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Sillen X1 oxychlorides, with the general formula MBiO2Cl (M = Ca, Sr, Ba, Cd), possess unique two-dimensional layered structures featuring self-induced internal electric fields that promote charge separation; however, their wide band gaps limit their applications in the visible portion of the solar spectrum. There is no straightforward cation-doping scheme to tune the optical bandgap into the visible region in Sillen X1 oxyhalides. In this study, we tuned the bandgap of CaBiO2Cl to the visible range by systematically incorporating Fe at the Bi site and synthesizing the samples via a modified gas-solid synthesis method. Substitution of up to 20 mol % Fe in CaBiO2Cl was successful, and the monoclinic symmetry (S.G.: P21/m) was retained. The Fe inclusion caused lattice contraction and local cationic disorder. Fe existed in mixed Fe2+/Fe3+ oxidation states, which led to the oxidation of some amount of Bi3+ to Bi5+. A significant red shift of the band edge with signatures of extended LMCT, intervalence charge transfer, and subtle d-d transitions (due to Fe2+) was observed in UV-visible spectra of the Fe-containing samples. The bandgap narrowed from 3.39-3.52 (CaBiO2Cl) to 2.02 eV (CaBi0.80Fe0.20O2Cl), indicating electronic band structure modification. A similar set of changes was observed when Bi in orthorhombic PbBiO2Cl (S.G.: Cmcm) was substituted with Fe, where the bandgap narrowing was limited (from 2.53-2.71 to 2.04 eV (PbBi0.80Fe0.20O2Cl)). The Fe-substituted samples catalyzed the decoloration of crystal violet dye within 120 min under visible-light irradiation, following pseudo-first-order kinetics. The reactive oxygen species involved in the photocatalytic decoloration were identified. Both catalysts demonstrated recyclability, with their crystal structures remaining intact after use. The demonstrated strategy for tuning the band gaps of the Sillen X1 phases, together with the enhancement of the visible-light photocatalytic properties by efficient charge migration via redox shuttling, qualifies them as sustainable photocatalysts.

The emergence of multidrug-resistant fungal pathogens from urinary tract infections (UTIs) poses a growing challenge in clinical settings. Here, we report a case of a complicated UTI caused by Nakaseomyces glabratus (Candida glabrata) that progressed to urosepsis, leading to the emergence of an isolate carrying simultaneous loss-of-function mutations in ERG3 and ERG11, and abrogated ergosterol biosynthesis. Together with a missense mutation in FUR1-likely responsible for 5-fluorocytosine resistance-this constellation confers resistance to all viable UTI antifungals: azoles, amphotericin B, and flucytosine. Engineered ERG3Δ + ERG11Δ strains recapitulated this multidrug resistance and revealed profound fitness costs that come with it, challenging the assumption that high-cost mutations are unlikely to persist during infection. Among fitness trade-offs, we detected collateral sensitivity to nitroxoline, a commonly used urinary tract antibiotic with potent antifungal activity and a unique mechanism of action. This study provides the first clinical evidence of an elusive mechanism of hyper-multidrug resistance in N. glabratus and highlights nitroxoline as a promising repurposing agent for treating multidrug-resistant fungal infections of the urinary tract. Evolutionary theory states that fitness determines survival. In a drug-treatment environment, resistance increases fitness, but it often comes at a cost, such as slower growth or reduced stress tolerance. If these costs are too severe, they can undermine virulence, making resistance unlikely to persist. Our study challenges this assumption. We describe the first clinical case of Nakaseomyces glabratus evolving multidrug resistance through loss-of-function mutations in ERG3 and ERG11, despite severe fitness trade-offs. This case suggests that certain infection niches, such as the urinary tract, can provide conditions where even highly impaired yet resistant strains persist under strong antifungal pressure. Importantly, we show that this extreme resistance induces collateral sensitivity to nitroxoline, a urinary tract infection antibiotic with potent antifungal activity and a unique mechanism of action. These findings open promising therapeutic avenues to counter multidrug-resistant fungal infections of the urinary tract.

