SARS-CoV-2 continues to circulate with the emergence of variants that evade existing immunity. However, all strains rely on conserved host factors for entry, making them attractive targets for host-directed antivirals. SARS-CoV-2 engages the ACE2 receptor and can enter cells through two alternative pathways depending on cell type: Spike cleavage at the plasma membrane by TMPRSS2, or within endocytic compartments by cathepsins. Cleavage triggers Spike-mediated membrane fusion and release of the viral genome. To discover small-molecule inhibitors of entry, we first screened compounds against live virus and active candidates were then tested using recombinant VSV expressing SARS-CoV-2 Spike, with VSV expressing its native glycoprotein serving as a control. This approach identified known and novel TMPRSS2 inhibitors, as well as Staurosporine and Retro-2.1. Both compounds inhibited infection in both TMPRSS2-dependent and -independent cell types. Entry bypass studies revealed that Staurosporine acts upstream of Spike cleavage, while Retro-2.1 functions downstream. Mechanistic studies in Calu-3 cells showed that Staurosporine, a pan-PKC inhibitor, blocks entry via PKCη, a pro-viral factor acting before Spike cleavage. Retro-2.1 targets NF1, which promotes infection downstream of Spike cleavage. Together, our screening pipeline identified inhibitors that block SARS-CoV-2 entry at distinct stages and revealed host factors that may inform the development of current and novel antiviral strategies.

DNA methylation is a widespread phenomenon in bacteria that can regulate gene expression, although the mechanisms underlying this epigenetic regulation are often poorly understood. In Clostridioides difficile, the orphan DNA methyltransferase CamA promotes sporulation, a process critical for the persistence and transmission of this nosocomial pathogen. However, the specific CamA target genes that drive this increased sporulation phenotype were unknown. Here, we show that methylation of a single CamA motif in the promoter region of spoIIE, which encodes a factor critical for activating the early-acting sporulation sigma factor, σF, is sufficient to promote spoIIE transcription, σF activation, and spore formation. Surprisingly, the CamA-dependent increase in spoIIE expression also increases the frequency with which cells prematurely activate σF prior to asymmetric division, resulting in miscompartmentalized σF activity. While this premature activation event triggers cell lysis in the well-studied spore-former Bacillus subtilis, we show that C. difficile cells retain developmental plasticity: predivisional cells that have prematurely activated σF can abort sporulation and resume vegetative growth, whereas cells that activate σF in the forespore after asymmetric division remain committed to sporulation. Thus, DNA methylation controls a critical cell fate decision in C. difficile without compromising its capacity to adapt to fluctuating environmental conditions. Finally, we show that CamA confers a significant fitness advantage during murine infection through mechanisms largely independent of its ability to promote sporulation. Since CamA is specific to C. difficile and epigenetically regulates multiple pathways critical for pathogen persistence, these analyses imply that CamA could be a promising antimicrobial target.

Invasive species can disrupt native epidemiological processes, potentially leading to the emergence of zoonotic pathogens. The relatively recent appearance and spread of the bank vole (Clethrionomys glareolus) in Ireland provides a unique model system to study these phenomena. The depauperate small mammal community in Ireland, combined with baseline data, allows us to identify the invasion gradient of C. glareolus and its effects on rodent-borne pathogens over time. A total of 498 C. glareolus and 584 Apodemus sylvaticus were sampled, via removal trapping, across nine sites in Ireland in 2016 and 2017, with six sites revisited in 2021 and 2022. 16S rRNA metabarcoding identified 10 putative pathogenic Operational Taxonomic Units (OTUs) present in these rodents, relating to four bacterial taxa, Bartonella spp., Mycoplasma coccoides, Mycoplasma haemomuris and Mycoplasma penetrans, and one family of protozoans, Sarcocystidae. Several epidemiological processes were found to be correlated with the C. glareolus invasion; firstly, C. glareolus in Ireland exhibited patterns consistent with the enemy release hypothesis, compared to native populations in France, and the native A. sylvaticus in Ireland. Secondly, a potential dilution effect, with reduced prevalence of M. haemomuris in A. sylvaticus at the C. glareolus invasion core compared to the invasion front, was observed. Finally, C. glareolus had increased prevalence of Sarcocystidae at the invasion front when compared to the invasion core, depending on the time and stage of invasion. These findings further our understanding of pathogen dynamics during biological invasions, demonstrating that invaders affect native host-pathogen communities differently as they advance through various stages of establishment.

