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Upon exposure to echinocandins, growing yeast cells begin to accumulate cell wall damage and eventually die, resulting in therapeutic effects. While resistance to echinocandins is well studied, tolerance and persistence mechanisms that may also contribute to clinical failures and relapses remain understudied. In time-kill assays with micafungin in vitro, the opportunistic pathogen Candida glabrata exhibited biphasic kinetics of cell death. Modeling with exponential decay equations distinguished a fast-dying major population from a slow-dying minor population, indicative of persistence. A genome-wide forward-genetic screen revealed dozens of genes that appeared to regulate persistence and/or tolerance, but not resistance. Several of those genes encoded calcineurin and its upstream regulators. Using individual gene knockout mutants and FK506, we show that calcineurin signaling increases the lifespans of most C. glabrata cells through a process that is largely independent of Crz1, one of its downstream effectors. The formation of long-lived persister-like cells (i.e., persistence) was strongly dependent on calcineurin signaling, independent of Crz1. Pre-activation of calcineurin using genetic or chemical stressors, such as manogepix, strongly increased tolerance and persistence in C. glabrata, suggesting antagonism of echinocandin efficacy by this new antifungal. Calcineurin signaling was also necessary for the induction of tolerance and persistence in Candida albicans. The findings suggest that short-term administration of FK506 during the earliest stages of echinocandin treatment may improve clinical outcomes while possibly avoiding long-term immunosuppression.

Importance: Treatment of fungal infections is often unsuccessful. Potential causes of antifungal failure include tolerance and persistence, which are poorly understood processes used by fungal pathogens to survive antifungal treatment. This study utilizes detailed experimental protocols and genome-wide screens to discover how Candida glabrata induces tolerance and persistence to a major class of antifungals. The findings suggest that a clinical immunosuppressant may be repurposed to combat tolerance and persistence in this pathogenic yeast, as well as Candida albicans and perhaps other species.

Entry of herpesviruses into cells requires coordinated action of multiple viral glycoproteins, including gH/gL and gB, which comprise the core fusion machinery conserved in herpesviruses. The gH/gL heterodimer activates the gB fusion protein, triggering its refolding from a prefusion to a postfusion form to drive membrane merger. The cytoplasmic tail domain (CTD) of gB is proposed to act as an inhibitory clamp that stabilizes the prefusion state, with interactions between gH and gB CTDs destabilizing this clamp. We previously found that herpes simplex virus 1 (HSV-1) and saimiriine herpesvirus 1 (SaHV-1) gB homologs are functionally interchangeable but mediate reduced fusion when coexpressed with heterotypic gH/gL. To map the regions of gB responsible for species-specific interactions, we generated HSV-1/SaHV-1 gB chimeras by swapping the ectodomain, membrane-proximal region (MPR), transmembrane domain (TMD), and CTD segments. Our results show that homotypic CTD interactions alone are insufficient to trigger fusion, suggesting that gH/gL contacts the ectodomain of gB. We show that the HSV-1 gB CTD is hyperfusogenic relative to the SaHV-1 CTD, whereas the HSV-1 MPR is hypofusogenic relative to the SaHV-1 MPR. Together, these findings suggest that a functional interaction between the gH/gL-gB ectodomains contributes to fusion and that gB maintains a balance between promotion and restraint of fusion through coordinated contributions of its domains.

Importance: Herpes simplex virus type 1 (HSV-1) entry requires the coordinated interaction of gD, gH/gL, and gB. Both gH/gL and gB are conserved herpesvirus proteins that are required for viral replication and are key targets of neutralizing antibodies. Despite their importance, how these proteins interact to mediate herpesvirus entry into cells remains poorly understood. In this study, we examined gB function by creating chimeras that swapped distinct domains between HSV-1 and saimiriine herpesvirus 1 (SaHV-1) homologs. Using these chimeras, we demonstrate that a species-specific interaction occurs in the gB ectodomain. Additionally, we found that the HSV-1 cytoplasmic tail domain (CTD) is hyperfusogenic compared to SaHV-1, suggesting that different gB domains can compensate for one another to balance fusion. This study provides new insight into how gB is regulated to mediate virus entry at the right time and place.