Soils harbor the most complex microbial diversity on Earth, in which bacteria are ubiquitously infected by temperate phages. While integrated prophages often enhance host fitness, active (inducible) prophages are traditionally perceived as "molecular time bombs" due to their intrinsic lysis threat. This dual nature has raised fundamental questions about the true contribution of temperate phages to microbial adaptation and ecosystem stability. To address this gap, we conducted a global-scale integrative analysis by synthesizing 123,207 high-quality bacterial genomes, 183 soil-specific viromic data sets, and 3,749 metagenomes. We established the Global Soil Active Prophage Database (GSAPD), comprising 21,397 high-confidence active prophages, which we found to represent 34.3% of the total soil viral population within our analytical framework. Our comparative genomic analysis reveals that active prophages possess significantly larger genomes and greater genetic complexity compared with their dormant counterparts. Crucially, by mapping phage-encoded auxiliary metabolic genes (AMGs) across diverse biomes, we found that active prophages are disproportionately enriched in key pathways for carbon, nitrogen, and sulfur cycling, as well as specialized resistance mechanisms against heavy metal toxicity. These findings suggest that active prophages act as dynamic reservoirs of functional diversity. We demonstrate that their lytic potential is not merely a survival risk, but a sophisticated mechanism underpinning host environmental adaptation and niche expansion. Ultimately, this study provides a comprehensive global catalog of soil viral pathways and redefines the role of temperate phages as pivotal drivers of microbial evolution and biogeochemical cycling in terrestrial ecosystems.IMPORTANCESoils contain immense microbial diversity, yet the ecological role of temperate phages-especially their active (inducible) forms-remains poorly understood. This study provides the first global-scale assessment of active prophages in soils, revealing that they are widespread and functionally distinct from dormant forms. By building a comprehensive database and integrating multi-omics data, we show that active prophages are enriched in genes linked to key biogeochemical processes and stress resistance. These findings challenge the traditional view of active prophages as purely harmful agents and instead highlight their role as dynamic contributors to microbial function and adaptation. Our work offers new insights into how viruses shape ecosystem processes and provides a valuable resource for future studies on soil microbial ecology and nutrient cycling.

Nakaseomyces glabratus (formerly Candida glabrata) is a leading cause of invasive candidiasis and rapidly develops antifungal drug resistance during treatment. An increasing number of clinical isolates show reduced susceptibility to echinocandins and azoles, leaving amphotericin B (AMB) as a last therapeutic option. Resistance of N. glabratus to this drug is rare, and its underlying mechanisms are still not fully understood. Here, we describe two independent multidrug-resistant bloodstream isolates displaying resistance to AMB and anidulafungin (ANF), as well as a reduced susceptibility to azoles. We performed whole-genome sequencing and sterol profiling on nine clinical N. glabratus isolates, which were resistant to ANF and displayed resistance or low susceptibility to fluconazole (FLU) and AMB. We identified loss-of-function mutations in the genes ERG3 and ERG4, which could be linked to ergosterol depletion and AMB resistance. The transcriptional response of the reference strain CBS138 and an AMBR + ANFR isolate was analyzed by RNA-seq, revealing that ergosterol depletion also contributed to upregulation of ERG and ABC transporter genes, which might explain the low FLU susceptibility. Surprisingly, the AMBR isolates displayed severe fitness defects, and one of them was fully virulent in a Galleria mellonella infection model. Our results indicate that ergosterol depletion in N. glabratus leads to AMB resistance without affecting fitness or virulence. The major human fungal pathogen Nakaseomyces glabratus is well known for its fast development of antifungal drug resistance, especially against commonly used azoles. However, it can also acquire resistance to echinocandins, leading to multidrug resistance (MDR) and leaving amphotericin B (AMB) as the last therapeutic option. AMB resistance is rare, mainly caused by ergosterol depletion, and is normally associated with severe fitness costs for the pathogen. However, we found N. glabratus bloodstream isolates with stable AMB resistance without apparent fitness and virulence defects. The underlying ergosterol depletion contributed to low azole susceptibility and was associated with anidulafungin resistance. These findings demonstrate how fast MDR can evolve in N. glabratus and underline the need for close resistance monitoring.

Zika virus (ZIKV) has multiple lineages and strains that cause a range of disease severity, underscoring the need to elucidate differential neuropathogenesis mechanisms. Here, we performed systematic, side-by-side comparisons of African, Asian, and American ZIKV lineage infections using cerebral organoids derived from human embryonic stem cells, a relevant human model experimental system. African lineage ZIKV strains, as well as the ancestral Asian Malaysia strain, persistently infected neural progenitor cells, causing apoptosis and severe disruption of ventricular cytoarchitecture. In contrast, contemporary Asian and American lineage viruses were cleared from ventricles, coinciding with low apoptosis and reduced neuropathology. Single-cell RNA sequencing demonstrated upregulated cell-type-specific antiviral signaling during American lineage infections, coinciding with viral clearance from ventricular progenitor cells. Conversely, pathogenic African lineage infections were associated with apoptosis, reduced STAT2 and IFIT1 protein levels, and enhanced activation of stress pathways. African lineage and ancestral Malaysian strain infections induced mitochondrial oxidative stress. Scavenging the reactive oxygen species improved ventricular cytoarchitecture and progenitor survival, but without reducing viral titers. Together, these findings suggest that lineage- and strain-specific host stress responses, rather than viral burden alone, contribute to ZIKV-induced neurodevelopmental damage. An implication of this study is that host-directed therapeutic strategies, used to improve host tolerance to viral infection, may benefit clinical outcomes. This study provides a systematic comparison of Zika virus (ZIKV) lineage infections in a relevant human model system to correlate mechanisms of host cellular responses with neuropathogenesis. By analyzing African, Asian, and American ZIKV infections side by side in cerebral organoids derived from human embryonic stem cells, we link persistent infection of neural progenitor cells to elevated cellular stress responses and structural disruption of organoid ventricles. Structural disruption was reduced by adding a hydroxyl radical scavenger, but without lowering viral titer. These data are important in strongly suggesting that host responses to viral infection can be of equal or greater importance than viral burden in determining pathogenesis. This study provides mechanistic insight into how closely related viral lineage infections result in divergent outcomes in the developing brain. The data have added importance for suggesting the potential value of host-directed therapeutics to improve ZIKV tolerance without addressing viral titer.

