Biology subjects

Sharan, R.

Publications and source records attributed to Sharan, R..

At least 19 recordsLinked to original sources

bioRxiv2026

Toward identification of common DNA repair process in mutational signatures

Mutational signatures are characteristic patterns of mutation frequencies assumed to be generated by specific mutagenic processes. A growing catalog of mutational signatures exists, but tools to systematically infer relationships between them remain limited. A mutational signature can be viewed as the combined outcome of two processes: DNA damage and DNA repair. Since cancer therapies often target DNA repair, inferring DNA repair pathways is important for treatment design, even when the mutagenic process is unknown. Here, we model the DNA repair step as a transformation, called RePrint, from damaged nucleotides to repair-related mutation patterns conditioned on the damage. We demonstrate that RePrint similarity is indicative of shared DNA repair mechanisms, enabling guilt-by-association prediction of DNA repair pathways. Using experimentally annotated signatures from environmental exposures and CRISPR gene knockouts as gold standards, we demonstrate that RePrint-based clustering consistently outperforms signature-based clustering across multiple evaluation metrics. We validate several guilt-by-association predictions with literature evidence, demonstrating RePrints ability to identify shared repair mechanisms even among signatures with divergent mutational profiles. RePrint provides the first approach to systematically transfer DNA repair information between signatures, opening doors to understanding signatures of unknown origin and informing therapeutic strategies. An open-source implementation is available at https://github.com/wojtowicz-lab/RePrintPy

cancer biology↗

bioRxiv2025

Planar cell polarity-directed cell crawling drives polarized hair follicle morphogenesis

During epithelial morphogenesis, cell polarity aligns individual cell behaviors into collective motions that shape developing tissues. Here, we combine experiments with computational modeling to investigate how cell-scale forces oriented by Planar Cell Polarity (PCP) direct the collective, counter-rotational cell flows that occur during hair placode morphogenesis. We rule out that PCP directs apical neighbor exchanges, as junctional myosin and PCP protein localization are not co-correlated with junction shrinkage. Instead, we find that PCP directs anterior-directed crawling of placode cells along the basal surface of the tissue through a mechanism that requires cell crawling regulator Rac1. Modeling the placode as a continuum viscoelastic fluid, we find that active forces from cell crawling at the basal surface is sufficient to generate the experimentally observed counter-rotational cell motion at the apical surface. Our results show an unexpected role for PCP in epithelial morphogenesis, centering the basal surface as the site of force generation.

developmental biology↗

bioRxiv2025

An adversarial scheme for integrating multi-modal data on protein function

In order to begin to decipher the structure of the cell, we need to integrate multiple types of data of different scales on subcellular organization. Such integration requires dealing with multiple data modalities and with missing data. To this end, we developed MIRAGE, a multi-modal generative model for integrating protein sequence, protein-protein interaction and protein localization data. Our approach successfully learns a joint embedding space that captures the complex relationships between these diverse modalities. We evaluate our models performance against existing methods, obtaining superior performance in several key tasks, including protein function prediction and module detection. MIRAGE source code is available at https://github.com/raminass/MIRAGE.

bioinformatics↗

bioRxiv2024

TGFb-ANGPT2-Tie2 axis in cancer-associated fibroblasts reprograms oral cancer cells to embryonic-like cell state with predictive significance of poor prognosis

