Table of Contents

As the research landscape evolves, scientific societies must adapt their programs to meet changing community needs. The American Academy of Microbiology (Academy or AAM) has recently developed a new model centered around scientific portfolios aimed at advancing its vision of becoming an effective scientific think tank. Here, we describe this transition and the process used to develop and implement a portfolio-based approach. We highlight the Climate Change and Microbes Scientific Portfolio as a case study, demonstrating its successes and its ability to guide the design of future portfolios.

Showing the data is the first rule of effective figures, yet this mandate is often ignored. Perhaps the signature offender is the “dynamite plot”—a bar graph showing mean and error. Here, we evaluate recent trends in the use of dynamite plots by analyzing 8,834 figures from 2,930 studies published between 2021 and 2025 in 18 journals from five fields of biology. We find that dynamite plots constitute ~25% of figures and are especially common in microbiology journals. However, the use of dynamite plots has declined substantially across fields and journals from 30% of figures in 2021 to 18% in 2025—evidence that biologists increasingly show the data in their figures. We advocate for authors, reviewers, and editors to continue this trend, suggest simple dot plots as an effective replacement for dynamite plots, and describe other options when space or sample size makes dot plots less feasible.

Beyond essential roles as central hubs integrating homeostatic cellular metabolism, mitochondria have emerged as critical determinants of infection outcomes. Mitochondrial activities, like MAVS signaling and the release of cytochrome c and mitochondrial DNA, drive host defenses. Across cell types, mitochondrial metabolism and antiviral responses are also increasingly being connected by evidence such as viral-encoded antagonists. Nonetheless, metabolic rewiring in infected cells is still largely viewed as a means to satisfy biosynthetic demands for both viral replication and the host response. However, perturbation of metabolic states within infected and bystander cells seemingly has consequences for outcomes, implying an incompletely understood metabo-immunoregulatory logic. Here, we consider roles for mitochondrial metabolism reprogramming as an active cue that licenses progressive immune states to adapt host responses. In the coming years, integration of mitochondrial biology and new methodologies, including spatial approaches, will illuminate the interplay of mitochondrial metabolism on primary antiviral responses.

Ecological guilds are groups of organisms that utilize the same class of resources and occupy similar niches, regardless of their taxonomic identities. Here we propose the Guild Model for Cystic Fibrosis Airway Microbial Ecology, which considers the ecological function and wider role of each microbe in the ecosystem. This model consists of four functional guilds: (i) “Brewers” metabolize host-derived substrates (e.g., mucins) and produce fermentation products; (ii) “Drunkards” exploit the metabolic niche built by Brewers, consuming fermentation products and secreting exopolysaccharides to build biofilms; (iii) “Putrifiers” produce toxic compounds causing inflammation and tissue necrosis; and (iv) “Nihilists” are specialist pathogens characterized by intracellular or lytic life cycles and cytotoxin production. By focusing on microbial function and the broader community context, this model offers a refined framework for interpreting cystic fibrosis airway ecology. Although developed for CF, the Guild Model is adaptable to other diseases influenced by microbial ecology.

In this study, we investigated the impact of G protein-coupled receptor (GPCR) signaling on the intracellular replication of the model pathogen Brucella neotomae. Building on a prior chemical genetics screen, we identified agonists of the Gαi-coupled adenosine A1 and dopamine D4 receptors as potent inhibitors of intracellular Brucella replication. In contrast, agonists of Gαs-coupled adenosine A2A or dopamine D1 receptors, as well as antagonists of A1 or D4 receptors, either failed to inhibit or enhanced intracellular replication. Wild-type B. neotomae induced a rapid, type IV secretion system-dependent increase in host-cell cAMP during early infection. ENBA and cilostamide prevented this infection-associated cAMP increase and completely inhibited intracellular growth; this effect was partially reversed by cell-permeable cAMP analogs. Using a real-time NanoBRET biosensor, we detected rapid Gαs activation within minutes of infection that was sustained during wild type but not ΔvirB4 infection and was abrogated by ENBA or cilostamide. Disruption of early Gαs-cAMP signaling redirected Brucella-containing vacuoles (BCVs) to replication-incompatible phagolysosomal and autophagy-associated compartments. Collectively, these data support a model in which early GPCR signaling dynamics, balancing Gαs and Gαi pathways, are critical for the establishment of productive intracellular Brucella infection.

IMPORTANCE

Brucella species cause chronic infections by surviving and multiplying inside immune cells. To do this, Brucella must remodel the membrane-bound compartment that surrounds it after uptake, steering it away from destructive lysosomes and toward a permissive niche where replication can occur. We found that Brucella rapidly triggers a host signaling response controlled by G protein-coupled receptors, leading to a rise in a common cellular messenger molecule (cAMP) within minutes of infection. This early signal depends on the bacterial type IV secretion system and is required to build the replication-permissive compartment. When we disrupted this signaling with small molecules, bacteria were rerouted into degradative, autophagy-associated compartments and failed to establish productive infection. These results reveal an early host signaling checkpoint that Brucella engages to establish its intracellular niche and suggest that targeting host signaling dynamics, rather than bacterial viability directly, may provide new strategies to block intracellular infection.

Coronaviruses (CoVs) replicate their RNA genomes with a higher degree of fidelity than other RNA viruses, a mechanism mediated by the proofreading and recombination activities of the exoribonuclease domain of replicase nonstructural protein 14 (nsp14-ExoN). Both murine hepatitis virus (MHV) and SARS-CoV tolerate nsp14-ExoN loss-of-function mutations (ExoN−) (D90A and E92A), but have impaired replication fidelity and pathogenesis; yet identical substitutions in MERS-CoV and SARS-CoV-2 have been reported to be lethal. Here, we report a saturation mutagenesis approach facilitating the recovery and analysis of several constellations of SARS-CoV-2 nsp14 ExoN-inactivating, loss-of-function substitutions, including the canonical D90A and E92A. Biochemical assays with purified WT or ExoN-nsp10-14 fusion proteins confirmed that active site substitutions abolished ExoN activity (ExoN−). SARS-CoV-2 ExoN− viruses exhibited impaired replication, RNA synthesis, and recombination, as well as decreased replication fidelity and loss of fitness in vitro. ExoN− viruses were significantly attenuated for replication in human primary airway epithelial cells and were attenuated for replication and pathogenesis in WT mice, as well as the highly susceptible K18 transgenic mice. In the absence of interferon signaling in vivo, SARS-CoV and SARS-CoV-2 ExoN− viral replication could be partially restored. These results demonstrate that SARS-CoV-2 ExoN− viruses are viable but highly impaired for replication, fitness, and fidelity in vitro, as well as innate immune antagonism and pathogenesis in vivo. Collectively, our results solidify the multiple critical roles of nsp14-ExoN across CoV genera and establish new approaches for rescuing and analyzing loss-of-function substitutions in studies of CoV replication, pathogenesis, and evolution.

IMPORTANCE

Coronaviruses (CoV) are important human pathogens causing hundreds of millions of infections and millions of deaths over the past 20 years. The study of how these viruses multiply and cause disease identifies points of attack for therapeutics. Using a high-throughput genetic approach, we systematically inactivated an essential enzyme CoV needs for replication called ExoN. We show that without ExoN, CoV replication fidelity and fitness are reduced in cell culture. Replication without ExoN in mice was diminished but could be partially restored in mice that lack key components of the immune response. Altogether, we reveal new insights into the complexities of CoV replication and virus and host interactions, which could be leveraged for the development of novel multifaceted therapeutics that attack the ever-expanding functions of the CoV replication complex in replication and pathogenesis

Klebsiella pneumoniae is a leading cause of global deaths due to antibiotic resistance. Of particular concern is the rapid expansion of resistance to beta-lactam antibiotics within K. pneumoniae lineages. The environmental factors that influence pathogen physiology and, subsequently, antibiotic resistance remain poorly understood. Here, we demonstrate that physiologically relevant reductions in pH increased K. pneumoniae beta-lactam resistance as much as 64-fold, with the most dramatic increase observed for beta-lactams that specifically inhibit cell division. We identified two genes that contribute to acid-dependent beta-lactam resistance: the class A penicillin-binding protein (PBP), PBP1b, and the paralogous class B PBP, PBP3_PARA. Loss of either PBP1b or PBP3_PARA increases K. pneumoniae susceptibility to beta-lactams at low pH. Altogether, these data emphasize the importance of functional redundancy among cell wall synthesis enzymes, which allows for specialization and ensures robust cell wall synthesis across a range of environmental conditions.

IMPORTANCE

Beta-lactams are the most prescribed class of antibiotics, but their effectiveness is threatened by a global rise in antimicrobial resistance. How the environment within a host or infection site shapes pathogen response to antibiotics is frequently overlooked in assessments of antibiotic effectiveness. We demonstrate that growth at physiologically relevant low pH substantially increases Klebsiella pneumoniae resistance to clinically important beta-lactams. An important finding of this study is that during growth in acidic pH, K. pneumoniae has a different repertoire of cell wall synthesis genes available than during growth at neutral pH due to the presence of acid-inducible paralogous copies of essential cell wall synthesis enzymes, PBP2 and PBP3. An additional functionally redundant enzyme, PBP1b, also contributes to acid-dependent beta-lactam resistance. Together, these findings expand our understanding of how bacteria maintain cell wall synthesis across diverse physicochemical environments and highlight potential new therapeutic targets.

Since the COVID-19 pandemic, several reverse genetics platforms for SARS-CoV-2 have been established. In general, a plasmid-based reverse genetics system is stable and easy to manipulate, distribute, and store. However, traditional methods for the assembly of a large viral genome in a plasmid rely on natural and artificially engineered restriction sites, which are inefficient, time-consuming, labor-intensive, and frequently not successful. Here, we developed a yeast-based homologous recombination system that allows the assembly of the SARS-CoV-2 genome as a cDNA in a bacterial artificial chromosome (BAC) plasmid in a single step. The entire protocol from cDNA construction to virus rescue is simple, rapid, accurate, highly efficient, and can be completed in 2 weeks. Using this system, we have quickly generated recombinant SARS-CoV-2 (rSARS-CoV-2) WA1, Omicron BA.2.86, and Omicron JN.1 viruses expressing mCherry, green fluorescent protein (GFP), and NanoLuc luciferase (Nluc) reporters. Insertion of these reporter genes does not significantly alter the replication of SARS-CoV-2 in cell culture. We also compared the replication kinetics of rSARS-CoV-2-WA1, BA.2.86, and JN.1 reporter viruses in ex vivo primary human nasal epithelial (HNE) and human bronchial epithelial (HBE) cultures. Omicron BA.2.86 replicated and spread more efficiently than JN.1, which spread much faster than SARS-CoV-2 WA1 in these cultures. In summary, we have developed a highly efficient yeast-based recombinant system for the construction of infectious cDNA clones of SARS-CoV-2, enabling rapid genetic manipulation of SARS-CoV-2. In addition, the reporter viruses generated in this study will be useful for monitoring SARS-CoV-2 infection in vitro and in vivo.

IMPORTANCE

Reverse genetics systems are an essential tool for probing the biology of viruses, testing antivirals, and developing live-attenuated vaccines. However, it has been a challenge to generate a rapid reverse genetics system for coronaviruses. Here, we developed a rapid, highly efficient reverse genetics system for SARS-CoV-2 that uses yeast homologous recombination. In this procedure, overlapping DNA fragments encompassing the entire SARS-CoV-2 and BAC plasmid fragments containing a yeast replication origin were mixed and transformed into yeast cells to assemble infectious cDNA clones in a single step. This system has enabled us to rapidly generate nine SARS-CoV-2 viruses: WA1, Omicron BA.2.86, and JN.1 viruses each expressing one of three reporters for tracking virus infection in vitro and in vivo. This method is easy, convenient, and highly efficient, generating infectious cDNA clones within 2 weeks. This system could readily be adapted to construct infectious cDNA clones for other large RNA viruses.

