of FFA. These findings identify a specific activation-independent route for the incorporation of exogenous FFAs into cyanobacterial specialized metabolites, expanding the known repertoire of bacterial FA utilization.</p><p><strong>Importance: </strong>Fatty acids (FAs) are essential building blocks of cell membranes and key metabolites in all living organisms. While bacteria synthesize FAs de novo, they can also incorporate exogenous FAs, saving the energetic cost of biosynthesis. Bacteria are generally thought to require their prior activation before they can be incorporated into metabolism. Here we show that a marine cyanobacterium bypasses this requirement through BrtB, which directly esterifies exogenous FAs onto specialized glycolipids <i>in vivo</i> without prior activation, likely at or near the cell envelope. This process occurs within minutes and is accompanied by minimal changes in gene expression, consistent with a pre-existing pathway. Our findings identify a specific activation-independent route for incorporation of exogenous FAs into cyanobacterial specialized metabolites and suggest that these glycolipids may transiently sequester FAs in response to environmental availability.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":" ","pages":"e0113226"},"PeriodicalIF":5.4,"publicationDate":"2026-10-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148912897","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Quantifying the metabolic heat from aerobic methanotrophs.","authors":"Tymon A Herzyk, Tania L Gomez-Borraz, Rebeca Gonzalez-Cabaleiro, William T Sloan","doi":"10.1128/mbio.00980-26","DOIUrl":"10.1128/mbio.00980-26","url":null,"abstract":"<p><p>Microorganisms must dissipate some energy as heat to keep energy-capturing reactions thermodynamically favorable. For methane-oxidizing bacteria (methanotrophs), circumstantial evidence, such as temperature increases within biofilters and landfills, suggests that the metabolic heat they generate might be valorized. However, the precise rate and yield of heat produced by different methanotrophic species have not been measured. Here, we measured heat flux from pure cultures of two methanotrophs, <i>Methylomonas methanica</i> S1 (type I) and <i>Methylosinus trichosporium</i> OB3b (type II). Isothermal calorimetry of sealed batch cultures at 30°C under methane concentrations of 8%, 4%, and 2% (vol/vol) in air showed that both species dissipated approximately 60% of the substrate energy as heat. Biomass-specific heat yields ranged from 1,473 to 2,000 kJ/Cmol for <i>M. methanica</i> S1 and from 2,638 to 3,728 kJ/C-mol for <i>M. trichosporium</i> OB3b. These values exceed experimentally reported heat yields for other aerobic microorganisms, suggesting that methane-oxidizing bacteria may represent the upper limit of metabolic heat production among aerobic microorganisms and are unique in their ability to dissipate significant metabolic heat at comparative biomass yields. This work provides the first direct quantification of heat generation during biological methane oxidation, establishing a necessary foundation to leverage this resource within methane mitigation biotechnologies and wider engineering applications.IMPORTANCEMethanotrophic bacteria dissipate substantial metabolic energy as heat while oxidizing methane, a trait that may have evolved to manage excess energy. If harnessed, this process positions these microorganisms as effective agents for passively mitigating methane emissions while providing an alternative source for energy recovery. Although field studies have suggested the potential for significant heat generation from biological methane oxidation, experimental quantification has been lacking. Here, we quantitatively measured the heat evolved from two species of methanotrophs across different methane concentrations. The observed heat yields are far higher than previously recorded for aerobic microorganisms, setting a new empirical boundary for aerobic metabolic heat production.