Biology subjects

Piya, D.

Publications and source records attributed to Piya, D..

8 recordsLinked to original sources

bioRxiv2026

Genome-wide characterization of host factors involved in single-stranded RNA and DNA phage infection pathways

Single stranded RNA (ssRNA) and single stranded DNA (ssDNA) bacteriophages represent a key component of the global virome, yet the host genetic networks supporting their infection cycles remain poorly understood. Here, we present a comprehensive, genome-wide mapping of the genetic landscape regulating infection cycles for F pilus-dependent ssRNA and ssDNA phages in Escherichia coli. Genetic screens across ssRNA phages spanning all four genogroups of the Leviviricetes revealed a highly conserved network of host dependencies, with the notable exception of the F plasmid gene traD. While primary structural receptor components and dsbA mediated disulfide bond formation are universally required across all lineages to ensure F pilus integrity, traD exhibits a strict genogroup-specific requirement during entry, showing variable essentiality across different viral groups despite sharing an identical primary receptor. Our gene dosage screens revealed that an elevated copy number of the hslU protease or the RNA chaperone stpA restricts infection, identifying clear genetic barriers that can perturb the viral life cycle. Parallel assays with filamentous ssDNA phages produced host factor profiles consistent with published literature, while revealing additional variations in host dependency. These screens confirmed that ssDNA phages strictly rely on the host TolQRA complex for entry downstream of pilus engagement. The assays tracked prominent negative fitness signatures across homeostatic clusters, highlighting how the physiological burden of continuous, non-lytic virion extrusion strains the host envelope. Finally, this comparative approach traced the selectivity of our isolation host (E. coli HSF) to a horizontally acquired capsule architecture from Klebsiella. This surface shield excludes a large panel of double stranded DNA phages isolated on diverse E. coli strains, while allowing virions from ssDNA and ssRNA phages to engage the extended F pilus and bypass the barrier via native pilus retraction. Together, this work provides a systematic, class-wide map of single stranded phage-host interactions, bridging classical genetics with modern viral discovery while establishing a robust host platform to access uncultured viral diversity and a functional blueprint to design next generation diagnostics, protein antibiotics, and biocontrol tools to halt horizontal gene transfer.

microbiology↗

bioRxiv2026

A comprehensive phage-bacteria interaction atlas links phage lineage and capsule serotype to genome-guided machine learning prediction in Klebsiella pneumoniae

Klebsiella pneumoniae is a WHO critical-priority pathogen for which strain-specific bacteriophages are being explored as precision antimicrobials, yet rapid phage-host matching remains a major barrier to therapeutic deployment. We constructed a comprehensive interaction atlas comprising 84 taxonomically diverse phages and 101 globally sourced, clinically representative K. pneumoniae strains, including multidrug-resistant isolates. Systematic pairwise profiling produced 8,484 interaction measurements, of which 2,656 (31.3%) scored positive for bacterial clearance. Genus was the dominant phage-side determinant of host range, while capsule K-serotype was the strongest host-side determinant of susceptibility; aggregate defense, prophage, plasmid, and antimicrobial-resistance features contributed comparatively little. A genome-guided machine learning model predicted interactions without curated host annotations (AUROC, 0.882; AUPR, 0.765), outperforming a model based only on phage genus and K-serotype and modestly exceeding a curated genomic baseline. The model recovered capsule- and lipopolysaccharide-biosynthesis genes, canonical receptors and defense-associated features as major predictors using SHAP analysis. Feasibility tests of expert- and model-selected cocktails exposed a translational constraint. Although all formulations suppressed growth in vitro, only the specific cocktail whose phages replicated robustly within the murine gut reduced colonization, suggesting in vivo amplification rather than predicted host range as the limiting factor for therapeutic efficacy. Together with the activity of a model-selected cocktail built for an isolate completely excluded from training, these results provide a species-wide resource for K. pneumoniae phage matching and support a hybrid workflow combining genome-based ranking with targeted phenotypic validation.

microbiology↗

bioRxiv2026

Surface architecture of the bacterial envelope determines phage adsorption route in pathogenic Escherichia coli O157:H7