Virus infection rapidly induces the production and secretion of interferons (IFNs), which amplify the antiviral responses in infected and neighboring uninfected cells. IFN regulatory factor 7 (IRF7), the "master transcription factor," is pivotal in IFN induction, particularly in myeloid cells. Ubiquitination of IRF7 is essential for its transcriptional activation; however, the underlying molecular mechanisms remain poorly understood. We hypothesized that deubiquitinases (DUBs) act as endogenous regulators of IRF7 activity, and conducted a genetic screen using an siRNA library of human DUBs. The screen identified USP2 as a positive regulator and OTUD5 as a negative regulator of IRF7 activity. OTUD5, an inducible DUB, interacted with IRF7 and inhibited its K63-linked ubiquitination, thereby suppressing IRF7 activation. Conversely, USP2 promoted IRF7 activity by binding to IRF7 and removing K27-linked ubiquitin chains, which we found to be inhibitory. Specifically, K27-linked ubiquitination impeded phosphorylation of IRF7, a critical step for its activation. Collectively, our independent lines of investigation, coupled with genetic screens and mechanistic studies, uncovered USP2 and OTUD5 as novel modulators of IRF7 function, providing novel insights into the regulation of antiviral immunity. IFN regulatory factor 7 (IRF7) is a central protein that launches type I interferon responses, and its timely activation is essential for antiviral immunity. Our study uncovers a mechanism by which IRF7 activation is controlled by enzymes that specifically remove small molecular tags, known as ubiquitin, from proteins. Through a focused screen, we identified two enzymes with opposing roles in modulating IRF7 activity. OTUD5, one of these enzymes, suppresses IRF7 activity by removing a ubiquitin tag that is essential for its transcriptional function. In contrast, USP2, the other enzyme, activates IRF7 by removing a ubiquitin tag that is inhibitory to IRF7 functions. These findings reveal previously unrecognized layers of IRF7 regulation and highlight how these enzymes can be targeted therapeutically in diseases driven by abnormal IRF7 functions.

Antiretroviral therapy suppresses HIV replication but fails to eliminate the virus due to the persistence of a transcriptionally silent reservoir, which remains the primary barrier to a cure. HIV latency is maintained through chromatin-mediated repression, making epigenetic regulators attractive therapeutic targets. To identify new modulators of latency, we screened a focused library of 84 chromatin-targeting small molecules. This screen identified BAY-299, a bromodomain inhibitor selective for TAF1 and BRD1, as a latency-modulating compound. BAY-299 reactivated HIV expression and enhanced the efficacy of established latency-reversing agents, including vorinostat, prostratin, and iBET-151, in cell line models. CRISPR/Cas9-mediated knockout experiments demonstrated that TAF1, but not BRD1, is essential for maintaining HIV latency and that TAF1 depletion selectively increases HIV transcription with minimal effects on host gene expression. Dual knockout of TAF1 and Tat revealed that reactivation of HIV in the absence of TAF1 is partially Tat-dependent. Cleavage Under Targets and Release Using Nuclease analysis further showed that TAF1 depletion increased histone acetylation at the viral promoter and across the HIV gene body, suggesting a chromatin-based mechanism. These findings identify TAF1 as a novel regulator of HIV latency and demonstrate the utility of targeted chemical screening to uncover therapeutic vulnerabilities within the latent reservoir. HIV remains incurable due to the persistence of a transcriptionally silent reservoir in infected cells that is not eliminated by antiretroviral therapy. This transcriptionally silent state, known as latency, is controlled by host cell factors that regulate access to the viral genome. In this study, we identified the host protein TAF1 as a key regulator that maintains HIV in a latent state in cell line models of latency. Using both genetic and chemical approaches, we demonstrate that reducing TAF1 levels selectively increases HIV gene expression without broadly disrupting host gene transcription. These findings highlight a previously unrecognized mechanism of HIV latency control and identify TAF1 as a potential therapeutic target for HIV. Understanding how host chromatin regulators contribute to latency is essential for developing strategies that aim to eliminate the persistent HIV reservoir.