In Pseudomonas aeruginosa, the sigma factor AlgU functions as a critical stress-responsive regulator governing essential physiological processes. This study assessed algU-dependent phenotypes through targeted gene knockout, including effects on alginate secretion and host cell invasion. Furthermore, an innovative fusion protein strategy addressed complex preparation challenges, enabling the determination of a high-resolution (2.101 Å) structure of the AlgU-MucA^cyto complex and revealing an exceptionally low backbone deviation of merely 0.01 Å relative to the reported 6IN7 complex. Structural analysis revealed an extensive mutation-resistant interaction network at the binding interface, stabilized primarily by salt bridges, hydrogen bonds, and hydrophobic interactions. Subsequent mutational validation confirmed that triple or sextuple alanine substitutions at critical interaction sites maintained complex binding integrity while significantly compromising thermal stability, indicating that although dispensable for binding, these residues are indispensable for complex stability. Crucially, pull-down assays identified SspB as the essential adaptor bridging the AlgU-MucA^cyto complex to the ClpX/ClpP protease; AlphaFold3 modeling and quantitative BLI measurements reveal that SspB engages AlgU via its N-terminal domain and binds ClpX through its disordered C-terminus, enabling substrate delivery and proteolysis-coupled AlgU release. Collectively, these findings elucidate a dual-safeguard regulatory mechanism: inherent mutation resistance at the binding interface prevents aberrant AlgU activation by common clinical mucA mutations, while SspB-mediated proteolysis ensures stimulus-responsive AlgU liberation. This evolutionary adaptation confers resistance against detrimental AlgU overactivation in P. aeruginosa and provides novel therapeutic targets.

IMPORTANCE

Deletion of the algU gene affected the production of pyoverdine, pyocyanin, and alginate in Pseudomonas aeruginosa, as well as its cell invasiveness. By fusing AlgU and MucA^cyto, we were able to obtain an AlgU-MucA^cyto protein complex with both high yield and purity, and successfully obtained a crystal structure of the AlgU-MucA^cyto complex. Structural analysis revealed an extensive and robust network of salt bridges, hydrogen bonds, and charge interactions between the two proteins. Multi-site alanine substitutions (up to sextuple mutations) in interfacial charged clusters of the AlgU-MucA^cyto complex fail to disrupt binding but severely impair thermal stability, revealing that these residues are binding-redundant yet stability-essential. Structural modeling and BLI measurements identify SspB as a dual-interface adaptor: its N-terminal domain recognizes the preassembled AlgU-MucA^cyto complex (via the AlgU hairpin surface), whereas its flexible C-terminal tail engages ClpX, thereby bridging AlgU-MucA^cyto to ClpXP and positioning MucA^cyto for proteolysis-coupled AlgU release.

The global regulator LaeA is widely recognized as a master activator of secondary metabolism and development in filamentous fungi. Yet its role under genetically buffered or metabolically stable conditions—where canonical phenotypes are masked—remains poorly understood. This study aimed to characterize the LaeA ortholog (ThlaeA) in Trichoderma hypoxylon and to elucidate its regulatory role in a ΔThtri5 background, where trichodiene synthase function is disrupted. We constructed single and double knockout mutants (ΔThlaeA, ΔThtri5, and ΔThlaeAΔThtri5) and performed integrated metabolomic and transcriptomic analyses to assess global regulatory effects. Additional assays evaluated oxidative stress responses and biocontrol activity. The deletion of ThlaeA alone had negligible effects on secondary metabolite production, whereas disruption of ThlaeA in the ΔThtri5 background restored biosynthesis of major terpenoid compounds abolished in ΔThtri5. Metabolomics revealed that ThlaeA regulates 48.4% of metabolites in the wild-type but 31.3% in ΔThtri5, while transcriptomics showed restoration of 16.7% of gene expression dysregulated in ΔThtri5. Phenotypically, ThlaeA deletion reinstated oxidative stress sensitivity and partially attenuated biocontrol efficacy. ThlaeA acts as a conditional repressor that counterbalances Thtri5-dependent perturbations to maintain metabolic homeostasis. This work redefines the LaeA paradigm and provides a framework for understanding the context-dependent regulation of secondary metabolism in fungi.

IMPORTANCE

LaeA plays compelling roles in secondary metabolism and development in filamentous fungi. However, related research also found that genetic operations of LaeA have no obvious effect on the metabolic spectrum in some fungal species. Many attempts have been made to decipher the phenomenon and to explain how about the function of LaeA in these cases. Here, we identified a ThlaeA in Trichoderma hypoxylon. The deletion of ThlaeA alone did not alter secondary metabolism but restored metabolite production in a ΔThtri5 background. Integrated metabolomic and transcriptomic analyses revealed that ThlaeA modulates metabolic homeostasis by compensating for Thtri5-related perturbations. ThlaeA-Thtri5 interaction regulates oxidative stress responses and membrane transport pathways, coupling secondary metabolism with physiological adaptation. This regulatory model broadens the understanding of the LaeA protein family. Fine-tuning this pathway can enhance the environmental adaptability and agricultural biocontrol potential of Trichoderma strains, while boosting bioactive secondary metabolite production and optimizing fungal cell factories. This study advances fundamental insights into fungal metabolic regulation and provides a rational basis for strain improvement and biotechnological applications in agriculture and industry.

Invasive mold diseases (IMDs) such as mucormycosis and aspergillosis carry high mortality despite optimal antifungal therapy. Adjunctive immunomodulation, including anti-PD-1 antibodies and interferon-γ, may help restore antifungal immunity in severely ill patients. We implemented multidisciplinary team meetings in France to assess and validate the use of adjunctive anti-PD-1 monoclonal antibodies and interferon-γ in combination with antifungal therapy for patients with life-threatening IMDs. Here, we report the characteristics and outcomes of the treated patients. Twelve cases were reviewed, and eight patients ultimately received adjunctive therapy. All presented with particularly severe IMDs, including seven mucormycosis (three cerebral, three intra-abdominal, and one extensive fasciitis) and one pulmonary aspergillosis. PD-1 expression on T cells was elevated in all patients. Following adjunctive treatment, six of the eight patients survived. Therapy was generally well tolerated; serious adverse events included one episode of cutaneous toxicity and two cases of acute respiratory distress syndrome, possibly related to immunotherapy. All resolved after treatment discontinuation. In our experience, adjunctive immunotherapy combined with antifungals was mostly associated with favorable outcomes in severe, refractory IMDs. These findings support further investigation of host-directed strategies as potential adjuncts in managing life-threatening fungal diseases.

IMPORTANCE

This study provides preliminary evidence supporting the clinical relevance of host-directed immunotherapy as an adjunct to antifungal treatment in severe invasive mold diseases (IMDs), which remain associated with high mortality despite optimized antifungal regimens. By targeting immune dysfunction, particularly T-cell exhaustion mediated through the PD-1 pathway, the combined use of anti-PD-1 monoclonal antibodies and interferon-γ aims to restore effective antifungal immune responses. The observed survival benefit in this small cohort supports the biological rationale that reversing immune paralysis can enhance pathogen clearance in patients with IMDs. Although limited by sample size, this work provides encouraging evidence supporting the feasibility, tolerability, and potential efficacy of combined immunomodulatory strategies, thereby contributing to the evolving paradigm of personalized and immune-guided management in IMDs. Future work should focus on biomarker-guided patient selection, rigorous monitoring, and determining the optimal timing for therapy initiation. Integrating immunological profiling into patient assessment may enable more precise, stratified therapeutic approaches.

Epstein-Barr virus (EBV) latent infection is causally linked to several epithelial cancers, including endemic forms of undifferentiated nasopharyngeal carcinoma (NPC), and to a subtype of gastric cancer (GC). EBNA1 is the virus-encoded sequence-specific DNA-binding protein required for episome maintenance but also contributes to host-cell survival through multiple mechanisms, including binding to the host chromosome. We previously developed small-molecule inhibitors of EBNA1 DNA-binding that block host cell cycle progression and growth of EBV+ cell lines and tumor models in vivo. However, the underlying molecular mechanisms of EBNA1 function and inhibition have not been completely elucidated. In this study, we employ VK1727 to inhibit EBNA1 DNA-binding to viral and cellular genomes in three EBV+ epithelial tumor-derived cell models (patient-derived xenograft C15, C666-1, and SNU719). We integrate EBNA1 ChIP-seq and transcriptomic RNA-seq analyses to identify the cell cycle-dependent kinase CDC7 and a stem cell transcription factor POU2F1 as direct functional targets of EBNA1 in each of these epithelial cancer models. EBNA1 binding to the CDC7 promoter and POU2F1 intron promotes RNA Pol II-pS5 to initiate transcription of these two genes. We show that CDC7 inhibitor simurosertib phenocopies VK1727, while Bcl-2 inhibitor venetoclax shows a synergistic effect with VK1727 for inhibition of EBV+ epithelial cancer cell proliferation and survival. Our study reveals new functional gene targets and pathways of VK1727 in EBV+ epithelial cancers that provide new biomarkers and combinatorial strategies to treat EBV-driven cancers.

IMPORTANCE

EBNA1 is essential for Epstein-Barr virus (EBV) latency and tumorigenesis, but its mechanism of action on host gene expression is not yet known. Small-molecule inhibitors of EBNA1 DNA-binding block cell cycle progression and inhibit the growth of EBV+ tumors. In this study, we use the EBNA1 small-molecule inhibitor VK1727 to identify cellular gene targets that are bound by EBNA1 and deregulated by its pharmacological inhibition in EBV+ epithelial cancer cell lines and an NPC patient-derived xenograft mouse model. We identify cell cycle-dependent kinase CDC7 and the stem cell transcription factor POU2F1 as EBNA1-bound and regulated genes important for EBV epithelial cancer proliferation. These findings not only decipher the molecular mechanism by which VK1727 blocks cell cycle progression and inhibits cell proliferation but also provide two new cellular gene targets and pathways for therapeutic intervention in EBV+ epithelial cancers.

Antiviral defenses at mucosal barriers are essential for preventing viral entry and systemic infection. Interferon epsilon (IFNε) is a unique type I IFN that, unlike other family members, is not induced by infection but is constitutively expressed in epithelial tissues. IFNε was initially characterized in the female reproductive tract (FRT), where it provides broad antiviral protection, but its roles outside the FRT remain poorly defined. Here, we used Ifnε^−/− mice and single-cell RNA sequencing to delineate IFNε function across distinct mucosal surfaces. In the FRT, Ifnε expression was restricted to specific epithelial subsets, was independent of estrous stage, and maintained basal ISG expression. IFNε was also retained intracellularly in primary human FRT-derived cells. Extending these analyses to the intestine, we found that IFNε is highly expressed in villous-tip enterocytes of the small intestine in vivo, where it sustains inflammatory enterocyte subsets and maintains type III IFN expression. Loss of Ifnε depleted these subsets and rendered mice more susceptible to enteric viral infection. Together, these findings establish IFNε as a constitutively expressed, spatially restricted IFN that coordinates mucosal antiviral defenses across both reproductive and gastrointestinal epithelial tissues.

IMPORTANCE

Interferon epsilon (IFNε) is a unique type I IFN that, unlike other family members, is not induced by infection but is constitutively expressed in epithelial tissues. In this manuscript, we define the epithelial cell types that constitutively express IFNε in the uterus and small intestine at a single-cell resolution. We show that mice lacking IFNε lose key antiviral defenses in a tissue-dependent manner; uterine epithelial cells have diminished basal ISG expression, and key populations of cytokine-expressing enterocytes are absent from the small intestine. In the intestine, this correlates with increased susceptibility to infection with an enteric virus in mice. These findings establish IFNε as a key contributor to mucosal immunity, sustaining antiviral defenses within tissue-specific epithelial cells of both the female reproductive tract and intestine, and broaden our understanding of its role beyond traditional pathogen-induced interferon responses.

Fungal balls (aspergillomas) are a debilitating complication of chronic pulmonary aspergillosis, but their functional biology as multi-kingdom ecosystems is poorly understood. Through integrated multi-omics analysis of 61 patient-derived fungal balls, we reveal their complex ecology. While Aspergillus fumigatus dominates the fungal niche (59% of patients), bacterial co-colonization is ubiquitous, primarily by Pseudomonas aeruginosa and Haemophilus influenzae. Metabolomics and metatranscriptomics unveil a structured division of labor and active warfare, including metabolic cross-feeding, competition for iron, and reciprocal antagonism via secondary metabolites, such as fumagillin and fumigaclavine C produced by A. fumigatus. Host metabolomics and transcriptomics revealed a potent but dysregulated human immune response, characterized by neutrophil activation and failed resolution. Our findings redefine aspergilloma not as a mere fungal aggregate, but as a resilient polymicrobial biofilm across kingdoms, in which synergistic and antagonistic inter-kingdom interactions drive pathogenesis and chronicity, suggesting new therapeutic strategies targeting the pathogenic consortium.

