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Precise and tunable genetic tools are essential for high-throughput functional genomics. To address this need in the important gram-negative pathogen Pseudomonas aeruginosa, we developed and characterized a tightly regulated CRISPR-interference (CRISPRi) system that enables precise and tunable repression of essential genes. The system utilizes a rhamnose-inducible promoter to control both the Streptococcus pasteurianus-derived dCas9 and gene-specific sgRNAs, each encoded on separate plasmids for modularity and efficiency. The combination of tight regulation and high conjugation efficiency facilitated the rapid and facile construction of strains with regulated depletion of 16 essential genes spanning diverse pathways. Comparison of phenotypes across the different genetically depleted strains, including growth rate, susceptibility to antibiotics, and changes in transcriptional programs, revealed novel aspects of gene function or small-molecule mechanism of action. Finally, the rhamnose-inducible CRISPRi system supports the generation and stable maintenance of pooled mutant libraries, thereby paving the way for future genome-wide, systematic assessment of individual gene vulnerabilities, which will provide critical insights for target prioritization in antibiotic discovery efforts against this recalcitrant pathogen.IMPORTANCECRISPR-interference (CRISPRi) has become an invaluable tool for studying genetics. In particular, the ability to knockdown (KD) genes enables the study of essential genes and their role in cell survival. However, a tightly regulated gene KD system is required to gain valuable insights into these vulnerable genes by virtue of their essentiality. We report a tightly regulated CRISPRi system to study the biology of essential gene perturbations in Pseudomonas aeruginosa, an important gram-negative pathogen that causes severe infections and is increasingly resistant to current antibiotics. This system enables characterization of both chemical genetic interactions between small molecules and specific gene depletions and the impact of genetic perturbations on transcriptional networks. Genetic perturbations using CRISPRi can thus further our understanding of basic biology with translation toward future antimicrobial development.

Antagonistic competition is a crucial survival strategy for microorganisms sharing ecological niches, playing a key role in shaping microbial communities and influencing biogeochemical cycles. Here, we report the first extracellular serine protease-dependent synergistic antagonism in archaea: a collaboration between an extracellular protease-producing strain and a precursor protein-producing strain. The serine protease secreted by the former cleaves the precursor protein released by the latter, generating an antibacterial effector molecule. This synergistic antagonism also occurs across domains (between halophilic bacteria and archaea), indicating broad ecological relevance. Using mass spectrometry and inhibition assays, we identified HFX_0892-from the model haloarchaeon Haloferax mediterranei ATCC 33500-as a key mediator of this process. Precursor protein HFX_0892 was cleaved by HlyR4 or other extracellular serine proteases, releasing the N-terminus of HFX_0892 (0892N), which displayed antagonistic activity against haloarchaea and bacteria. Disruption of the α-helical structure in 00892 via point mutations abolished the antagonistic activity. Furthermore, fusing the 0892N to HlyR4 did not interfere with HlyR4's proteolytic function but conferred antibacterial activity. Gene knockout experiments revealed that HFX_0892 is not the sole antagonistic precursor protein in H. mediterranei ATCC 33500. This study uncovers a modular proteolytic activation mechanism that can be harnessed for antimicrobial agent development. The potential prevalence of HFX_0892-like precursors among extremophiles provides a feasible strategy for exploring structurally novel antimicrobial agents.IMPORTANCEAntagonistic interactions are key drivers of microbial community dynamics in hypersaline environments. Here, we report, for the first time, a fan-shaped growth inhibition zone-an atypical phenotypic signature-resulting from synergistic antagonism between two halophilic archaeal species against a sensitive haloarchaeal strain. Using the model haloarchaeon Haloferax mediterranei, we identified a secreted precursor protein (HFX_0892) that is cleaved by a serine protease (such as HlyR4) to release an active antagonistic peptide (0892N). This novel form of archaeal interaction is defined as synergistic antagonism. The antagonistic activity of HFX_0892 is mediated by two α-helical motifs in its N-terminus, and this region can confer antimicrobial function when fused to other proteins. Notably, H. mediterranei encodes additional precursor proteins with potential antagonistic functions beyond HFX_0892. Our work identifies and elucidates a previously uncharacterized antagonistic interaction among archaea, providing critical insights into the complex interspecific interactions and microbial community assembly in hypersaline ecosystems.