Spring viremia of carp virus (SVCV), an aquatic rhabdovirus, causes lethal disease and major economic losses in carp aquaculture, yet no licensed antivirals are available. In this study, we identify the plant-derived diterpenoid quinone cryptotanshinone (CPT) as a potent inhibitor of SVCV that acts by inhibiting clathrin-mediated endocytosis (CME) and attenuating early mitochondrial apoptosis. CPT was well tolerated by epithelioma papulosum cyprinid cells (50% cytotoxic concentration [CC50] = 303.2 µM) and suppressed SVCV replication in a dose-dependent manner (50% inhibitory concentration [IC50] = 3.2 µM), reducing nucleoprotein (SVCV-N) gene expression and infectious virion production by >99% at 16 μM. Time-of-addition/removal and virion pre-incubation assays revealed that CPT acts predominantly at early entry or early post-entry steps: CPT did not impair viral attachment but selectively blocked internalization, causing DiO-labeled SVCV particles to remain trapped at the plasma membrane. Mechanistically, CPT inhibited CME, as evidenced by reduced transferrin uptake and redistribution of clathrin heavy chain from the cytosol to the cell surface. CPT also preserved mitochondrial integrity by limiting Bcl-2-associated X protein translocation, cytochrome c release, and caspase-9/3 activation. In vivo, therapeutic bath treatment with 4 μM CPT significantly lowered viral loads, decreased cumulative mortality, and reduced horizontal transmission in juvenile common carp, whereas prophylactic oral CPT primed type I interferon and interferon-stimulated gene expression, and markedly improved survival after SVCV challenge. Together, these findings define CME-dependent internalization as a druggable entry checkpoint for an aquatic rhabdovirus and highlight CPT as a promising antiviral lead for integrated control of SVCV.IMPORTANCEViral diseases are a major bottleneck to sustainable aquaculture, yet most control strategies still depend on husbandry and culling rather than on mechanism-based antiviral therapy. For spring viremia of carp virus (SVCV), a notifiable rhabdovirus of global concern, it remains unclear whether host entry pathways in fish are realistically "druggable" and how small molecules can be integrated into practical farm interventions. Here, we use cryptotanshinone (CPT) as a chemical probe to demonstrate that clathrin-mediated endocytosis in fish cells can be selectively perturbed to block SVCV internalization, and that this strategy is compatible with immersion and oral delivery in juvenile carp. At the same time, CPT reshapes host responses by stabilizing mitochondrial function and sustaining antiviral signaling, thereby linking a defined entry pathway to cell-death control and innate immunity in vivo. These findings open a conceptual route toward host-targeted, entry-focused antivirals for aquatic rhabdoviruses and provide a translational framework for developing environmentally compatible antivirals in aquaculture.

Acinetobacter baumannii is a critical nosocomial pathogen that causes infections in diverse host niches, including the respiratory tract, bloodstream, and urinary tract. Considering the contemporary landscape of antibiotic resistance, alongside targeting resistance determinants, there is a need to identify novel and alternative targets for therapeutic intervention. Central metabolism plays a major role in the success of a pathogen and represents a pool of plausible drug targets. However, metabolic networks are inherently complex and often employ redundant pathways to ensure the supply of essential metabolites. In our efforts to delineate the metabolic pathways contributing to the pathophysiological fitness of A. baumannii, we focused on biosynthesis and uptake mechanisms that maintain intracellular cysteine homeostasis. Cysteine is one of the least abundant amino acids in proteins; however, it is crucial for the folding and catalytic activity of enzymes involved in diverse pathophysiological pathways. We show that A. baumannii possesses two partially redundant serine acetyltransferases involved in cysteine biosynthesis. Disruption of cysteine biosynthesis affects the intracellular cysteine pool, leading to metabolic dysregulation. The metabolic dysregulation further leads to a defect in cellular bioenergetics, oxidative stress mitigation, and antibiotic stress survival in vitro and in vivo. Combinatorial disruption of cysteine biosynthesis and cystine transport results in synthetic lethality and fitness attenuation within the host. Our work provides insights into the metabolic complexities and vulnerabilities of A. baumannii and underscores the therapeutic potential of targeting pathways that sustain cysteine homeostasis, including combination strategies, to combat infections caused by this priority pathogen.IMPORTANCEAcinetobacter baumannii is a critical nosocomial pathogen, and the rising prevalence of antibiotic resistance in this organism necessitates the identification of novel therapeutic targets. Central metabolism, particularly amino acid metabolic pathways, represents a promising yet underexplored area for such interventions. Cysteine, a sulfur-containing amino acid, is essential for the activity of different enzymes involved in key physiological processes. In this study, we demonstrate that the cysteine biosynthesis pathway plays a critical role in maintaining metabolic homeostasis and survival under antibiotic stress while exhibiting functional redundancy with the cystine uptake system in sustaining in vivo fitness in A. baumannii . Simultaneous disruption of cysteine biosynthesis and uptake results in synthetic lethality and a marked fitness defect in a murine pneumonia model. Our work reveals metabolic complexities and highlights the metabolic vulnerabilities of A. baumannii, which can be further explored for therapeutic interventions to curb infections caused by this priority pathogen.