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":" ","pages":"e0098026"},"PeriodicalIF":5.4,"publicationDate":"2026-10-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148990504","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Enantiomer-dependent biodistribution and exposure determine the <i>in vivo</i> behavior of a pleurocidin-derived antimicrobial peptide.","authors":"Janis Romanopulos, Paul Cressey, Charlotte K Hind, Seong-Heun Kim, Zayed A Shalim, Nadiya Mohamed Akram, Hala Alnasif Alhadi, Amelia Claxton, Yuang You, Anna V Kelis, Bailey Ramsden, Siham Memdouh, Ana C Jacob, Tam T Bui, Simon J Cleary, Maya Thanou, Richard T Amison, Vincenzo Abbate, J Mark Sutton, A James Mason","doi":"10.1128/mbio.01718-26","DOIUrl":"10.1128/mbio.01718-26","url":null,"abstract":"<p><p>Antimicrobial peptides (AMPs) display rapid bactericidal activity <i>in vitro</i>, but have largely failed as systemically administered therapeutics, indicating that intrinsic potency alone does not predict <i>in vivo</i> efficacy. Here, we examine how pharmacokinetics, stereochemistry, and host context shape early <i>in vivo</i> activity using pleurocidin-derived peptides. The D-amino-acid analog D-P-KR rapidly eradicates priority bacterial pathogens within minutes <i>in vitro</i>, achieving this at low multiples of the minimum inhibitory concentration (MIC) for <i>Staphylococcus aureus</i> but requiring higher multiples for Gram-negative species. Near-infrared imaging of fluorescently labeled enantiomers reveals that stereochemistry strongly influences systemic behavior, with D-enantiomers showing reduced renal and hepatobiliary clearance and increased exposure following intravenous administration. Despite these favorable properties, systemic D-P-KR does not reduce pulmonary bacterial burden within 2 h of treatment in mouse models of <i>S. aureus</i> or <i>Klebsiella pneumoniae</i> pneumonia. In contrast, increasing exposure through short intravenous infusion produces a rapid, exposure-dependent reduction in pulmonary neutrophil recruitment in <i>S. aureus</i> infection, without corresponding early changes in bacterial burden. This effect is not observed in the disseminating <i>K. pneumoniae</i> model. These findings demonstrate a dissociation between rapid <i>in vitro</i> killing, early bacterial clearance, and host-response modulation, and show that early <i>in vivo</i> AMP activity is governed primarily by systemic exposure and infection context. More broadly, this work highlights the need to integrate pharmacokinetics, dosing strategy, and host response when evaluating antimicrobial peptides for systemic use.IMPORTANCEAntimicrobial peptides can kill bacteria far more rapidly than most conventional antibiotics, yet very few have succeeded as systemically administered drugs. This study shows that differences in peptide stereochemistry profoundly influence how antimicrobial peptides distribute within the body and how long effective exposure can be maintained. Our findings demonstrate that strong antibacterial activity <i>in vitro</i> does not necessarily translate into early bacterial clearance <i>in vivo</i>, but that delivery strategies can still affect host responses during infection. By linking peptide stereochemistry, systemic exposure, and biological outcomes, this work highlights why many antimicrobial peptides struggle to translate clinically and underscores the need to prioritize pharmacokinetics, formulation, and dosing strategy alongside antibacterial potency during antimicrobial peptide development.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":" ","pages":"e0171826"},"PeriodicalIF":5.4,"publicationDate":"2026-10-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148840475","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Laccase-mediated biotransformation potential for fluorinated compounds by geographically diverse human gut microbiota.","authors":"Milo R Schärer, Yaochun Yu, Aaron Grawe, James K Christenson, Serina L Robinson, Nicholas A Bokulich","doi":"10.1128/mbio.01026-26","DOIUrl":"10.1128/mbio.01026-26","url":null,"abstract":"<p><p>The growing prevalence of synthetic organofluorine substances in agrochemicals, food packaging, and consumer products has led to increasing gastrointestinal exposure, with potential consequences for human health. Despite the extreme stability of fluorinated compounds, several microbial pathways for their transformation are known, including those involving laccases, a type of multicopper oxidase. However, the functionality of laccases in the gut microbiome, a natural contact point between food-associated chemicals and microbial biotransformation pathways, is poorly defined. Through a multi-study analysis of 1,578 human gut metagenomes spanning a global gradient from hunter-gatherer societies to industrialized