Klebsiella pneumoniae is one of the most common causes of nosocomial infections, and the rise of drug-resistant K. pneumoniae strains is complicating treatment and contributing to a mounting global health crisis. K. pneumoniae has two pathotypes: classical (cKp) and hypervirulent (hvKp). CKp typically causes opportunistic infections in immunocompromised individuals in healthcare settings and often is multi-drug resistant. HvKp can be community-acquired and cause high-mortality infections in immunocompetent individuals. Concerningly, antibiotic-resistant cKp strains with hypervirulence-associated genes and traits have recently emerged. Determining if and how hv-associated genes contribute to increased virulence of cKp strains is essential to addressing this growing threat. The rmpADC operon is an hv-associated locus that confers hypermucoviscosity (HMV), a key virulence phenotype, and rmp genes are often found in convergent strains. In this study, we aimed to determine if the rmp genes alone could increase the virulence of cKp strains in the absence of other hv-associated genes. We introduced genetically distinct rmp loci from different lineages into a broad array of cKp isolates and found that, while many isolates became HMV positive, only a subset of these strains showed an increase in virulence in a mouse model of pneumonia. Sequence type and capsule type were not predictive of how rmp acquisition impacted the clinical isolates. Our results indicate that HMV is likely necessary but not sufficient for hypervirulence and that rmp sequence can influence virulence potential in cKp strains.IMPORTANCEKlebsiella pneumoniae is a global pathogen, and gene exchange between hypervirulent (hvKp) and classical (cKp) strains is a rising threat. It is essential to understand how hvKp genes impact virulence phenotypes and identify the cKp strain backgrounds most amenable to enhanced virulence. Hypermucoviscosity (HMV) is a critical virulence factor in hypervirulent K. pneumoniae, conferred by the rmpADC locus. The rmp genes are encoded on mobile genetic elements and have been detected in convergent antibiotic-resistant K. pneumoniae strains of concern. In this study, we explored the impact of rmp acquisition in a broad set of classical clinical isolates. We observed that HMV appears necessary, but not sufficient, for increased virulence. Sequence type, capsule type, and HMV capacity could not predict which classical isolates gain an rmp-dependent colonization benefit. These insights increase our understanding of the distinctions between cKp and hvKp and further our ability to identify and treat new strains of concern.

Compared with lethal betacoronaviruses, there is limited knowledge of how human alphacoronaviruses HCoV-NL63 (NL63) and HCoV-229E (229E) interact with host innate immune responses. We compared NL63 and 229E infections in human lung-derived cell lines, A549^ACE2 and MRC-5, and primary nasal epithelial air-liquid interface (ALI) cultures. We measured the infection rates and viral replication kinetics. Additionally, we assessed the activation of three dsRNA-induced pathways, interferon (IFN) production and signaling, oligoadenylate synthetase-ribonuclease L (OAS/RNase L), and protein kinase R (PKR), following infection with each virus. Although both 229E and NL63 replicated efficiently in nasal ALI cultures, NL63 replicated minimally in A549^ACE2 or MRC-5. In lung-derived cell lines, significant IFN mRNA induction as well as PKR activation was observed during NL63 but not during 229E infection. In contrast, in nasal ALI cultures, significant induction of both the IFN and PKR pathways was observed during 229E and NL63 infection. Notably, there was no evidence of RNase L activation during infection with either virus in cell lines or nasal ALI cultures. Infection with a recombinant 229E expressing an inactivated nsp15 endoribonuclease U (EndoU) led to increased dsRNA levels, stronger induction of all three antiviral pathways, and attenuation of replication relative to wild-type 229E. This indicates that 229E nsp15 EndoU regulates host dsRNA responses, as shown previously for porcine epidemic diarrhea virus (PEDV) and pathogenic betacoronaviruses. These findings demonstrate that NL63 and 229E differentially modulate host dsRNA-induced innate immune pathways and highlight the critical role of nsp15 EndoU in suppressing antiviral responses to facilitate efficient viral replication.