Valley fever, a disease caused by Coccidioides spp., is a fungal respiratory disease with an expanding range. Methods to culture the pathogen from soil, especially Coccidioides posadasii, are very challenging, limiting the genomics knowledge of environmental strains. In this study, we designed and tested a targeted DNA capture and enrichment system for the characterization of Coccidioides genomes without the need to culture. In this system, RNA probes are hybridized to Coccidioides DNA in a complex sample, followed by DNA amplification, sequencing, and analysis. Our enrichment system was targeted toward coding region sequences in C. posadasii str. Silveira and tested on control DNA spiked into soil; DNA hybridized to probes was then sequenced and correctly placed into a reference phylogeny, based on the known placement of the whole-genome sequence. We then applied the enrichment system to a range of sample types (soil, air filters, rodent tissue) from a site in Mesa, Arizona, USA. The enriched samples were sequenced and placed into the C. posadasii phylogeny to understand the phylogenetic diversity within the Mesa site over time. The results demonstrate that low DNA signal in most sample types was boosted after enrichment. Enriched sequences from air filters collected at multiple time points from the Mesa site linked two different isolates collected from fatal cases of Coccidioidomycosis in a pig-tailed macaque colony housed at the Mesa site. This represents the first time that environmental C. posadasii DNA was directly linked to Coccidioidomycosis and demonstrates the power of this approach for genomic epidemiology.IMPORTANCEAll human cases of Valley fever are acquired through environmental exposure, so surveillance and characterization of the pathogen in soil are critical for risk mitigation efforts. Current databases are biased toward human clinical isolates, and little is known about the genomics of environmental strains of Coccidioides posadasii. In this study, we designed, tested, and validated a probe enrichment system that amplifies trace DNA in a complex sample. Sequenced DNA can be used to link environmental exposure with human cases, directing public health agencies to interventions that limit human exposure. This use case was demonstrated in this study, as trace DNA trapped on air filters was linked to a fatal case of primate Coccidioidomycosis at a site in Arizona. The probe enrichment system described in this study represents a powerful tool to better understand the genomic composition of environmental C. posadasii strains, which can aid in public health investigations.

Small DNA tumor viruses such as polyomaviruses have evolved persistent, in some cases lifelong infections despite their compact genomes and host immune pressure. This review synthesizes historical and recent insights into the mechanisms underlying polyomavirus persistence and shedding, including dynamic host cell cycle regulation, viral non-coding control region modulation, and viral microRNA-mediated repression. We highlight modes of shedding consistent with concurrent latent/lytic and smoldering infections, discuss emerging evidence of reversible latency, and identify unresolved questions in viral-host interplay. Understanding these strategies is critical for managing viral reactivation and disease in immunocompromised patients and exemplifies the remarkable evolutionary success of polyomaviruses.

Bacteriophages are ubiquitously present in bacterial communities; however, phage-bacteria interactions in complex environments like the gut remain poorly understood. Although antibiotic resistance is driving a renewed interest in phage therapy, most studies have been conducted in in vitro systems, offering limited insight into the complexity of such dynamics in physiological contexts. Here, we use the mouse-restricted enteric pathogen Citrobacter rodentium (CR), a well-established model for human enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) infections, to investigate phage-pathogen interactions in a murine model with a complex microbiota. We isolate and characterize Eifel2, a novel lytic phage infecting CR, and generate anti-phage-specific antibodies that enable the visualization of phage infections in vitro. In a murine model of CR infection, oral administration of Eifel2 led to robust phage replication in the gut without reducing the bacterial burden or infection-associated inflammation, confirming the establishment of a stable co-existence in the gut. Despite the emergence of a sub-population of phage-resistant CR mutants in vivo, they did not undergo clonal expansion, indicating that additional selective pressures impaired their widespread dissemination in the gut. Together, our findings demonstrate that imaging approaches can capture key infection stages in vitro, although in vivo models are essential for capturing the complexity of phage-bacteria interactions. This work highlights the importance of studying phage therapy in host-pathogen contexts that include a normal microbiota and a suitable host environment, where dynamic co-existence rather than eradication may define therapeutic outcomes.IMPORTANCEBacteriophages, or phages, are viruses that can either kill or persist inside bacteria. Current interests in phage biology are in part ignited by the fact that they could be used to treat infections caused by antibiotic-resistant bacteria. However, most of our understanding of phage-bacterial interactions comes from in vitro models and/or in vivo gut models relying on altering the endogenous microbiota. Here, we report the finding of a novel phage, Eifel2, which specifically targets Citrobacter rodentium (CR), the mouse equivalent of human diarrheagenic E. coli pathogens. Despite effectively killing CR in vitro, CR and Eifel2 develop a co-existence relationship in mice with an intact microbiota. Although CR phage-resistant mutants emerge, host and microbial factors constrain their expansion. This work highlights the importance of studying phage therapy in host-pathogen contexts that include the complete microbiota, where therapeutic outcomes may rely on dynamic co-existence and containment rather than eradication.