Myofibroblastic cancer-associated fibroblasts (CAFs) in tumor stroma is identified as poor-prognostic indicator in oral cancer; however, biological mechanisms are largely unexplored. Here, we discovered the role of autocrine or exogenous transforming growth factor beta (TGF{beta}) in inducing Tunica Interna Endothelial cell kinase 2 (Tie2) -signaling through histone deacetylase-mediated downregulation of Tie2-antagonist, Angiopoietin-2 in CAFs, responsible for induction and maintenance of myofibroblastic differentiation. To understand the influence of CAF-specific Tie2-signaling on cancer cell properties, we performed CAF-Cancer cell co-culture and its single-cell RNA sequencing (scRNA-Seq). Distinct clustering of CAFs suggested their transcriptional heterogeneity, driven by TGF{beta}-Tie2 activation. Interestingly, CAF-specific Tie2-signaling was responsible to reprogram cancer cells, producing embryonic-like cell state with increased stemness and EMT signatures. Importantly, both the Tie2-specific gene expression signature as well as reprogrammed cancer cell specific gene expression modules were validated respectively in fibroblasts clusters and malignant cell clusters in two independent earlier reported scRNAseq studies of HNSCC tumors. Highlighting the translatability of our study, the gene expression signature derived from reprogrammed cancer cells showed significant association with poor prognosis in HNSCC patient of TCGA cohort. Pharmacological inhibition of Tie2-signaling in CAFs, significantly abrogated the tumor initiating ability of co-cultured oral cancer cell lines. Overall, combining our molecular and computational analysis, we may propose Tie2 as a novel factor responsible for CAF mediated cancer cell plasticity, associated with aggressive nature of oral cancer. TeaserTie2-signaling is activated in myofibroblasts which impacts the behaviour of malignant cells by inducing cancer cell plasticity to acquire stemness.

cancer biology↗

bioRxiv2023

Region-specific reversal of epidermal planar polarity in the fancy rosette mouse

The planar cell polarity (PCP) pathway collectively orients thousands of cells with respect to a body axis to direct cellular behaviors that are essential for embryonic morphogenesis. Hair follicles of the murine epidermis provide a striking readout of PCP activity in their uniform alignment along the entire skin surface. Here, we characterize, from the molecular to tissue-scale, PCP establishment in the rosette fancy mouse, a natural variant with posterior-specific whorls in its fur, to understand how epidermal polarity is coordinated across the tissue. We find that embryonic hair follicles of rosette mutants emerge with reversed orientations specifically in the posterior region, creating a mirror image of epidermal polarity. The rosette trait is associated with a missense mutation in the core PCP gene Fzd6, which alters a consensus site for N-linked glycosylation and inhibits its membrane localization. Unexpectedly, this defect in Fzd6 trafficking, observed across the entire dorsal epidermis, does not interfere with the ability of other core PCP proteins to localize asymmetrically. Rather, the normally uniform axis of PCP asymmetry is disrupted and rotated in the posterior region such that polarity is reflected on either side of a transition zone. The result is a reversal of polarized cell movements that orient nascent follicles, specifically in the posterior of the embryo. Collectively, our multiscale analysis of epidermal polarity reveals PCP patterning can be regionally decoupled to produce the unique posterior whorls of the fancy rosette mouse. SummaryRegion-specific rotation of the Planar Cell Polarity axis reverses posterior hair follicles in the fancy rosette mouse.

developmental biology↗

bioRxiv2022

Vangl facilitates mesenchymal thinning during lung sacculation independently of Celsr

The planar cell polarity (PCP) complex orients cytoskeletal and multicellular organization throughout vertebrate development. PCP is speculated to function in formation of the murine lung, where branching morphogenesis generates a complex tree of tubular epithelia whose distal tips expand dramatically during sacculation in preparation for gas exchange after birth. Here, using tissue-specific knockouts, we show that the PCP complex is dispensable in the airway epithelium for sacculation. Rather, we find a novel, Celsr1-independent role for the PCP component Vangl in the pulmonary mesenchyme: loss of Vangl1/2 inhibits mesenchymal thinning and expansion of the saccular epithelium. Further, loss of mesenchymal Wnt5a mimics the sacculation defects observed in Vangl2-mutant lungs, implicating mesenchymal Wnt5a/Vangl signaling as a key regulator of late lung morphogenesis. By mathematically modeling sacculation, we predict that the process of sacculation requires a fluid mesenchymal compartment. Finally, lineage-tracing and cell-shape analyses are consistent with the pulmonary mesenchyme acting as a fluid tissue, and suggest that loss of Vangl1/2 likely impacts the ability of mesenchymal cells to exchange neighbors. Our data thus uncover an explicit function for Vangl and the pulmonary mesenchyme during late lung morphogenesis to actively shape the saccular epithelium.

