Free fatty acids (FAs) secreted by the sebaceous glands are critical components of the skin barrier. Here, we investigate the activity of the major FA species present in the skin against Streptococcus pyogenes (group A Streptococcus [GAS]), a major cause of skin infections, such as impetigo, erysipelas, cellulitis, and necrotizing fasciitis. Several relevant FAs had antimicrobial activity against GAS, and linoleic acid was found to be therapeutic when applied in a murine skin infection model. However, sapienic acid, a FA abundant only in human sebum, significantly worsened disease severity despite possessing antimicrobial activity. Transcriptional profiling and molecular genetic analysis showed that sapienic acid, but not other structurally similar FAs, induced the sag operon. The sag operon encodes streptolysin S (SLS), and sapienic acid induction of this cytolytic toxin significantly increased hemolysis of human red blood cells by GAS. Screening of additional FAs identified forms with combined antimicrobial and anti-lytic activity useful as therapeutics. Taken together, we report a species- and tissue-specific trigger for GAS virulence and limitations to the use of FAs as therapeutics against infectious disease.IMPORTANCEGAS naturally only infects humans. Here, we report that the human-specific sebum lipid sapienic acid induces production of streptolysin S (SLS), the hemolysin responsible for the hallmark β-hemolytic phenotype of GAS. Reliance on detection of a human-specific FA for expression of a critical virulence factor exposes a vulnerability of GAS and suggests a potentially variable role for SLS at different infection sites. Furthermore, it details a limitation of existing infection models, which all lack sapienic acid, for understanding the role of SLS in disease.
Due to the scarcity of cell-adapted strains and limitations of current reverse genetics techniques, the factors determining cell tropism of infectious bronchitis virus (IBV), a prototype gammacoronavirus, remain unclear. Here, we demonstrated that the expanded cell tropism of CEA, a chicken embryo fibroblast-adapted strain derived from tl/CH/LDT3/03, was related to its enhanced attachment capacity. Then, a reverse genetics platform for IBV based on circular polymerase extension reaction (CPER) was established, enabling the successful recovery of rCEA and rLDT, which retained the biological properties of their respective parental viruses. Using this platform, we constructed a series of chimeric viruses based on the CEA strain by incorporating the entire S gene, the S1 and S2 subunits, and substitutions at differential sites derived from the tl/CH/LDT3/03 strain. Our findings indicated that subunit S1, rather than S2, was associated with viral adaptation in DF-1 cells, with the amino acid residue at position 413 in the C-terminal domain of the S1 protein as a key determinant. Evaluation with recombinant S1 proteins and chimeric viruses revealed that the histidine residue at position 413 (His413) promoted enhanced attachment of both S1 proteins and viruses to cells, while exerting no direct effect on viral entry. Mechanistically, His413 facilitated efficient viral binding to α-2,3-linked sialic acids presented on gangliosides rather than glycoproteins. This interaction activated Src kinase and triggered caveolae-mediated endocytosis, initiating viral entry and subsequent replication. Collectively, the CPER-based reverse genetics platform established in this study represents a significant technical advancement for IBV, and our findings provide novel insights into the cell adaptation and entry mechanisms of IBV.IMPORTANCEAlthough infectious bronchitis virus (IBV) was the first discovered coronavirus, the cellular receptors and cofactors that mediate successful infection, as well as the specific pathways and mechanisms of viral entry into host cells, remain to be elucidated. In this study, we established a reverse genetics platform for IBV using circular polymerase extension reaction for the first time. A series of chimeric viruses were subsequently recovered, and a specific amino acid substitution at position 413 in the C-terminal domain of the S1 subunit was identified as a key determinant for the expanded DF-1 cell tropism of IBV. The histidine residue at this site facilitated viral binding to α-2,3-linked sialic acids on gangliosides, thereby activating the caveola-mediated endocytosis and enabling viral entry into DF-1 cells. This study provides novel insights into the receptor-binding function of the IBV S protein and the strategies employed by the virus for cell adaptation. Additionally, our findings offer new perspectives for developing cell-culture-based vaccines.