IMPORTANCE

Chronic pulmonary aspergillosis (CPA) and its hallmark fungal balls (aspergillomas) represent a debilitating and difficult-to-treat respiratory disease, affecting millions worldwide. Here, we provide the first integrated multi-omics profile of surgically resected fungal balls from 61 CPA patients, revealing these structures not as mere fungal colonies, but as resilient, cross-kingdom biofilms teeming with bacterial co-colonizers, particularly Pseudomonas aeruginosa and Haemophilus influenzae. Our findings uncover a dynamic battlefield where fungi and bacteria engage in metabolic cross-feeding, chemical warfare, and competition for nutrients such as iron. We demonstrate that the host mounts a potent but dysregulated immune response characterized by chronic neutrophilic inflammation and failed resolution, driving tissue damage and disease persistence. Our data provide a foundation for novel therapeutic strategies aimed at disrupting microbial synergy, modulating host inflammation, and breaking the cycle of chronic infection, an approach that could significantly improve outcomes for patients with this refractory disease.

Chlamydia trachomatis (C.t.) infections can lead to severe complications due to the pathogen’s ability to evade the host immune response, often resulting in asymptomatic infections. The mechanisms underlying this immune subversion remain incompletely understood, but likely involve specific bacterial effector proteins. Here, we identify CT181 as a novel effector that binds to Mcl-1, a key regulator of neutrophil survival. While a C.t. CT181 mutant exhibited only modest defects in epithelial cell replication and inclusion development, it was required for C.t. survival in neutrophils, which correlated with elevated Mcl-1 levels in cells infected with wild-type C.t. Using a murine infection model, we demonstrate that CT181 contributes to C.t. colonization and inflammatory cytokine production in vivo. Our findings establish CT181 as the first bacterial effector protein known to bind Mcl-1 and show that it is associated with prolonged neutrophil survival, revealing a novel strategy by which C.t. promotes immune dysregulation, facilitating bacterial persistence while driving C.t. pathogenesis.

IMPORTANCE

Chlamydia trachomatis is an obligate intracellular pathogen that must evade early immune defenses to establish infection. This study identifies CT181 as a previously undescribed secreted effector that associates with the host pro-survival protein Mcl-1 and is linked to prolonged neutrophil survival during infection. Neutrophils, which normally undergo rapid apoptosis, persist longer when infected with wild-type C. trachomatis, whereas loss of CT181 reduces bacterial survival in these cells. In a mouse model of infection, the CT181 mutant exhibits reduced bacterial burden and diminished inflammatory responses, including neutrophil recruitment and cytokine production. Together, these findings highlight CT181 as a bacterial factor that contributes to host cell survival and immune modulation during C. trachomatis infection, underscoring the complex strategies used by intracellular pathogens to persist within the host.

DNA phosphorothioate (PT) modification is an epigenetic mark that enables bacteria to discriminate self from non-self DNA, directing restriction effectors to cleave unmodified foreign DNA. In the PT-dependent Ssp system, SspE acts as the restriction effector that recognizes PT marks to block phage propagation. While the mechanism of the Streptomyces homolog (StSspE) is known, the basis for the exceptional potency of Escherichia coli (E. coli) 3234/A SspE (EcSspE) remained unclear. Here, we combine cryo-electron microscopy (cryo-EM), biochemistry, and functional assays in vivo to define its mechanism. The cryo-EM structure reveals that EcSspE forms a dynamic homotetramer with a side-by-side assembly, featuring a substantially reduced inter-subunit interface compared to the intertwined StSspE tetramer. A hydrophobic cavity harboring Y63 specifically recognizes the 5′-C_PSCA-3′ PT motif. This recognition triggers GTP hydrolysis via the essential residue R133. Hydrolysis, in turn, drives an asymmetric allosteric rearrangement that licenses the flexible C-terminal HNH nuclease domain for DNA cleavage. Disrupting PT sensing (Y63A), GTP hydrolysis (R133A), or nuclease activity (N724A) completely abolishes anti-phage defense, confirming strict functional coupling. Our work establishes a conserved “recognize–hydrolyze–activate” paradigm for SspE proteins, wherein PT-stimulated GTPase activity licenses the nuclease via an allosteric switch. The distinct tetrameric architecture of EcSspE likely underlies its enhanced activity by facilitating conformational dynamics. This study elucidates the precise molecular logic of a potent bacterial immune system and provides a framework for engineering phage resistance.

IMPORTANCE

Bacterial antiphage defense systems must precisely destroy invaders while avoiding self-harm. This study provides a high-resolution molecular blueprint of the exceptionally potent PT-dependent Ssp system from E. coli 3234/A. We elucidate its conserved “recognize–hydrolyze–activate” mechanism: the effector EcSspE integrates PT recognition, GTP hydrolysis, and allosteric signaling to license DNA cleavage. Beyond this paradigm, we reveal that subtle evolutionary refinements in its quaternary architecture—a streamlined, side-by-side assembly with a reduced interface—amplify defensive output by enhancing conformational dynamics. This insight bridges structural biophysics and immunity. The system’s strict PT-dependence ensures biosafety, and its defined mechanistic logic and key molecular switches (Y63, R133, N724) establish a framework for engineering programmable phage resistance, advancing both our understanding of host-virus conflict and our ability to harness it.

Carbapenems are last-resort antibiotics against gram-negative pathogens, including multidrug-resistant Campylobacter. However, carbapenem-resistant Campylobacter strains are emerging and have been isolated in patients treated with carbapenems. This emerging resistance mechanism may be underpinned by multiple genetic determinants, yet it has not been systematically characterized or elucidated until now. In this study, two candidate mutations identified via resistance evolution and whole-genome sequencing were validated through genetic and functional assays. De novo structural modeling and molecular dynamics simulations elucidated the impact of mutations on protein dynamics. Furthermore, carbapenem hydrolysis and binding-pocket interactions were characterized using ultra-high-performance liquid chromatography, MM/PBSA calculations, and alanine substitution experiments. Our findings reveal that carbapenem resistance in Campylobacter is mediated by synergistic mutations in porA, encoding the major outer membrane protein (MOMP), and bla_OXA-61, encoding a non-carbapenemase β-lactamase OXA-61. Specifically, the D157H substitution in MOMP induces structural and functional remodeling, thereby hindering meropenem translocation across the outer membrane, while a G → T transversion in the bla_OXA-61 promoter leads to overexpression of the enzyme that sequesters meropenem instead of hydrolyzing it. Notably, either porA alteration or bla_OXA-61 overexpression alone confers only a modest increase in carbapenem resistance (2- to 8-fold), whereas their synergy yields a high-level resistance (16- to 128-fold). Together, these results define a novel carbapenem resistance mechanism mediated by reduced porin permeability coupled with antibiotic sequestration acted by a non-carbapenemase β-lactamase. This synergistic mechanism explains clinical carbapenem resistance phenotypes in Campylobacter, facilitates diagnosis and surveillance, and likely represents a general carbapenem resistance strategy across other bacterial species.

IMPORTANCE

Campylobacter ranks among the leading causes of bacterial gastroenteritis worldwide. In recent years, carbapenem-resistant Campylobacter strains have been emerging in clinical settings and have been increasingly reported from patients following carbapenem treatment. Despite the importance of carbapenems in therapeutic treatment of multidrug-resistant Campylobacter, the molecular basis of this resistance phenotype remains poorly understood. Additionally, non-carbapenemase β-lactamases have been implicated in carbapenem resistance in many bacterial species, but how they contribute to the resistance without hydrolyzing the antibiotic is unknown. Our study defines a new mechanism for carbapenem resistance and accounts for the carbapenem-resistant phenotype observed in clinical isolates. They also provide timely information useful for the diagnosis and treatment of infections caused by antibiotic-resistant Campylobacter. Equally important, this study provides a mechanistic explanation for carbapenem resistance mediated by non-carbapenemase β-lactamases, which exist in many gram-negative pathogens.

Inorganic polyphosphate (polyP) is a linear polymer composed of three to several hundred orthophosphate units linked by high-energy phosphoanhydride bonds and is found in both prokaryotes and eukaryotes. In Trypanosoma cruzi, the causative agent of Chagas disease, polyP plays important roles in osmoregulation and persistence within host tissues and is synthesized by a polyP polymerase known as the vacuolar transporter chaperone (VTC) complex. This complex, localized to acidocalcisomes, is composed of Vtc1 and the catalytic subunit Vtc4. Using CRISPR/Cas9-mediated genome editing, we generated Vtc1 knockout (Vtc1-KO), Vtc4 single knockout (Vtc4-SKO), and conditional knockout lines for both subunits (Vtc1-CKO and Vtc4-CKO). Analysis of these mutants revealed essential roles for Vtc1 and Vtc4 in parasite proliferation, differentiation, and egress from mammalian host cells. Moreover, co-immunoprecipitation and proteomic analyses identified a novel component of the complex, termed TcVtc6, which associates with Vtc1 and Vtc4, forms part of the VTC complex, and is involved in polyP synthesis in Trypanosoma cruzi.

IMPORTANCE

Chagas disease affects millions of people across the Americas and remains a major unmet medical challenge. Here, we investigate the essentiality and molecular composition of the vacuolar transporter chaperone (VTC) complex in Trypanosoma cruzi, the causative agent of the disease. We identify a previously unrecognized component of this complex, which we term TcVtc6, and show that it is involved in polyphosphate synthesis. Functional analyses reveal that the VTC complex is indispensable for parasite differentiation and host cell egress, two processes critical for infectivity. Although the VTC complex is conserved in trypanosomatids, apicomplexans, fungi, and algae, it is absent from mammalian cells. This evolutionary divergence, together with the essential role of the pathway in infectious stages of T. cruzi, highlights the VTC complex as a promising and selective therapeutic target for the treatment of Chagas disease.

Lipid droplets (LDs) and small extracellular vesicles (sEVs) are classically known for lipid metabolism and intercellular communication, respectively. Here, we reveal a mechanistic connection between LD dynamics and sEV-mediated non-lytic release of Japanese encephalitis virus (JEV) from neuronal cells. Using Neuro2A, SHSY-5Y, N9 microglia, and primary cortical neurons, we show that JEV is packaged within sEVs (~200 nm) through an ESCRT-independent, neutral sphingomyelinase 2 (nSMase2)/ceramide-dependent pathway. Virions inside sEVs display a higher JEV premature membrane/membrane protein (PrM/M) ratio compared to those released via the conventional secretory pathway. Although containing a higher proportion of premature virions than mature ones, sEV-associated JEV virions gain an evolutionary advantage by evading immune detection and delivering multiple virions to recipient cells, thereby increasing overall infection efficiency. Temporal profiling showed early cytoplasmic LD enrichment (from 6 hpi), followed by a surge in sEV release from 14 hpi, suggesting sequential roles for LDs and sEVs. nSMase2 inhibition decreased sEV-mediated egress without affecting viral replication but increased cytoplasmic LD abundance, consistent with LD underutilization in multivesicular bodies (MVB) biogenesis. Our findings identify LDs as facilitators of MVB formation and nSMase2 as a key driver of sEV-mediated viral exit, revealing parallel yet coordinated pathways in JEV’s stealthy egress.

IMPORTANCE

Lipid droplets and sEVs are traditionally regarded as regulators of lipid homeostasis and intercellular communication. We propose that this LD–sEV connection represents a key mechanism enabling JEV egress from neuronal cells. Japanese encephalitis virus (JEV) exploits small extracellular vesicles (sEVs) for non-lytic viral release from neuronal cells. sEV-containing JEV (~200 nm) is released via an ESCRT-independent, nSMase2/ceramide-dependent pathway. A higher precursor membrane protein (PrM)-to-membrane protein (M) ratio in MVBs and sEVs suggests packaging of immature JEV via a non-secretory pathway. LDs facilitate MVB formation, facilitating sEV-mediated JEV release. sEV release drives LD utilization for MVB formation; nSMase2 knockdown blocks sEV-mediated egress and causes cytoplasmic LD accumulation. JEV enters neuronal cells, releases its RNA from late endosomes, and replicates in the cytoplasm. Virions are subsequently released either through the conventional secretory pathway or by packaging into multivesicular bodies (MVBs) and secretion within small extracellular vesicles (sEVs). Maturation of JEV requires cleavage of the premature membrane protein (PrM) into the membrane protein (M). Notably, the PrM/M ratio is higher in virions released via the secretory pathway compared to those packaged within sEVs, where a greater proportion of immature virions are enclosed. Lipid droplets (LDs), derived from the endoplasmic reticulum (ER), interact with MVBs and contribute to JEV release inside sEVs. This process is driven by a ceramide-dependent, ESCRT-independent pathway regulated by nSMase2. Together, the model highlights the coordinated role of the LD–sEV axis in mediating JEV non-lytic egress.