developmental biology↗

bioRxiv2022

Identification of factors mediating the signaling convergenceof multiple receptors following cell-cell interaction

MotivationCell-cell crosstalk involves simultaneous interactions of multiple receptors and ligands, followed by downstream signaling cascades working through receptors converging at dominant transcription factors which then integrate and propagate multiple signals into a cellular response. Single cell RNAseq of multiple cell subsets isolated from a defined microenvironment provides us with a unique opportunity to learn about such interactions reflected in their gene expression levels. ResultsWe developed the FLOW framework to map the potential ligand-receptor interactions between different cell subsets based on a maximum flow computation in a network of protein-protein interactions (PPIs). The maximum flow approach further allows characterization of the intracellular downstream signal transduction from differentially expressed receptors towards dominant transcription factors, therefore, enabling the association between a set of receptors and their downstream activated pathways. Importantly, we were able to identify key transcription factors toward which the convergence of multiple receptor signaling occurs. These identified factors have a unique role in the integration and propagation of signaling following specific cell-cell interactions.

bioinformatics↗

bioRxiv2022

A mutation-level covariate model for mutational signatures

Mutational processes and their exposures in particular genomes are key to our understanding of how these genomes are shaped. However, current analyses assume that these processes are uniformly active across the genome without accounting for potential covariates such as strand or genomic region that could impact such activities. Here we suggest the first mutation-covariate models that explicitly model the effect of different covariates on the exposures of mutational processes. We apply these models to test the impact of replication strand on these processes and compare them to strand-oblivious models across a range of data sets. Our models capture replication strand specificity, point to signatures affected by it, and score better on held-out data compared to standard models that do not account for mutation-level covariate information.

bioinformatics↗

bioRxiv2021

Spontaneous emergence of non-convertible cell states with CD24-High phenotype results in phenotypic heterogeneity that associates with poor prognosis in oral cancer