urban populations, we found that laccase-coding gene homologs are widely distributed in the human gut microbiome. We identified a significant association between both the abundance and phylogenetic diversity of laccase homologs and the degree of urbanization. As human gut microbial laccase activity has not been experimentally demonstrated, eight gut metagenome-derived laccases were heterologously expressed and screened for activity with a redox mediator system. Six of the eight laccases demonstrated activity. One of these gut microbial laccases, derived from <i>Veillonella</i>, and three previously characterized laccases were then tested for their capacity to deplete 11 different food-associated chemicals. The <i>Veillonella</i> laccase depleted the agrochemicals cyflumetofen and fluazinam, as well as the industrial chemical bisphenol AF, to a lesser extent. By linking global gut metagenomes with activity assays, this work demonstrates the untapped potential of mining human gut metagenomes for laccases and other microbial enzymes that can actively modify various agricultural and industrial chemicals.IMPORTANCEAs adverse effects of fluorinated compounds on human health are emerging, the responsible enzymes from the human gut microbiome of geographically diverse human cohorts mediating interactions with fluorinated compounds in the gastrointestinal tract remain poorly characterized. In a multi-study analysis of publicly available microbiome sequencing data, we linked the abundance, diversity, and phylogeny of laccases within the gut microbiome to the degree of urbanization of human cohorts and experimentally demonstrated the ability of these laccases to deplete a range of food-associated fluorochemicals. Another significant contribution of our study is in the integration of rural catchment areas as a quantitative metric of urbanization in gut microbiome metagenomics and enzyme activity surveys.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":" ","pages":"e0102626"},"PeriodicalIF":5.4,"publicationDate":"2026-10-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148865638","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Redox control and mechanisms of transmembrane signaling in CSS domain c-di-GMP phosphodiesterases that control biofilm formation in <i>Escherichia coli</i>.","authors":"Katharina Pressler, Martin Lorkowski, Regine Hengge","doi":"10.1128/mbio.01325-26","DOIUrl":"10.1128/mbio.01325-26","url":null,"abstract":"<p><p>Bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) control of biofilm formation in <i>Escherichia coli</i> K-12 is balanced by multiple diguanylate cyclases (DGCs) and c-di-GMP-specific phosphodiesterases (PDEs). Five of the 13 PDEs feature a periplasmic CSS domain with two conserved cysteines, flanked by transmembrane (TM) regions, and an enzymatically active cytoplasmic EAL domain. One of these (PdeC) was previously shown to be redox-regulated by DsbA/DsbB-mediated disulfide bond (DSB) formation in the cysteine serine serine (CSS) domain. Comparing all five CSS domain PDEs, we found them to fall into two groups with similar biochemical features, resulting in different consequences for PDE activity. PdeB, PdeC, and PdeG are more active when lacking the periplasmic DSB, resulting in diminished biofilm formation, while PdeN and PdeD are active in their oxidized forms. Using PdeB and PdeN as prototypes for the two groups, not only the periplasmic DSB but also differently charged amino acid motifs close to the transmembrane (TM) domains and a putative additional DSB in PdeN were identified as important for transmembrane signaling. All these elements, including its stable structural DSB in the periplasm, which can form independently of DsbA, maintain PdeN in a rigid, proteolysis-resistant active conformation. By contrast, for PdeB, the more rigid DSB-containing conformation is inactive, with mutations in the stabilizing elements leading to a structurally less constrained, more active enzyme. Notably, low PdeN levels are post-transcriptionally upregulated at acidic pH, resulting in less biofilm formation. Overall, the five CSS domain PDEs enable <i>E. coli</i> to adapt to diverse environmental niches.IMPORTANCESensing environmental cues and transmembrane signal transduction via membrane-embedded proteins is a process of key importance in all living cells. To investigate the molecular mechanisms involved, we performed