Vaccination is the most effective way to prevent infectious diseases and safeguard public health. Yet, most new vaccines fail in late clinical trials, and even established ones often underperform in populations apart from those in which they were initially tested. This can lead to reduced vaccine responsiveness, breakthrough infections, and prevent or delay herd immunity. While the causes of vaccine hyporesponsiveness remain difficult to identify, quantify, and therefore address, numerous reports indicate a predominant role of environmental factors. This has notably been demonstrated by a reduction in the immunogenicity and efficacy of various vaccines when transitioning from urban to rural human populations. Here, we tested whether and, if so, how the environment can cause vaccine hyporesponsiveness. We hypothesised that if the leading causes of vaccine hyporesponsiveness were environmental, then environmentally driven hyporesponsiveness would be exacerbated when individuals are under nutritional stress; specifically predicting that high-quality diet supplementation would increase vaccine responsiveness. Finally, we predicted that parasitic helminth infections, which are more common in rural populations, would degrade vaccine responsiveness, e.g., due to their ability to modulate host immunity, and that anthelmintic treatment could rescue vaccine responsiveness in infected individuals. To test these hypotheses, we coupled lab and field experiments with structural causal modelling, and quantified diphtheria toxoid-specific IgG1 optical density (OD) in paired conspecific cohorts of laboratory-reared and wild wood mice (Apodemus sylvaticus) given a single or two doses of diphtheria toxoid vaccine formulated with alum, with and without diet supplementation. We found that anti-toxoid IgG1 OD was ∼ 47 % lower in thewildwoodmice compared to the laboratory-reared population. We also demonstrated that, across both habitats (wild and lab), substantial variation in vaccine responsiveness was caused by diet. However, contrary to our predictions, this high-quality dietary supplementation resulted in lower vaccine responsiveness. Further, once the effects of habitat, diet, and sex were adjusted for, increasing helminth infection burdens negatively affected anti-toxoid IgG1 OD. Counterfactual predictions from our structural causal model suggested that targeting anthelmintic treatment at heavily infected individuals could have improved their anti-toxoid IgG1 OD responses by approximately 2 to 4-fold. Our results indicated that the wild environment and access to a high-quality diet played a dramatic role in shaping the immune system's response to immunisation. Further, we showed that laboratory settings, even when using a genetically diverse, non-traditional model, systematically yielded higher IgG1 OD than was observed in free-living conspecifics on the same protocol. We provide a causally explicit modelling approach to quantify how habitat, diet, and parasites jointly shape anti-diphtheria toxoid IgG1 levels in a focal population, and to prioritise adjunct interventions such as deworming where model assumptions hold.

Drosophila has been established as a powerful genetic model to study not only blood cell development but also innate cellular immunity. Lamellocytes are the most distinct blood cell type which are large key effector cells in the anti-parasitoid immune response in Drosophila. Findings on cell morphology and function of lamellocytes are based on pioneering studies conducted in the early 1980s. Here, we analyzed their remarkable cell cytoskeleton and cell morphology using high-resolution microscopy combined with a lamellocyte-specific candidate RNAi approach to identify key regulators of the lamellocyte morphology and functions. Similar to disc-shaped rigid platelets in our bloodstream, non-migratory lamellocytes only passively circulate in the hemolymph. Once attached to parasitic wasp egg, lamellocytes undergo a marked reorganization of their cortical actin cytoskeleton promoting cell spreading and encapsulation of parasitic wasp eggs. Atomic force microscopy (AFM) indeed showed that lamellocytes become significantly softer while the cortical actin cytoskeleton reorganizes and forms lamellipodia-like protrusions to spread. Cortical actin cytoskeleton reorganization does not depend on WAVE-Arp2/3-branched actin filament nucleation but rather requires the formin Frl (also known as FMNL) downstream of Rac2 and Cdc42 signaling. Supporting this notion, RNAi-mediated depletion of either Frl/FMNL or Rac2 and Cdc42 but not Rac1 results in prominent changes in lamellocyte morphology and immune dysfunction. Our data further suggest a pathway in which Rac2/Cdc42 recruits Frl/FMNL to the cell cortex controlling lamellocyte spreading and encapsulation of parasitoid wasps.