The outermost surface layers of Gram-negative bacteria determine phage access to terminal receptors, yet their genetic basis has been mapped almost exclusively in laboratory strains that lack them. Here we apply genome-wide RB-TnSeq fitness profiling to four Escherichia coli O157:H7 strains from distinct phylogenetic clades sharing the O157 O-antigen, using 38 phages with terminal receptors previously mapped in E. coli K-12 strain. RB-TnSeq fitness landscapes across all four pathogenic backgrounds were mostly similar, and dominated by surface-associated loci, including the gfc-etk group 4 capsule operon, O-antigen biosynthesis genes, LPS core assembly genes and outer membrane proteins. Disruption of gfc-etk abolished infection in 11 genetically diverse myoviruses, establishing the O-antigen capsule as a widespread required primary recognition substrate. O-antigen loci generated two classes of fitness score patterns. For 10 phages, disruption increased infectivity, indicating it is a barrier to receptor access; for 3 others, disruption abolished infectivity, demonstrating it can also be a primary recognition substrate. Outer membrane protein receptor identity was conserved across laboratory and pathogenic backgrounds, with the same proteins recognized in both K-12 and O157:H7, while glycan layer state determines whether these receptors are reached. These results demonstrate that outer surface glycan layers can act as primary and optional recognition substrates for phage infection, or as physical barriers preventing terminal receptor access. Extending the ability to probe phage-targeted receptors beyond outer membrane proteins provides a framework for incorporating glycan layer state into predictive models of phage-host interactions.

microbiology↗

bioRxiv2026

Comprehensive interaction profiling and machine learning prediction of bacteriophage infectivity across clinically diverse Pseudomonas aeruginosa

The rise of antibiotic-resistant bacterial infections has driven renewed interest in bacteriophage therapy, where viruses that specifically kill bacteria are used as targeted antimicrobials. Pseudomonas aeruginosa, a WHO critical-priority pathogen that causes severe infections in hospitalized and immunocompromised patients, presents a major challenge for phage therapy because of its extraordinary genetic diversity. Phages effective against one bacterial strain often fail against others, and existing cross-resistance-profiling approaches require iterative empirical testing of each new patient isolate. To establish a genome-based framework for rapid phage-isolate matching, we assembled a collection of 95 genomically diverse P. aeruginosa phages representing 20 genera and tested each against 99 genetically diverse clinical isolates, generating 9,405 infection outcome measurements. Bacterial O-antigen serotype emerged as the dominant determinant of strain susceptibility, while defense systems, anti-defense systems, and prophage burden contributed smaller strain-specific effects. The full curated multivariate model explained 47% of strain-susceptibility variance. Machine-learning models integrating these features and pangenome-derived gene clusters reached a per-strain AUROC of 0.86. In an in vivo proof-of-concept test against a single held-out strain, the ML-designed cocktail produced a [~]12-fold greater median CFU reduction than the expert-designed cocktail (q = 0.045), with both cocktails substantially reducing burden relative to the untreated control ([~]113-fold for ML, [~]9-fold for CG; both q < 10{square}3). SHAP analysis of the model identified bacterial surface-architecture genes (LPS biosynthesis, outer membrane proteins, type IV pili) as the dominant predictors, with defense-system content modulating which specific phages succeed against a strain rather than uniformly damping susceptibility. Together, these results establish a genome-based framework for predicting phage susceptibility in genetically diverse clinical isolates.

microbiology↗

bioRxiv2025

Surviving phage attack dynamically regulates bacterial immunity to defeat counterdefenses

Transcriptional repressors with ligand-responsive WYL domains control diverse bacterial defense systems, yet the natural cues driving derepression and the physiological rationale for dynamic regulation in native host-phage contexts remain unknown. Here, we show that restricted phage infection, where defense clears a primary attack, serves as a natural cue for WYL-mediated derepression in Vibrio cholerae. Although not required to block the initial infection, this response increases defense protein abundance, shifting host-phage stoichiometry and priming surviving bacteria to overwhelm phage-encoded counterdefenses in subsequent attacks. We also find that restricted infection co-induces an unlinked anti-plasmid defense via its own WYL repressor, showing that parallel WYL sensors coordinate a broader immune response without regulatory crosstalk. In contrast, productive phage infection triggers horizontal transfer of the defense-encoding mobile element, ensuring its persistence in the population. Together, our work reveals that infection fate dictates divergent outcomes for the expression and dissemination of bacterial immunity.

microbiology↗

bioRxiv2023

Genome-wide CRISPRi knockdown to map gene essentiality landscape in coliphages {lambda} and P1