Canonical partner-switching systems (PSSs) regulate sigma factor activity through reversible phosphorylation, but their established roles have been largely limited to stress responses and sporulation in Firmicutes. Whether this regulatory mechanism also controls developmental cell fate decisions in other bacterial phyla has remained unknown. Here, we identify a canonical PSS that governs heterocyst differentiation in the multicellular cyanobacterium Anabaena sp. PCC 7120. This system comprises the anti-sigma factor All2284 (NfsS) and the anti-anti-sigma factor All2283 (NfsR). Structural predictions and biochemical assays showed that NfsS phosphorylates NfsR on a conserved serine residue, whereas bacterial two-hybrid and co-purification assays demonstrated that NfsS binds the developmental sigma factors SigC and SigE. Deletion of nfsR abolished heterocyst formation and diazotrophic growth, and transcriptomic analysis revealed broad failure to induce late heterocyst genes, including nitrogen fixation functions such as nifHDK and fdxH. Phylum-wide comparative genomics further showed that PSS genes and putative functional clusters are strongly enriched in filamentous and heterocyst-forming taxa, indicating an association between the expansion of these signaling modules and the emergence of multicellularity and developmental specialization. Together, these findings establish a PSS as a direct regulator of terminal cell differentiation in a gram-negative bacterium and reveal partner switching as a conserved regulatory principle linking environmental signaling to developmental fate in a major bacterial phylum. IMPORTANCE:While partner-switching systems are classically associated with stress responses and sporulation control in Firmicutes, whether this regulatory logic governs developmental decisions in other bacterial phyla has remained unknown. Here, we establish that a related partner-switching mechanism operates in a distinct bacterial lineage, the cyanobacteria, where it controls a major developmental transition involving terminal cell differentiation. Specifically, we show that a phosphorylation-dependent checkpoint involving the anti-sigma factor NfsS and the anti-anti-sigma factor NfsR directly regulates heterocyst formation. Disruption of this switch abolishes cell differentiation and diazotrophic growth, revealing that this system is an obligate gatekeeper for terminal differentiation. Conceptually, these findings substantially extend the known functional repertoire of partner-switching circuits: rather than controlling stress adaptation or spore dormancy, this module has been co-opted to govern a complex, multicellular developmental program in an organism that underpins global carbon and nitrogen cycles. This work, therefore, establishes a new paradigm for phosphorylation-based control of developmental sigma factors and provides a tractable model for dissecting how conserved signaling modules are rewired to drive lineage-specific innovations across the bacterial domain.
ABSTRACT Methane oxidation coupled to sulfate reduction is the dominant process restricting methane release from cold seeps. Methane oxidation can also be coupled to nitrate/nitrite reduction, thereby linking the cycles of CH 4 , CO 2 , and N 2 O. However, how this coupling shifts during seep development and its impact on N 2 O production remains poorly understood. Here, we integrated geochemical profiling, 14 C-based labeling, and omics-based analyses to quantify the process rates, identify the key microbial mediators, and assess the net N 2 O production across the developmental stages of cold seeps in the South China Sea. In an early-stage, polychaete-dominated seep, the depth-integrated, potential nitrate/nitrite-coupled methane oxidation rate reached 21.9 ± 3.0 mmol C/m 2 /day, accounting for 36%–73% of the total methane oxidation. By contrast, in a typical epifauna-prevalent mature seep, this contribution diminished to ~1% (0.62 ± 0.16 mmol C/m 2 /day), while sulfate-dependent methane oxidation became dominant. Omics data identified the predominant methane-oxidizing bacteria Methyloprofundus and QPIN01 (relative abundances of 7%–24%) as the potential mediators of nitrate/nitrite-coupled methane oxidation in the early-stage seep sediments. Consistent with the absence of N 2 O-reduction gene nosZ in these lineages, simultaneous nitrate and methane amendments significantly stimulated net N 2 O production by 14%–46% in the early-stage seep sediments. This increase correlated strongly with the potential nitrate/nitrite-coupled methane oxidation rate and was estimated to offset 41% ± 9% of the climate benefit gained through methane oxidation. These findings revealed the previously unrecognized successional dynamics of dominant methane oxidation pathways and necessitate the incorporation of nitrate/nitrite-coupled methane oxidation into assessments of greenhouse-gas release from these globally distributed ecosystems. IMPORTANCE Cold seep biota are widely regarded as a critical “biofilter” that restricts seafloor methane emission, mainly through coupled methane and sulfur cycling. This study refines this prevailing view by demonstrating a major shift in the dominant methane oxidation pathway during seep ecosystem development. Contrasting to the dominance of sulfate-coupled methane oxidation in mature seep systems, the early-stage seep is dominated by nitrate/nitrite-coupled methane oxidation, a process primarily mediated by methane-oxidizing bacteria. Crucially, these organisms possess a truncated denitrification pathway that leads to the production of nitrous oxide (N 2 O), a greenhouse gas with nearly 10 times the warming potential of methane. The resulting net N 2 O production thereby substantially offsets the climate benefit achieved by methane consumption. This study highlights the dual role of cold seep microbiota in regulating climate-active gases, underscoring that accurate assessment of their environmental impact requires a holistic understanding of temporal changes in coupled carbon and nitrogen cycles.
Bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) control of biofilm formation in Escherichia coli K-12 is balanced by multiple diguanylate cyclases (DGCs) and c-di-GMP-specific phosphodiesterases (PDEs). Five of the 13 PDEs feature a periplasmic CSS domain with two conserved cysteines, flanked by transmembrane (TM) regions, and an enzymatically active cytoplasmic EAL domain. One of these (PdeC) was previously shown to be redox-regulated by DsbA/DsbB-mediated disulfide bond (DSB) formation in the cysteine serine serine (CSS) domain. Comparing all five CSS domain PDEs, we found them to fall into two groups with similar biochemical features, resulting in different consequences for PDE activity. PdeB, PdeC, and PdeG are more active when lacking the periplasmic DSB, resulting in diminished biofilm formation, while PdeN and PdeD are active in their oxidized forms. Using PdeB and PdeN as prototypes for the two groups, not only the periplasmic DSB but also differently charged amino acid motifs close to the transmembrane (TM) domains and a putative additional DSB in PdeN were identified as important for transmembrane signaling. All these elements, including its stable structural DSB in the periplasm, which can form independently of DsbA, maintain PdeN in a rigid, proteolysis-resistant active conformation. By contrast, for PdeB, the more rigid DSB-containing conformation is inactive, with mutations in the stabilizing elements leading to a structurally less constrained, more active enzyme. Notably, low PdeN levels are post-transcriptionally upregulated at acidic pH, resulting in less biofilm formation. Overall, the five CSS domain PDEs enable E. coli to adapt to diverse environmental niches.IMPORTANCESensing environmental cues and transmembrane signal transduction via membrane-embedded proteins is a process of key importance in all living cells. To investigate the molecular mechanisms involved, we performed a systematic functional comparison of the five CSS domain phosphodiesterases of Escherichia coli, which degrade the bacterial second messenger c-di-GMP in response to redox and other signals. With a sensory domain in the periplasm linked to a cytoplasmic enzymatic domain, these proteins represent minimal devices for transmembrane signaling. We demonstrate that these signal-transducing enzymes fall into two functional classes with a similar periplasmic redox biochemistry resulting in opposite states of cytoplasmic enzymatic activity. Several characteristic sequence elements convey redox and structural information in the periplasmic and transmembrane protein segments to their ability to dimerize into an enzymatically active form in the cytoplasm. Comparing the five enzymes also shows that evolution has played with these elements to facilitate adaptation to various environmental niches.
Soil bacteria play a central role in global biogeochemical cycles and are critical for soil health and agricultural productivity. The dynamic nature of soil hydration status affects bacterial habitats by changing the energy state of soil water and disrupting aqueous connections critical for nutrient diffusion. To study how soil bacteria respond to desiccation, we used the rhizobacterium Pseudomonas synxantha 2-79 as a model organism and quantified its response to co-occurring water and nutrient limitations at the single-cell level. We hypothesized that the relative importance of osmolyte synthesis and starvation responses to desiccation tolerance is context dependent, with the optimal strategy determined by the trajectory of nutrient and water deprivation. We constructed a transcriptional reporter to track P. synxantha's expression of biosynthesis genes for the osmolyte N-acetylglutaminylglutamine amide (NAGGN) and collected extensive single-cell growth rate, cell size, and reporter expression data through experiments that mimicked different rates and extents of soil drying. Only actively growing cells responded to an osmotic shock by synthesizing NAGGN; this response was not observed for pre-starved bacteria. Despite the lack of osmolyte NAGGN synthesis, prior starvation enhanced P. synxantha's ability to recover from osmotic stress once water and nutrients were restored. In line with our observation that prior starvation prevented cell lysis upon rewetting, starved cells had more rigid membranes. Together, our results indicate that diverse cellular properties contribute to soil bacterial desiccation tolerance, whose relative response and fitness are tuned to different challenges imposed by soil drying dynamics.IMPORTANCESoil bacteria are critical to agriculture, but it is unclear how these organisms respond to desiccation, a common and worsening stress. Desiccation both dehydrates bacterial cells and eliminates the liquid water connections between soil pores that bacteria use to access nutrients. We studied how a model soil bacterium responds to (co)-occurring starvation and water stress at the single-cell level, focusing on osmolyte synthesis and physiological adjustments that take place under starvation. We describe the desiccation and regrowth trajectories in these conditions at single-cell resolution. We find that starvation restricts synthesis of a dipeptide osmolyte but rigidifies the membrane, enabling bacteria to withstand more severe water stress. Distinct cellular factors thus contribute differentially to desiccation tolerance along a drying trajectory.
