Streptomyces bacteria are prolific producers of clinically essential natural products, yet high-throughput tools to systematically interrogate their genomes remain underdeveloped. By establishing a robust CRISPRi-seq platform for en masse functional screening in Streptomyces albidoflavus, our work closes a critical technological gap in Streptomyces functional genomics. Our study not only identifies a small subset of transporter operons essential for fitness but also introduces a scalable, generalizable approach for dissecting gene function. This platform will accelerate systems-level understanding of an industrially and medically important genus.

Candida albicans causes severe mucosal and systemic infections, with hypha formation playing a key role in its virulence. Hyphal invasion via endocytosis is mediated predominantly through interactions between Als3p and the epidermal growth factor receptor (EGFR). Subsequent EGFR activation by candidalysin, a hyphal-secreted cytolytic peptide toxin encoded by the ECE1 gene, induces receptor signaling and immune responses. While EGFR ubiquitination critically regulates receptor trafficking and signaling, its involvement during C. albicans infection has remained unexplored. Here, we demonstrate that C. albicans induces EGFR ubiquitination, leading to altered trafficking and lysosomal degradation in an ECE1- and ALS3-dependent manner. This correlates with changes in EGFR ligand expression, adaptor recruitment, and protein ubiquitination in oral epithelial cells. In a mouse model of oropharyngeal candidiasis, wild-type C. albicans and ece1Δ/Δ and als3Δ/Δ mutant strains were found to differentially regulate Egfr expression, ubiquitin pathway-associated genes, and protein ubiquitination. Furthermore, conditional EGFR knockout was protective during infection. Together, our findings reveal that C. albicans infection modulates the host ubiquitin system, including direct effects on EGFR, highlighting a novel aspect of host-fungal interactions.IMPORTANCECandida albicans is a common fungal pathogen that causes both mucosal infections, such as thrush, and life-threatening systemic diseases. A key step in infection is the fungus invading epithelial tissues and activating the host epidermal growth factor receptor (EGFR). We discovered that C. albicans alters how EGFR is regulated by inducing its ubiquitination, a modification that leads to receptor degradation. This process depends on two major fungal virulence factors: the adhesin Als3p and Ece1p, the polypeptide that contains the candidalysin toxin. The fungus also broadly increases protein ubiquitination in oral epithelial cells. In a mouse model of oral infection, loss of EGFR in epithelial tissues reduced disease severity, suggesting that the receptor helps the fungus establish infection. These findings reveal a previously unrecognized strategy by which C. albicans manipulates protein ubiquitination and regulation in epithelial cells, offering new insights into fungal pathogenesis and potential therapeutic approaches that target host pathways.

Human metapneumovirus (HMPV) causes acute respiratory disease worldwide and is the second leading cause of lower respiratory infection and hospitalization in young children in the USA. There is no licensed vaccine or therapeutic. HMPV mutates rapidly; however, the specific genomic features that explain strain dominance remain undefined because there is limited routine genomic surveillance of HMPV. We analyzed prospectively collected nasal specimens and medical data from 8,000 pediatric acute respiratory infection cases and sequenced 219 HMPV whole genomes from Pittsburgh, PA, between 2016 and 2021. Only A2, B1, and B2 subgroups were detected. The dominant subgroup varied between seasons. Variants with an in-frame 111- or 180-nucleotide (nt) insertion that nearly duplicates the preceding flanking region in the 660-nt G gene (encodes the attachment protein) were the predominant A2 viruses detected by 2016-2017. Among B2 viruses, variants with smaller in-frame insertions in the same location of the G gene became dominant by 2017-2018. Each insertion length formed a distinct phylogenetic clade. The insertions are in the ectodomain and contain positively charged residues or predicted O-glycosylation sites. Epidemiological analysis revealed that HMPV infection was independently associated with age, insurance type, and comorbidities. Elevated disease severity was independently associated with age and comorbidities, although not with HMPV subgroup. To our knowledge, in the USA, this is the earliest detection of the A2 insertion variants and the first report of the B2 insertion variants. It is the largest population-based genomic HMPV study that provides a detailed phylodynamics and epidemiological analysis of prospectively collected clinical specimens.IMPORTANCEHuman metapneumovirus (HMPV) is a leading cause of lung infection and pediatric hospitalizations worldwide for which there is no licensed vaccine or therapeutic. Because HMPV mutates rapidly, understanding which mutations enhance its ability to multiply and spread is important for the development of interventions and treatments. We prospectively collected patient data and nasal specimens from children with symptoms of acute respiratory illness. The predominant A2 and B2 HMPV variants circulating in the population contained insertions in the attachment protein, which suggests that these insertions may be advantageous to the virus. Furthermore, our analysis suggests that age, insurance type, and underlying health conditions were associated with HMPV infection. Age and underlying health conditions were associated with elevated HMPV disease severity, whereas HMPV subgroup was not. This large HMPV genomic epidemiological study provides insight into patient factors associated with disease and the emergence of the dominant variants in the USA.