Quercetin is recognized for diverse pharmacological activities. However, the mechanism underlying its broad-antiviral effects has not been elucidated. Herein, we identified quercetin as a potent inhibitor of both double-stranded DNA virus Bombyx mori nucleopolyhedrovirus (BmNPV) and single-stranded RNA virus porcine reproductive and respiratory syndrome virus (PRRSV). Surface plasmon resonance (SPR) revealed that quercetin targets host ubiquitin-activating enzyme 1 (Uba1) homologs. Uba1 knockdown reduced viral proliferation and enhanced the antiviral effect of quercetin, whereas Uba1 overexpression functioned oppositely. Quercetin bound Uba1 homologs with high affinity. Notably, mutation of two binding residues, Q977 and G978, significantly disrupted the binding between BmUba1 and quercetin, and abolished quercetin's antiviral activity. Quercetin obstructed the transfer of ubiquitin from Uba1 to the E2 enzyme Ubc6, impairing the ubiquitination process. Similarly, quercetin inhibited PRRSV proliferation via targeting Uba1 in mammals. These findings elucidate the molecular mechanism underlying the pharmacological effects of quercetin, providing a theoretical basis for the development of novel antiviral agents against both DNA and RNA viruses.

2'-O-methylation (Nm) is a key epitranscriptomic mark across diverse RNAs, yet its role in antiviral defense remains largely unexplored. To systematically investigate the landscape and functional significance of internal Nm modifications on enterovirus RNAs, a sensitive sequencing method termed RACE-Nm was established, in combination with the machine learning-based predictor NanoNm, to map Nm sites on low-abundance RNAs at single-nucleotide resolution. A conserved Nm site was identified at nucleotide 307C within the viral internal ribosome entry site (IRES), which was catalyzed by the methyltransferase FBL. The 2'-O-methylation at 307C caused steric hindrance that disrupted PCBP2 recruitment to the IRES and repressed cap-independent translation. Consequently, FBL-mediated suppression of IRES activity limited the production of the viral RNA polymerase 3D, which in turn prevented the upregulation of a set of pro-viral host genes, including FOSL1, ITGB3, and KCNQ4. The physiological importance of this defense mechanism was further demonstrated in vivo, as EV71 mutant lacking the 307C site showed markedly enhanced replication, aggravated tissue damage, and increased lethality in mouse model. Our study establishes FBL-mediated internal Nm modification as a novel host defense pathway that directly targets cap-independent translation to restrict enteroviral infection, highlighting the epitranscriptomic regulation of host-virus interactions.

Biofilm formation by the fungus Candida albicans is a central virulence trait that enables colonization of implanted medical devices and mucosal surfaces. Biofilm formation reflects a complex regulatory network, and depends upon multiple master regulators that include transcription factors Efg1 and Ndt80. It is well established that efg1Δ/Δ and ndt80Δ/Δ single gene mutants are defective in biofilm formation. We report here that an efg1Δ/Δ ndt80Δ/Δ double mutant of reference strain SC5314 is able to form a robust biofilm in vitro and in vivo. We refer to the efg1Δ/Δ ndt80Δ/Δ biofilm as an emergent biofilm because this phenotype could not have been predicted from the phenotypes of efg1Δ/Δ or ndt80Δ/Δ single gene mutants. In four additional strain backgrounds, efg1Δ/Δ ndt80Δ/Δ mutants do not form biofilms, but in all strain backgrounds the efg1Δ/Δ ndt80Δ/Δ mutants can form filamentous cells, which are components of biofilms. Emergent biofilm formation is especially pronounced in YPD + FBS medium at 30°C, and RNA-seq under those conditions reveals altered expression in the efg1Δ/Δ ndt80Δ/Δ double mutant of biofilm-related genes: upregulation of BCR1, UME6, and HGC1, and downregulation of ALS3, BRG1, and HWP1. These gene expression changes suggest that the emergent biofilm program is partially distinct from the conventional biofilm program. This inference is supported by functional analysis: emergent biofilm formation is independent of Brg1, Rob1, Tec1, and Wor3, all of which have positive roles in conventional biofilm formation. Emergent biofilm formation depends upon the hyphal cyclin Hgc1, the biofilm transcription factors Bcr1 and Ume6, and the Bcr1/Ume6-activated adhesin gene FLO9. The seemingly simple emergent biofilm program may represent a primordial surface colonization strategy.