a systematic functional comparison of the five CSS domain phosphodiesterases of <i>Escherichia coli</i>, which degrade the bacterial second messenger c-di-GMP in response to redox and other signals. With a sensory domain in the periplasm linked to a cytoplasmic enzymatic domain, these proteins represent minimal devices for transmembrane signaling. We demonstrate that these signal-transducing enzymes fall into two functional classes with a similar periplasmic redox biochemistry resulting in opposite states of cytoplasmic enzymatic activity. Several characteristic sequence elements convey redox and structural information in the periplasmic and transmembrane protein segments to their ability to dimerize into an enzymatically active form in the cytoplasm. Comparing the five enzymes also shows that evolution has played with these elements to facilitate adaptation to various environmental niches.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":" ","pages":"e0132526"},"PeriodicalIF":5.4,"publicationDate":"2026-10-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148865595","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"KSHV promotes leukocyte adhesion and transendothelial migration via induction of VCAM1.","authors":"Zhigang Zhang, Kurtis M Host, Jason Wong, Penny Anders, Blossom Damania","doi":"10.1128/mbio.01499-26","DOIUrl":"10.1128/mbio.01499-26","url":null,"abstract":"<p><p>Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus are gammaherpesviruses associated with multiple human cancers. Kaposi's sarcoma (KS) is a human malignancy associated with KSHV infection. KS lesions are characterized by proliferating endothelial-derived spindle cells, leaky blood vessels, and inflammatory infiltrating leukocytes. The inflammatory leukocytes secrete cytokines and growth factors that may contribute to the survival and proliferation of tumor cells. Unless exposed to inflammatory cytokines, resting endothelial cells usually do not express adhesion molecules. Here, we identify a novel mechanistic link demonstrating that KSHV-infected latent endothelial cells express vascular cell adhesion molecule (VCAM1) and induce leukocyte adhesion and transendothelial migration (TEM) in a VCAM1-dependent manner. Inhibition of VCAM1 function using siRNA knockdown or antibody blockade impairs leukocyte adhesion and TEM, suggesting that cell adhesion and TEM are mediated by VCAM1. VCAM1 expression in KSHV-infected endothelial cells involves the non-canonical NF-кB (NF-κB2) pathway because depletion of NIK, IKKα, RelB, or NF-κB p52 via siRNA results in a decrease of VCAM1 levels, as well as a corresponding impairment of cell adhesion and TEM. Thus, KSHV-infected endothelial cells likely promote the infiltration of immune cells by modulating the expression of adhesion factors like VCAM1 on their surface. Moreover, we report that KSHV vFLIP, a latency protein encoded by the virus, directly induces VCAM1 expression and promotes leukocyte adhesion and transendothelial migration. We further demonstrate that endothelial cells infected with a vFLIP-deleted virus had reduced VCAM1 expression and reduced leukocyte adhesion and transendothelial migration.IMPORTANCEKaposi's sarcoma-associated herpesvirus (KSHV) is associated with the development of Kaposi's sarcoma (KS). KS tumors are characterized by proliferating endothelial-derived spindle cells and infiltrating leukocytes, which secrete cytokines and growth factors that may contribute to the proliferation of tumor cells. Unless exposed to inflammatory cytokines, resting endothelial cells usually do not express adhesion molecules. Here, we demonstrate that KSHV-infected latent endothelial cells induce the expression of vascular cell adhesion molecule, which promotes leukocyte adhesion and transendothelial migration.