Pathogenicity, the ability to cause infectious diseases in multicellular eukaryotes, is widespread among bacteria and eukaryotes but has not been reliably identified in archaea. To explore possible reasons for this disparity, we perform comparative analyses of thousands of bacterial and archaeal isolates. Our results show that isolated bacterial pathogens possess the ability to use organic compounds as sources of energy, electrons, and carbon (chemo-organo-heterotrophy or COH), suggesting that COH is a metabolic prerequisite for pathogenicity. Moreover, we find that isolated archaea capable of COH do not inhabit multicellular eukaryotes and instead predominantly inhabit extreme environments that preclude such eukaryotes. In contrast, all isolated archaea that inhabit multicellular eukaryotes are incapable of COH metabolism. This metabolism-habitat covariation in isolated archaea, together with COH as a potential prerequisite for pathogenicity in bacteria, suggests that the absence of archaeal pathogens may be due to a combination of the two factors: COH-capable archaea lack the environmental opportunity to inhabit multicellular eukaryotes, while non-COH archaea lack the metabolic prerequisite for pathogenicity.IMPORTANCEPathogenicity-the ability to cause infectious disease-is widespread among bacteria and eukaryotes but conspicuously absent from archaea, the third domain of life. To understand why archaea are non-pathogenic, we performed comparative analyses of thousands of bacterial and archaeal isolates. We found that the absence of pathogenic archaea can be explained by a combination of metabolic and environmental factors. Specifically, archaea living within eukaryotic, multicellular hosts lack a metabolic capability correlated with pathogenicity-chemo-organo-heterotrophy-whereas those possessing this metabolism typically live under extreme conditions, such as high temperature, and therefore lack the environmental opportunity to interact with eukaryotic hosts. These findings advance our understanding of microbes by revealing important links between their metabolism, habitats, and pathogenicity.
The persistence of latent HIV-1 reservoirs remains the primary barrier to a cure. Shock and kill strategies aim to reactivate these reservoirs and eliminate them via effector cells, such as natural killer (NK) cells. However, chronic infection leaves NK cells exhausted. In this study, we investigated the interplay between cytokine-mediated NK cell activation and antibody-dependent cellular cytotoxicity (ADCC) across diverse HIV-1 subtypes. We demonstrated that while cytokine stimulation enhanced natural cytotoxicity, it simultaneously induces shedding of the Fc receptor CD16 via the metalloproteinase enzyme ADAM17. However, restoring CD16 expression through ADAM17 inhibition (TAPI-1) did not improve ADCC, suggesting that CD16 surface levels in NK cells are not the only limiting factor. On the other hand, Fc-engineered broadly neutralizing antibodies (bNAbs) with increased CD16 affinity, in particular LPLIL and GASDALIE, significantly enhance ADCC across different HIV-1 subtypes, regardless of NK cell activation or CD16 downregulation. These findings suggest that enhancing receptor affinity of bNAbs can bypass viral immune evasion and NK cell exhaustion, supporting their potential incorporation into HIV cure strategies.IMPORTANCEEradicating latent HIV reservoirs remains a global health priority as it would liberate millions of people living with HIV (PLWH) from the economic and physiological burdens of lifelong antiretroviral therapy (ART). By utilizing a primary human cell model that resembles in vivo conditions across diverse viral strains, we identified that simply preventing CD16 receptor shedding from natural killer (NK) cells is insufficient to improve HIV-1 clearance. Instead, we demonstrated that enhancing the binding of antibodies to the CD16 receptor enables NK cells to overcome viral immune evasion and the loss of CD16 expression. This allows for potent elimination of infected cells through antibody-dependent cellular cytotoxicity (ADCC). These findings provide a clear strategy for designing more effective "kill" components in future therapeutic strategies.
Zika virus (ZIKV) infection during pregnancy can result in severe fetal outcomes, yet the mechanisms of transplacental dissemination remain incompletely defined. We previously showed that ZIKV induces tunneling nanotubes (TNTs), actin-rich intercellular conduits that enable direct cell-to-cell transfer of viral components. Here, we investigated the in vivo role of TNTs in ZIKV maternal-fetal transmission using complementary pregnancy models. A TNT-deficient ZIKV mutant (ZIKVΔTNT), harboring a change between residues 40 and 52 of the nonstructural protein 1 (NS1), showed markedly reduced viral dissemination to maternal and fetal tissues across all models tested, whereas the TNT-competent ZIKV established a robust infection. ZIKVΔTNT infection was associated with reduced placental pathology, altered junctional-to-labyrinth architecture, improved placental efficiency, and protection from fetal growth restriction. Loss of TNT-forming capacity also limited viral persistence despite maternal type III interferon (IFN-λ) responses, suggesting a role for TNTs in immune evasion. Together, these findings provide in vivo evidence that TNTs are a key mechanism by which ZIKV enhances dissemination, promotes placental dysfunction, and drives fetal pathogenesis.IMPORTANCEZika virus infection during pregnancy can cause severe fetal abnormalities, yet how the virus overcomes the placenta remains incompletely understood. Here, we show that ZIKV exploits direct intercellular connections called tunneling nanotubes (TNTs) to facilitate cell-to-cell transmission and impact placental infection and fetal outcomes. Using multiple pregnancy models, we demonstrate that viruses capable of forming these structures disseminate more efficiently, damage the placenta, and lead to fetal growth restriction, whereas TNT-deficient viruses show reduced infection and milder disease. Importantly, TNT-mediated dissemination is associated with viral persistence despite maternal interferon responses, suggesting a role in immune evasion. These findings identify TNTs as a previously underappreciated pathway of viral transmission during pregnancy and suggest new therapeutic targets. More broadly, TNTs may contribute to the pathogenesis of other vertically transmitted or emerging viral infections.