Importance: Seasonal human coronaviruses (HCoVs) are the causative agents of more than 15% of common cold cases each year. However, compared with more virulent HCoVs such as SARS-CoV-2, there has been limited research on these viruses. We compared the replication of HCoV-NL63 (NL63) and HCoV-229E (229E). Additionally, we examined their interactions with interferon signaling and related innate immune pathways in lung-derived cell lines and primary nasal epithelial cultures. 229E replicates efficiently in each of these culture systems, with significant dsRNA-induced pathway induction only in nasal cells. In contrast, NL63 replicates efficiently only in nasal cell cultures but induces innate immune pathways in all three culture systems. Moreover, the conserved CoV innate immune antagonist endoribonuclease U aids in evading these responses in 229E infection. This study expands our understanding of common-cold HCoV-host interactions and provides insight into differences between seasonal and lethal HCoVs.

Respiratory viruses typically exhibit seasonal patterns, posing ongoing public health challenges. The coronavirus disease 2019 pandemic altered these patterns dramatically, with many common respiratory viruses disappearing from circulation for extended periods. Here we analyzed over three million diagnostic tests from a metropolitan healthcare system in New York City over 7 years, tracking severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and eight common respiratory viruses before and during the pandemic. Following the initial SARS-CoV-2 wave in the spring of 2020, influenza A/B, respiratory syncytial virus, seasonal coronaviruses, parainfluenza, and human metapneumoviruses were absent for months to years, a phenomenon that we termed the "pandemic gap." This disruption likely resulted from public health measures and SARS-CoV-2-induced antiviral immune responses resembling trained immunity. These findings suggest that the pandemic has temporarily reshaped respiratory virus epidemiology, potentially affecting immune development and increasing susceptibility to future respiratory virus epidemics.

Importance: In this retrospective study using millions of diagnostic tests over 7 years from patients at the Mount Sinai Health System in New York City, we show that when the coronavirus disease 2019 pandemic began in early 2020, many but not all common respiratory viruses disappeared from circulation. We observed prolonged absences ranging from 10 months to nearly 3 years for viruses such as influenza A/B viruses, respiratory syncytial viruses, seasonal coronaviruses, parainfluenza, and human metapneumoviruses. This unusual decline in enveloped respiratory RNA virus activities may have been linked to public health interventions like social distancing, wearing of masks, and lockdowns. Additionally, the rapid spread of severe acute respiratory syndrome coronavirus 2 may have triggered broad, pathogen-agnostic immune responses and the imprinting of antiviral signatures in innate immune cells that conferred temporary protection against other viruses. This phenomenon resembles "trained immunity," a form of enhanced innate immune memory observed after certain infections or vaccinations.

Antagonistic competition is a crucial survival strategy for microorganisms sharing ecological niches, playing a key role in shaping microbial communities and influencing biogeochemical cycles. Here, we report the first extracellular serine protease-dependent synergistic antagonism in archaea: a collaboration between an extracellular protease-producing strain and a precursor protein-producing strain. The serine protease secreted by the former cleaves the precursor protein released by the latter, generating an antibacterial effector molecule. This synergistic antagonism also occurs across domains (between halophilic bacteria and archaea), indicating broad ecological relevance. Using mass spectrometry and inhibition assays, we identified HFX_0892-from the model haloarchaeon Haloferax mediterranei ATCC 33500-as a key mediator of this process. Precursor protein HFX_0892 was cleaved by HlyR4 or other extracellular serine proteases, releasing the N-terminus of HFX_0892 (0892N), which displayed antagonistic activity against haloarchaea and bacteria. Disruption of the α-helical structure in 00892 via point mutations abolished the antagonistic activity. Furthermore, fusing the 0892N to HlyR4 did not interfere with HlyR4's proteolytic function but conferred antibacterial activity. Gene knockout experiments revealed that HFX_0892 is not the sole antagonistic precursor protein in H. mediterranei ATCC 33500. This study uncovers a modular proteolytic activation mechanism that can be harnessed for antimicrobial agent development. The potential prevalence of HFX_0892-like precursors among extremophiles provides a feasible strategy for exploring structurally novel antimicrobial agents.IMPORTANCEAntagonistic interactions are key drivers of microbial community dynamics in hypersaline environments. Here, we report, for the first time, a fan-shaped growth inhibition zone-an atypical phenotypic signature-resulting from synergistic antagonism between two halophilic archaeal species against a sensitive haloarchaeal strain. Using the model haloarchaeon Haloferax mediterranei, we identified a secreted precursor protein (HFX_0892) that is cleaved by a serine protease (such as HlyR4) to release an active antagonistic peptide (0892N). This novel form of archaeal interaction is defined as synergistic antagonism. The antagonistic activity of HFX_0892 is mediated by two α-helical motifs in its N-terminus, and this region can confer antimicrobial function when fused to other proteins. Notably, H. mediterranei encodes additional precursor proteins with potential antagonistic functions beyond HFX_0892. Our work identifies and elucidates a previously uncharacterized antagonistic interaction among archaea, providing critical insights into the complex interspecific interactions and microbial community assembly in hypersaline ecosystems.