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":" ","pages":"e0149926"},"PeriodicalIF":5.4,"publicationDate":"2026-10-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148906735","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The cellular protein TIAR mediates rapid initiation of West Nile virus genome RNA synthesis.","authors":"Jessica L Siemer, Hsuan Liu, Jin Zhang, Dan Cui, Jeenah Jung, Philip J Santangelo, Markus W Germann, Margo A Brinton","doi":"10.1128/mbio.01947-26","DOIUrl":"10.1128/mbio.01947-26","url":null,"abstract":"<p><p>During the intracellular replication cycle of West Nile virus (WNV), genome RNA synthesis is initially inefficient but increases exponentially as viral replication complexes are sequestered in invaginations in the endoplasmic reticulum. In this study, we investigated the functional role of the cellular protein TIAR (T-cell intracellular antigen-related protein) in the transcription of WNV genome RNA. Close colocalization of cytoplasmic TIAR with viral double-stranded RNA was detected by a proximity ligation assay in WNV-infected cells. TIAR binds specifically to the WNV 3'(-) SL but not to the complementary WNV 5'(+) SL in <i>in vitro</i> RNA binding assays. Only the 3' end of the WNV minus-strand RNA was enriched by immunoprecipitation of infected cell lysates with anti-TIAR antibody. Stable overexpression of TIAR in clonal A549 cells increased the ratio of intracellular viral plus-strand to minus-strand RNA in a dose-dependent manner. TIAR contains three RNA recognition motifs (RRMs). Biophysical data indicated that only RRM2 directly contacts RNA and that up to three TIAR molecules can bind cooperatively to the WNV 3'(-) SL RNA. These data provide additional evidence that TIAR functions as a proviral host factor facilitating exponential amplification of WNV genome production in infected cells.IMPORTANCEWest Nile virus (WNV) is a mosquito-borne orthoflavivirus associated with increasing global human disease incidence. The molecular mechanisms underlying viral replication are not fully understood. In early stages of infection, viral genome transcription is inefficient; however, in late stages, viral genome transcription increases exponentially. T-cell intracellular antigen-related (TIAR) protein is a cellular protein that has been shown to interact with the 3' end of the WNV negative-sense antigenomic RNA. We obtained data showing colocalization of cellular TIAR with viral replication complexes in infected cells and an increased ratio of intracellular genomic to antigenomic viral RNA in TIAR-overexpressing cells, and confirmed preferential binding of TIAR to the 3' end of the WNV antigenome both <i>in vitro</i> and in infected cell extracts. We also demonstrated that multiple TIAR proteins can bind cooperatively to the WNV 3'(-) stem-loop RNA. These data provide supporting evidence for a model of TIAR-mediated rapid initiation of nascent genome RNA synthesis in infected cells.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":" ","pages":"e0194726"},"PeriodicalIF":5.4,"publicationDate":"2026-10-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148906790","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Monitoring kinetic changes and restriction of influenza A virus RNA species during infection using a Flu-Stranded CRISPR platform.","authors":"Anh-Tu Tran, Wen-Fang Tang, Chung-Fan Hsieh, Ya-Fang Chiu, Chung-Guei Huang, Kuo-Ming Lee, Rei-Lin Kuo, Kuei-Ching Hsiung, Cheng-Hsun Chiu, Jim-Tong Horng","doi":"10.1128/mbio.01789-26","DOIUrl":"10.1128/mbio.01789-26","url":null,"abstract":"&lt;p&gt;&lt;p&gt;Influenza A virus (IAV) generates three closely related RNA species: viral RNA (vRNA), complementary RNA (cRNA), and messenger RNA (mRNA), whose strand-specific quantification remains limited by sensitivity and quantitative dynamic range, particularly at low RNA abundance. Here, we developed Flu-Stranded CRISPR-Cas12a, a strand-specific detection platform integrating tagged reverse transcription, segment-specific PCR, and Cas12a collateral cleavage to support quantitative analysis of all three RNA species across a broad dynamic range. The assay enables reliable detection down to 10&lt;sup&gt;2&lt;/sup&gt; copies per reaction, extending the lower quantitative boundary relative to both SYBR Green and TaqMan reverse transcription quantitative PCR (RT-qPCR) under matched conditions. Validated in infected cell lines, murine lung tissues, and clinical nasopharyngeal specimens, the platform enabled subtype-discriminating, strand-resolved detection, including samples near or below the quantitative range of SYBR Green RT-qPCR. Using finely resolved infection time-course analyses in NP and NA segments, we identified a reproducible early vRNA decline within the early post-infection phase. This decline was partially attenuated in RIG-I knockout A549 cells, while subsequent vRNA accumulation was enhanced, consistent with a modulatory rather than essential role for RIG-I in early viral RNA dynamics. Subcellular fractionation localized this decline to cytoplasmic incoming genomes. In contrast, importazole-mediated inhibition of nuclear import abolished vRNA recovery without affecting the early decline, indicating that nuclear entry functionally separates early genome reduction from subsequent productive replication. These findings establish Flu-Stranded CRISPR-Cas12a as a strand-resolved framework for monitoring IAV RNA dynamics and reveal an early window of genome vulnerability during cytoplasmic transit that shapes infection outcome.