Intestinal microbiota are essential for maintaining the host's immune homeostasis, but the mechanism is not fully understood. While microbial metabolite desaminotyrosine (DAT) is recognized for its protective role in viral immunity, its potential involvement in anti-parasitic defense remains unexplored. Here, we demonstrate that DAT orchestrates tuft cell hyperplasia and subsequent type 2 immunity, establishing critical defense against helminth infection. Mechanistically, DAT-mediated intestinal epithelial remodeling requires histone deacetylase 3 (HDAC3), as pharmacological inhibition of this epigenetic regulator abrogates both tuft cell expansion and impairs type 2 immune responses. Collectively, our findings not only explore DAT novel effects in anti-parasitic defense but also reveal a pathway whereby the small molecule metabolites calibrate intestinal type 2 immunity.IMPORTANCEA small molecule metabolite DAT drives tuft cell hyperplasia and type 2 immunity in the small intestine. DAT-mediated tuft cell hyperplasia depends on HDAC3 and an intact microbiota; our findings reveal how small molecule metabolites fine-tune intestinal type 2 defenses against parasites.

Integrative and conjugative elements (ICEs) are major mediators of horizontal gene transfer in bacteria. However, the role of recipient cells in their acquisition has received little attention. Using the ruminant pathogens Mycoplasma agalactiae and Mycoplasma bovis as minimal models, we combined genome-wide transposon mutagenesis with high-throughput mating assays to identify recipient factors required for ICE acquisition. The surface lipoprotein P48 emerged as the primary determinant of ICE uptake in both species. Structural and functional analyses revealed that P48 is the substrate-binding component of an ABC transporter with nucleoside-binding capacity. A single-point mutation that abolished nucleoside binding drastically reduced ICE acquisition, demonstrating that P48-mediated nucleoside recognition is essential for conjugative transfer. However, ICE uptake did not require nucleoside transport, as inactivation of the transporter permease blocked nucleoside analog toxicity but not ICE invasion. Loss of P48 also triggered transcriptional activation of vestigial ICE genes, suggesting that surface recognition affects the intracellular state of the recipient. Remarkably, ICE transfer from recipient-derived donors was unaffected by P48 loss, underscoring its acquisition-specific role. Together, these results reveal a previously unrecognized, surface-exposed recipient factor critical for efficient ICE transfer in mycoplasmas and identify nucleotide binding as a central function in conjugation. By demonstrating that recipient-encoded functions can directly control ICE dissemination, this work challenges the donor-centric paradigm of bacterial conjugation and suggests new strategies to restrict horizontal gene flow in pathogenic and synthetic mycoplasmas.IMPORTANCEIntegrative and conjugative elements (ICEs) are mobile DNA elements that drive bacterial conjugation, a major process by which bacteria exchange genes. Although conjugation has been studied for decades, the focus has been almost exclusively on donor cells and the ICE itself, leaving the role of recipient cells largely overlooked. Using the wall-less ruminant pathogens Mycoplasma agalactiae and Mycoplasma bovis as minimal models, we discovered that a single recipient lipoprotein is required for efficient ICE uptake. Our data show that nucleoside recognition by P48, but not transport, is critical for conjugation, revealing an unexpected mechanistic link between nutrient sensing and gene acquisition. These findings shift the paradigm of conjugation from a donor-driven process to one jointly determined by donor and recipient functions. By identifying a recipient-encoded determinant of ICE transfer, this work opens new avenues to control horizontal gene flow in both pathogenic and engineered bacteria.

Rapid and accurate diagnostics of bacterial infections are necessary for efficient treatment of antibiotic-resistant pathogens. Cultivation-based methods, such as antibiotic susceptibility testing (AST), are limited by bacterial growth rates and seldom yield results before treatment needs to start, increasing patient risk and contributing to antibiotic overprescription. Here, we present a deep-learning method that leverages patient data and available AST results to predict antibiotic susceptibilities that have not yet been measured. After training on three million AST results from 30 European countries, the method achieved an average accuracy of 93% across bacterial species and antibiotics. It predicted susceptibility with an average major error rate below 5% for quinolones, cephalosporins, and carbapenems, and below 8% and 14% for aminoglycosides and penicillins, respectively. Furthermore, the model predicted resistance with an average very major error rate below 10% for cephalosporins, carbapenems, and aminoglycosides, but with higher very major error rates for penicillins and quinolones. We combined the method with conformal prediction and demonstrated accurate estimation of the predictive uncertainty at the patient level. Our results suggest that artificial intelligence-based decision support may offer new means to meet the growing burden of antibiotic resistance.IMPORTANCEImproved diagnostic tools are vital for maintaining efficient treatment of antibiotic-resistant bacteria and for reducing antibiotic overconsumption. In our research, we introduce a new deep learning-based method capable of predicting untested antibiotic resistance phenotypes. The method uses transformers, a powerful artificial intelligence (AI) technique that efficiently leverages both antibiotic susceptibility tests (AST) and patient data simultaneously. The model produces predictions that can be used as time- and cost-efficient alternatives to results from cultivation-based diagnostic assays. Significantly, our study highlights the potential of AI technologies to address the increasing prevalence of antibiotic-resistant bacterial infections.