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Phage-plasmids (P-Ps) are temperate phages that replicate as plasmids during lysogeny. Despite their high diversity, they carry genes similar to phages and plasmids. This leads to gene exchanges and to the formation of hybrid or defective elements, which limits accurate detection of P-Ps. To address this challenge, we developed tyPPing, an easy-to-use method that efficiently detects and types P-Ps with high accuracy. It searches for distinct frequencies and sets of conserved proteins to separate P-Ps from plasmids and phages. tyPPing's strength comes from both its precise predictions and its ability to systematically type P-Ps, including the assignment of confidence levels. We tested tyPPing on several databases and a collection of incomplete (draft) genomes. While predictions rely on the quality of assemblies, we detected high-quality P-Ps and experimentally proved them to be functional. Compared to other classification methods, tyPPing is designed to detect distinct P-P types and surpasses other tools in terms of sensitivity and scalability. P-Ps are highly diverse, making the systematic identification of new types a difficult task. By combining tyPPing with other tools, however, we show a valuable foundation for addressing this challenge. How to use tyPPing and other approaches is documented in our GitHub repository: github.com/EpfeiferNutri/Phage-plasmids/.

Importance: Mobile genetic elements, such as phages and plasmids, are diverse and drive bacterial evolution through horizontal gene transfer. Phage-plasmids, of which many carry antibiotic resistance genes or virulence factors, are both phages and plasmids and have life cycles of temperate phages and plasmids. This makes accurate classification difficult as current computational tools typically classify them as one or the other. We addressed this problem by developing tyPPing, a new and highly precise method, to systematically identify, separate, and catalog phage-plasmids. We demonstrated that tyPPing is highly accurate and broadly compatible. It provides a reliable foundation for all future studies involving phages and plasmids, ranging from agriculture environments to pathogenic strains of clinical settings.

Cryptococcus neoformans (Cn) is the main cause of fungal meningitis in people living with HIV. Perturbations in normal immunoglobulin (Ig) levels are observed in these individuals, but their association with Cn pathogenesis is unclear. Here, we investigated the physical and biological effects of normal (not elicited by known cryptococcal infection) human immunoglobulins (Igs), IgM, IgG, and IgA on Cn (strain H99). Each isotype affected the growth, surface morphology, and proteome of Cn. However, IgA had the most prominent effect. It induced growth inhibition after 24 h of co-culture with Cn, altered the structural organization of capsular fibers, and significantly reduced protein synthesis and proteins associated with intracellular glucuronoxylomannan (GXM) synthesis, such as those mediating transport of sugar precursors to Golgi and the cyclic AMP pathway. Together with prior data showing an association between reduced plasma IgA and HIV-associated cryptococcal meningitis (CM), our findings suggest that the influence of human IgA on Cn pathogenesis warrants further investigation.IMPORTANCECryptococcal meningitis (CM) causes approximately 1,200,000 deaths annually in people living with HIV and is also a threat to individuals with non-HIV-associated immune-compromising conditions, such as organ transplant recipients and other patients receiving immunosuppressants. Prior work has shown that normal human immunoglobulins (Igs) bind Cryptococcus neoformans (Cn) and that plasma levels of IgM, IgG, and IgA differ as a function of CM status. We investigated how human IgM, IgG, and IgA affect Cn growth, morphology, and protein synthesis. We found that IgA has major effects on these aspects of Cn biology and lends plausibility to the hypothesis that previously reported reductions in IgA levels in HIV-associated CM may influence Cn pathogenesis. Overall, our findings show that antibody immunity to Cn is more complex than previously thought.

Human cytomegalovirus (HCMV) establishes latency in CD34⁺ hematopoietic progenitor cells (HPCs), where reactivation is intimately linked to cellular differentiation. We demonstrate that the Notch signaling pathway, a key regulator of stem cell maintenance and differentiation, functions as a barrier to HCMV reactivation. Two viral gene products, UL8 and miR-UL36, modulate this pathway during reactivation. UL8 promotes degradation of the Notch3 receptor via the endosomal/lysosomal pathway, dependent on two tyrosine-based motifs (Y305/314) in its cytoplasmic tail. A UL8 mutant lacking these motifs fails to degrade Notch3, resulting in sustained Notch signaling and impaired reactivation in vitro and in humanized mice. Similarly, miR-UL36 reduces the expression of Notch3 and the Notch transcription factor recombination signal binding protein for immunoglobulin kappa J region (RBPJ), suppressing Notch signaling. Deletion of miR-UL36 inhibits reactivation, but this defect, like that of the UL8 mutant, can be rescued by pharmacologic Notch inhibition. Thus, HCMV employs multiple gene products to suppress Notch signaling and promote conditions conducive to reactivation. These findings reveal how HCMV manipulates host differentiation pathways to control latency and suggest therapeutic strategies to prevent viral recurrence in immunocompromised patients.IMPORTANCEHuman cytomegalovirus (HCMV) establishes lifelong latency, posing significant risks to transplant recipients and other immunocompromised individuals. Reactivation depends on progenitor cell differentiation; however, the viral mechanisms governing this process remain unclear. We identify Notch signaling as a major inhibitory pathway to reactivation and show that HCMV uses UL8 and miR-UL36 to suppress this pathway. UL8 degrades Notch3, while miR-UL36 downregulates Notch3 and RBPJ, together reducing Notch signaling and enabling reactivation. Mutant viruses lacking these regulators fail to reactivate efficiently, but this can be reversed by pharmacological inhibition of Notch. These findings establish Notch pathway suppression as a critical viral strategy for reactivation and highlight potential therapeutic targets for preventing HCMV disease.