Drought has consequences for microbial decomposition rates, including indirect effects through changes in plant litter chemistry. Here, we studied the impact of a decade-long drought on plant litter chemistry and microbial decomposition traits in a semi-arid ecosystem during an 18-month litter bag experiment. We investigated litter sourced from four conditions: grass and shrub vegetation under ambient and reduced precipitation. We hypothesized that litter chemistry drives microbial decomposition capabilities and enzyme activity due to vegetation differences and drought effects on litter chemistry. We found that carbohydrate-rich grass litter had a higher abundance of decomposition genes detected using metagenomics and enzyme activity than more recalcitrant shrub litter, which was richer in lignin and lipids; these patterns were related to substrate supply. Drought decreased some carbohydrate fractions in grass litter but did not change the lignin fraction in grass and shrub litter, suggesting that drought does not make litter more recalcitrant. Most decomposition genes and enzyme activities were not significantly affected by drought, thereby maintaining decomposition rates. Microbial community succession patterns—decreasing fungal abundance and increasing bacterial abundance with time—corresponded with decreasing chitin gene abundance and increasing peptidoglycan gene abundance over time, indicating microbial necromass recycling. We demonstrate minimal litter chemistry-mediated effects of drought but show significant changes in community composition and their decomposition capabilities over time, highlighting that complex microbial-chemical interactions under climate change can influence ecosystem-scale processes.

IMPORTANCE

Climate change is causing more severe and frequent droughts in semi-arid ecosystems, affecting soil microbes breaking down plant litter. Our research focuses on understanding the less studied pathway of drought impact on microbes via changes in plant litter chemistry. Drought can alter the plant litter chemistry by changing the composition and physiology of plants, which can alter microbial decomposition and ecosystem-level carbon cycling. We investigated litter decomposition traits of microbial communities in grass and shrub litter under long-term drought. There were significant changes in litter chemistry under drought but no increase in lignin fraction. Despite this, microbial communities maintained their decomposition capabilities under drought, highlighting the ability of microbes to adapt and continue functioning. We also demonstrate unique microbial community succession patterns and dead biomass recycling, which can have implications for carbon cycling rates in the ecosystem. This study sheds light on the complex microbial interactions that affect ecosystem functioning under climate change.

Biofilms are structured microbial communities that thrive on diverse surfaces in natural, industrial, and host environments. The biofilm lifestyle underpins microbial survival, shapes ecosystem function, and drives persistent infections; yet, for many microbes, the molecular determinants of biofilm development remain poorly defined. Here, we introduce “label-free analysis of biofilms” (LFAB), an imaging method that integrates time-lapse, low-magnification brightfield microscopy with regional optical density measurements to quantify biofilm biomass. Unlike conventional assays, LFAB enables real-time, non-perturbative, and high-throughput monitoring of biofilm dynamics. We validated LFAB across diverse microbial species and observed a strong correlation with traditional biofilm quantification methods. Applying LFAB to Streptococcus pneumoniae, a major human pathogen whose biofilm lifecycle underpins colonization and infection, we uncovered reproducible patterns of microcolony biofilm expansion and growth. LFAB-enabled screening of a transposon mutant library revealed that biofilm formation in S. pneumoniae is shaped by genes spanning carbohydrate metabolism, cell wall synthesis, adhesion, and surface interactions. Further analysis identified choline-binding protein A (CbpA) and its associated two-component regulator, as well as the peptidoglycan hydrolase LytB, as key drivers of microcolony biofilm dynamics. Together, these findings establish LFAB as a broadly applicable platform for dissecting biofilm biology and reveal new regulators of biofilm development in a clinically important pathogen.

IMPORTANCE

Biofilms are structured communities of microorganisms that attach to surfaces and persist within a self-produced matrix. The biofilm lifestyle underlies microbial survival in nature, contributes to industrial biofouling, and drives many chronic infections. Despite the importance of biofilms, high-throughput measurements of biofilm growth dynamics are challenging using existing tools, which are often disruptive or are not scalable. To overcome this limitation, we developed “label-free analysis of biofilms” (LFAB), a brightfield-based imaging platform that enables real-time, non-perturbative, and scalable quantification of biofilm biomass. LFAB is broadly applicable across species and correlates strongly with traditional assays. Applying LFAB to Streptococcus pneumoniae, a major human pathogen, we performed a mutagenesis screen, uncovering new genetic regulators of biofilm formation in this organism. These findings advance understanding of S. pneumoniae pathogenesis and establish LFAB as a powerful approach for dissecting the molecular basis of microbial community growth.

Protection against pathogens relies heavily on the adaptive immune response, whose key regulators are CD4 T cells. CD4 T cells, notable for their complex repertoire and functional potential, can most easily be dissected by identifying, quantifying, characterizing, and isolating epitope-specific cells. In the study reported here, we present a systematic and unbiased strategy that has enabled the identification of highly immunogenic peptide epitopes derived from influenza virus and SARS-CoV-2, presented by human HLA-DR proteins. Coupling the use of HLA-DR transgenic mice with infection and vaccination and highly sensitive epitope-specific cytokine ELISpot assays, we have narrowed the potential epitopes from 450 to 600 peptides to 5–15 peptides for each allele by an iterative process of elimination and selection, which we have termed a funnel approach. These epitopes have been validated in HLA-DR-typed human CD4 T cells directly ex vivo and enabled the derivation and implementation of HLA-DR peptide tetramers. Tetramer staining of human PBMCs enriched for CD4 T memory populations from healthy adult subjects, highlighted this approach as a sensitive and specific method for identifying novel epitopes, and subsequent CD4 T-cell responses to human viral infections.

IMPORTANCE

Tracking single epitope-specific CD4 T cells enables sophisticated analyses of the human response to infectious pathogens, vaccines, and probing the human CD4 T-cell immune memory compartment. The studies presented here provide an unbiased strategy for accomplishing this goal and provide a verified compilation of candidate HLA-DR-restricted CD4 T-cell peptide epitopes for future studies by researchers in the field of human immunology.

The atmosphere contains thousands to millions of bacterial cells per cubic meter. However, it remains unclear if microbes are at all active or growing in situ or whether they are merely being transported in an inactive state. Based on the analyses of 32 overland radiation fog events over a 2-year period, we show that fog waters, with bacterial concentrations similar to those in continental or marine bodies of water, contain microbiomes well differentiated in composition from those in the dry aerosol microbiomes that occur locally before, during, or after fog events. They are consistently and strongly enriched in photoheterotrophic Methylobacterium species, suggesting that fog populations may be metabolizing volatile C1 compounds in situ, although phototrophy seems much less important. Indeed, metabolically active bacteria in the fog, and representative isolates of the main field populations, can degrade formaldehyde at unprecedently high rates; most of this activity seems to play a detoxification role. The increase in bacterial aerobiome counts upon intervening fog events, the dependence of microbial concentration on ambient temperature, the increases in cell size and frequency of dividing cells in fog water with respect to cells in interstitial aerosols of fogs, in addition to their metabolic capacity, all suggest that the fog water microbiome is actually growing. Consequently, droplets of atmospheric water should be considered a potential aquatic microhabitat. Our results highlight the fog microbiome’s role in atmospheric chemistry and have implications for fog harvesting as a source of fresh water for human use.

IMPORTANCE

While bacteria are common in the atmosphere, their activity in situ has remained unclear. Using stagnant radiation fogs as new study systems where sampling is optimal, the dynamics, composition, cellular characteristics, and metabolic rates of fog water microbiomes, dominated by Methylobacterium sp., show that they are a hub of active detoxification of atmospheric formaldehyde and likely growing in situ on the basis of heterotrophic or photoheterotrophic metabolism of volatile C1 compounds, with implications for atmospheric chemistry and fog harvesting as sources of freshwater.

Bacteroides spp. are a key immune-programming microbe in healthy individuals—these bacteria have been shown to be reduced in abundance across a variety of disease states. Our study investigated the systemic and region-specific responses to Bacteroides colonization in the gut, including sex-related differences, in mice. Utilizing C57BL/6 mice, we administered Bacteroides to conventional, antibiotic-treated mice, then assessed this microbe’s influence on the gut microbiota composition and inflammatory responses following an airway lipopolysaccharide challenge to assess effects on the gut-lung axis. We found that Bacteroides successfully colonizes the intestinal tract of antibiotic-treated mice, particularly the colon lumen of the large intestine, as evidenced by 16S rRNA amplicon gene sequencing and culturing. Differential gene expression analysis using NanoString technology revealed significant immune response variations across the gut regions, with notable differences in adaptive immune response genes. A striking sex-dependent outcome was noted in the regulation of atg12 in the cecum, potentially enhancing autophagic function, particularly in female mice. Additionally, Bacteroides intestinal colonization was associated with altered expression of macrophage markers such as cd163, cd84, and ms4a4a, which may reflect shifts in the macrophage profile within the cecum. These findings pave the way for novel therapeutic approaches that leverage microbial impacts on gut and systemic health, offering a deeper understanding of Bacteroides’ role in human health and disease. Our study highlights the necessity for further research to elucidate the intricate relationships between gut microbiota, host immunity, biological sex, and their interplay.

IMPORTANCE

This research marks an investigation into how specific microbiota, like Bacteroides, regulate host responses across different gut regions to influence systemic health. By dissecting the impact of Bacteroides across multiple regions of the intestinal tract, this study offers new insights into the localized and whole-body effects of this important immune-programming microbe. Such an understanding is crucial as it helps in unraveling the complex interplay between gut microbes and the host’s immune system. This research helps bridge the gap between local intestinal ecology and overall systemic health, addresses important questions relevant to the gut-lung axis, and helps pave the way for innovative therapies.

Coccidioides is an endemic fungus that is increasing in prevalence and causes life-threatening diseases in immunocompetent people. In the environment, the spores (arthroconidia) develop into hyphae; however, when they are inhaled by a mammalian host, they develop into a unique form called the spherule. The transition to spherule can be triggered in vitro with elevated temperatures and high CO₂ levels, but how the host triggers Coccidioides spherulation is not known. We used live imaging to investigate how macrophages affect the fate of Coccidioides arthroconidia. Under tissue culture conditions, arthroconidia quickly developed into hyphae. Remarkably, the addition of macrophages promoted spherule development and delayed hyphal formation. Macrophage supernatants were not sufficient to promote spherule development, and chemical blockade of phagocytosis inhibited spherule formation. Transcriptomics analysis of Coccidioides co-cultured with macrophages revealed a signature concordant with spherules grown in vitro and allowed the identification of a core set of 143 spherule-specific transcripts. Additionally, we identified 229 Coccidioides transcripts with significantly higher abundance in the presence of macrophages compared to in vitro generated spherules, suggesting that these factors may be needed for growth in the presence of innate immune cells; 48 induced transcripts were predicted to encode secreted proteins, suggesting a function at the host-pathogen interface. Taken together, this work highlights the capacity of macrophages to promote development of the parasitic form of Coccidioides and lays a foundation for uncovering host-pathogen signaling as well as Coccidioides factors that are critical for pathogenesis.

IMPORTANCE

Valley Fever is a disease caused by inhalation of the spores of the fungus, Coccidioides spp. It can present like the flu, pneumonia, bone infections, or meningitis. Once inhaled, the spores change into a pathogenic form that allows the fungus to spread throughout the body and cause disease. How the spores make this transition in the body is not well understood. We investigated how immune cells affected this transition. We found that engulfment of spores by innate immune cells stimulated the transition to the pathogenic form of the fungus. We determined which fungal genes are induced during interactions with innate immune cells, potentially identifying genes that may be critical for the development of the pathogenic form. This work helps us understand how this pathogen is taking advantage of our immune system to survive and cause disease.

Many Gram-negative pathogens, including Pseudomonas aeruginosa, use a type III secretion system (T3SS) to intoxicate eukaryotic cells. The T3SS is an important virulence factor linked to increased morbidity and mortality in infections, yet its expression slows bacterial growth and activates innate immune receptors. T3SS genes are expressed heterogeneously, with T3SS-ON cells arising from “primed” bacteria that express the T3SS transcriptional activator ExsA and respond immediately to T3SS activating signals. However, the mechanistic basis for priming is not known. ExsA is part of a complex protein-sequestration network, positively regulating its own expression, as well as that of its anti-activator (ExsD), its anti-anti-activator (ExsC), and ExsC’s binding partner (ExsE). These four proteins create a bistable regulatory network. We hypothesized that transcription from a cAMP-dependent promoter upstream of ExsA could drive cells into the primed state, and tested this at the single-cell level. Exogenous cAMP increased the proportion of primed, ExsA-expressing cells, with whole-cell cryo-electron tomography demonstrating the assembly of T3SS injectisomes under these conditions. Interstrain variation in endogenous cAMP levels correlated with strain-specific proportions of primed bacteria, while genetic manipulation of cAMP levels altered primed population size. This work demonstrates how endogenous and exogenous cAMP inputs into a bistable regulatory switch generate subpopulations of T3SS-primed cells poised to respond to activating signals.