Conservons are operons that encode unusual regulatory systems found in bacteria of the phylum Actinomycetota. These regulatory systems are composed of four core proteins: a sensor histidine kinase-like protein (CvnA homolog), an MglB-type roadblock protein (CvnB homolog), a protein containing a domain of unknown function (CvnC homolog), and a small Ras-like GTPase (CvnD homolog). Based on their conserved small GTPase components and their phylogenetic distribution, we propose that the systems encoded by conservons should be known as actinobacterial G protein systems (AGPSs). The signal transduction path through AGPSs remains poorly understood, and some AGPSs have additional accessory proteins (CvnE and CvnF homologs) of unknown function. In this work, we show that AGPS accessory proteins are present when the cognate histidine kinase protein (CvnA homolog) lacks an extracytoplasmic sensory domain. It was previously shown that the Cvn8 AGPS of Streptomyces coelicolor controls the expression of multiple pathways for specialized metabolism. The Cvn8 AGPS also contains an accessory protein, CvnF8. Through protein modeling, we found that CvnF8 may share an interaction interface with the histidine kinase CvnA8, prompting the hypothesis that CvnF8 may act as a modulator of CvnA8 activity. Consistent with this hypothesis, we found that when co-expressed in a heterologous host, CvnA8 and CvnF8 were purified as a stable complex. In a purified system, CvnF8 strongly stimulated the ATPase activity and autophosphorylation of CvnA8. Taken together, these findings indicate that CvnF family accessory proteins likely serve as sensors and/or modulators of histidine kinases of AGPSs found broadly in Actinomycetota. Many lineages of bacteria in the phylum Actinomycetota contain conserved operons (conservons) that encode an unusual type of regulatory system whose function is poorly understood. These lineages include pathogens such as Mycobacterium tuberculosis and members of the genus Streptomyces that produce valuable natural products. These regulatory systems are composed of four proteins, including a sensor histidine kinase and a small Ras-like GTPase. We propose that these regulatory systems be known as actinobacterial G protein systems (AGPSs). We show that some AGPSs include accessory proteins that are only found with partner histidine kinases that lack sensory domains. We demonstrate that one such accessory protein can control the activity of its cognate histidine kinase. Our findings indicate that these CvnF-family accessory proteins likely serve as sensory inputs for AGPSs found broadly in Actinomycetota. This work sheds light on the initial steps of signal transduction within these unusual regulatory systems.

Iron-sulfur [Fe-S] clusters are ubiquitous cofactors of a wide array of structurally and functionally diverse proteins. Acyl carrier protein (ACP) is the universal factor required for fatty acid (FA) synthesis. In this study, in E. coli, we demonstrated that [Fe-S] and FA biosynthesis pathways are coordinated processes, driven by a physical interaction between ACP and the ISC [Fe-S] biogenesis machinery. Using bacterial two-hybrid assays, co-purification, and biochemical analyses, we demonstrated a molecular interaction between ACP and IscS, the ISC machinery cysteine desulfurase that provides sulfur for [Fe-S] cluster formation. Structural modeling and directed mutagenesis pinpointed the ACP-binding site in a region of IscS shared for interactions with other components of the ISC [Fe-S] biogenesis system, mainly Fdx and CyaY. At the cellular level, ACP depletion was found to disrupt ISC-dependent [Fe-S] cluster biogenesis, diminishing the activity of key [Fe-S]-dependent regulators (IscR, FNR, and NsrR) and enzymes (aconitase and biotin synthase). Moreover, FASII inhibition by triclosan, which perturbs ACP species homeostasis, also compromised the assembly of the [Fe-S]-dependent FNR regulator. Our findings underscore a functional link between [Fe-S] cluster biogenesis and fatty acid metabolism, with far-reaching unexplored intricacies of metabolic coordination and cellular homeostasis. Comparison with eukaryotic systems highlights a strong evolutionary driving force toward a link between [Fe-S] cluster and fatty acid biosynthesis in all living systems.IMPORTANCECellular functions rely on interconnected metabolic pathways; however, many regulatory links remain unexplored. Iron-sulfur [Fe-S] clusters are cofactors of proteins driving fundamental cellular processes, from respiration to gene regulation. Our study uncovers a direct connection between [Fe-S] cluster biogenesis and fatty acid biosynthesis. We demonstrate the molecular connection between these two essential cellular processes to lie within the interaction between the acyl carrier protein (ACP), a shuttle of fatty acid biosynthetic intermediates, and IscS, the source of sulfur for [Fe-S] cluster assembly. Intriguingly, similar interactions between ACP and [Fe-S] building cysteine desulfurase have been observed in yeast and human models; however, these mechanisms rest on different molecular determinants. This points out the existence of a strong evolutive driving force toward establishing a link between [Fe-S] cluster and fatty acid biosynthesis in all living systems, with far-reaching implications for metabolic coordination and cellular homeostasis.