Parasitic nematodes infect billions of humans and livestock worldwide, causing major health and economic burdens, while the spread of anthelmintic resistance threatens current control strategies. A critical step in parasite infection is the resumption of development of infective third-stage larvae (iL3) upon host entry, a process controlled by the nuclear receptor DAF-12. Activation of DAF-12 by dafachronic acids promotes developmental progression and reproductive maturation, making this receptor an attractive therapeutic target. However, the molecular mechanisms governing DAF-12 activation, particularly transcriptional coactivator recruitment, remain poorly understood. Here, we combined biophysical, cellular, structural, and bioinformatic approaches to investigate coactivator recognition by DAF-12 from the parasitic nematodes Brugia malayi and Haemonchus contortus. Crystal structures of ligand-bound DAF-12 ligand-binding domains in complex with coactivator-derived peptides reveal conserved features of ligand-dependent coactivator recruitment shared with mammalian nuclear receptors. In addition, we uncover previously unrecognized interaction features, including motif-specific contacts that extend beyond the canonical LXXLL binding mode of coactivators and distinct patterns of DAF-12 conservation across nematode clades. Structure-guided analyses redefine the interaction motif of the only described parasite-specific coactivator DIP-1 and suggest novel candidate motifs for DAF-12-interacting proteins. Together, these findings establish the structural basis of coactivator binding to nematode DAF-12 and provide mechanistic insight into the transcriptional regulation underlying parasite development. These results expand current understanding of nuclear receptor signaling in parasitic nematodes and provide a framework for the future design of strategies aimed at disrupting DAF-12 activation as a potential antiparasitic approach.

N6-methyladenosine (m6A) RNA methylation is one of the most prevalent reversible post-transcriptional RNA modifications and has been recognized as a crucial regulator of host immune responses. Intestinal epithelial cells (IECs) constitute an important component of gastrointestinal mucosal immunity. Interferons (IFNs) play a central role in maintaining intestinal homeostasis, and m6A methylation status influences IFN-mediated cell-intrinsic defense. In this study, we investigated the potential role of m6A RNA modifications in IFN-γ-stimulated IEC-intrinsic defense. We observed significant alterations in the topology of the m6A mRNA methylome in murine IECs following IFN-γ stimulation. A subset of IFN-γ-stimulated immune gene transcripts exhibited increased m6A RNA methylation, including several members of the immunity-related GTPase family M (IRGM) genes. In addition, IFN-γ-responsive long non-coding RNAs may modulate the m6A methylation levels of multiple IFN-γ-stimulated immune transcripts. Enhanced m6A methylation of the Irgm2/3 transcripts was associated with strengthened cell-intrinsic defense against infection by the protozoan parasite Cryptosporidium. Notably, Cryptosporidium infection altered the host m6A mRNA methylome in IECs, thereby counteracting the IFN-γ-mediated defense response. Although the RNA levels of Irgm2/3 genes were upregulated, their m6A RNA methylation levels and protein expression were reduced in infected cells. This effect was associated with host delivery of dsRNAs derived from Cryptosporidium parvum virus 1, a virus harbored in the parasite. Collectively, our findings suggest that m6A methylation of RNA transcripts enhances IFN-γ-mediated IEC-intrinsic antiparasitic defense, while Cryptosporidium has evolved mechanisms to evade this response by suppressing m6A RNA methylation of IFN-γ-stimulated immune genes.

Plants and their associated bacteria engage in complex bidirectional interactions mediated by a diverse array of signaling molecules of both plant and microbial origin. Indole-3-acetic acid (IAA) is a central phytohormone regulating plant growth and development and is increasingly recognized as an intra- and inter-kingdom signaling molecule that modulates diverse bacterial processes relevant during plant-microbe interactions. While IAA biosynthesis is widespread among plant-associated bacteria, the mechanisms through which this auxin regulates bacterial physiology and virulence, as well as those controlling its production, remain poorly understood. Here, we show that IAA synthesis deficiency in the global phytopathogen Dickeya dadantii triggers transcriptional reprogramming and results in reduced virulence and fitness during plant infection. Endogenous IAA was found to regulate the expression of the AaeXAB efflux pump, which mediates endogenous IAA secretion, confers resistance to plant defense-related phytohormones, and contributes to plant virulence in D. dadantii. Consistent with its role in plant-bacteria interactions, phylogenetic analyses revealed that AaeXAB-encoding genes are commonly present among Pseudomonadota isolated from plant-associated environments. Moreover, IAA deficiency altered the expression of a previously uncharacterized indole-responsive MarR-type regulator, DDA3937_RS07305, suggesting cross-talk between IAA- and indole-mediated signaling networks. Our data also uncover a complex regulatory circuit coordinating IAA production in D. dadantii, involving the ExpIR and Vfm quorum-sensing systems and the transcriptional regulators TyrR, TrpR, and LrhA. Collectively, our findings provide new insights into the role of IAA as a bacterial signal promoting plant adaptation and virulence. Targeting IAA biosynthesis and efflux pump activity may offer promising avenues for the development of anti-virulence strategies in phytopathogenesis.

