Biology subjects

Gupta, A.

Publications and source records attributed to Gupta, A..

At least 109 records · Page 6Linked to original sources

bioRxiv2025

Native epitranscriptome sequencing reveals Hepatitis B virus RNA stability protected by heavy m6A modification

Although the RNA of Hepatitis B virus (HBV) is known to be regulated by the epitranscriptomic modification N6-methyladenosine (m6A), it remains controversial whether if m6A is beneficial or detrimental for viral replication. Here, we employ Nanopore direct RNA sequencing (DRS) to analyze the HBV m6A epitranscriptome on a single molecule level and elucidate how m6A regulates the HBV replication cycle. We found more than 60% of all HBV RNA in cells are m6A methylated, [~]3x more methylated than host mRNAs. Previously unreported m6A sites were found in pol gene, which when mutated reduced viral production. A sizable proportion of HBV RNAs carry poly(A) tails at least twice as long (>120nt) as cellular mRNAs, with the shorter-tailed population more pronounced among m6A-free transcripts. In contrast, encapsidated RNAs have shorter poly(A) tails and less m6A. Concordantly, pharmacological reduction of viral m6A using the m6A methyltransferase inhibitor STM2457, shortens viral poly(A) tails and reduces viral RNA stability. Our findings thus suggest that the abundance of m6A on viral RNAs protects the surprisingly long viral poly(A) tails and thus enhances RNA stability. Overall, m6A strongly enhances HBV viral replication, in support of RNA modification machinery as a promising class of antiviral targets.

microbiology↗

bioRxiv2025

Active Learning for Protein Structure Prediction

Accurate protein structure prediction is challenging especially for families and types that are not well-represented in current training data. Active learning selects candidates for labeling with the aim of most rapidly improving model performance. In a general, many labeling strategies have been proposed. However, with protein structure prediction, most of these strategies dont apply or are difficult due to the high-dimensional, variable-dimensional regression target and the inherent complexity of the models involved. We applied a novel active learning strategy, DEW-DROP, to protein structure prediction on two different protein datasets: VHH-only antibodies (Nanobodies), and Mycobacterial proteins. We introduce a domain-specific fine-tuned Equifold model for VHH structures and apply DEWDROP to generate ensembles of predictions using Monte Carlo dropout. Using the statistics of these we select batches with high information content for labeling. We show that DEWDROP (1) improves model training efficiency through batch optimization outper-forming baselines, and (2) selects data with relevant high information content.

molecular biology↗

bioRxiv2025

Non-canonical regulation of the plasma membrane copper transporter CTR1 through modulation of membrane mechanical properties

We describe a non-canonical, membrane receptor-like regulation of the human copper transporter CTR1 in response to copper stimuli. CTR1 is the sole high-affinity trimeric plasma-membrane copper-importing channel that self-regulates by undergoing endocytosis to limit copper uptake. We observed that preceding copper-induced endocytosis, CTR1 forms clusters on the plasma membrane, a phenomenon that is typically observed in membrane receptors. We deciphered the mechanism of CTR1 clustering and studied its ramifications on plasma membrane physical properties harboring the clusters that favors endocytosis. Membrane tension and fluctuation are fundamental regulators of pre- and post-endocytic events. Using coarse-grain MD simulation and coupled Interference Reflection Microscopy-Total Internal Reflection Fluorescence Microscopy we demonstrated that CTR1 clusters induce positive membrane curvature, increase in local membrane tension and decrease in local membrane fluctuation; alterations that favors formation of endocytic pits. Clustering is facilitated by copper-sequestering Methionine rich extracellular amino-terminus of CTR1. MD-simulations and IRM-TIRF imaging revealed that CTR1 clustering is facilitated by membrane cholesterol, depletion of which delays CTR1 endocytosis. CTR1 clustering promotes clathrin-coated pit formation that engages recruitment of adapter protein AP-2. To summarize, we report a hitherto unknown pre-endocytic receptor-like phenomenon of ligand-induced clustering of a metal channel, that in-turn regulates self-endocytosis by modulating membrane properties.

cell biology↗

bioRxiv2025

The differential mechanisms of eIF4A1-mediated translational activation instructed by distinct RNA features

All mRNAs require eukaryotic translation initiation factor (eIF) 4A1 for translation through its different functions: loading of the pre-initiation complex onto mRNAs and unwinding of RNA structure. eIF4A1 is the catalytic subunit of the cap- binding eIF4F complex and presumed to select the mRNAs for translation through these activities, instructed by signalling pathways driving cell fate. The mechanisms underlying activation of the distinct eIF4A1 functions and establish translational selectivity are unknown. Here, we have unravelled the complexity of mRNA selection by eIF4A1. We have mechanistically characterised the biological and atomic basis of inhibition by gain- and loss-of-function eIF4A1-inhibitors eFT226 and hippuristanol, and used machine learning to model the mRNA features associated with specific inhibition. This uncovered the eIF4A1 function - mRNA sequence relationship: 5UTRs containing C/CG-rich require efficient mRNA loading by eIF4F which is specifically targeted by hippuristanol, while 5UTRs harbouring alternatives starts sites together with AG-rich motifs utilise eIF4A1 for start site selection, specifically perturbed by eFT226. Our model is validated through a massively parallel reporter assay using 5UTRs from a distinct evolutionary origin. This prompted us to examine the conservation between mRNA sequence and eIF4A1 function, and revealed their co- development. Our findings highlight opportunities for novel therapeutic strategies targeting eIF4A1 and for improved design of mRNA-based therapeutics.