PurposeCellular dynamics between phenotypically heterogeneous subpopulations of cancer cells within individual tumor is shown to be responsible for drug tolerance and overall poor prognosis; however, evidences were largely missing in oral cancer. Therefore, this study was undertaken to describe the dynamic phenotypic states among oral cancer cells, its influence on transcriptomic heterogeneity as well as its clinical significance. Experimental DesignWe multiplexed phenotypic markers of putative oral-stem-like cancer cells (SLCCs) and characterized diversity among CD44-positive oral cancer cell subpopulations with respect to distinct expression of CD24 and aldehyde dehydrogenase (ALDH)-activity in multiple cell lines. Population trajectories were characterized by Markov model and cell states were defined based on the population specific RNA sequencing (RNAseq). ssGSEA based gene expression signatures were explore for prognostic significance. ResultsOral cancer cells followed two distinct patterns of spontaneous repopulation dynamics with stochastic inter-conversions on ALDH-axis, however a strict non-interconvertible transition on CD24-axis. Interestingly, plastic ALDH-axis was harnessed to enrich ALDHHigh subpopulations in response to Cisplatin treatment, to adapt a drug tolerant state. Phenotype-specific RNAseq results suggested the organization of subpopulations into hierarchical structure with possible maintenance of intermediate states of stemness within the differentiating oral cancer cells. Further, survival analysis with each subpopulation-specific gene signature strongly suggested that the cell-state dynamics may act as possible mechanism to drive ITH, resulting in poor prognosis in patient. ConclusionsOur results emphasized the prognostic power of the population dynamics in oral cancer. Importantly, we have described the phenotypic-composition of heterogeneous subpopulations critical for global tumor behaviour in oral cancer; which is a prerequisite knowledge important for precision treatment, however largely lacking for most solid tumors. Graphical AbstractWe have characterized diversity among CD44-positive oral cancer cells lines with respect to distinct expression of CD24 and ALDH-activity. Subpopulations showed stochastic inter-conversions on ALDH-axis but a strict non-interconvertible transition of CD24Low to CD24High phenotype, even in response to chemotherapy-induced stress. RNAseq study suggested the organization of subpopulations into hierarchical structure with possible maintenance of intermediate alternate states of stemness within the differentiating oral cancer cells. The described population dynamics demonstrtaed influence tumor behaviour possibly by increasing intratumoral heterogeneity in aggressive oral tumors. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/457509v3_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1dc6a7org.highwire.dtl.DTLVardef@dd0948org.highwire.dtl.DTLVardef@18c9603org.highwire.dtl.DTLVardef@cca8fb_HPS_FORMAT_FIGEXP M_FIG C_FIG Translational relevanceIntratumoral heterogeneity (ITH) has been the clinically important factor, impacting aggressive cancer behaviour, drug tolerance and overall poor prognosis. Recent high-throughput studies have provided better cellular and molecular resolution of ITH; however, the prerequisite knowledge which defines the composition of subpopulations critical for global tumor behaviour is majorly lacking for most of the solid tumors. By combining phenotypic markers, we have defined four subpopulations of oral cancer cells. These subpopulations showed stochastic inter-conversions as well as a strict non-interconvertible transition among them to acheive heterogeneity. Importantly, transcriptional states of each subpopulations indicated a clinically relevant signatures for patient prognosis. Also, we observed interconversions of these subpopulations in response to Cisplatin to accumulate drug-tolerant cell state, as rapid and reversible strategy to respond to chemotherapy induced stress. Thus, the characteristics of described phenotypic subgroups may be translated to the clinic for estimating the extent of intratumoral heterogeneity in oral cancer patients.

cancer biology↗

bioRxiv2021

Proteomic landscape of multi-layered breast cancer internal tumor heterogeneity

Despite extensive research, internal tumor heterogeneity presents enormous challenges to achieve complete therapeutic responses. Changes in protein expression are central determinants of cancer phenotypes that reflect potential therapeutic targets. However, previous proteomic studies did not address internal heterogeneity, therefore, masked the necessary spatial resolution to achieve a comprehensive understanding of cancer complexity. Here we present the first large-scale multi-focal breast cancer proteomic study of 330 tumor regions which associated cancer cell function, pathological parameters, and spatial localization of each tumor region. We found marked internal proteomic heterogeneity even within tumors presenting homogeneous receptor expression. Additionally, analysis of the internal heterogeneity, based on coexisting receptor expression or histological patterns in single tumors, showed significant functional differences between homogeneous and heterogeneous tumors related to cancer metabolism, immunogenicity, and proliferation. We anticipate that this study will serve as a starting point towards the development of improved cancer therapy and diagnostics.

cancer biology↗

bioRxiv2021

Effect of SARS-CoV-2 proteins on vascular permeability

SARS-CoV-2 infection leads to severe disease associated with cytokine storm, vascular dysfunction, coagulation, and progressive lung damage. It affects several vital organs, seemingly through a pathological effect on endothelial cells. The SARS-CoV-2 genome encodes 29 proteins, whose contribution to the disease manifestations, and especially endothelial complications, is unknown. We cloned and expressed 26 of these proteins in human cells and characterized the endothelial response to overexpression of each, individually. Whereas most proteins induced significant changes in endothelial permeability, nsp2, nsp5_c145a (catalytic dead mutant of nsp5) and nsp7 also reduced CD31, and increased von Willebrand factor expression and IL-6, suggesting endothelial dysfunction. Using propagation-based analysis of a protein-protein interaction (PPI) network, we predicted the endothelial proteins affected by the viral proteins that potentially mediate these effects. We further applied our PPI model to identify the role of each SARS-CoV-2 protein in other tissues affected by COVID-19. Overall, this work identifies the SARS-CoV-2 proteins that might be most detrimental in terms of endothelial dysfunction, thereby shedding light on vascular aspects of COVID-19.