HIV-1 viral core transport to the nucleus, an early infection event, triggers the redistribution of cleavage and polyadenylation specificity factors (CPSF) 5 and CPSF6 to nuclear speckles, forming puncta-like structures. CPSF5 and CPSF6 regulate alternative polyadenylation (APA), which governs approximately 70% of gene expression. APA alters the lengths of mRNA 3'-untranslated regions (3'-UTRs), which contain regulatory signals influencing RNA stability, localization, and function. We investigated whether HIV-1 infection-induced changes in CPSF5 and CPSF6 subcellular localization are accompanied by APA changes. Using two independent methodologies to assess APA in human cell lines and primary CD4+ T cells, we found that HIV-1 infection regulates APA, shaped by the interaction of CPSF6 with the viral capsid, recapitulating the APA phenotype observed in CPSF6 knockout cells. Our study demonstrates that HIV-1 infection leverages the interaction between the viral capsid and CPSF6 to co-opt cellular processes, alter gene expression, and potentially contribute to viral pathogenesis.IMPORTANCEThe interaction between HIV-1 and the cellular protein CPSF6 has been known for over 15 years; however, depletion of CPSF6 does not impair productive infection. An alternative possibility is that the virus exploits this protein to modulate cellular processes. This study demonstrates that HIV-1 infection alters the cellular function of CPSF6, an essential regulator of alternative polyadenylation-a mechanism that controls 70% of gene expression. Here, we show that HIV-1 regulates gene expression by disrupting the alternative polyadenylation function of CPSF6 through direct interaction. Overall, this reveals a novel strategy employed by the virus to modulate cellular gene expression.

Streptomyces specialized metabolites account for over half of all clinically used antibiotics, as well as numerous antifungal, anticancer, and immunosuppressant agents. Two-component systems, which are widespread in bacteria, are key regulators of antibiotic production in Streptomyces species, yet their activating signals remain poorly understood. CutRS was the first two-component system identified in the genus Streptomyces, and deletion of cutRS in Streptomyces coelicolor was shown to enhance antibiotic production, although its CutR regulon does not include biosynthetic genes. Here, we used Streptomyces venezuelae NRRL B-65442 to further investigate CutRS function. We show that deletion of cutRS increases growth rate and a reversal of the glucose-mediated carbon catabolite repression typically observed in Streptomyces species. We also demonstrate that CutR DNA binding is glucose-dependent, but CutR does not directly regulate genes involved in growth, antibiotic biosynthesis, or glucose metabolism. The only CutR targets conserved in both S. coelicolor and S. venezuelae are the foldase genes htrA3 and htrB, which are involved in the protein secretion stress response. Consistent with this, we show that CutS homologs all contain two conserved cysteine residues in their extracellular sensor domains and that changing these residues to serine constitutively activates S. venezuelae CutRS. We propose that failure of a disulfide bond to form between these cysteine residues indicates secretion stress and leads to activation of the CutRS system and the secretion stress response.IMPORTANCEStreptomyces bacteria are the primary source of clinically useful antibiotics. While many two-component systems have been linked to antibiotic biosynthesis in Streptomyces species, few have been well characterized. Here, we characterize a secretion stress-sensing two-component system called CutRS and propose a model for how the sensor kinase detects extracellular protein misfolding via two highly conserved cysteine residues. Importantly, we also show that deletion of cutRS triggers antibiotic overproduction in the presence of glucose. Since glucose normally represses antibiotic biosynthesis in Streptomyces species through carbon catabolite repression, this finding reveals a simple genetic route to bypass this barrier. This has significant implications for antibiotic discovery pipelines and industrial production, where glucose-rich media are preferred for cost and scalability. Our results position CutRS as a key target for future strain-improvement strategies.