The Gram-negative bacterial cell envelope comprises an outer membrane (OM) with an asymmetric arrangement of lipopolysaccharides and phospholipids (PLs), protecting them from both physical and chemical threats. To build the OM, PLs must be transported across the cell envelope; this process has remained elusive until recently, where three collectively essential AsmA-superfamily proteins-YhdP, TamB, and YdbH-are proposed to function as anterograde PL transporters in Escherichia coli. Here, we identify the cell wall-binding protein DedD as a novel interacting partner of YhdP and discover that all three AsmA-superfamily proteins are recruited to and strongly enriched at the cell poles. Our observation raises the possibility that anterograde PL transport could be spatially restricted to the cell poles and highlights the importance of understanding the spatial-temporal regulation of OM biogenesis in coordination with cell growth and division.IMPORTANCEThe outer membrane (OM) of Gram-negative bacteria serves as an effective permeability barrier and confers intrinsic antibiotic resistance. This barrier function requires distinct distribution of lipids across the bilayer, yet how phospholipids, the most basic building block, get transported and assembled into the OM is not well understood. In this study, we describe the observation revealing that three putative phospholipid transporters are mostly present at the cell poles in Escherichia coli, highlighting possible polar localization of lipid transport to ultimately support OM biogenesis during growth and division. Our work sets the stage for studying how phospholipid transport impacts OM stability, lipid asymmetry, and/or function, thus informing future strategies for antibiotics development against these processes.

Pathogenic mycobacteria encounter acidic environments during host invasion, necessitating sophisticated acid resistance mechanisms. Here, we identify the GntR family regulator DasR as a conserved cyclic di-AMP (c-di-AMP) receptor in Mycobacterium tuberculosis that orchestrates acid adaptation through a multilayer network. Biochemical analyses demonstrated that DasR binds c-di-AMP with 20-fold higher affinity under acidic conditions than under neutral conditions, as evidenced by a Kd shift from 226 μM to 11.4 μM. This pH-sensitive binding aligns with acidified host niches during infection. ChIP-seq revealed that DasR directly targets nucleotide second-messenger metabolism genes, dynamically balancing intracellular pools of (p)ppGpp, cyclic AMP (cAMP), and c-di-AMP via positive feedback regulation. Concurrently, DasR upregulated the expression of the molecular chaperone HtpG, which stabilizes the DasR complex under acid stress. Functionally, c-di-AMP enhances DasR-DNA binding capacity at low pH, whereas HtpG-mediated thermostability amplifies signal output. This integrated axis coupling pH sensing, transcriptional reprogramming of stress metabolites, and chaperone reinforcement confers robust acid resistance. These findings establish the c-di-AMP-DasR pathway as an evolutionarily optimized strategy for mycobacterial persistence in hostile environments and suggest that this axis could be targeted to disrupt M. tuberculosis resilience.IMPORTANCEThe findings identified a regulatory axis central to mycobacterial acid adaptation, and DasR was found to be a conserved c-di-AMP receptor in Mycobacterium tuberculosis. A key feature is its highly pH-sensitive binding to c-di-AMP, which exhibits a 20-fold increase in affinity under acidic conditions, indicating that environmental cues are linked to the transcriptional response. DasR directly targets genes governing (p)ppGpp, cyclic AMP (cAMP), c-di-AMP metabolism, and acid adaptation, creating a feedback loop that dynamically balances stress signaling pathways. The concurrent upregulation of the chaperone HtpG stabilizes the DasR complex, increasing signal output under stress. This integrated system, which combines allosteric enhancement of DNA binding with chaperone-mediated stabilization, constitutes an evolutionarily refined strategy for acid resistance. The c-di-AMP-DasR pathway is therefore a promising target that could enable researchers to address the persistence of M. tuberculosis.