IMPORTANCE

Type III secretion systems (T3SS) are specialized protein secretion systems that allow bacteria to inject toxins into eukaryotic cells. T3SS are important virulence factors, but their expression carries a fitness cost: they slow bacterial growth and make bacteria vulnerable to detection by the innate immune system. Some pathogens, like Pseudomonas aeruginosa, balance the costs and benefits of T3SS expression by restricting T3SS expression to a subset of cells. T3SS-ON cells arise from “primed” bacteria that express the transcriptional activator ExsA and respond immediately to T3SS activating signals. However, the mechanistic basis for priming is unknown. In this study, we tested whether expression of ExsA from a cAMP-dependent promoter could drive cells into the primed state and found this to be true. Whole-cell cryo-electron tomography demonstrated that primed bacteria assembled T3SS injectisomes. This work demonstrates how cAMP inputs into a bistable regulatory switch generate subpopulations of T3SS-primed cells.

Vancomycin is a widely prescribed antibiotic used in the treatment of gram-positive bacterial infections. We previously showed that this antibiotic disrupted protective antifungal immune responses via microbiome dysbiosis, enhancing susceptibility to invasive candidiasis. Antibiotics are an independent risk factor for developing this life-threatening fungal infection, but whether microbiota-independent mechanisms also drive this association is not clear. Here, we show that vancomycin directly impairs macrophage responses to Candida albicans, the main causative agent of invasive candidiasis. Vancomycin-treated macrophages were less able to kill C. albicans despite normal phagocytosis rates and were hyper-inflammatory and more likely to die during infection. Using a fluorescently labeled vancomycin, we observed vancomycin uptake by macrophages in vivo and within close proximity to the mitochondrial outer membrane. Vancomycin treatment led to a significant depolarization, reduced respiratory capacity, and a hyper-fragmented morphology of mitochondria, as well as increased cellular ROS production. Taken together, this work demonstrates direct effects of vancomycin on mammalian immune cells, helping us to understand the pro-inflammatory effects of this drug and how it promotes susceptibility to life-threatening fungal infection.

IMPORTANCE

Antibiotics are widely prescribed drugs used to treat bacterial infections; however, their use may increase the likelihood of developing life-threatening fungal infections in vulnerable patients. Candida albicans is a commensal fungus in humans but may cause serious disease in patients with defined risk factors, including antibiotic exposure. We find that the antibiotic vancomycin significantly impairs the ability of macrophages to kill C. albicans yeast. Vancomycin-induced defects in fungal killing were associated with changes to mitochondria in antibiotic-exposed macrophages, which also exhibited enhanced oxidative stress and reduced survival during fungal infection. This work identifies a direct mechanism by which antibiotics may impair antifungal immunity.

The emergence of multidrug-resistant fungal pathogens from urinary tract infections (UTIs) poses a growing challenge in clinical settings. Here, we report a case of a complicated UTI caused by Nakaseomyces glabratus (Candida glabrata) that progressed to urosepsis, leading to the emergence of an isolate carrying simultaneous loss-of-function mutations in ERG3 and ERG11, and abrogated ergosterol biosynthesis. Together with a missense mutation in FUR1—likely responsible for 5-fluorocytosine resistance—this constellation confers resistance to all viable UTI antifungals: azoles, amphotericin B, and flucytosine. Engineered ERG3Δ + ERG11Δ strains recapitulated this multidrug resistance and revealed profound fitness costs that come with it, challenging the assumption that high-cost mutations are unlikely to persist during infection. Among fitness trade-offs, we detected collateral sensitivity to nitroxoline, a commonly used urinary tract antibiotic with potent antifungal activity and a unique mechanism of action. This study provides the first clinical evidence of an elusive mechanism of hyper-multidrug resistance in N. glabratus and highlights nitroxoline as a promising repurposing agent for treating multidrug-resistant fungal infections of the urinary tract.

IMPORTANCE

Evolutionary theory states that fitness determines survival. In a drug-treatment environment, resistance increases fitness, but it often comes at a cost, such as slower growth or reduced stress tolerance. If these costs are too severe, they can undermine virulence, making resistance unlikely to persist. Our study challenges this assumption. We describe the first clinical case of Nakaseomyces glabratus evolving multidrug resistance through loss-of-function mutations in ERG3 and ERG11, despite severe fitness trade-offs. This case suggests that certain infection niches, such as the urinary tract, can provide conditions where even highly impaired yet resistant strains persist under strong antifungal pressure. Importantly, we show that this extreme resistance induces collateral sensitivity to nitroxoline, a urinary tract infection antibiotic with potent antifungal activity and a unique mechanism of action. These findings open promising therapeutic avenues to counter multidrug-resistant fungal infections of the urinary tract.

The post-translational lipoprotein modification pathway is conserved in bacteria, in which prolipoprotein phosphatidylglycerol diacylglyceryl transferase (Lgt) catalyzes the first and committed step. Due to its essentiality for cell viability in Proteobacteria, its membrane localization, and relative accessibility, Lgt is proposed as a promising target for the development of novel antibiotics. To answer the question of the degree of conservation between Lgt homologs of WHO-listed pathogenic species, we performed evolutionary, structural, and functional analyses. Our data show that Lgt is present in all bacteria and absent from archaea. AlphaFold structural models are similar to the X-ray structure of Lgt from E. coli with most variability and less conserved residues in the arm and head domains between Lgt homologs. Lgt of diderm bacteria, but not of monoderm bacteria, restores growth and viability of an Lgt depletion strain in E. coli. Sequence alignments and site-directed mutagenesis demonstrate that unique conserved residues on arm-2 together with histidine 103 and the periplasmic head domain, determine protein substrate specificity. This large-scale analysis led to the definition of an Lgt motif and an alternative catalytic mechanism. Our results highlight similarities in catalytic mechanism and differences in substrate specificity between Lgt homologs from pathogenic species, with an impact on strategies to develop narrow-spectrum antibiotics targeting Lgt.

IMPORTANCE

Antimicrobial resistance is a major threat to public health, for which the identification of novel targets and the development of new therapies are urgently needed. The bacterial lipoprotein modification pathway is promising for the exploration of new antibiotics since it is unique to bacteria, essential for bacterial viability and virulence, and accessible to drugs due to the exposed domains of the modification enzymes. In this study, we explored large-scale sequence analysis, structural modeling, and functional assays of the first enzyme in the pathway. Our findings show that the enzyme is highly conserved across distant phyla, that homologous enzymes have similar structures and contain a signature motif composed of invariant essential residues, but that functional conservation divides monoderm and diderm pathogenic bacteria. This correlates with structural variation and differences in substrate specificity, illustrating the potential for the development of narrow-spectrum antibiotics targeting the lipoprotein modification pathway.

Aim2-like receptors (ALRs) play crucial roles in innate immune signaling pathways and demonstrate strong positive selection likely driven by pathogens. IFI207, an ALR found in all Mus species, enhances interaction with and stabilization of STING, contributing to the control of murine leukemia virus (MLV) infection. We show here that IFI207 enhances the type 1 interferon response by inhibiting activation-induced K63-linked ubiquitination of STING, thereby preventing its recognition by hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), a key component of the ESCRT complex, and its subsequent degradation in lysosomes. IFI207 promotes downstream signaling in the STING pathway in multiple cell types and moreover enhances the STING-dependent response to herpes simplex virus 1 infection ex vivo and in vivo. We also show that IFI207 likely functions in dendritic cells to suppress MLV infection. Our study reveals that IFI207 acts as a modulator in the STING pathway, strengthening the host’s defense against viral infections and suggesting that the expansion of the Alr locus in mice may have occurred in response to endemic viruses.

IMPORTANCE

The innate immune system serves as a first line of defense by utilizing a variety of pattern recognition receptors (PRRs) that detect various nucleic acids generated during virus infection, many of which activate the STING pathway, leading to interferon production. One family of PRRs, the Aim2-like receptors (ALRs), is thought to contribute to innate immunity by this mechanism. However, the Alr locus is highly polymorphic at the sequence and copy number level. We found that at least one member in mice, IFI207, contributes to innate immunity by preventing the degradation of STING that normally occurs after its activation, defining a novel mechanism for sustaining immune response. Several ALRs have been implicated in adipogenesis, autoimmune disease, and inflammation, and identification of the pressures that shaped the genes in the locus is thus important for understanding the biological processes in which the ALRs function.

The cell-to-cell communication process called quorum sensing enables bacteria to synchronize collective behaviors. Quorum sensing relies on the production, release, and detection of signaling molecules called autoinducers. In Vibrio cholerae, the VqmA transcription factor, following binding of the DPO autoinducer, activates the expression of the gene encoding the VqmR small regulatory RNA. VqmR controls traits including biofilm formation. Here, we identify repressors of DPO-VqmA-VqmR signaling. We focus on one identified repressor, the LuxT transcription factor. We show that LuxT represses vqmR transcription. VqmR post-transcriptionally represses luxT translation. This arrangement forms a double-negative feedback loop between the two regulators. Reciprocal control hinges on the N-terminal eight amino acids of LuxT. The nucleotide sequence encoding this LuxT region serves as the VqmR binding site in the luxT mRNA and the amino acids specified by this same N-terminal region are required for LuxT to bind the vqmR promoter. This same LuxT N-terminal region also expands the DNA motifs to which LuxT can bind. We show this regulatory circuit is unique to V. cholerae and closely related species and absent from other vibrios. We define the set of LuxT-controlled genes in V. cholerae and show that LuxT promotes biofilm formation, a key requirement for successful colonization of eukaryotic hosts.

IMPORTANCE

Bacterial quorum sensing enables control of collective behaviors. In Vibrio cholerae, the DPO-VqmA-VqmR quorum-sensing circuit governs key processes, including biofilm formation. Here, we identify a double-negative feedback loop between the transcription factor LuxT and the small RNA VqmR. This regulatory circuit depends on an eight amino acid N-terminal region that exists only in V. cholerae LuxT and LuxT from its close relatives. This short peptide sequence confers three distinct functions: it enables LuxT to repress vqmR, renders luxT mRNA susceptible to VqmR repression, and governs which DNA motifs LuxT can bind. Our findings reveal a pathogen-specific regulatory module that links small RNA targeting of mRNAs to transcription factor DNA binding specificity. The results show how evolution tailors bacterial regulatory circuits to adapt to different environments.

In gram-negative bacteria, the outer membrane (OM) acts in conjunction with the peptidoglycan (PG) cell wall as a barrier against physical, osmotic, and chemical environmental stressors, including antibiotics. SanA, an inner membrane protein in Escherichia coli K-12, is required for vancomycin resistance at high temperatures (>42°C) and impacts sodium dodecyl sulfate (SDS) resistance during the stationary phase reached from carbon limitation. However, its function remains unknown. Here, we show that ΔsanA has a synthetic genetic interaction with ΔwecA, a mutation that increases the availability of the isoprenoid carrier for PG synthesis. Specifically, the ΔsanA ΔwecA strain demonstrated heightened SDS-EDTA sensitivity, activation of the Rcs stress response, and increased cell length. Further investigation tied the SDS-EDTA sensitivity to increased lipid II available for PG synthesis. Spontaneous suppressor mutants of this phenotype harbored point mutations in prc, which encodes tail-specific protease, or ftsI, which encodes the cell division DD-transpeptidase, a target of Prc. We focused on the ftsI mutations and demonstrated that the ftsI mutations increased cell length but nevertheless enhanced PG incorporation at the septum compared to the ΔsanA mutant, returning PG incorporation to wild-type levels. Moreover, other mutations affecting septal PG synthesis, but not divisome assembly, also suppressed the SDS-EDTA sensitivity. These findings suggest that in the absence of SanA, increased lipid II availability perturbs the balance between septal PG synthesis, lateral PG elongation, and other envelope biogenesis pathways, leading to increased OM permeability.

IMPORTANCE

The gram-negative cell envelope is a barrier that protects the cell from environmental stress. Therefore, the synthesis of each layer of this envelope needs to be closely coordinated throughout growth and division. Here, we investigated SanA, a protein in Escherichia coli K-12 that affects envelope permeability under cellular stress, including nutrient limitation and high temperature. We found that SanA plays a key role in maintaining the permeability barrier when precursor levels for peptidoglycan (PG) synthesis are elevated, linking envelope integrity to balanced septal PG production during cell division. Our results suggest that SanA modulates substrate availability to preserve envelope function, and that in its absence, imbalanced substrate flux to septal PG synthesis disrupts septum formation and compromises barrier integrity.