Th1 cells are viewed as a cornerstone of immunity to fungi and other intracellular pathogens. Despite the widely accepted role of Th1 cells in antifungal resistance, the development of protective strategies harnessing them is stunted by a limited understanding of how best to promote their development. We and others have reported a requisite role for Th17 cells in resistance to fungi. We have long been puzzled about how to reconcile seminal roles for both Th1 and Th17 subsets. Here, we report that Th17 cells convert into polyfunctional Th1 cells producing multiple cytokines, including IFN-γ, TNF, and GM-CSF, when we used adjuvant formulations that include glucopyranosyl lipid adjuvant (GLA) to enhance antifungal immunity. GLA-induced plastic Th17 cells that convert into polyfunctional Th1 memory cells.IMPORTANCEVaccines have furnished major improvements in public health worldwide. An understanding of the immune mechanisms that underpin the development of vaccines and strategies to improve them thus represents important goals. We employed a mouse model of blastomycosis to study an experimental vaccine against the causative fungus Blastomyces dermatitidis. By using a protein antigen-based vaccine, we protected the animals against experimental infection. Analysis of the types of cells and features of immunity that shaped vaccine immunity revealed that the cells are ideally endowed with the capacity to protect when they are "polyfunctional"; that is, they evolve to produce several protein products-cytokines-that recruit and activate other immune cells. We found that we could optimally induce polyfunctional cells by adding a special adjuvant, or vaccine enhancer, to the vaccine antigen. Our results establish an important role for the adjuvant and polyfunctional cells in promoting effective vaccination.

The ability to sense, import, and detoxify copper (Cu) has been shown to be crucial for microbial pathogens to survive within an infected host. Previous studies conducted with the opportunistic human fungal pathogen Cryptococcus neoformans (Cn) have revealed two extreme Cu environments encountered during infection: a high Cu environment within the lung, and a low Cu environment within the brain. However, how Cn senses these different host Cu microenvironments and the consequences of a blunted Cu stress adaptation for pathogenesis are not well understood. In contrast to ascomycete model fungi, the basidiomycete Cn has a single transcription factor (TF), CnCuf1, to regulate adaptive responses to both high and low Cu stress. Sequence comparison with other fungal Cu-responsive TFs identified three conserved cysteine (Cys)-rich motifs located within the CnCuf1 N-terminal domain, which were therefore predicted to play a role in Cu sensing. Mutation of these conserved Cys-rich motifs demonstrated that the 1st Cys-rich motif is functionally relevant for CnCuf1 transcriptional activity during high Cu stress, while it is dispensable for low Cu stress adaptation. An inhalation model of murine infection showed that strains with defective high Cu stress regulation present a distinct and anatomically constrained pattern of yeast distribution within the infected lungs compared to a more widespread infection observed in lungs infected with the wild-type strain. Based on these findings, we hypothesize that Cuf1-driven high Cu responses modulate not absolute fitness, but the containment of Cn cells at the initial site of infection within the lung. Copper is an essential micronutrient required for survival in all kingdoms of life, as it is used as a catalytic cofactor for many essential processes in the cell. In turn, this reactivity of copper ions makes elevated levels of free copper toxic to the cell. This dual nature of copper-essential for life but toxic at elevated levels-is used by our innate immune system in a process called nutritional immunity to combat and kill invading pathogens. In this work, we explore how the fungal human pathogen Cryptococcus neoformans senses high copper stress, a copper microenvironment encountered within the host lung. We identified a specific cysteine-rich motif within the copper-responsive transcription factor Cuf1 to be essential for high copper stress sensing. Mutation of this motif led to an impaired high copper stress adaptation, which did not affect the fitness of the yeast but did impact the containment and distribution of yeast cells inside the host lung.

Type 3 secretion (T3S) systems assemble bacterial nanomachines, including the flagellum and virulence-associated injectisomes, by exporting distinct classes of substrates in a defined temporal order. In both systems, completion of an early assembly intermediate triggers an irreversible switch from early to late substrate secretion. In the flagellar system, this switch is controlled by the secreted molecular ruler FliK, which acts on the core T3S component FlhB, but the molecular mechanism governing this transition has remained unclear. Here, we show that removal of two components, Fluke and the cleaved C-terminal domain of FlhB (FlhBCCD), locks the secretion apparatus in a constitutive late secretion state. In these mutants, secretion specificity no longer requires completion of the hook-basal body or the FliK ruler, indicating that Fluke and FlhBCCD function to maintain the apparatus in early secretion mode. Consistent with this model, synchronized flagellar gene expression experiments reveal that FlhBCCD is retained during early assembly and is lost coincident with hook-basal body completion and activation of σ28-dependent late gene expression of flagellin and chemosensory genes. Mutants in fliK and the core secretion apparatus protein genes fliP, fliQ, and fliR that slow the rate of FliK secretion allow FliK to flip the secretion-specificity switch independent of the assembly of a hook-basal body structure. Structural modeling of the FliK C-terminal switch domain and FlhBCCD supports a mechanism in which secretion of FliK promotes destabilization and ejection of FlhBCCD from the secretion apparatus. Disruption of a folded region within the FliK switch domain uncouples secretion from switching, indicating that the timing of FliK unfolding during secretion is critical for activation of the specificity switch. These findings show that secretion-specificity switching is driven by FliK-dependent removal of inhibitory components, rather than passive sensing of assembly completion. Type III secretion (T3S) systems build complex bacterial nanomachines, including the flagellum and virulence-associated injectisomes, by exporting distinct classes of substrates in a defined temporal order. How these systems switch secretion specificity during assembly has remained a long-standing question. We demonstrate that the flagellar T3S-specificity switch requires removal of two inhibitory components that actively maintain the apparatus in an early secretion state. Their FliK-dependent ejection irreversibly triggers the transition to late substrate export, revealing that secretion switching is controlled by active inhibitory regulation rather than passive sensing of assembly completion. These results define a molecular mechanism for hierarchical control in bacterial secretion systems and provide insights likely relevant to other T3S nanomachines.