Fluconazole (FLC)-resistant Candidozyma auris isolates have reduced drug accumulation compared to azole-susceptible isolates. Of 119 C. auris isolates, 83 out of 87 resistant isolates (~95%) had extremely low fluconazole uptake, whereas 30 out of 32 susceptible isolates (~93%) had high fluconazole uptake. In search of a genetic explanation for this phenomenon, we compared metadata for TAC1B and CDR1 single-nucleotide polymorphisms (SNPs) and found overlap with many but not all isolates that are FLC resistant. We found that CDR1 mutations are common in resistant isolates from Clade 1, and TAC1B mutations are commonly found in resistant isolates from clades 1 and 3. There is clearly an association between FLC resistance and certain CDR1 and TAC1B polymorphisms, but mutations in these genes do not account for all mechanisms of resistance in this species and do not account for the difference in FLC accumulation. However, when ERG11 SNPs were included in the analysis, there is a clear correlation between low FLC accumulation and isolates that have one of five ERG11 variants and also high FLC accumulation and isolates that have non-variant ERG11 sequences. The ERG11 mutations F126L, K143R, V125/F126L, Y132F, or Y501H are correlated to fluconazole resistance and reduced fluconazole accumulation. This is a unique characteristic of C. auris, suggesting mutations in ERG11 can cause changes in the ergosterol biosynthesis pathway and membrane composition, organization, and permeability.IMPORTANCECandidozyma auris is a global human health threat because of its near-universal resistance to the antifungal fluconazole as well as a predisposition to multidrug resistance among clinical isolates. The underlying mechanisms of antifungal drug resistance in this species are still largely under investigation, and these efforts are significantly supported by research that increase our understanding of unique aspects of C. auris biology. We have identified a correlation between C. auris isolates' susceptibility to fluconazole and intracellular drug accumulation in which drug-resistant isolates have significantly reduced intracellular fluconazole compared to isolates that are susceptible to fluconazole. We have proposed a mechanism for this phenomenon and demonstrated important roles for mutations in ERG11, TAC1B, and CDR1 gene sequences for drug resistance.

Using a selective plating strategy for staphylococci, we surveyed the local community wastewater and purified 16 independent isolates representing the following seven species of Staphylococcus: S. cohnii, S. equorum, S. lentus, S. nepalensis, S. sciuri, S. shinii, and S. xylosus. Staphylococcus aureus was not detected. The wastewater also served as a source to identify a bacteriophage (phage), referred to here as JS1, that could infect all these species of Staphylococcus, as well as a range of clinical S. aureus strains, including methicillin-resistant isolates. The class Caudoviricetes are tailed phages, and classification systems recognize the following three major morphotypes: the Myo-like (medium-to-long, straight, contractile tails), Sipho-like (long, flexible, non-contractile tails), and Podo-like (very short, rigid tails). Electron microscopy showed that JS1 virions have 252 nm long, curved, contractile tails. Curvature analysis showed that this represented a range with a 1/R value of 7.6 ± 1.3 μm^-1, where R is the radius of curvature. Phage JS1 also encodes hydrolases that are assembled onto the phage virions. One of these hydrolases, JS1_0224, was biochemically characterized and found to etch regions from the Staphylococcal cell wall. The possibility that these on-board hydrolases and the curvature of the long contractile tails are advantageous to the phage for navigating through the cell wall of these various species of Staphylococcus is discussed.IMPORTANCEPast work has seen over-representation of Staphylococcus aureus clinical isolates in genome and biology studies on staphylococci. Here, we show by a selective plating analysis of municipal wastewater that independent isolates representing seven other species of Staphylococcus were recovered (S. cohnii, S. equorum, S. lentus, S. nepalensis, S. sciuri, S. shinii, and S. xylosus), as readily identified in the samples. Genome sequence analysis revealed some species-specific antibiotic resistance profiles across the strains, and a bacteriophage was isolated that had a cross-species host range. Using this broad biological approach to analyze staphylococci has identified a phage with a broad killing range, and this phage is morphologically distinct from the three known types of tailed phages.

Overlapping genes-wherein two different proteins are translated from alternative reading frames of the same DNA sequence-provide a means to stabilize an engineered gene by directly linking its evolutionary fate with that of an overlapping gene. However, creating overlapping gene pairs is challenging, as it requires redesigning both protein products to accommodate overlap constraints. Here, we present a new "overlapping, alternate-frame insertion" (OAFI) method for creating synthetic overlapping genes by inserting an "inner" gene, encoded in an alternate frame, into a flexible region of an "outer" gene. Using OAFI, we create new overlapping gene pairs of genetic reporters and bacterial toxins within an antibiotic resistance gene. We show that both the inner and outer genes retain function despite redesign, with translation of the inner gene influenced by its overlap position in the outer gene. Importantly, we show that, despite these inner gene sequences not contributing to outer gene function, selection for the outer gene alters the permitted inactivating mutations in the inner gene, and that overlapping toxins can restrict horizontal gene transfer of the antibiotic resistance gene. Overall, OAFI offers a versatile tool for synthetic biology, expanding the applications of overlapping genes in gene stabilization and biocontainment.