Porcine reproductive and respiratory syndrome virus (PRRSV) infection often causes severe immunosuppression in pigs. However, the mechanisms by which PRRSV antagonizes host antiviral immune responses remain incompletely understood. We found that granulocyte-macrophage colony-stimulating factor (CSF2) was significantly upregulated in porcine alveolar macrophages (PAMs) upon PRRSV infection. CSF2 upregulation inhibits PRRSV replication by promoting IL15 expression, and Fos-related antigen 1 (FRA1) enhances the antiviral activity of the CSF2-IL15 axis via transcriptional regulation. Further investigation revealed that the PRRSV N protein directly interacts with HSPA1B and activates HSPA1B-mediated chaperone-mediated autophagy (CMA) to degrade FRA1, thereby antagonizing host antiviral immunity. Our study reveals that the PRRSV N protein promotes FRA1 autophagic degradation by upregulating heat shock protein HSPA1B, thereby suppressing the host CSF2-IL15 antiviral pathway. This study provides new insights into the arms race between PRRSV and the host, as well as novel perspectives for the development of anti-PRRSV infection strategies. IMPORTANCE:Focusing on the core scientific issue of PRRSV immune evasion, this study identifies a novel pathway by which host FRA1 restricts viral infection through regulating the CSF2-IL15 cytokine axis. It reveals that the PRRSV N protein hijacks HSPA1B-mediated chaperone-mediated autophagy to target and degrade the transcription factor FRA1, thereby dismantling the host CSF2-IL15 antiviral defense and advancing the understanding of PRRSV-related immunosuppression. These findings elucidate a new paradigm in the host-pathogen tug-of-war, whereby viruses exploit autophagy machinery to eliminate critical immune regulators. This work also provides mechanistic insights for the development of intervention strategies targeting CMA and cytokine homeostasis.
Enterococci appear to have originated in the guts of early terrestrializing arthropods and invertebrates over 425 million years ago-hosts that are now highly diverse and widespread in nature today. Yet most knowledge of the genus comes from human infection-associated lineages with genomes swollen by the recent accretion of foreign DNA conveyed by mobile elements. Because invertebrates dominate terrestrial animal diversity and biomass, they would be predicted to constitute a major but little-explored reservoir of enterococcal diversity. We therefore systematically examined Enterococcus association and species diversification in invertebrate hosts of the comparatively natural, isolated, but well-characterized environment of the Azorean island of Terceira. Over 100 invertebrate specimens were examined for associated enterococci, which were taxonomically classified by whole-genome sequencing. Supporting the existence of a large pool of uncharacterized enterococci and Enterococcus-adapted genes, 40% (eight of 20) of the Enterococcus species identified were either undescribed, including four candidate new species described here, or very recently discovered. In contrast, control isolates from vertebrates were exclusively of known species typical of sampling elsewhere, discounting geographic isolation as a main driver of the novelty observed. Further, because of the abundance of E. casseliflavus and E. flavescens in this collection, we obtained the resolution necessary to quantify the divergence and decipher the drivers of speciation in the controversial division between these naturally vancomycin-resistant species. These findings provide robust support for the existence of a large pool of new species and unexplored adaptive traits in invertebrate-associated enterococci-diverse environmental survival traits optimized for expression in an enterococcal background, and well positioned for transmission into human-associated enterococcal strains.IMPORTANCEEnterococci are auxotrophic gut-associated bacteria that co-evolved with their terrestrial hosts over many eons. In the last 75 years-the "antibiotic era"-E. faecalis and E. faecium gained genes for antibiotic resistance and enhanced virulence, emerging as leading causes of multidrug-resistant infection. Little is known about the source of those genes or the pathway by which they entered human-associated strains. A recent global survey suggested a potentially large repository of uncharacterized genetic diversity in the enterococci of invertebrates. We directly tested this prospect by examining enterococci of invertebrate hosts in a largely natural and pastoral environment. Our findings provide clear evidence that invertebrates naturally harbor vast unexplored enterococcal diversity. Moreover, associations are likely driven by intrinsic host selection factors rather than geographic isolation. This expands our knowledge of Enterococcus biodiversity, including the identification of four novel species, identifying a vast reservoir of enterococcal genes available to species that colonize and infect humans.