Paramyxoviral transmission between hosts may be, in part, attributed to the ability of the viral envelope-displayed receptor-binding protein (RBP) to bind to cell surface receptors of different host species. We sought to elucidate the architecture of the receptor-binding head region of the RBPs presented by jeilongviruses, a group of emerging and genetically unique paramyxoviruses belonging to the genus Jeilongvirus, family Paramyxoviridae. Structure determination of J and Beilong jeilongvirus RBPs reveals that the proteins exhibit a prototypical six-bladed β-propeller fold, present a binding site with residues associated with sialic acid recognition and hydrolysis, and bear a close structural relationship with sialic acid binding hemagglutinin-neuraminidase (HN)-type paramyxoviral RBPs. Additionally, unlike other paramyxoviruses, jeilongviruses encode an RBP with an unusually long C-terminal extension. In our dimeric Beilong virus RBP structure, we find that the C-terminal extension exchanges a hat-like domain with the central region of the β-propeller of the opposing protomer through domain-swapping. The hat-like domain occludes residues putatively associated with sialic acid binding and hydrolysis, providing a structural rationale for the absence of observed hemadsorption and neuraminidase activity. The insights gleaned from this analysis expand our appreciation of the structural palette available to the plastic paramyxoviral RBP and how their architectures may be adapted to regulate host-cell interactions at the cell surface.

Importance: The paramyxovirus receptor-binding protein (RBP) plays a primary role in determining cell and species tropism. Here, we study the RBPs of jeilongviruses, a group of paramyxoviruses that present a distinctive RBP that encodes an elongated C-terminal region. While the jeilongviral RBP structurally categorizes with paramyxoviral RBPs that interact with sialic acid during host-cell entry, the unusually long C-terminal domain was found to sterically occlude the associated binding site, suggesting that the molecule has developed strategies for autoinhibition of receptor interactions. These data expand our understanding of the architectural space occupied by paramyxoviral RBPs and the structural elaborations that may be incorporated into the paramyxovirus genome to modulate native functionality.

Metallo-β-lactamases, particularly New Delhi metallo-β-lactamase (NDM), threaten antibiotic therapy by disseminating across diverse bacteria. The rise of NDM-producing carbapenem-resistant Acinetobacter baumannii (Ab) highlights the risk of global spread of this pathogen. NDM-mediated resistance depends on periplasmic proteostasis, which regulates the levels of folded, metalated, and active proteins. In Escherichia coli, zinc limitation-common at infection sites-causes loss of the essential metal cofactors in NDM, leading to protein degradation via a specific periplasmic quality control system. Our study reveals that the mechanisms regulating NDM-1 stability under zinc starvation are highly host-dependent. Notably, we identify the proteases DegP and CtpA as responsible for NDM-1 degradation in Ab, differing from the process in E. coli. In-cell stability of NDM in Ab is highly variable depending on minor mutations in CtpA from clinical strains, as well as by mutations in the allelic variants of this β-lactamase. Particularly, NDM-5 displays a higher stability and confers an enhanced resistance phenotype that may help Ab thrive under zinc-limiting conditions. These results reveal selective pressures driving NDM adaptation to each bacterial host. Understanding how pathogens engage their periplasmic metabolism to regulate NDM levels offers insights into the overlooked role of host-specific adaptation of resistance mechanisms. These findings highlight the importance of developing host-directed therapeutic interventions based on the understanding of protein cell homeostasis. In this regard, exploiting host-specific proteases as targeted tools to destabilize resistance enzymes represents a novel therapeutic avenue for curbing the spread of NDM in different pathogens.IMPORTANCEThe alarming rise of Acinetobacter baumannii producing New Delhi Metallo-β-lactamase (NDM) threatens last-line antibiotic therapies. While β-lactamase dissemination is often accounted for the underlying genetics, the biochemical mechanisms involved in the adaptation of these enzymes within specific bacterial hosts are scarcely known. Here, we show that the stability of NDM differs significantly between A. baumannii and Escherichia coli, due to the varying roles of periplasmic proteases involved in NDM degradation in each host. Variant NDM-5 exhibits enhanced stability and confers increased antibiotic resistance in A. baumannii under zinc-limited conditions (common in infection sites). These findings underscore the role of host-specific proteostasis in shaping the adaptation of resistance determinants and suggest new strategies to combat antibiotic resistance.