Herpesviruses, such as Epstein-Barr virus (EBV), are thought to potentially play a significant role in multiple disease processes, including neoplasia, multiple sclerosis (MS), and more recently, Alzheimer's disease (AD). Animal models remain vital tools for understanding these diseases and developing therapeutics. Callitrichine herpesvirus 3 (CalHV-3) was identified in the early 2000s in the common marmoset (Callithrix jacchus). Although phylogenetically related to human EBV, the biological similarities between CalHV-3 and EBV have not been thoroughly characterized. Over 450 marmosets from five biomedical research colonies in the United States were screened for CalHV-3 using droplet digital PCR (ddPCR). Peripheral blood mononuclear cells (PBMCs) were magnetically separated to determine viral loads in B-cell selected and B-cell depleted populations. A CalHV-3 infected cell line was reactivated to determine gene expression profiles using quantitative-Reverse Transcription PCR (q-RT-PCR). Archived cases of lymphoma in the marmoset were immunophenotyped by immunohistochemistry (IHC). In the neoplastic tissue, CalHV-3 viral loads were measured by ddPCR, and viral transcripts were visualized using RNAscope. The prevalence of CalHV-3 in these research colonies ranged from 19-63%. The virus was detected longitudinally in PBMCs and saliva. Infected marmosets had CalHV-3 viral loads enriched in B-cells. All cases of B-cell lymphoma in the marmoset were positive for CalHV-3 DNA, with transcripts of EBV latent and lytic gene homologs detected in neoplastic tissue. Like EBV, CalHV-3 is characterized by persistent infection, shedding in saliva, B-cell tropism, latent and lytic gene expression profiles, and lymphomagenesis in a subset of infected animals. These results further suggest that CalHV-3 in the common marmoset may serve as a translational model of EBV infection and associated diseases.

Apicomplexan parasites encounter the complement system during extracellular stages of infection, where it imposes a major barrier to survival. Complement activation promotes pathogen elimination through opsonization, inflammation, and membrane attack complex (MAC) formation. To persist, apicomplexans deploy mechanistically distinct but functionally convergent strategies that modulate complement activity instead of fully inhibiting it. These include rapid invasion to limit extracellular exposure, recruitment of host complement regulatory proteins, surface shielding to prevent MAC insertion, and direct interference with complement activation. A central feature of these strategies is the regulation of C3 deposition and processing, which determines downstream effector outcomes. Here, we define a unifying framework for complement evasion across apicomplexans based on these four mechanisms.

We recently identified the FACT-ETS-1 Antiviral Response (FEAR) pathway as an interferon-independent innate immune response that restricts DNA virus replication and that is antagonized by poxvirus-encoded A51R proteins (Rex et al., 2024, Nature Microbiology). The human FEAR pathway is mediated by the FACT complex, consisting of hSpt16 and SSRP1 subunits, that remodels chromatin to activate expression of the antiviral transcription factor, ETS-1. To counter this pathway, poxvirus A51R proteins tether SUMOylated hSpt16 subunits to microtubules to prevent ETS-1 expression. While these observations indicate a role for the FEAR pathway in DNA virus restriction, it was unclear if RNA viruses interact with this pathway. Here, we show that the RNA virus, vesicular stomatitis virus (VSV; Rhabdoviridae), is also restricted by the FEAR pathway but has evolved a distinct mechanism to block this antiviral response. Through in vitro assays and cell culture studies, we demonstrate that the VSV encoded matrix (M) protein directly interacts with SUMOylated hSpt16 to promote its ubiquitination and proteasome-dependent degradation. Using deletion mapping, we discovered a N-terminal motif in VSV M that is both required for interaction with host hSpt16 proteins and for their proteasomal degradation. This degradation effectively blocks ETS-1 induction and suppresses FEAR pathway activation during infection. Strains encoding mutant M proteins that cannot antagonize the FEAR pathway exhibit replication defects in human cells that can be rescued by hSpt16 or ETS-1 depletion, suggesting FEAR pathway evasion is critical for efficient VSV replication. Moreover, we show that the inability of VSV M to interact with and degrade SUMOylated Spt16 in lepidopteran cells results in an abortive infection, suggesting VSV-Spt16 interactions can influence virus host range. Collectively, our study illustrates that DNA and RNA viruses have independently evolved diverse mechanisms to antagonize SUMOylated host Spt16 proteins, underscoring the physiological importance of the FEAR pathway to antiviral immunity.