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Importance: &lt;/strong&gt;The early fate of incoming influenza virus genomes remains unclear, limiting our understanding of how infection is established or aborted in host cells. We developed Flu-Stranded CRISPR-Cas12a, a strand-specific platform for sensitive and quantitative analysis of influenza viral RNA (vRNA), complementary RNA (cRNA), and messenger RNA (mRNA) across experimental and clinical samples. Using high-resolution time-course analysis, we identified a reproducible early decline in vRNA during the post-entry phase. Our data suggest that this early genome loss arises from multiple processes, with RIG-I acting as a modulatory factor rather than a primary driver. Subcellular fractionation localized this effect to cytoplasmic incoming genomes, whereas importin-β-mediated nuclear entry was required for subsequent vRNA recovery. These findings support a model of an early cytoplasmic phase of genome attrition that is distinct from replication and provide a framework for understanding early influenza RNA kinetics and for","PeriodicalId":18315,"journal":{"name":"mBio","volume":" ","pages":"e0178926"},"PeriodicalIF":5.4,"publicationDate":"2026-10-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148963515","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The <i>Chlamydia trachomatis</i> effector CT226 directly engages LRRFIP1 to recruit a multiprotein complex that limits inclusion size and progeny production.","authors":"Cherilyn A Elwell, Amy Diallo, Barbara S Sixt, Jessica Sherry, Khavong Pha, Raphael H Valdivia, Oren Rosenberg, Kliment Verba, Joanne Engel","doi":"10.1128/mbio.01953-26","DOIUrl":"10.1128/mbio.01953-26","url":null,"abstract":"<p><p><i>Chlamydia</i> species secrete a unique class of effector proteins termed inclusion membrane proteins (Incs) that are translocated into the inclusion membrane and reprogram host cell processes to support intracellular growth. Using affinity purification-mass spectrometry, we previously identified an interaction between the early expressed Inc CT226 and a human multiprotein complex comprising leucine-rich repeat Flightless-I-interacting protein 1 (LRRFIP1), LRRFIP2, Flightless-I (FLII), and tropomodulin 3 (TMOD3). Here, we define the molecular basis and functional consequences of this interaction during <i>Chlamydia trachomatis</i> infection. We show that CT226 is both necessary and sufficient to recruit the LRRFIP1/2:FLII:TMOD3 (LFT) complex to the inclusion membrane. <i>In vitro</i> reconstitution with purified proteins demonstrates that the predicted coiled-coil region of CT226 directly binds the N-terminal helix of LRRFIP1. Using <i>in silico</i> structural modeling with AlphaFold-Multimer coupled with mutational validation, we identify key residues that mediate an electrostatic CT226:LRRFIP1 interaction required for LFT recruitment to the inclusion. Unexpectedly, genetic disruption of CT226 or its interaction with the LFT complex results in modest increases in inclusion size and recovery of infectious progeny late in infection, demonstrating that the CT226:LFT interaction modulates <i>C. trachomatis</i> developmental progression. Together, these findings establish the molecular basis of CT226-mediated recruitment of the LFT complex to the inclusion membrane and uncover a previously unrecognized role for this effector-host interaction at late timepoints of the <i>C. trachomatis</i> intracellular developmental cycle.IMPORTANCE<i>Chlamydia trachomatis</i> is a major cause of sexually transmitted infections and blindness worldwide. This bacterium survives by replicating inside host cells within a membrane-bound compartment, where it deploys specialized proteins to manipulate host processes. These proteins, known as inclusion membrane proteins (Incs), help organize the host-pathogen interface, but the molecular mechanisms