Human fungal infections, especially those caused by Aspergillus fumigatus, pose a significant global health threat, particularly in immunocompromised individuals. Azole antifungals are the primary treatment for this pathogen; however, the prevalence of azole-resistant A. fumigatus strains is steadily increasing. Mutations in cyp51A, which encodes an enzyme involved in ergosterol biosynthesis and the molecular target of the azoles, are well established to confer resistance in this fungal species. However, additional mechanisms governing resistance to this antifungal class remain understudied and poorly characterized, despite growing recognition of their importance in clinical resistance. In this study, we investigated the genetic basis of azole resistance in A. fumigatus isolates from clinical settings worldwide, with a particular focus on mechanisms independent of cyp51A (non-canonical). Using a combination of genomic and functional approaches, including whole-genome sequencing and transcriptomic analysis, we identified novel genetic variants and characterized population structure, advancing our understanding of the genetic diversity and evolutionary dynamics of resistance in A. fumigatus. By expanding our understanding of the complex genetic and molecular factors underlying azole resistance in this important human fungal pathogen, this research is poised to inform the development of novel antifungal strategies and contribute to global efforts to combat fungal infections. IMPORTANCE:Azole antifungals are the frontline therapy for infections caused by the opportunistic mold Aspergillus fumigatus, yet resistance to these drugs is rapidly increasing worldwide. Most studies have focused on mutations in cyp51A, the canonical target of azoles; however, a growing proportion of resistant clinical isolates lack these mutations, indicating that alternative resistance mechanisms are emerging. Here, we integrate population genomics, transcriptomics, and functional analyses across a global collection of isolates to define the architecture of cyp51-independent (non-canonical) azole resistance. We show that this resistance phenotype is strongly associated with a distinct population lineage and is driven by a highly polygenic network of metabolic, mitochondrial, and regulatory adaptations rather than single target site mutations. These isolates exhibit extensive transcriptional rewiring and metabolic remodeling under azole stress, suggesting distinct survival strategies beyond canonical resistance. Our findings reveal that azole resistance in A. fumigatus can evolve through diverse evolutionary routes and emphasize the need to monitor and therapeutically target non-canonical pathways that may increasingly contribute to antifungal treatment failure.
Environmental conditions strongly influence interactions between bacteria and bacteriophages (phages). Here, we examined how osmolality (solute concentration) shapes the in vitro co-evolution of T4 phage and its host Escherichia coli during serial passage. When evolved independently, we observed substantial fitness gains in both bacteria and phages, particularly in high-osmotic conditions. During co-evolution, however, fitness gains were limited, bacterial populations consistently evolved phage resistance, and several phage populations went extinct. Furthermore, the resistance mechanisms varied by osmolality. In lower osmolalities, mutations disrupted phage-binding sites, conferring strong resistance. In higher osmolalities, mutations led to increased colonic acid production, producing a mucoid phenotype with weaker resistance. Because mucoidy has been associated with increased bacterial virulence, these findings suggest that gut-relevant osmotic conditions may constrain evolutionary trajectories, favoring resistance strategies that are less effective against phage but potentially more virulent, with important implications for phage therapy design.Phages offer a promising alternative to antibiotics, but their safety and efficacy strongly depend on the environmental conditions where the bacteria and phages interact. In the human gut, for instance, solute concentrations can vary widely due to factors such as food intolerances or laxative use. In this study, we show that such variations significantly impact how bacteria and phages co-evolve. In particular, we find that in higher osmolalities, bacteria evolve phage resistance through mucoidy-a phenotype linked with increased bacterial virulence-rather than receptor loss. This highlights the need to consider environmental factors when developing phage therapies.