cell biology↗

bioRxiv2021

Long Reads Capture Simultaneous Enhancer-Promoter Methylation Status for Cell-type Deconvolution

MotivationWhile promoter methylation is associated with reinforcing fundamental tissue identities, the methylation status of distant enhancers was shown by genome-wide association studies to be a powerful determinant of cell-state and cancer. With recent availability of long-reads that report on the methylation status of enhancer-promoter pairs on the same molecule, we hypothesized that probing these pairs on the single-molecule level may serve the basis for detection of rare cancerous transformations in a given cell population. We explore various analysis approaches for deconvolving cell-type mixtures based on their genome-wide enhancer-promoter methylation profiles. ResultsTo evaluate our hypothesis we examine long-read optical methylome data for the GM12787 cell line and myoblast cell lines from two donors. We identified over 100,000 enhancer-promoter pairs that co-exist on at least 30 individual DNA molecules per pair. We developed a detailed methodology for mixture deconvolution and applied it to estimate the proportional cell compositions in synthetic mixtures based on analyzing their enhancer-promoter pairwise methylation. We found our methodology to lead to very accurate estimates, outperforming our promoter-based deconvolutions. Moreover, we show that it can be generalized from deconvolving different cell types to subtle scenarios where one wishes to deconvolve different cell populations of the same cell-type. AvailabilityThe code used in this work to analyze single-molecule Bionano Genomics optical maps is available via the GitHub repository https://github.com/ebensteinLab/Single_molecule_methylation_in_EP. Contactuv@post.tau.ac.il (Y.E), roded@tauex.tau.ac.il (R.S)

bioinformatics↗

bioRxiv2020

SARS-CoV-2 infection leads to acute infection with dynamic cellular and inflammatory flux in the lung that varies across nonhuman primate species

There are no known cures or vaccines for COVID-19, the defining pandemic of this era. Animal models are essential to fast track new interventions and nonhuman primate (NHP) models of other infectious diseases have proven extremely valuable. Here we compare SARS-CoV-2 infection in three species of experimentally infected NHPs (rhesus macaques, baboons, and marmosets). During the first 3 days, macaques developed clinical signatures of viral infection and systemic inflammation, coupled with early evidence of viral replication and mild-to-moderate interstitial and alveolar pneumonitis, as well as extra-pulmonary pathologies. Cone-beam CT scans showed evidence of moderate pneumonia, which progressed over 3 days. Longitudinal studies showed that while both young and old macaques developed early signs of COVID-19, both groups recovered within a two-week period. Recovery was characterized by low-levels of viral persistence in the lung, suggesting mechanisms by which individuals with compromised immune systems may be susceptible to prolonged and progressive COVID-19. The lung compartment contained a complex early inflammatory milieu with an influx of innate and adaptive immune cells, particularly interstitial macrophages, neutrophils and plasmacytoid dendritic cells, and a prominent Type I-interferon response. While macaques developed moderate disease, baboons exhibited prolonged shedding of virus and extensive pathology following infection; and marmosets demonstrated a milder form of infection. These results showcase in critical detail, the robust early cellular immune responses to SARS-CoV-2 infection, which are not sterilizing and likely impact development of antibody responses. Thus, various NHP genera recapitulate heterogeneous progression of COVID-19. Rhesus macaques and baboons develop different, quantifiable disease attributes making them immediately available essential models to test new vaccines and therapies.

immunology↗

bioRxiv2020

Identification of essential genes and fluconazole resistance genes in Candida glabrata by profiling of Hermes transposon insertions.