Importance: Genetically engineered microbes promise to improve human health and help solve global climate crises. However, the widespread adoption of these microbes is often hindered by genetic instability caused by mutations and by the unpredictable spread of synthetic genes in the environment. We present a simple but effective method for creating synthetic overlapping genes to stabilize genes against mutations and prevent their spread in the environment. This method is broadly useful for constructing stable genetically engineered microbes and studying how they evolve in the environment.

Taxonomic classification alone fails to capture the ecological and functional diversity of vaginal microbiomes, particularly those dominated by Gardnerella species. Using the expanded VIRGO2 gene catalog, we developed the vaginal inference of subspecies and typing algorithm (VISTA), a novel ortholog-based framework that defined metagenomic subspecies and 25 metagenomic community state types (mgCSTs), including six distinct Gardnerella-dominated profiles. The mgCSTs exhibit marked differences in species composition, functional gene content, transcriptional activity, and host immune responses. These findings reveal that Gardnerella predominance does not uniformly equate to dysbiosis and underscore the importance of functional context in shaping host-microbiome interactions. VISTA provides scalable classifiers and an interactive application to support mechanistic studies of vaginal microbiome function and its implications for reproductive health.IMPORTANCEThe vaginal microbiome plays a central role in reproductive and gynecologic health, yet its functional diversity and ecological organization remain poorly understood. Traditional 16S rRNA approaches provide only a partial view of this complexity, overlooking the strain-level variation that often determines microbial behavior and host outcomes. By applying metagenomic sequencing and scalable computational modeling, we developed the vaginal inference of subspecies and typing algorithm, a framework that defines gene-based subspecies and community state types across diverse populations. These classifications reveal new insights into the genomic and ecological foundations of vaginal community structure and offer a standardized resource for comparative and translational microbiome research. This work establishes the foundation for functionally informed diagnostics and precision interventions targeting women's reproductive health.

The intracellular pathogen Legionella pneumophila has evolved multiple effector proteins delivered into host cells by the Dot/Icm Type IVb secretion system that prevents recognition of the vacuole in which it resides by the host autophagy pathway. The number of effectors involved in this process remains unclear. Thus, we conducted a screen in Saccharomyces cerevisiae to identify Legionella effectors that were sufficient to block autophagy. This screen identified the Legionella protein Lem26 as an effector capable of autophagy inhibition. Lem26 production inhibited the recruitment of core autophagy proteins to autophagic targets and prevented the proteolytic processing of autophagy substrates in both yeast and mammalian systems. The Lem26 protein encodes an ADP-ribosyltransferase (ART) domain that was found to be essential for anti-autophagy activity. In vitro studies showed that purified Lem26 was inactive in solution, but the addition of pre-autophagosomal membranes obtained from fractionated mammalian cell lysates stimulated Lem26 ART activity. The addition of synthetic membranes containing lipid-conjugated ATG8 proteins was sufficient to stimulate Lem26 activity in vitro. An ATG8-interacting motif identified in Lem26 was critical for the activation of Lem26. These data establish that Lem26 is a Legionella effector that is recruited and activated upon interaction with autophagic membranes, and this promotes the posttranslational modification of proteins on the autophagic membrane to arrest the autophagy pathway.IMPORTANCEBacterial pathogens have evolved intricate mechanisms to specifically avoid detection by the host autophagy pathway, which is a cell-autonomous innate immune pathway conserved in all eukaryotic organisms. The intracellular pathogen Legionella pneumophila has co-evolved with evolutionarily diverse protozoan hosts for over 100 million years. Thus, these bacteria have devised multiple strategies for evading host autophagy. In this study, we analyzed roughly 300 different Legionella effector proteins for their ability to disrupt autophagy in yeast. The Legionella effector protein Lem26 was found to specifically block autophagy in both yeast and mammalian cells. Biochemical studies revealed that this protein is tightly regulated and is activated upon binding to autophagosomal membranes, which stimulates Lem26 ADP-ribosyltransferase activity and results in the modification of critical autophagy proteins colocalized to these membranes. Thus, Lem26 has evolved the capacity to disrupt host autophagy by proximity labeling of host determinants on autophagosomal membranes, which represents a unique strategy for autophagy inhibition.