cell biology↗

bioRxiv2025

TENT5C extends Odf1 poly(A) tail to sustain sperm morphogenesis and fertility

Changes in the poly(A) tail length of Odf1 and other transcripts critical for male fertility have been linked to translational activation during sperm formation 1-3. The mRNA poly(A) polymerase TENT5C is required for fastening the flagellum to the sperm head, but its role in shaping the poly(A) tail profile of the spermatid transcriptome remains limited 4,5. Here, we comprehensively document how changes in mRNA poly(A) tail length across the transcriptome reflect transcript metabolism in spermatids. In the absence of TENT5C polymerase activity, the poly(A) tail length of Odf1 transcripts is reduced, and the local distribution of ODF1 proteins in spermatids is disrupted. We show that mice expressing a catalytically inactive TENT5C produce headless spermatozoa with outer dense fibers detached from the axoneme, and other flagellar abnormalities associated with ODF1 deficiency 6. We propose that TENT5C poly(A) polymerase activity regulates the spatial translation of Odf1 mRNAs during spermiogenesis, a process critical for sperm morphogenesis and fertility. These findings highlight the power of poly(A) tail profiling to identify abnormal mRNA processing causative of infertility.

developmental biology↗

bioRxiv2025

Commensal-derived Trehalose Monocorynomycolate Triggers γδ T Cell-driven Protective Ocular Barrier Immunity

Commensals shape host physiology through molecular crosstalk with host receptors. Identifying specific microbial factors that causally influence host immunity is key to understanding homeostasis at the host-microbe interface and advancing microbial-based therapeutics. Here, we identify trehalose monocorynomycolate (TMCM) from Corynebacterium mastitidis (C. mast) as a potent stimulator of IL-17 production by {gamma}{delta} T cells at the ocular surface. Mechanistically, TMCM-driven IL-17 responses require both IL-1 signals and {gamma}{delta} TCR signaling, which also supports endogenous {gamma}{delta} T cell IL-1R1 expression. Notably, synthetic TMCM alone is sufficient to mimic the effect of C. mast in inducing {gamma}{delta} T cell immunity and protect against pathogenic corneal infection. Our findings establish TMCM as a key mediator of commensal-driven immune defense, highlighting its potential as a {gamma}{delta} T cell adjuvant and a microbiome-informed therapeutic to enhance IL-17-driven protection at barrier sites such as the ocular surface. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/643820v1_ufig1.gif" ALT="Figure 1"> View larger version (68K): org.highwire.dtl.DTLVardef@63139forg.highwire.dtl.DTLVardef@9d6978org.highwire.dtl.DTLVardef@775e37org.highwire.dtl.DTLVardef@ca84b6_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LICorynomycolates enable ocular C. mast colonization and protective IL-17 immunity C_LIO_LITMCM drives IL-17 from {gamma}{delta} T cells through TCR and IL-1R signaling C_LIO_LI{gamma}{delta} TCR signaling maintains the expression of endogenous IL-1R1 C_LIO_LISynthetic TMCM mimics the ability of C. mast to induce {gamma}{delta} T cell immunity in the eye C_LIO_LITMCM protects against P. aeruginosa keratitis, highlighting its therapeutic potential C_LI

immunology↗

bioRxiv2025

Endothelin-converting enzyme 2 differentially regulates kappa opioid receptor trafficking and function

Following activation by endogenous opioid peptides, mu and delta opioid receptors have been shown to undergo differential internalization and recycling; the rate and extent of recycling but not internalization was found to be regulated by the endocytic peptide converting enzyme, ECE2. This study focuses on kappa opioid receptors (KOR) and the ability of endogenous opioid peptides released by post-translational processing of prodynorphin and proenkephalin to induce KOR and ECE2 internalization/recycling, and how ECE2 modulates these processes. Using a proximity-based ligation assay we show that KOR and ECE2 are in close proximity to facilitate co-internalization. In addition, we find that treatment with longer opioid peptides induces fast and robust internalization and recycling of ECE2 at a rate and extent comparable to that of KOR. Next, we directly examined the role of ECE2 in modulating KOR recycling. We find that ECE2 inhibition significantly attenuates KOR recycling. Finally, we examined the role of ECE2 in modulating KOR signaling and find that resensitization of KOR by peptides that are substrates of ECE2 are attenuated by ECE2 inhibition. Taken with the differential expression of ECE2 in the brain (relatively high expression in midbrain & hypothalamus and low expression in the striatum & hippocampus), these results highlight a pivotal role for ECE2 in differentially modulating KOR function. Significance StatementThis study highlights a role for ECE2 in agonist mediated regulation of KOR function by select prodynorphin and proenkephalin derived peptides. Collectively our studies suggest that ECE2 inhibitors could be developed as therapeutics for pathologies involving dysregulations in KOR signaling.