Within the budding yeasts, the opportunistic pathogen Candida glabrata and other members of the Nakaseomyces clade have developed virulence traits independently from the CTG clade that includes Candida albicans. To begin exploring the genetic basis of C. glabrata virulence and its innate resistance to antifungals, we launched the Hermes transposon from a plasmid and obtained more than 500,000 different semi-random insertions throughout the genome. Using machine learning, we identify up to 1278 protein-encoding genes (25% of total) that cannot tolerate transposon insertions and are thus essential for C. glabrata fitness in vitro. Interestingly, genes involved in mRNA splicing were less likely to be essential in C. glabrata than their orthologs in S. cerevisiae, whereas the opposite is true for genes involved in kinetochore function and chromosome segregation. Insertions in several known genes (e.g. PDR1, CDR1, PDR16, PDR17, UPC2A, DAP1) caused hypersensitivity to the first-line antifungal fluconazole, and we identify 12 additional genes that also contribute to innate fluconazole resistance (KGD1, KGD2, YHR045W, etc). Insertions in 200 other genes conferred significant resistance to fluconazole, two-thirds of which function in mitochondria and likely down-regulate Pdr1 expression or function. These findings show the utility of transposon insertion profiling in genome-wide forward-genetic investigations of fungal pathogens. IMPORTANCEPathogenic yeasts cause mucosal and systemic infections in millions of people each year. The innate resistance of Candida glabrata to fluconazole and its ability to acquire resistance to 2 other antifungals are contributing to its rise in incidence. Our understanding of C. glabrata biology has been hampered by inefficient genetic and genomic tools. This study addresses those deficiencies by developing powerful transposon mutagenesis strategies for the first time in this pathogen. We identify nearly all essential genes of C. glabrata that could be targeted for development of new antifungals. We generate large pools of random insertion mutants that can be easily monitored en masse with deep sequencing, thus enabling identification of genes involved in any number of biological processes. We identify dozens of new genes that increase or decrease innate resistance of clinical isolate BG2 to fluconazole and provide resources for further exploration of C. glabrata genetics and genomics.

genetics↗

bioRxiv2019

Evolution of metastases-associated fibroblasts in the lung microenvironment is driven by stage-specific transcriptional plasticity

Mortality from breast cancer is almost exclusively a result of tumor metastasis, and lungs are one of the main metastatic sites. Cancer-associated fibroblasts (CAFs) are prominent players in the microenvironment of breast cancer. However, their role in the metastatic niche is largely unknown. In this study, we profiled the transcriptional co-evolution of lung fibroblasts isolated from transgenic mice at defined stage-specific time points of metastases formation. Employing multiple knowledge-based platforms of data analysis provided powerful insights on functional and temporal regulation of the transcriptome of fibroblasts. We demonstrate that fibroblasts in lung metastases are transcriptionally dynamic and plastic, and reveal stage-specific gene signatures that imply functional tasks, including extracellular matrix remodeling, stress response and shaping the inflammatory microenvironment. Furthermore, we identified Myc as a central regulator of fibroblast rewiring and found that stromal upregulation of Myc transcriptional networks is associated with worse survival in human breast cancer.Competing Interest StatementThe authors have declared no competing interest.View Full Text

cancer biology↗

bioRxiv2019

Comparing the utility of in vivo transposon mutagenesis approaches in yeast species to infer gene essentiality