Viroids are small, circular non-coding RNAs that autonomously replicate in plants, exploiting host cellular machinery for replication and spread. Recent studies reveal that viroid-like agents can infect filamentous fungi, suggesting cross-kingdom interactions. In this study, we report the discovery and the characterization of TsvlRNA1 in Trichoderma spirale. TsvlRNA1 is a 712-nt-long viroid-like RNA containing a hammerhead ribozyme in one polarity strand. Bioinformatic data, molecular validation, and reverse genetics experiments demonstrate that TsvlRNA1 is circular with an active ribozyme essential for replication. TsvlRNA1 replicates autonomously and transmits between Trichoderma species, eliciting 20-22 nt viroid-derived small RNAs consistent with RNA silencing targeting. The biocontrol capacity of Trichoderma against Rhizoctonia solani is variably modulated by TsvlRNA1, with effects ranging from positive to negative depending on host strain. In T. spirale, data suggest that TsvlRNA1 negatively affects antagonistic potential. TsvlRNA1 transmission via horizontal routes is prevalent, and the viroid-like RNA fails to infect plant hosts experimentally. These results highlight the as-yet underappreciated ecological and functional diversity of viroid-like agents in fungi, with implications for fungal biology, biocontrol, and genotype-phenotype relationships in eukaryotes.IMPORTANCESpecies of the fungal genus Trichoderma play a central role in sustainable agriculture by controlling fungal plant pathogens and supporting plant growth. For this reason, Trichoderma-based products represent a substantial share of the global market for microbial biofungicides. Viroids are the smallest known infectious agents, and their presence in filamentous fungi has only recently been discovered. Consequently, little is known about their biology, transmission, or interactions with fungal hosts. In this study, we describe TsvlRNA1, a viroid-like RNA associated with T. spirale, representing only the second viroid-like RNA to be biologically characterized in fungi. We show that TsvlRNA1 can influence the ability of Trichoderma to inhibit Rhizoctonia solani, a major plant pathogen, demonstrating its biological relevance. Unexpectedly, TsvlRNA1 can be transmitted between different Trichoderma species. This finding raises concerns about the possible transfer of genetic traits between fungi, including potentially those related to fungicide resistance, with important implications for agricultural biocontrol.

Antibiotic tolerance paves the way for acquired resistance in bacterial pathogens. However, the mechanisms of tolerance and its evolutionary role in acquired resistance in pathogenic fungi, and particularly in filamentous fungi, remain elusive. Here, we identified an Inhibitor of Growth domain-containing protein (IngB) as a novel epigenetic regulator of azole tolerance in Aspergillus fumigatus. The loss of ingB promotes supra-MIC growth on agar surfaces despite susceptible MICs in standardized assays. Moreover, established ΔingB biofilms are also less susceptible to azoles in vitro. In a murine model of invasive pulmonary aspergillosis, loss of ingB results in higher pulmonary fungal levels when animals are treated with voriconazole compared to the wild-type control. Subsequent exposure of the ΔingB-tolerant strain to high azole concentrations in vitro resulted in rapid acquired resistance, most notably driven by a frameshift mutation in a putative 20S proteasome maturation protein-encoding gene, umpA, while the susceptible wild-type strain failed to acquire adaptive mutations. The data suggest that loss of IngB provides an epistatic background for the emergence of azole resistance. Our work shows that drug tolerance in a critical fungal pathogen can facilitate azole resistance emergence.IMPORTANCEWhile antimicrobial drug resistance causes adverse effects on human health, drug tolerance can also lead to insufficient pathogen clearance, resulting in infection relapse. However, the mechanisms of antifungal drug tolerance and its evolutionary role in acquired drug resistance in pathogenic fungi, particularly the molds, remain elusive. We identified IngB as a novel regulator of azole tolerance in Aspergillus fumigatus. In a murine model of invasive pulmonary aspergillosis treated with voriconazole, loss of ingB facilitated higher fungal burden levels than the wild-type control, suggesting the observed in vitro tolerance translates to the murine pulmonary environment. Importantly, loss of IngB leads to rapid azole drug resistance under azole-selective pressure in vitro and led to the discovery of a new gene associated with azole resistance, umpA. Our work identifies a novel regulator of antifungal tolerance in a critical human fungal pathogen and suggests that drug tolerance can pave the way for resistance emergence.