The bioRxiv and medRxiv preprint servers brought preprinting to the life sciences and played a critical role in disseminating COVID research during the pandemic. Here, I reflect on the birth of bioRxiv and medRxiv and the crucial role so many members of the community played, our experience during the pandemic, and the launch of the new non-profit organization set up to oversee the servers. The pandemic was a stress test for bioRxiv and medRxiv that demonstrated their value and robustness. Under the umbrella of openRxiv, they are now poised to become long-term infrastructure underpinning a new publishing ecosystem.

Liquid-liquid phase separation (LLPS) is a phenomenon of growing interest in cell biology. It is a part of the replication cycles of diverse viruses, but our understanding of the molecular basis that underpins the mechanism of phase separation is incomplete. We previously demonstrated that the virus factories of the birnavirus IBDV, a major agricultural pathogen, are biomolecular condensates formed through LLPS. In this study, we discovered that VP3 was necessary but not sufficient for condensates to form, and the minimal components of these structures were VP3, VP1, and likely vRNA. We also discovered that the C-terminal 36 amino acid region of IBDV VP3 encoded a highly dynamic intrinsically disordered region that promoted the formation of the cytoplasmic puncta and modulated their physical properties. This work contributes to a more detailed understanding of birnavirus replication at the molecular level and to the study of LLPS as a phenomenon.

Heteroresistance is a transient resistance phenotype characterized by the presence of small subpopulations of bacterial cells with elevated antibiotic resistance within a susceptible main population. In gram-negative pathogens, heteroresistance is frequently caused by tandem amplification of genes encoding resistance proteins with low activity toward the antibiotic, a process commonly mediated by homologous recombination between flanking repeated sequences. However, the specific roles of individual recombination proteins in this mechanism remain largely undefined. In this study, we systematically evaluated the contribution of 19 recombination-associated genes to tandem amplification-driven heteroresistance in Escherichia coli. A clinical plasmid causing tobramycin heteroresistance by tandem amplification of the aac(3)-IId gene was conjugated into recombination gene-deficient mutants and the wild-type parental strain. While heteroresistance was observed with all mutants, the frequency of resistant subpopulations was decreased in recA and recB mutants, and a shift in resistance mechanism toward increased plasmid copy number and resistance mutations was observed. Partially reduced frequencies of tandem amplifications and a shift toward other heteroresistance mechanisms were also observed with recC, recJ, ruvA, and ruvC mutants, whereas other deletions of recombination genes had no or little impact on tandem amplifications. These findings identify RecABC as a key pathway in heteroresistance via tandem amplification, but even when these genes are deleted, resistant subpopulations can still be generated by other mechanisms.IMPORTANCEHeteroresistance poses a threat to efficient antibiotic treatment, as the rare resistant subpopulations often go undetected by standard laboratory tests. In Escherichia coli, heteroresistance often arises by tandem gene amplification of a resistance gene with low activity toward the specific antibiotic. This amplification is thought to be mediated by homologous recombination between repeat sequences. However, the specific roles of individual recombination proteins in this process are unclear. Here, we systematically determined the specific roles of individual recombination proteins in this process by the individual deletion of 19 recombination-associated genes. The RecABC pathway was identified as a major contributor to amplification-driven heteroresistance, but even when this pathway was disrupted, resistant subpopulations still emerged through alternative mechanisms, revealing the remarkable adaptability of bacterial populations under antibiotic stress. These findings advance our understanding of the molecular flexibility underlying heteroresistance and highlight that strategies aimed at preventing gene amplification to reduce heteroresistance are unlikely to succeed.

Undecaprenyl phosphate (C55-P) is a critical lipid carrier required for the transport of cell envelope precursors across the cytoplasmic membrane in bacteria. Recent studies have identified proteins of the DedA family and DUF368 domain family as C55-P flippases in both Gram-positive and Gram-negative organisms. However, their roles remain undefined in many clinically relevant pathogens. Here, we screened for DedA and DUF368 proteins in Pseudomonas aeruginosa and assessed their functional importance. We show that PA4029, a DedA family membrane protein, is involved in C55-P recycling. Deletion of PA4029 sensitizes cells to fosmidomycin and limits the emergence of spontaneous colistin-resistant mutants. Using native mass spectrometry, we demonstrate that PA4029 binds C55-P with high affinity and selectivity over membrane phospholipids, and that this interaction is disrupted by the C55-P targeting antibiotic amphomycin. We also show that a DUF368 protein, found in some Pseudomonas species lacking PA4029 orthologs, can functionally substitute for PA4029 in P. aeruginosa, suggesting divergent strategies for C55-P recycling in this genus. Together, these findings position PA4029 within the conserved DedA-mediated lipid carrier pathway and highlight its importance for cell envelope homeostasis and antibiotic resistance in P. aeruginosa.IMPORTANCEBacteria use lipid carrier undecaprenyl phosphate (C55-P) to build and maintain their cell envelope, which is necessary for survival and is the target of many antibiotics. Recent studies have implicated DedA family proteins in C55-P transport, but how these proteins function in important pathogens like Pseudomonas aeruginosa remains uncharacterized. In this work, we uncover a specific DedA protein, PA4029, and show its involvement in C55-P recycling and importance for bacteria's ability to develop resistance to the last-resort antibiotic colistin. These findings extend the relevance of DedA-mediated lipid transport to one of the most dreaded human pathogens.