ABSTRACTIn vivo transposon mutagenesis, coupled with deep sequencing, enables large-scale genome-wide mutant screens for genes essential in different growth conditions. We analyzed six large-scale studies performed on haploid strains of three yeast species (Saccharomyces cerevisiae, Schizosaccaromyces pombe, and Candida albicans), each mutagenized with two of three different heterologous transposons (AcDs, Hermes, and PiggyBac). Using a machine-learning approach, we evaluated the ability of the data to predict gene essentiality. Important data features included sufficient numbers and distribution of independent insertion events. All transposons showed some bias in insertion site preference because of jackpot events, and preferences for specific insertion sequences and short-distance vs long-distance insertions. For PiggyBac, a stringent target sequence limited the ability to predict essentiality in genes with few or no target sequences. The machine learning approach also robustly predicted gene function in less well-studied species by leveraging cross-species orthologs. Finally, comparisons of isogenic diploid versus haploid S. cerevisiae isolates identified several genes that are haplo-insufficient, while most essential genes, as expected, were recessive. We provide recommendations for the choice of transposons and the inference of gene essentiality in genome-wide studies of eukaryotic haploid microbes such as yeasts, including species that have been less amenable to classical genetic studies.Competing Interest StatementThe authors have declared no competing interest.View Full Text

bioinformatics↗

bioRxiv2019

Network-based approaches elucidate differences within APOBEC and clock-like signatures in breast cancer

Studies of cancer mutations typically focus on identifying cancer driving mutations. However, in addition to the mutations that confer a growth advantage, cancer genomes accumulate a large number of passenger somatic mutations resulting from normal DNA damage and repair processes as well as mutations triggered by carcinogenic exposures or cancer related aberrations of DNA maintenance machinery. These mutagenic processes often produce characteristic mutational patterns called mutational signatures. Understanding the etiology of the mutational signatures shaping a cancer genome is an important step towards understanding tumorigenesis. Considering mutational signatures as phenotypes, we asked two complementary questions (i) what are functional pathways whose gene expression profiles are associated with mutational signatures, and (ii) what are mutated pathways (if any) that might underlie specific mutational signatures? We have been able to identify pathways associated with mutational signatures on both expression and mutation levels. In particular, our analysis provides novel insights into mutagenic processes in breast cancer by capturing important differences in the etiology of different APOBEC related signatures and the two clock-like signatures. These results are important for understanding mutagenic processes in cancer and for developing personalized drug therapies.

bioinformatics↗

bioRxiv2019

Phage-encoded cationic antimicrobial peptide used for outer membrane disruption in lysis

Spanins are required for the last step in bacteriophage lysis: the disruption of the outer membrane. Bioinformatic analysis has shown that ~15% of phages lack a spanin gene, which suggests an alternate mechanism of outer membrane disruption. To address this, we selected virulent podophage {phi}KT as a spaninless exemplar and tested {phi}KT genes for outer membrane disruption during lysis. Hypothetical novel gene 28 causes outer membrane disruption when co-expressed with {phi}KT lysis genes and complements the lysis defect of a {lambda} spanin mutant. Gp28 is a 56 aa cationic peptide with predicted amphipathic helical structure and is associated with the particulate fraction after lysis. Urea and KCl washes did not release gp28 from the particulate, suggesting a strong hydrophobic interaction with the membrane. Super high-resolution microscopy supports a primarily outer membrane localization for the peptide. Additionally, holin function is not required for gp28-mediated lysis. Gp28 is similar in size, charge, predicted fold, and membrane association to the human cathelicidin antimicrobial peptide LL-37. In standard assays to measure bactericidal and inhibitory effects of antimicrobial peptides on bacterial cells, synthesized gp28 performed equivalently to LL-37. The studies presented here suggest that {phi}KT Gp28 disrupts bacterial outer membranes during lysis in a manner akin to antimicrobial peptides.\n\nSignificanceHere we provide evidence that {phi}KT produces an antimicrobial peptide for outer membrane disruption during lysis. The disruptin is a new paradigm for phage lysis, and has no similarities to other known lysis genes. Many mechanisms have been proposed for the function of antimicrobial peptides, however there is not a consensus on the molecular basis of membrane disruption. Additionally, there is no established genetic selection system to support such studies. Therefore, the {phi}KT disruptin may represent the first genetically tractable antimicrobial peptide.

microbiology↗