by which many Incs engage host factors remain poorly understood. Here, we show that the Inc protein CT226 recruits a host multiprotein complex to the bacterial compartment by directly binding one of its components. Using structural modeling and targeted mutations, we define the molecular interface required for this interaction. Disruption of CT226 or its ability to engage this host complex results in modest increases in inclusion size and production of infectious progeny, suggesting a role for the CT226:host interaction in regulating <i>Chlamydia</i> intracellular development. These findings reveal how a bacterial effector can engage a host protein complex to influence the outcome of infection.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":" ","pages":"e0195326"},"PeriodicalIF":5.4,"publicationDate":"2026-10-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148990506","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Experimental versus clinical studies capture distinct types of relapse in tuberculosis.","authors":"Christian T Michael, Maral Budak, Philana Ling Lin, Denise Kirschner","doi":"10.1128/mbio.01977-26","DOIUrl":"10.1128/mbio.01977-26","url":null,"abstract":"<p><p>Tuberculosis (TB) remains a global health concern, as <i>Mycobacterium tuberculosis</i> (Mtb) currently infects a quarter of the world's population. Though many TB patients cure infection with short-course treatment, regimens shorter than 4 months risk post-treatment relapse. This is facilitated by the presence of granulomas, spatially heterogeneous immune structures that form during infection that harbor a caseated core that sequesters Mtb in a non-replicating state and pharmacokinetically impedes antibiotics. Typically, clinical relapse is described as TB recurrence <2 years after a misdiagnosis of cure upon treatment completion (MDxC). Comparatively, many experimental models cannot (or do not) screen for cure and examine relapse ~2 months after treatment completion. Understanding how relapse occurs, and whether it is impacted by experimental versus clinical study designs, may improve treatment. Here, we examine two hypothesized mechanisms of relapse: (i) reservoir, where treatment kills all Mtb except those in a non-replicating, diagnostically inaccessible host reservoir; and (ii) threshold, where replicating Mtb remain alive below detectable levels within a test-accessible reservoir. We use our computational model capturing whole-host Mtb infection dynamics, HostSim. Our simulations uncover rates of reported relapse correlating with how studies are performed. Experimentally, relapse is likely driven by incomplete sterilization of replicating bacteria, whereas clinical relapse is most likely driven by gradual expansion of non-replicating Mtb from caseum into granuloma cellular areas. This suggests that TB patients who relapse may best be re-treated with regimens including caseum-penetrating antibiotics, such as rifamycin-class antibiotics, and that experimental animal relapse models better translate to clinical when \"cure\" is established at treatment completion.IMPORTANCEIncomplete treatment of TB leads to risks relapse, which may occur years later. The threat of relapse is the main reason TB treatment takes 4-9 months. Predictors of relapse are not well-defined given variability in technical definitions of relapse and nuances of study designs. Here, we use biologically based computation to simulate both clinical and experimental TB relapse studies, employing multiple diagnostic tests and relapse definitions. We find that two hypothesized types of relapse, each possibly requiring different re-treatment strategies, are simultaneously at play. Simulations suggest that relapse after a \"cure\" diagnosis (most often clinical studies) is caused by reactivation of non-replicating bacteria hidden from treatment within necrotic granuloma tissue or other sites, whereas experimental systems that cannot or do not test for cure post-treatment are likely to report relapse caused by incomplete sterilization of replicating Mtb. Predictions also depend on the currently unclear relationship between bacterial burden and clinical symptoms.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":" ","pages":"e0197726"},"PeriodicalIF":5.4,"publicationDate":"2026-10-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148990552","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}