Many pathogens target the host actin cytoskeleton through the delivery of actin depolymerizing toxins, including mono-ADP-ribosyltransferases (mART), ultimately triggering host cell death. Despite the importance of mARTs in pathogen virulence, it remains unclear whether actin ribosylation is required for mART-dependent cell death, and how actin depolymerization leads to cell death. Using the non-typhoidal Salmonella enterica Typhimurium-encoded mART, SpvB, we report that cell death is induced exclusively through ribosylation of actin. We found cell death to be morphologically and mechanistically distinct from apoptosis as well as any previously reported mode of cell death. Instead, our data identify the Hippo signaling MAP4Ks as the essential host cell sensors of actin depolymerization signaling through JNK to facilitate vacuolization and host cell death. Cell death following treatment of cells with the actin depolymerizing agent latrunculin A followed the same pathway, identifying a conserved mechanism of cell death. Therefore, we identify MAP4K family members as key regulators of an atypical caspase-independent cell death induced by actin depolymerization, building on our understanding of host-cell death signaling and mechanisms of bacterial virulence.

IMPORTANCE

Host cell death plays a critical role as an intrinsic defense mechanism against infection and disease. However, many pathogens subvert cell death signaling to enhance their replication and survival. Here, we show that the mono-ADP ribosyl transferase family of toxins encoded by pathogens of global importance, including Salmonella spp., Neisseria spp. and C. difficile induces actin depolymerization leading to MAP4K activation and JNK-dependent cell death. Through mechanistically characterizing this atypical cell death pathway, our study identifies and positions key components of a previously undescribed cell death pathway and broadens our understanding of bacterial pathogenesis and virulence.

Obesity has been identified as an independent risk factor for increased morbidity and mortality during pandemic H1N1 influenza and SARS-CoV-2 infections. Various studies have determined that obese hosts have dysregulated antiviral responses driven by systemic low-grade inflammation, resulting in prolonged viral replication, induction of proinflammatory cytokines during the disease resolution phase, and profound lung parenchymal damage. However, the impact of obesity on disease outcomes in the context of other viral respiratory infections remains unclear. In this study, we sought to understand how obesity affects disease progression during respiratory syncytial virus (RSV) infection using an obese mouse model in which male C57BL/6 mice were fed a high-fat diet (HFD). Mice fed a standard chow diet served as controls. We found that HFD mice exhibited reduced weight loss, illness scores, and histopathological changes after intranasal inoculation with RSV A2. Investigation of potential mechanisms underlying this reduction in severity revealed reduced viral load in obese mice, along with altered innate and adaptive immune responses. Specifically, HFD mice had reduced proinflammatory cytokines in the lungs early in infection and increased CD4⁺ T cells late in infection. HFD mice also had reduced M2 macrophages in the lungs at 7 days post-infection. Notably, HFD mice infected with RSV did not exhibit aberrant proinflammatory cytokine secretion late in infection, as seen with influenza, likely due to effective viral control. In conclusion, HFD mice exhibited reduced disease severity during RSV infection, associated with decreased viral load and an attenuated but sufficient antiviral response.

IMPORTANCE

Obesity has been shown to induce dysregulated antiviral responses during influenza infections, resulting in extensive morbidity and mortality. No studies to date have investigated how obesity-induced immune dysregulation affects respiratory syncytial virus (RSV) disease progression. RSV has a high global burden, inflicting millions of infections and tens of thousands of deaths yearly, most notably among the very young, the elderly, and those with comorbidities. It is essential to understand how risk factors, such as obesity, affect disease progression to ensure appropriate protection and care for patients. Here, we demonstrate that male C57BL/6 mice fed a high-fat diet had lower viral loads and attenuated inflammatory responses during RSV infection, resulting in reduced morbidity and immunopathology. This pilot study advances our understanding of how obesity affects pulmonary antiviral immunity to RSV and, concurrently, further elucidates RSV pathogenesis.

Quorum sensing is a cell-to-cell communication process bacteria use to orchestrate collective behaviors. Quorum sensing involves the production, release, and detection of extracellular signal molecules called autoinducers. Some temperate phages can monitor bacterial autoinducers, enabling them to track the abundance of potential host cells in the vicinity. Quorum-sensing-responsive phages can preferentially launch the transition from lysogeny to lytic replication at high cell density, presumably maximizing transmission. Once the phage lytic program is enacted, if nearby host cells are already lysogens, infections initiated by released virions could be nonproductive due to homoimmunity or superinfection exclusion mechanisms, posing a conundrum for temperate phages, including those that surveil quorum-sensing autoinducers. Here, we define host and phage components that influence the transmission of the first-discovered quorum-sensing-responsive phage, phage VP882, in populations of its host, Vibrio parahaemolyticus. We show that phage VP882 uses the K-antigen of serotype O3:K6 as its receptor. We demonstrate that host cells can prevent phage access to the O3:K6 K-antigen via quorum-sensing control of the export of polysaccharides that shield the K-antigen from the phage at high cell density. We discover that phage VP882 can superinfect and superlysogenize V. parahaemolyticus, overcoming the challenge of detecting whether or not potential hosts are lysogens. Following superlysogenization, recombination of the resident and newly infecting phage genomes can occur, possibly promoting phage VP882 genome diversification.

IMPORTANCE

A longstanding mystery is how temperate phages optimally time the launch of their lytic cascades to maximize spread. Quorum-sensing-responsive phages can preferentially execute their lytic replication programs at high host cell density, which in principle should foster transmission. However, if nearby host cells are already lysogens, infections initiated by released virions could be nonproductive due to homoimmunity or superinfection exclusion. We define host and phage components influencing the transmission of the quorum-sensing-responsive phage VP882 in Vibrio parahaemolyticus populations. Phage VP882 uses the O3:K6 K-antigen as its receptor. Host cells prevent phage infection via quorum-sensing-controlled export of polysaccharides that shield the K-antigen at high cell density. We discover that phage VP882 can superinfect and superlysogenize V. parahaemolyticus, overcoming the challenge of detecting whether or not potential hosts are lysogens. These findings reveal how phages can capitalize on interception of host quorum-sensing cues to maximize their reproductive success.

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has led to significant global morbidity and mortality. The severe disease outcomes are often associated with a hyperinflammatory response known as a “cytokine storm.” The mechanisms underlying this exaggerated immune response remain incompletely understood. This study aimed to investigate the molecular pathways contributing to the severe inflammatory damage and mortality associated with COVID-19. SARS-CoV-2 hijacks host lipid metabolism, particularly the phospholipase A2 (PLA2) pathway, leading to the production of bioactive lipid mediators, including 12-lipoxygenase (12-LOX)-derived lipid mediators in platelets, and in lung and vascular cells. We hypothesized that 12-LOX drives the hyperinflammatory response and disease severity, and that its inhibition could reduce inflammation and improve outcomes. Analysis of autopsy lung samples from COVID-19 decedents and SARS-CoV-2-infected K18-hACE2 transgenic mice revealed increased 12-LOX expression. We evaluated VLX-1005, a selective small-molecule 12-LOX inhibitor, in infected mice. Treatment initiated 48 h post-infection significantly improved survival, reduced body weight loss, and decreased lung inflammation compared to controls. Notably, male mice showed higher survival rates than females. VLX-1005 treatment also suppressed key chemokines and cytokines associated with the cytokine storm, and reduced lung damage. These findings identify 12-LOX as a critical mediator of the hyperinflammatory response in severe COVID-19 and support its inhibition as a promising therapeutic strategy to mitigate inflammatory damage and reduce mortality.

IMPORTANCE

This study provides critical insights into the mechanisms underlying severe COVID-19, identifying 12-lipoxygenase (12-LOX) as a key driver of the hyperinflammatory response that contributes to disease severity and mortality. By demonstrating that SARS-CoV-2 hijacks host-lipid metabolism to elevate proinflammatory lipid mediators, the research uncovers a novel pathogenic pathway that exacerbates lung inflammation. The use of VLX-1005, a selective 12-LOX inhibitor, significantly improved survival and reduced inflammatory damage in a mouse model, highlighting its therapeutic potential. These findings not only deepen our understanding of COVID-19 pathogenesis but also position 12-LOX as a promising intervention target, offering a new avenue to mitigate the effects of cytokine storms in severe cases.

Intestinal protists are emerging as key modulators of host immunity and microbial ecology, yet their impact on mammalian hosts remains poorly defined. Here, we investigated the role of two distinct protists, the amoeba Entamoeba muris, and the parabasalid, Tritrichomonas, to determine how they shape gut immunity in vivo individually and together. Unlike the well-characterized inducer of type 2 immunity, Tritrichomonas, which activates the tuft cell–IL-25–ILC2 circuit in the small intestine, E. muris failed to elicit robust immune responses in the intestine or colon. However, the introduction of E. muris into mice naturally colonized by Tritrichomonas spp., or co-infection with E. muris and Tritrichomonas spp. reduced Tritrichomonas-induced type-2 response in the small intestine and Tritrichomonas-dependent immune activation in the colon. Our data suggest that E. muris may limit the abundance of Tritrichomonas spp., with reduced protist loads in the cecum specifically correlating with diminished tuft cell activation. We also identified sex-specific differences in the intestinal response to Tritrichomonas spp., which have not previously been reported. Taken together, these findings reveal that the presence of E. muris reduces Tritrichomonas-dependent activation of type 2 immunity in the small intestine, even when both protists can be detected in the cecum, without triggering overt inflammation. This work provides a framework for understanding how protists interact within the gut ecosystem and shape mucosal immunity in the absence of pathogenicity.

IMPORTANCE

Single-cell parasites called protists are common in mammalian intestinal tracts, yet their modulation of the host immune response and interactions with each other remain poorly defined. Here, we investigated the role of two protists, Entamoeba and Tritrichomonas, to determine how they shape gut immunity individually and together. Unlike the well-characterized inducer of type 2 immunity, Tritrichomonas, which activates the tuft cell circuit, Entamoeba failed to elicit a robust immune response. The introduction of Entamoeba into mice naturally colonized by Tritrichomonas, or co-infection with Entamoeba and Tritrichomonas, reduced the Tritrichomonas-induced immune response. Our data suggest that Entamoeba limits the abundance of Tritrichomonas, correlating with diminished tuft cell activation. We also identified sex-specific differences in the intestinal response to Tritrichomonas. These findings show that Entamoeba reduces Tritrichomonas-dependent activation of type 2 immunity without triggering much inflammation. It helps our understanding of how protists interact within the gut and shape immunity without disease.

Staphylococcus aureus is a leading cause of skin and soft tissue infections (SSTIs), which can escalate into systemic disease. While innate immune responses play a critical role in bacterial clearance, the bacterial components themselves can exacerbate inflammation. Here, we demonstrate that S. aureus lipoproteins (Lpp) and polymeric peptidoglycan (PG) synergistically induce skin abscesses in mice, in a process that requires both the lipid moiety of Lpp and the intact polymeric structure of PG. This synergy is mediated by Toll-like receptor 2 (TLR2) and nucleotide-binding oligomerization domain-containing protein 2 (NOD2) and depends on infiltrating neutrophils and monocytes. Co-administration of Lpl1 and PG results in a 5-fold to 10-fold increase in macrophage inflammatory protein-2 (MIP-2) levels in the skin compared to either ligand alone, indicating a clear synergistic effect. Furthermore, we show that local alteration in coagulation and fibrinolysis contributes to the inflammatory response, as fibrinogen depletion significantly reduced lesion size. To extend these findings to a clinically relevant model, we employed an S. aureus double mutant that lacked both lipidation (Δlgt) and peptidoglycan O-acetyltransferase (ΔoatA). This strain exhibited markedly attenuated virulence in a murine skin infection model. Importantly, this attenuation was fully reversed by neutrophil depletion, indicating that neutrophils are essential mediators of the host responses to these bacterial structures. Our findings reveal a cooperative mechanism through which S. aureus cell wall components drive skin lesion development, and we identify potential therapeutic targets for reducing the severity of SSTIs.

IMPORTANCE

Staphylococcus aureus is a bacterium that often causes skin infections, including painful abscesses. We discovered that two components of S. aureus, lipoproteins on its surface and peptidoglycan in its cell wall, collaborate to drive the formation of skin abscesses. This combination triggers potent immune responses by activating the receptor Toll-like receptor 2 (TLR2) and nucleotide-binding oligomerization domain-containing protein 2 (NOD2) on host cells. As a consequence, high numbers of neutrophils and monocytes swarm the infection site. The resulting immune overreaction, together with activation of the coagulation system, produces intense inflammation. We confirmed the importance of these bacterial components using mutant S. aureus strains in a skin infection model. These mutants generated much smaller abscesses in our experiments. Our findings highlight a cooperative mechanism that exacerbates staphylococcal infections. Targeting this synergy could be a valuable strategy to reduce disease severity.