Phages are one of the key ecological drivers of microbial community dynamics, function and evolution. Despite their importance in bacterial ecology and evolutionary processes, phage genes are poorly characterized, hampering their usage in a variety of biotechnological applications. Methods to characterize such genes, even those critical to the phage life cycle, are labor-intensive and are generally phage-specific. Here, we develop a systematic gene essentiality mapping method scalable to new phage-host combinations that facilitate the identification of non-essential genes. As proof of concept, we use a catalytically inactive Cas12a mediated genome-wide CRISPRi assay to determine the essential genes in the canonical coliphages {lambda} and P1. Results from a single panel of CRISPRi probes largely recapitulate the essential gene roster determined from decades of genetic analysis for lambda and provide new insights into essential and nonessential loci in P1. We present evidence of how CRISPRi polarity can lead to false positive gene essentiality assignments and recommend caution towards interpreting CRISPRi data on gene essentiality when applied to less studied phages. Finally, we show that we can engineer phages by inserting DNA barcodes into newly identified inessential regions, which will empower processes of identification, quantification and tracking of phages in diverse applications.

microbiology↗

bioRxiv2023

Geochemical constraints on bacteriophage infectivity in terrestrial environments

Lytic phages can be potent and selective inhibitors of microbial growth and can have profound impacts on microbiome composition and function. However, there is uncertainty about the biogeochemical conditions under which phage predation can proceed and modulate microbial ecosystem function, particularly in terrestrial systems. Ionic strength is known to be critical for infection of bacteria by many phages, but there is limited quantitative data on ion thresholds for phage infection that can be compared with environmental ion concentrations. Similarly, while carbon composition varies in terrestrial environments, we know little of which carbon sources favor or disfavor phage infection and how these higher order interactions impact microbiome function. Here, we measured the half-maximal effective concentrations (EC50) of 80 different inorganic ions for the infection of E. coli with two canonical dsDNA and ssRNA phages, T4 and MS2, respectively. We found that many alkaline earth metals and alkali metals enabled successful lytic infection but that the ionic strength thresholds varied for different ions between phages. Additionally, using a freshwater nitrate reducing microbiome, we found that the ability of lytic phage to influence nitrate reduction end-products was dependent on the carbon source as well as the ion concentration. For all phage:host pairs we tested, the ion EC50s for phage infection we measured exceed the ion concentrations found in many terrestrial freshwater systems. Thus, our findings support a model where the influence of phages on terrestrial microbial functional ecology is greatest in hot spots and hot moments such as metazoan guts, drought influenced soils, or biofilms where ion concentration is locally or transiently elevated and carbon source composition is of a sufficiently low complexity to enrich for a dominant phage susceptible population. SignificanceViral-prokaryote dynamics greatly influence microbial ecology and the earths biogeochemical cycles. Thus, identifying the key environmental controls on phage predation is critical for predictive microbial ecology. Here we conduct laboratory experiments that implicate ionic strength and carbon composition as major controls on phage interactions with bacterial hosts in terrestrial microbiomes. We propose a model in which terrestrial phage predation is most favored in drought impacted soils and in higher ionic strength environments such as metazoan guts or between adjacent cells in biofilms.

microbiology↗

bioRxiv2020

High-throughput mapping of the phage resistance landscape in E. coli

Bacteriophages (phages) are critical players in the dynamics and function of microbial communities and drive processes as diverse as global biogeochemical cycles and human health. Phages tend to be predators finely tuned to attack specific hosts, even down to the strain level, which in turn defend themselves using an array of mechanisms. However, to date, efforts to rapidly and comprehensively identify bacterial host factors important in phage infection and resistance have yet to be fully realized. Here, we globally map the host genetic determinants involved in resistance to 14 phylogenetically diverse double-stranded DNA phages using two model Escherichia coli strains (K-12 and BL21) with known sequence divergence to demonstrate strain-specific differences. Using genome-wide loss-of-function and gain-of-function genetic technologies, we are able to confirm previously described phage receptors as well as uncover a number of previously unknown host factors that confer resistance to one or more of these phages. We uncover differences in resistance factors that strongly align with the susceptibility of K-12 and BL21 to specific phage. We also identify both phage specific mechanisms, such as the unexpected role of cyclic-di-GMP in host sensitivity to phage N4, and more generic defenses, such as the overproduction of colanic acid capsular polysaccharide that defends against a wide array of phages. Our results indicate that host responses to phages can occur via diverse cellular mechanisms. Our systematic and high-throughput genetic workflow to characterize phage-host interaction determinants can be extended to diverse bacteria to generate datasets that allow predictive models of how phage-mediated selection will shape bacterial phenotype and evolution. The results of this study and future efforts to map the phage resistance landscape will lead to new insights into the coevolution of hosts and their phage, which can ultimately be used to design better phage therapeutic treatments and tools for precision microbiome engineering.

microbiology↗