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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.

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.

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.

Oropouche virus (OROV), a neglected arbovirus, has historically been considered a self-limiting infection associated with febrile illness. However, the recent surge in cases since late 2023 has been marked by atypical outcomes, highlighting its underestimated clinical impact. Gastrointestinal symptoms such as diarrhea have also been reported, but the prevalence and mechanistic insight remain largely elusive. Here, through a meta-analysis of 12 identified clinical studies, we revealed a pooled prevalence of diarrhea as 15% (95% CI, 10%-20%) among the Oropouche patient population. In primary human intestinal organoid-based experimental models, we demonstrated productive infection by both a recent patient isolate (OROV-2024) and a historical strain (Be An19991). This is shown by the accumulation of intracellular OROV RNA, release of infectious particles, and immunostaining of OROV glycoprotein Gc. Interestingly, OROV infection mildly triggered the expression of type III interferons, but this endogenous response was insufficient to limit viral replication. In contrast, exogenous treatment with type I and III interferons strongly inhibited OROV replication, with interferon-alpha completely abolishing infectious virus production. Together, these results suggest the human intestine as a potential target organ for OROV infection and highlight interferons as potential therapeutic candidates.IMPORTANCEOropouche virus (OROV) is an emerging arbovirus with rapidly increasing incidence and recent reports of severe disease outcomes. While gastrointestinal symptoms have been described, the intestinal tropism of OROV has not been experimentally explored. By combining meta-analysis of clinical data with human intestinal organoid infection models, we demonstrate that OROV can replicate in intestinal epithelial cells. We further show that, in a human intestinal organoid model, endogenous interferon responses are insufficient to restrict replication, while treatment with interferons exerts potent antiviral activity. These findings highlight the susceptibility of intestinal epithelial cells to OROV infection and the therapeutic potential of interferons.

Treatment with latency-reversing agents (LRAs) alone has been ineffective in reducing HIV-1 reservoirs in people living with HIV-1 (PLWH) who are on antiretroviral therapy (ART), due to inefficiencies in reservoir reactivation and adaptive immune responses. However, NK cells activated with cytokines may be able to target HIV-1 reservoirs more effectively. To explore the therapeutic potential of NK cells, we expanded blood NK cells from multiple donors ex vivo into CD56^bright CD16⁺ "eNK" cells using artificial antigen-presenting cells (aAPCs) expressing membrane-bound IL21. eNK cells express multiple activating receptors and are highly cytotoxic against specific target cells. They can also kill HIV-infected CD4+ T cells via antibody-dependent cell-mediated cytotoxicity (ADCC) using broadly neutralizing antibodies (bNAbs) against HIV-1 Env gp120/gp41. Notably, eNK cells from PLWH on ART efficiently killed autologous HIV-1+ T cells reactivated by a combination of vorinostat (SAHA) and IL-15 or an IL-15 superagonist (N-803), as evidenced by declines in proviral load, inducible HIV-1 mRNA, and virus release. Adoptive immunotherapy with eNK cells combined with LRA treatment thus presents a promising strategy to reduce the latent HIV-1 reservoir in PLWH.IMPORTANCEAntiretroviral therapy (ART) lowers HIV levels in the blood to nearly undetectable amounts, but stopping therapy almost always leads to HIV rebounding in the bloodstream. DNA and RNA tests show that most people living with HIV (PLWH) on ART retain long-lasting HIV reservoirs that remain hidden from the immune system when no HIV is being produced. Eradicating HIV might look like "drug-free remission," where HIV reservoirs are kept under control by the immune system even if ART is stopped indefinitely. Current strategies for this potential eradication include using HIV latency-reversing agents (LRAs), ex vivo expansion of natural killer (NK) cells, and improving the ability to kill infected cells with broadly neutralizing antibodies against HIV. Here, we demonstrate that NK cells from PLWH can be expanded outside the body into "eNK" cells that specifically attack HIV-infected cells without harming uninfected ones, significantly reducing HIV reservoirs in vitro after LRA treatment.

The HerA-NurA complex reportedly functions in DNA end resection in archaea. End resection is important to start homologous recombination by forming a single-stranded DNA region with an overhanging 3'-end, which invades double-stranded DNA (dsDNA) with a homologous sequence to form a D-loop. Here, we studied the structure and functions of HerA-NurA from the hyperthermophilic archaeon, Thermococcus kodakarensis. Our analyses demonstrated that NurA is a non-directional and single-stranded specific nuclease, but the HerA-NurA complex cleaves both strands of dsDNA in an exonucleolytic manner, regardless of the structure of the DNA end. The 3D structures of HerA-NurA and its complex with dsDNA revealed the detailed molecular mechanisms of these nuclease reactions. These results suggest that HerA-NurA may not be involved in the end resection process but instead performs other functions, such as exerting an antiviral function by degrading the dsDNA of foreign viruses, similar to recent studies in bacteria.

Importance: To understand the specific function of the HerA-NurA complex, which is believed to function in the end resection process to create a 3'-overhanging DNA for the following strand invasion in homologous recombination, we performed biochemical and structural analyses of this complex from a hyperthermophilic archaeon, Thermococcus kodakarensis, inhabiting a harsh environment where DNA is easily damaged. We found that the HerA-NurA complex cleaves both strands of double-stranded DNA in an exonucleolytic manner, regardless of the structure of the DNA end. Our structural analysis revealed the detailed characteristics of the nuclease activity exhibited by the HerA-NurA complex. Based on the presented information, it is unlikely that the HerA-NurA complex directly functions in end resection, but rather is involved in other functions, possibly in defense against viral infections.