Polymorphic toxins mediate interbacterial antagonism among competitors in the gut microbiome. Nuclease effectors, distantly related to the type VI-secreted Bacteroidales Tde, are enriched in human gut Bacillota. Tde mediates antagonism among Bacillota, and expression of the cognate immunity, Tdi, in recipients is protective. Crystal structures of Tde/Tdi complexes from two Bacillus spp. and Enterococcus quebecensis highlight a conserved mechanism of immunity. Tdi engages Tde with high-affinity, specific binding at an interface that features predominantly polar amino acids. A separate Tdi interface has a very highly conserved P(Φ)4GG motif that structurally mimics and displaces a short helix in Tde's active site, which contains the critical catalytic residues. An isolated P(Φ)4GG motif peptide is sufficient for Tde nuclease activity inhibition at high concentrations. However, key residues at both the polar interface and P(Φ)4GG are required for complete inhibition of nuclease activity and protection against toxicity. We propose a multivalent Tde/Tdi neutralization mechanism where an initial high-affinity interface increases the local concentration of Tdi's P(Φ)4GG motif, enabling it to displace the Tde active site through structural mimicry. The resulting conformational rearrangement of Tde increases its flexibility in solution and susceptibility to proteolysis, which may aid in eliminating the toxic effector.IMPORTANCEBacteria in the gut microbiome compete using toxin secretion systems. Prior research has emphasized the importance of secretion systems in gram-negative bacteria. We describe a class of secreted nuclease effectors (toxins) and protective immunity proteins that are enriched in gram-positive Bacillota in human gut microbiomes. These effector/immunity pairs mediate antagonism among Bacillus and Enterococcus spp. The immunity proteins neutralize the nuclease effector through a unique mechanism of enzymatic active site mimicry. The immunity proteins bind effectors with very high-affinity at an interface with polar residues. The effector undergoes a large conformational change. A very highly conserved motif on the immunity surface competitively displaces an active site short helix and loop that contains the key catalytic residues. This rearrangement of the effector renders it inactive and susceptible to elimination by proteolysis.
End-elongation of amyloid fibrils is a prevailing theory to explain prion replication, but direct experimental evidence for this phenomenon is limited by the lack of research tools. To serve as molecular probes for prion fibril termini, here, we designed protein binders against high-resolution structures of infectious prion fibrils using a diffusion-based design model. By generating protein scaffolds around short β-strand segments from terminal prion rungs, we designed β-hairpin-interfacing proteins that cap prion fibrils, which we termed PRICAPs. We validated that PRICAPs exhibit binding to prion fibril termini and confirmed the role of prion fibril ends in replication by demonstrating that PRICAPs inhibit prion seeding activity and attenuate prion replication in organotypic cerebellar slice cultures. Collectively, these findings describe a class of prion-capping protein that can be used to probe prion fibril termini and verify that these surfaces contribute to prion replication and infectivity. IMPORTANCE:Prions replicate by templating the misfolding of native proteins; however, direct evidence identifying the precise sites of replication has remained limited. Here, we address this gap by developing a new class of rationally designed protein tools that specifically bind and cap the ends of infectious prion fibrils. Using these probes, we demonstrate that fibril termini are replication-competent surfaces required for prion seeding and propagation. By inhibiting these sites, our designed proteins markedly reduce prion replication in a disease-relevant ex vivo system, providing functional validation of the end-elongation model. Beyond resolving a fundamental question in prion biology, this work establishes a generalizable strategy for targeting amyloid fibril ends with high specificity. These findings have broad implications for understanding protein aggregation in neurodegenerative diseases and open new avenues for the development of therapeutics that selectively disrupt pathogenic amyloid propagation.
The class Bunyaviricetes encompasses several highly pathogenic viruses that cause lethal hemorrhagic fevers. Due to their limited prevention and treatment options and high pathogenicity, these viruses require handling in biosafety level-4 facilities. Within the bunyaviruses, arenaviruses are particularly notable for their pathogenicity and ability to cause severe hemorrhagic disease in humans. The cap-dependent endonuclease (CEN) is a unique and crucial enzyme involved in the replication cycle of these viruses. As humans do not possess a similar enzyme, CEN represents an ideal target for antiviral drug development with reduced risk of side effects. Recently, we identified a promising CEN inhibitor (CENi) demonstrating potent inhibition of virus replication. In this manuscript, we demonstrate the successful therapeutic efficacy of CENis against Lassa fever and Argentine hemorrhagic fever virus infections in guinea pig models of lethal hemorrhagic fever. In addition, we identified several CENis with antiviral activity against other highly pathogenic arenaviruses. These findings further support the potential of CENis as therapeutic agents for arenavirus infections that cause severe and often lethal hemorrhagic fever. Collectively, our results suggest that CENis are promising candidates for pan-arenavirus therapy and may also have broader utility against other CEN-containing viruses for which no approved antiviral treatments currently exist.IMPORTANCEArenaviruses, such as Lassa virus and Junin virus, cause severe hemorrhagic fevers in humans and are associated with high mortality rates and limited treatment options. Because of their pathogenicity and potential for outbreaks, these viruses represent an important global health threat. The viral cap-dependent endonuclease (CEN) plays a critical role in arenavirus replication by enabling the cap-snatching process required for viral mRNA synthesis. Notably, mammalian cells lack a comparable enzyme, making CEN an attractive target for antiviral drug development. In this study, we demonstrate that inhibitors targeting the arenavirus CEN effectively suppress viral replication and provide therapeutic protection in animal models of lethal Lassa virus and Junin virus infections. We also identify compounds with antiviral activity against multiple highly pathogenic arenaviruses. These findings establish CEN inhibition as a promising strategy for the development of broad-spectrum antivirals against arenaviruses that cause life-threatening hemorrhagic fevers.