Toxoplasmosis is a disease of worldwide distribution, causing high morbidity and mortality in humans, as well as heavily impacting animal health and the economy. Toxoplasma gondii, the causative agent, is an intracellular parasite with a complex life cycle whose completion entails asexual, pre-sexual, and sexual stage conversions. Pre-sexual and sexual differentiation take place only within the intestinal epithelium of felines. Recently, several transcriptional factors and epigenetic components crucial to trigger parasite stage transitions within the cat have been identified, allowing, through precise genetic manipulation, obtaining pre-sexual stages known as merozoites in vitro. Through conditional depletion of two pre-sexual stage-specific gene silencing transcription factors, AP2XII-1 and AP2XII-2, we have characterized the interplay between cell division and the sequence of events leading up to differentiation of tachyzoites into merozoites. We explored genome duplication, assembly of daughter cells, karyokinesis, and cytokinesis, characterizing the differential cell division modes and kinetics undergone by critical structures along the differentiation axis. Building onto the pre-existing body of knowledge, primarily describing the underpinnings of these forms of division by transmission electron microscopy, our work contributes previously unexplored temporal and spatial resolution to the transitions between endodyogeny and endopolygeny, providing a conceptual framework for understanding and exploring T. gondii’s route of sexual differentiation.

IMPORTANCE

Sexual development in Toxoplasma gondii is essential for transmission, but remains poorly understood, largely because pre-sexual stages are restricted to the feline intestine and have only recently become experimentally accessible. Here, we leverage an in vitro differentiation system to resolve how parasites transition toward merozoite formation at the cellular level. By combining expansion microscopy, stage-specific markers, and quantitative analyses, we define the temporal sequence of nuclear division and daughter cell assembly during merogony, addressing longstanding ambiguity regarding division modes in these stages. Our findings reveal that parasites can adopt alternative division strategies emerging from a polyploid intermediate, highlighting an unexpected degree of flexibility in how cell division is executed during differentiation. Beyond refining this developmental framework, this work establishes a foundation for future mechanistic studies of pre-sexual biology and provides broader insight into the diversity of eukaryotic cell division strategies.

Nakaseomyces glabratus (formerly Candida glabrata) is a leading cause of invasive candidiasis and rapidly develops antifungal drug resistance during treatment. An increasing number of clinical isolates show reduced susceptibility to echinocandins and azoles, leaving amphotericin B (AMB) as a last therapeutic option. Resistance of N. glabratus to this drug is rare, and its underlying mechanisms are still not fully understood. Here, we describe two independent multidrug-resistant bloodstream isolates displaying resistance to AMB and anidulafungin (ANF), as well as a reduced susceptibility to azoles. We performed whole-genome sequencing and sterol profiling on nine clinical N. glabratus isolates, which were resistant to ANF and displayed resistance or low susceptibility to fluconazole (FLU) and AMB. We identified loss-of-function mutations in the genes ERG3 and ERG4, which could be linked to ergosterol depletion and AMB resistance. The transcriptional response of the reference strain CBS138 and an AMB^R + ANF^R isolate was analyzed by RNA-seq, revealing that ergosterol depletion also contributed to upregulation of ERG and ABC transporter genes, which might explain the low FLU susceptibility. Surprisingly, the AMB^R isolates displayed severe fitness defects, and one of them was fully virulent in a Galleria mellonella infection model. Our results indicate that ergosterol depletion in N. glabratus leads to AMB resistance without affecting fitness or virulence.

IMPORTANCE

The major human fungal pathogen Nakaseomyces glabratus is well known for its fast development of antifungal drug resistance, especially against commonly used azoles. However, it can also acquire resistance to echinocandins, leading to multidrug resistance (MDR) and leaving amphotericin B (AMB) as the last therapeutic option. AMB resistance is rare, mainly caused by ergosterol depletion, and is normally associated with severe fitness costs for the pathogen. However, we found N. glabratus bloodstream isolates with stable AMB resistance without apparent fitness and virulence defects. The underlying ergosterol depletion contributed to low azole susceptibility and was associated with anidulafungin resistance. These findings demonstrate how fast MDR can evolve in N. glabratus and underline the need for close resistance monitoring.

Streptococcus agalactiae, or group B Streptococcus (GBS), is an opportunistic pathogen that asymptomatically colonizes the vaginal tract of up to 30% of healthy individuals. However, during pregnancy, it is associated with adverse pregnancy outcomes, and GBS can be transmitted to the fetus in utero or the newborn during vaginal birth, resulting in invasive neonatal disease. Previously, we identified that Akkermansia muciniphila increases GBS vaginal persistence in a cohort of human vaginal microbiome samples collected throughout pregnancy and promotes GBS vaginal colonization in a murine model. However, the mechanisms responsible for these observations are unknown. Here, we analyze additional vaginal shotgun metagenomic data sets and show that across independent studies with diverse populations, A. muciniphila-positive samples had higher GBS abundance. We determined that A. muciniphila aggregates with human vaginal isolates of GBS across all serotypes and promotes GBS attachment to human vaginal epithelial cells (hVECs). RNA-sequencing analysis reveals that A. muciniphila changed the expression of 281 unique GBS genes during hVEC co-colonization, many of which are involved in cell wall/membrane/envelope biogenesis. We demonstrate the importance of the GBS capsule and pili for direct interaction with A. muciniphila and increased attachment to hVECs, respectively. Lastly, we found that A. muciniphila promoted GBS aggregation in the murine vaginal lumen and that continual treatment with A. muciniphila reduced GBS vaginal persistence. Our results provide mechanistic insights and further evidence of the impact of A. muciniphila on GBS vaginal colonization and also demonstrate a beneficial potential of A. muciniphila treatment in the vaginal environment.

IMPORTANCE

Group B Streptococcus (GBS) is a frequent colonizer of the vaginal tract of healthy people; however, during pregnancy, maternal colonization is associated with adverse pregnancy outcomes. GBS is a leading cause of neonatal sepsis and meningitis, with transmission to neonates occurring either during vaginal delivery or through ascension into the uterus during pregnancy. The influence of the vaginal microbiota on GBS pathogenesis remains greatly underappreciated. We have found that GBS is associated with the mucin-degrading intestinal commensal Akkermansia muciniphila, a newly identified colonizer of the vaginal tract. Our research identifies the mechanistic impact of this commensal organism on GBS aggregation, cell adherence, and gene expression, as well as its therapeutic potential during GBS vaginal colonization. Unraveling relationships between GBS and the vaginal microbiota will improve maternal-fetal health and may facilitate the development of alternative methods to reduce GBS in utero complications and neonatal disease.

Bacteria produce high-affinity, iron-chelating secondary metabolites called siderophores to access insoluble Fe(III) in their environments. Genome mining has revealed many predicted siderophore biosynthetic gene clusters (BGCs) in bacterial genomes; however, the structures of their siderophore products remain mostly undetermined. This limits our molecular-level understanding of how bacteria acquire iron. Here, we apply inverse stable isotope labeling (InverSIL) to rapidly connect predicted siderophore BGCs to their products. With InverSIL, bacteria are grown on ¹³C-substituted carbon sources and then fed predicted biosynthetic precursors at their natural isotopic abundance to identify BGC products by mass spectrometry, removing issues with the availability of isotopically substituted precursors. We use InverSIL to determine the structures of the siderophore products of predicted BGCs from the methylotrophic genera Methylophilus and Methylorubrum, as well as the siderophores produced by the opportunistic pathogen Chromobacterium violaceum, which were previously shown to be essential for virulence yet remained structurally uncharacterized. We next use this approach to reveal the unexpected production of enterobactin by the genera Kushneria and Paracoccus, which was difficult to predict from genome sequences due to the distributed nature of the biosynthetic genes within the genomes. Finally, we use InverSIL to discover new siderophores, the cellulochelins, from the cellulose-degrading plant symbiont Cellulomonas sp. strain Leaf334. These findings demonstrate the utility of InverSIL for functional BGC characterization and expand our molecular understanding of bacterial iron acquisition strategies.

IMPORTANCE

Iron acquisition is important for microbial survival, and bacteria produce secondary metabolites called siderophores to scavenge iron from the environment. While bacterial genome sequences show many predicted genes for making siderophores, most remain unlinked to their metabolic products. Understanding which siderophores bacteria produce is critical for elucidating microbial iron acquisition strategies, ecological interactions, and potential roles in host-microbe interactions. Here, we demonstrate how inverse stable isotope labeling (InverSIL) can rapidly link predicted siderophore gene clusters to their corresponding metabolites. By applying InverSIL to diverse bacterial strains, we validate known siderophore products and uncover unexpected products, highlighting the limitations of current in silico predictions. This study highlights the value of combining experimental approaches with genome mining to advance our understanding of how bacteria acquire iron from their environment.

The complex structure of fungal biofilms generates microenvironments that impact the fitness of cells within the biofilm community. Contributions to fitness include the development of emergent properties resulting in the tolerance or resistance to external stressors, such as rapid environmental changes and, in the context of an infection, antifungal drug exposure. The biofilm developed by the filamentous fungal pathogen Aspergillus fumigatus develops zones of low oxygen, which contribute to a reduction in antifungal drug susceptibility. The genes and mechanisms involved in driving this biofilm-specific emergent property are ill-defined. In this study, we utilized a transcriptomic approach to probe the biofilm structure in comparison to drug-susceptible planktonic cultures to identify transcriptional patterns and genes unique to the A. fumigatus biofilm. Importantly, we utilized two phenotypically diverse strains that allowed us to identify biofilm-specific gene co-expression networks. One of these networks was highlighted by a gene encoding a ceramide synthase, designated barA, with a striking increase in barA transcript abundance specifically in the biofilm. Null mutants of barA in two strain backgrounds display a stunted biofilm morphology, with some strain-specific differences in the impact of biofilm biomass. Importantly, barA has a role in regulating susceptibility to the ergosterol-targeting antifungal drugs voriconazole and amphotericin B. These data identify biofilm-specific genes in A. fumigatus for further study and highlight the importance of fungal ceramide synthases in mediating antifungal drug susceptibility in infection-relevant biofilms.

IMPORTANCE

Biofilms are problematic structures in the context of microbial infections due to their ability to resist both host- and drug-mediated attempts at tissue sterilization. Consequently, it is imperative to identify mechanisms underlying the development of these structures and the emergent properties they develop. The filamentous fungal pathogen Aspergillus fumigatus forms robust-structured biofilms that are resistant to contemporary antifungal drug treatments, although the mechanisms are ill-defined. In this study, we compared the transcriptional landscape of two A. fumigatus reference strains grown as biofilms and in planktonic culture conditions to identify biofilm-specific genes and pathways. These analyses and subsequent genetic and phenotypic studies revealed that a ceramide synthase is important for biofilm development and is involved in antifungal drug susceptibility of the biofilm. Consequently, these data support the rationale for targeting fungal lipid homeostasis for antifungal therapeutic development, particularly in the context of biofilm-mediated infections.

In fungi, the continuous biosynthesis and remodeling of the cell wall are crucial for growth, division, and development. A hallmark of fungal cell walls is their layered structure, which includes several carbohydrate polymers, such as β-glucans, and a large number of associated cell wall proteins. The fungal-specific family of SUN domain proteins has been implicated in cell wall remodeling and cell separation, but detailed structure-based analyses revealing precise molecular functions have been lacking until now. In this study, we determined high-resolution crystal structures of the SUN domains from two paralogs of the SUN family in budding yeast. We find that their bilobal architecture consists of a domain with high structural similarity to the Sushi/SCR/CCP domain (INTERPRO family IPR000436), and an intimately associated thaumatin-like domain (IPR001938). Together, these domains form a highly conserved canyon fitted to accommodate both single- and triple-helical β-glucan polymers. Within this canyon, we identify 12 conserved polar residues that are crucial for the function of SUN domains in mediating cell separation. We further demonstrate that SUN domains are functionally interchangeable between paralogs in budding yeast, as well as between orthologs from budding yeast and phylogenetically distant fission yeast or filamentous fungi. We conclude that the fungal SUN domain family represents a unique class of β-1,3-glucan-binding proteins involved in cell wall remodeling and separation, whose successful evolution was enabled by the fusion of ancestral sushi- and thaumatin-like domains.