Human papillomaviruses (HPVs) establish persistent infections in stratified epithelia and rely on host DNA damage and repair factors to support their replication. The E2 protein is central to viral genome replication and maintenance and depends heavily on its interaction with the host factor TOPBP1 for these functions. Here, we define the E2 and TOPBP1 interactomes in differentiating keratinocytes and identify nucleolin (NCL) as a critical differentiation- and TOPBP1-dependent E2 partner required for episomal genome stability. These findings expand the understanding of how HPV16 coordinates viral replication with host chromatin and DNA repair networks, uncovering a cooperative E2-TOPBP1-NCL axis that may represent a new target for antiviral intervention.

Oropouche virus (OROV) is reemerging in the Americas, along with a growing threat to global public health. Recent outbreaks have witnessed the first reported fatalities, vertical transmissions, and intercontinental importations of OROV, underscoring its expanding risk. Despite this, no vaccines or specific therapeutics are available, and fundamental research on OROV vaccinology and antigenicity remains limited. Here, we show that co-expression of the M polyprotein and nucleocapsid protein (NP) drives the assembly of OROV virus-like particles (VLPs) with high immunogenicity. Using the prototype strain OROV/sloth/Brazil/PA-UG-BeAn19991/1960, we developed an mRNA vaccine, M/N-vac, encoding these VLPs. Immunization with M/N-vac in mice elicited robust OROV-specific IgG and pseudovirus-neutralizing antibodies that cross-reacted with a contemporary circulating strain, hOROV/Brazil/AM-UKY-AM0088/2024. The vaccine also induced a durable, antigen-specific Th1-biased cellular immune response characterized by high-level interferon-gamma secretion. Additionally, we identified a highly conserved potential immunodominant epitope in BALB/c, N2-3, within the nucleocapsid protein. Furthermore, the VLP-encoding mRNA vaccine induced stronger OROV-specific humoral and cellular immune responses than the VLP protein vaccine. In vivo results based on immunization with M/N-vac demonstrate VLP-based vaccines to be a promising broad-spectrum strategy against OROV while providing novel insights into their antigenicity and design.IMPORTANCEOropouche virus (OROV) is a reemerging pathogen with no approved countermeasures, and it poses a growing public health threat. In response, we have developed a virus-like particle-based mRNA vaccine that elicits potent and durable neutralizing antibodies against both historical and circulating OROV strains, alongside a robust Th1-biased cellular immune response. This study reports the design and development of a critically needed vaccine candidate and provides fundamental insights into OROV antigenicity, thus demonstrating the utility of the mRNA platform for rapid response to emerging viral threats.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is an emerging bat-origin alphacoronavirus causing severe disease in neonatal piglets, with significant economic losses to the swine industry. The virus exhibits a broad species tropism, infecting cells derived from pigs, humans, and mice, highlighting its potential for cross-species transmission. Due to drawbacks associated with the use of young piglets, there is a need for an appropriate small animal model to study SADS-CoV biology. Here we established a mouse infection model based on a murinized mutant of the virus, mSADS-CoV, in which the ectodomain of the viral spike protein was replaced by that of the murine coronavirus mouse hepatitis virus. This chimeric virus, generated through targeted RNA recombination, replicated efficiently in murine cell cultures and exhibited an age-dependent infection in neonatal mice that was lethal in 2-day-old BALB/c mice, affecting various organs, notably the intestine. We validated our infection model by successfully verifying the efficacy of the RNA-dependent RNA polymerase inhibitor remdesivir. The model will serve as a valuable tool for studying SADS-CoV pathogenesis and for elucidating the roles of host factors in viral replication as well as for preclinical evaluation of antiviral compounds targeting the viral replication machinery.IMPORTANCESwine acute diarrhea syndrome coronavirus (SADS-CoV) poses a threat to the swine industry and public health because of its broad species tropism and potential for cross-species transmission. The emergence of other bat-derived coronaviruses, including severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-2, and Middle East respiratory syndrome coronavirus, underscores the need for robust models to study these pathogens. The successful rescue of mSADS-CoV and the development of a mouse infection model represent significant advancements in SADS-CoV research. This model not only enables the evaluation of antiviral therapeutics such as remdesivir but also provides a powerful platform for investigating viral replication mechanisms and host-pathogen interactions, offering critical insights for pandemic preparedness.