Neurotropic coronaviruses can invade the central nervous system (CNS) and cause severe neurological disease, but the mechanisms underlying virus-induced neuroinflammation remain incompletely understood. Here, we report that infection with porcine hemagglutinating encephalomyelitis virus (PHEV), a neurotropic betacoronavirus, is associated with mitochondrial dysfunction, including increased reactive oxygen species (ROS) production and loss of the mitochondrial membrane potential (ΔΨm). Our data identify the NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome as an important component connecting PHEV infection, mitochondrial stress, and PANoptosis. The viral nucleocapsid (N) protein interacted with the pyrin domain (PYD) of NLRP3 through its C-terminal region and facilitated inflammasome assembly without detectably disrupting the NLRP3-NIMA-related kinase 7 (NEK7) interaction. Pharmacological inhibition of NLRP3 or caspase-1, as well as genetic knockout of NLRP3, significantly reduced viral load, attenuated neuroinflammation, and delayed disease progression in PHEV-infected mice. Moreover, NLRP3 deficiency shifted the cell-death modality from PANoptosis to necroptosis, thereby mitigating CNS immunopathology. Together, these findings demonstrate that PHEV hijacks the NLRP3-mitochondrial axis to exacerbate neuroinflammatory injury and support further evaluation of inflammasome- and PANoptosis-related pathways as therapeutic targets for neurotropic coronavirus infections.IMPORTANCEPorcine hemagglutinating encephalomyelitis virus (PHEV) is a neurotropic betacoronavirus that primarily invades the central nervous system (CNS) and induces fatal encephalomyelitis in young piglets. However, the host and viral determinants that contribute to PHEV-induced brain injury remain incompletely defined. In this paper, we show that PHEV infection activates the host NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammatory pathway and that this response contributes to CNS immunopathology. Loss of NLRP3 shifts the infection-associated cell death program from PANoptosis to necroptosis. Blocking this pathway reduces viral load, attenuates neuroinflammation, and improves survival in infected mice. These findings expand our understanding of host responses to neuroinvasive coronaviruses and highlight inflammasome signaling as a candidate therapeutic target for controlling coronavirus-associated encephalitis in swine.
In chronic, recalcitrant infections, Pseudomonas aeruginosa forms biofilm aggregates that are encapsulated in a protective extracellular matrix made of exopolysaccharides, extracellular DNA, and matrix proteins. Biofilm formation is a major cause of antibiotic clearance failure in P. aeruginosa, and there is growing pressure to elucidate biofilm preventative measures and develop novel anti-biofilm treatments. Toward this goal, much work has been done to understand the genetic adaptations P. aeruginosa undergoes in chronic respiratory infections, but how the mucosal environment, found in the lung, impacts P. aeruginosa behavior is unclear. Mucus is a protective hydrogel lining wet epithelial cells and is mainly composed of water and its structural component, the glycoprotein mucin. Using commercially purified porcine gastric mucin, which is missing its end tail regions and has modified glycosylation, as a model of structurally altered mucus, we show that P. aeruginosa rapidly increases surface exploration via twitching motility, depositing trails of the exopolysaccharide Psl and elevating total Psl levels. This increased Psl production occurs on short timescales (<1 h), is independent of transcriptional or translational regulation, and enhances tolerance to select antimicrobials, including hydrogen peroxide and ciprofloxacin. Together, these findings indicate that structurally altered mucus environments function as contextual signals that elicit rapid, non-genetic bacterial adaptive behaviors relevant to persistence in chronic infection.IMPORTANCEUnderstanding how the host environment impacts pathogen behavior is crucial for developing effective treatments for infections. Here, we provide evidence that the mucosal glycoprotein, mucin, and the O-glycans that decorate the mucin protein backbone alter surface exploration by stimulating twitching motility in the opportunistic pathogen Pseudomonas aeruginosa. This increase in twitching then results in an extensive network of Psl trails, which are a precursor to microcolony formation. Mucin additionally promotes Psl production in a novel post-translational manner across a small cohort of isolates, suggesting Psl production by mucin is conserved. This increase in Psl production was found to also protect cells from killing by antimicrobials. Independent of mucin, we present evidence that the secondary messenger, c-di-GMP, can also post-translationally regulate Psl production, which is the first evidence of post-translational regulation for this exopolysaccharide.