IMPORTANCE

Fungal cell walls are dynamic extracellular structures essential for growth and morphogenesis, making them prime targets for antifungal drugs and the host immune system. Although many protein families involved in the synthesis, crosslinking, and degradation of cell wall polymers are known, the molecular functions and structural evolution of most cell wall proteins remain poorly understood. Our in-depth structural, functional, and phylogenetic analysis of the fungal SUN domain protein family sheds light on a central question: how specific protein families have evolved structurally to enable dynamic cell wall remodeling during growth and division. Moreover, this work identifies precise structural targets within the fungal cell wall that could guide the development of novel diagnostics and therapeutics against life-threatening fungal infections.

Invasive infections caused by Aspergillus fumigatus have high mortality rates, even when treated with the first-line agent voriconazole. The global emergence of azole resistance further increases treatment failure, underscoring the urgent need for antifungals with novel mechanisms of action. The fungal cell cycle is essential for viability and represents an attractive but underexplored target. In A. fumigatus, progression from G2 to M phase requires interaction between the phosphatase NimT and cyclin-dependent kinase NimX. Here, we characterize the role of this interaction and its inhibition by 2-fluoro-4-hydroxybenzonitrile (compound 1), a small molecule that targets the human Cdc25B–Cdk2 interface. Using mutagenesis, we show that NimT residues Arg438 and Arg442 are critical for NimT–NimX binding and confirm they are essential for viability. A co-immunoprecipitation assay demonstrates that compound 1 disrupts the interaction, while live-cell imaging shows that this inhibition arrests cell cycle progression. Our findings provide mechanistic insight into fungal mitosis and highlight cell cycle regulators as promising antifungal drug targets.

IMPORTANCE

Invasive aspergillosis has high mortality and limited treatment options, threatened by rising drug resistance. Targeting the fungal cell cycle represents an unexplored strategy for antifungal drug development. The dynamic interaction between NimT and NimX is critical to the fungal duplication cycle. Here, we show evidence that, unlike in Schizosaccharomyces pombe, relocation of NimT from the nucleus to the cytoplasm mid-interphase is the switching event that causes activation of NimX and allows the cell cycle to progress. We also show that disruption of the NimT–NimX interaction can be achieved using a reversible small-molecule inhibitor that arrests the fungal duplication cycle, highlighting mitotic regulators as promising antifungal drug targets.

To establish effective infection, viral pathogens employ diverse strategies through encoded proteins to interfere with host antiviral responses. While previous studies have predominantly focused on elucidating the mechanisms by which individual viral proteins regulate type I interferon (IFN) responses, this study presents the first demonstration that Japanese encephalitis virus (JEV)-encoded NS1 and NS4B proteins cooperatively target the TLR3 receptor signaling pathway to suppress IFN production. Here, we first discovered JEV-encoded multifunctional glycoprotein NS1, which inhibits TLR3-mediated IFN-β production by targeting TLR3 and TRIF. Mechanistically, NS1 interacts with these host factors and may induce their degradation via the autophagy pathway. Building on these findings, we further demonstrated that NS1 and NS4B act synergistically to suppress type I IFN production by enhancing TLR3 and TRIF degradation, thereby facilitating JEV replication. Structural simulation of the NS1-NS4B-TLR3 complex unveiled a dynamic mechanism that NS4B binding induces conformational changes in dimerized NS1, which leads to a tighter binding posture between NS1 and TLR3. Notably, NS4B binding expands the interface between NS1 and TLR3 by 1.5-fold that results in enhanced TLR3 degradation and downstream IFN-β suppression. Functional analysis confirmed that the C291/K293/R314 triple mutation in NS1 synergistically impairs TLR3 degradation. Collectively, using JEV as a model, this study reveals a novel mechanism by which two viral components can act synergistically to evade the host antiviral response. Given the common coexistence and functional interplay of viral-encoded proteins in naturally infected cells, this study establishes a framework for investigating cooperative interactions among multiple viral proteins.

IMPORTANCE

Viruses evade host immunity through encoded viral proteins, while previous research has predominantly focused on single protein mechanisms. This study reveals a novel cooperative immune evasion strategy, demonstrating for the first time that Japanese encephalitis virus NS1 and NS4B proteins act synergistically to degrade the host’s TLR3 and TRIF adaptor, thereby suppressing type I interferon production. Structural simulations show that NS4B induces conformational changes in NS1, enhancing its binding to TLR3 and accelerating its degradation. This work establishes a new paradigm for how multiple viral components can function cooperatively to subvert antiviral defenses. Given that viral proteins naturally coexist and interact, this study provides a crucial framework for investigating complex viral protein interplay, which is fundamental to understanding viral pathogenesis.

Rhizobia live as free-living microorganisms in the soil and in association with legume hosts. Both environments exert selective pressures on rhizobia, influencing the reproductive success of individual strains (e.g., fitness). The soil, a heterogeneous and fluctuating environment, is often overlooked, and little is known about whether selection in the soil influences the outcomes of the rhizobium-legume mutualism. We exposed a mixture of 68 Sinorhizobium meliloti strains to soil-mediated selection using eight different treatments (temperature, osmotic, and texture perturbations) and host-mediated selection with two Medicago species as hosts. We found that cold (4°C) and warm (32°C) temperatures, as well as salt addition, had the strongest effects on diversity, population composition, or population size. Strain relative fitness was strongly positively correlated among soil treatments, except cold, warm, and salinity, suggesting strains undergo similar selection in the soil. Genome-wide association analysis revealed a complex genetic architecture for soil fitness, characterized by numerous loci of small effect that did not show significant associations. In contrast, when comparing rhizobial fitness between soil and host environments, we found minimal strain fitness correlations, suggesting an independent genetic basis and habitat-specific adaptations. Finally, by examining the relationship between rhizobial fitness in soil and their benefits to the host plant, we found that soil selection influenced the relative abundance of high- and low-quality strains; however, whether these effects were positive or negative for the plant was host-dependent. Our results suggest that rhizobial evolution in soil and host are largely independent, but soil selection can alter mutualism benefits.

IMPORTANCE

Rhizobium-legume mutualism is crucial for introducing nitrogen into agricultural and natural ecosystems, and rhizobial persistence in the soil is an important component of agroecosystems. However, we know little about how populations of rhizobia persist and adapt to this environment, especially in the context of the soil's spatial and temporal variations (temperature, moisture, and soil texture). We found that rhizobia are similarly selected across abiotic soil conditions, but their reproductive success in the soil is independent from their reproductive success in the host. Intriguingly, we found that certain soil conditions increase (or decrease) the relative abundance of more beneficial strains. Understanding how rhizobia adapt to diverse environments is crucial for developing effective bioinoculants that persist in the soil while remaining highly competitive for host colonization and beneficial to the plant.

Parainfluenza virus is a significant respiratory pathogen affecting both humans and animals, capable of causing acute respiratory tract infections in infants and immunocompromised individuals. Recently, parainfluenza virus 5 (PIV5) has been frequently detected in swine diarrheal samples. However, experimental infections revealed that PIV5 alone induces only mild clinical symptoms. Further analysis suggested that co-infection with porcine sapelovirus (PSV) may underlie the observed disease severity. This hypothesis was confirmed through animal studies. Co-infection was shown to occur intracellularly, where PSV significantly enhanced PIV5 replication. Mechanistically, this effect was primarily mediated by PSV 3D, an RNA-dependent RNA polymerase (RdRp). PSV 3D directly interacted with PIV5 nucleoprotein and genomic RNA. Mini-genome assays demonstrated that PSV 3D substantially increased PIV5 genomic activity. Using an infectious cDNA clone of PIV5 and a series of mutants, we further confirmed that PSV 3D promoted transcription of both plus- and minus-strand PIV5 RNAs. Notably, RdRp (3D) proteins from two other picornaviruses also enhanced PIV5 RNA synthesis, suggesting a conserved function across picornaviruses. In summary, this study provides the first evidence that PIV5 hijacks the RdRp of a co-infecting virus from a different viral family to support its own replication. These findings advance our understanding of virus–virus interactions and offer a novel perspective in virology.

IMPORTANCE

The gastrointestinal tract harbors a vast and diverse community of microorganisms, making it an ideal environment for exploring microbial interactions. While virus–bacteria interactions have been widely studied, virus–virus interactions remain largely uncharacterized. In this study, we demonstrated that porcine sapelovirus (PSV) and parainfluenza virus 5 (PIV5) co-infect cells and directly interact within the host. Specifically, the RNA-dependent RNA polymerase protein (3D) of PSV significantly promoted PIV5 replication by interacting with key components of the PIV5 ribonucleoprotein complex and enhancing the synthesis of both plus- and minus-strand viral RNAs. Similar effects were observed with the 3D proteins from two additional picornaviruses, suggesting a shared mechanism among picornaviruses in facilitating co-infecting virus replication. This work uncovers a novel cross-family polymerase hijacking event and provides important insights into virus–virus interactions, highlighting new potential targets for the control and prevention of swine enteric diseases.

Dietary supplementation with prebiotics such as inulin has been associated with a broad range of health benefits. However, the effects of inulin on the opportunistic fungal pathogen Candida albicans, which resides as a commensal in the gut, have not been characterized. Here, RNA sequencing revealed that inulin affects the expression of C. albicans genes associated with cell wall construction, adhesion, and yeast-hypha morphogenesis. Consistent with these changes in gene expression, inulin inhibited hyphal development, increased adhesion to human Caco-2 and A431 cells, decreased the thickness of the inner layer of the C. albicans cell wall, reduced the exposure of cell wall pathogen-associated molecular patterns [β-(1,3)-glucan and chitin], and affected antifungal drug sensitivity. These changes impacted host immune recognition and cytokine responses, ultimately attenuating the virulence of C. albicans in an invertebrate infection model. Therefore, dietary supplementation with inulin is likely to influence host-fungus interactions.

IMPORTANCE

The benefits of prebiotic dietary supplements, such as inulin (a natural plant dietary fiber), are thought to include a healthier gut microbiome, a reduced risk of colon cancer, and lower cholesterol levels. Unsurprisingly, prebiotic usage is increasing rapidly. However, while the effects of prebiotics upon gut bacteria have been characterized, the impacts upon Candida albicans, an opportunistic fungal pathogen that resides in the human gut, have remained obscure. We show that inulin affects the expression of virulence-related phenotypes and antifungal drug sensitivity in Candida. Furthermore, we show that inulin reduces the virulence of this fungus in an invertebrate model, consistent with the idea that inulin may lower the risk of fungal infection in healthy individuals.

The actinomycete Actinoplanes missouriensis grows by extending branched hyphae and forms terminal sporangia containing a few hundred spores. A sortase-dependent spore surface protein, SspA, has recently been shown to be important for sporangium maturation in A. missouriensis. In this study, we functionally characterized all five sortase candidates in A. missouriensis and revealed that only a class E sortase (SrtE) is required for sporangium formation. The srtE null mutant strain scarcely produced sporangia, in contrast to the sspA null mutant, which produced fragile sporangia, indicating that there are other SrtE substrate proteins besides SspA. Unexpectedly, ΔsrtE colonies displayed a significantly increased rate of surface area expansion on nutrient-rich solid media compared with wild-type colonies. The growing hyphae of the ΔsrtE strain raised the pH of the surrounding environment, possibly by emitting basic volatile compounds. This growth mode shares several defining characteristics with the exploratory growth (or exploration) of Streptomyces. We identified two SrtE-dependent cell wall-anchored proteins (CwpA and CwpB) required for the suppression of latent exploration in the wild-type strain; colonies of single-gene mutants of cwpA and cwpB showed exploration. While the ΔcwpA strain formed normal sporangia, severe defects in the initiation of sporangium formation were observed in the ΔcwpB strain, indicating that CwpB, but not CwpA, is essential for sporangium formation. Based on these results, we propose that SrtE suppresses latent exploration and promotes the initiation of sporangium formation by making the cell surface hydrophobic through the attachment of the hydrophobic proteins CwpA and CwpB to the cell wall.

IMPORTANCE

Exploration has recently been established as a new growth mode in the filamentous actinobacterial genus Streptomyces, where rapidly expanding mycelia lead to extensive surface colonization on nutrient-rich solid media. However, the phylogenetic prevalence and genetic basis of this unique growth mode remain unclear. Here, we report the discovery of exploration in Actinoplanes missouriensis, a filamentous actinomycete that forms terminal sporangia and zoospores. Through extensive genetic experiments, we demonstrated that a class E sortase suppresses latent exploration via covalent anchoring of two surface hydrophobic proteins to the cell wall in A. missouriensis. This study revealed that exploration capabilities are prevalent among filamentous actinomycetes beyond Streptomyces and that the hydrophobic nature of the cell surface appears to be crucial for suppressing latent exploration in A. missouriensis. The hydrophobic nature of the cell surface also appears to be important for initiating sporangium formation on nutrient-poor solid media in A. missouriensis.