Antifungal tolerance, a transient survival state distinct from genetic resistance, poses a significant challenge to antifungal therapy. However, the factors that induce antifungal tolerance and their underlying mechanisms remain poorly understood. Here, we demonstrate that iron starvation exerts a paradoxical effect on azole susceptibility in the human pathogenic filamentous fungus Aspergillus fumigatus by specifically increasing azole tolerance in hyphae while reducing the minimal inhibitory concentration against both conidia and hyphae. This hyphal tolerance could be quantified using a newly developed protoplast-based time-kill assay. The minimum duration required to kill 99% of hyphal cells (MDK99) following azole treatment was significantly prolonged under iron-starved conditions. Further investigations reveal that iron starvation impairs mitochondrial function by reducing the activity of mitochondrial electron transport chain (ETC) complexes I and III. This attenuation leads to a marked reduction in two key hallmarks of azole-induced hyphal death, including reactive oxygen species accumulation and carbohydrate patch formation. Supporting this mechanism, genetic deletion of mba1, a critical mitochondrial assembly factor essential for the proper biogenesis of ETC complexes I and III, confers azole tolerance in A. fumigatus hyphae even under iron-replete conditions. Importantly, this iron starvation-mediated azole tolerance could be restored through supplementation with the mitochondrial cofactor coenzyme Q10. Overall, these findings identify iron availability as a key environmental modulator of azole fungicidal effect and reveal potential therapeutic targets to counteract this antifungal tolerance in invasive fungal infections.IMPORTANCEAspergillus fumigatus undergoes an obligate life cycle with distinct morphotypes, and hyphae represent the predominant morphological form of the fungus during invasive pulmonary aspergillosis. However, owing to the multinucleate nature and pronounced physiological heterogeneity of hyphae, it is challenging to achieve quantitative and effective assessment of their drug susceptibility. In this study, by developing a protoplast release-based time-kill assay, we uncovered that iron starvation confers azole tolerance in A. fumigatus hyphae. Importantly, mechanistic investigations further revealed that supplementation with the mitochondrial cofactor coenzyme Q10 restores fungicidal activity of azoles against A. fumigatus hyphae under iron-starved conditions. Overall, this study underpins the elucidation of environmental factor-antifungal drug tolerance associations and offers targeted strategies against this tolerance.

The enteric bacteriome of Anopheles mosquito vectors has been linked with vectorial competence; however, its influence on insecticide resistance is poorly understood. We found that antibiotic treatment-administered either through sugar feeding (penicillin/streptomycin and gentamicin) or via a blood meal (amoxicillin)-which depleted the bacterial microbiome in susceptible Anopheles strains, led to greater than 50% insecticide deltamethrin tolerance compared to untreated mosquitoes. Simultaneous inhibition of cytochrome P450 activity reverted the antibiotic-induced tolerance phenotype, indicating that the antibiotic-induced deltamethrin tolerance is P450-dependent. We found that the antibiotic treatment, while suppressing most enteric bacterial taxa, allowed proliferation of a particular antibiotic-tolerant Aeromonas taxon, most closely related to Aeromonas hydrophila. Increasing the abundance of this taxon in mosquitoes not treated with antibiotics phenocopied the tolerance phenotype, converting deltamethrin-susceptible Anopheles mosquitoes to deltamethrin-tolerant mosquitoes. Collectively, these results highlight a mechanistic interplay in Anopheles mosquitoes between antibiotic-induced enteric dysbiosis and cytochrome P450-mediated detoxification that promotes insecticide tolerance. This effect could influence mosquito vectorial capacity, especially in Africa, where antibiotic self-medication is highly prevalent.IMPORTANCEOur findings highlight an unexpected link between antibiotic use and the effectiveness of mosquito control strategies. It shows that disrupting the natural gut bacteria of malaria-carrying mosquitoes can make them significantly more tolerant to insecticides commonly used in public health programs. This occurs because antibiotic treatment alters the microbial balance, allowing certain antibiotic-resistant bacteria to thrive and enhance the mosquito's internal detoxification systems. As a result, mosquitoes that would normally be killed can survive exposure. These findings are important because they suggest that widespread antibiotic use-especially in regions heavily affected by malaria-could unintentionally reduce the impact of insecticide-based interventions such as bed nets and indoor spraying. This adds a new layer of complexity to vector control efforts and highlights the need to consider microbial and environmental factors alongside traditional approaches. Understanding this interaction could help improve strategies to combat insecticide resistance and better control mosquito-borne diseases.