neuroscience↗

bioRxiv2025

Macrophages of diabetes and tuberculosis co-morbid conditions show perturbed differentiation and metabolic signatures

Diabetes negatively impacts the host immune system, leading to allergies, recurring infections, and lameness. Its impact on the distribution and molecular phenotypes of the bone marrow-derived macrophages (BMDMs) needs attention and, more importantly, in co-morbid conditions like diabetes and tuberculosis (DM-TB). In this study, total bone marrow cells were harvested at 3 w.p.i., from the low aerosol dose (100-120 CFU) of Mycobacterium tuberculosis H37Rv infected control and NA-STZ induced diabetic C57BL/6 mice. The bone marrow cells were differentiated into BMDMs, and CD11c, CD11b and F4/80 marker expressions were monitored using flow cytometry. BMDMs stimulated with Mtb H37Rv lysate were subjected to global transcriptome profiling to identify the perturbed metabolic and molecular landscape. The BMDMs of the DM-TB comorbid mice showed low CD11c, CD11b and higher F4/80 expression. The macrophages of the DM group showed a skewed pro-inflammatory response, characterized by perturbed complement response, NF-kB pathway and fatty acid metabolism, whereas macrophages of the DM-TB group showed deregulated ceramide and amino acid metabolism. Macrophages from the DM and DM-TB group stimulated with Mtb H37Rv lysate, showed a deregulated transcript signature that impacts humoral response. The dampened immune response observed in the DM-TB group macrophages highlights the importance of identifying novel targets for developing tailored drugs for comorbid conditions.

immunology↗

bioRxiv2025

An immunologist, ecologist and clinician walk into Plato's cave to discuss infections

If the immune system is an interconnected network, then the evolution of an archetype that is ideal for fighting one pathogen should result in tradeoffs decreasing its ability to fight others. How many archetypes are there in an immune system? We infected diverse mice with Plasmodium chabaudi, and identified five distinct archetypes of responses based on the hosts position in microbial load, immune activity, and host damage space. To better understand the nature of these archetypes, we developed a mathematical model of a generalized host-pathogen system. This model explains the number, and distribution of archetypes across a population of diverse hosts. Mice resilient to P. chabaudi exhibited poor outcomes when challenged with influenza, SARS-CoV-1, or Mycobacterium tuberculosis, and vice versa, supporting our tradeoff hypothesis.

systems biology↗

bioRxiv2025

Development of Bioisosteric Iboga-alkaloids as Antinociceptive and Anxiolytic Agents with Neuroprotective Effects

The clinical importance of iboga alkaloids lies in their efficacy in reversing drug addiction and modulating drug tolerance. However, due to safety concerns, their use is restricted to appropriate medical supervision. These alkaloids often cause severe hallucinogenic effects due to differential binding to various brain receptors and cardiotoxicity by blocking the human ether-a-go-go-related gene (hERG) potassium channel. To create safer analogs, our group previously synthesized various benzofuran-containing iboga analogs with good opioid binding selectivity and excellent antinociceptive property. However, the present manuscript disclosed a step-economical and cost-effective synthesis of modified ibogaine/ibogamine analogs (C1, C2, C3 & C4) with bio-isosteric replacement of the indole scaffold with a benzofuran moiety, and comparing their antinociceptive/anxiolytic activity with their natural counterparts. Among the synthesized iboga analogs, the Endo-iboga analogs (C2 & C4, epimers of C1 and C3, respectively) not only exhibited superior anti-inflammatory and oxidative stress-relieving activity, but also effectively improved restricted locomotor activity in a formalin-induced acute pain model in mice. These Endo-iboga analogs significantly elevated the levels of inhibitory neurotransmitters (GABA and dopamine) and brain-derived neurotrophic factor (BDNF) compared to their Exo-counterparts or previously published benzofuran-containing iboga analogs lacking the tetrahydroazepine ring. Amongst, C2 and C4, the latter exhibited superior cardiac safety profile in C2C12 cells (IC50 = 235 {micro}M) and showed no adverse effects on rat hearts during in vivo ECG tests, indicated by no significant QTc prolongation. Overall, the development of bioisosteric iboga analogs, particularly C4, demonstrated significant potential for acute pain management without notable cardiotoxicity, representing a breakthrough in pain therapy innovation.

physiology↗

bioRxiv2025

β-Amyloid Induces Microglial Expression of GPC4 and APOE Leading to Increased Neuronal Tau Pathology and Toxicity

To elucidate the impact of A{beta} pathology on microglia in Alzheimers disease pathogenesis, we profiled the microglia surfaceome following treatment with A{beta} fibrils. Our findings reveal that A{beta}-associated human microglia upregulate Glypican 4 (GPC4), a GPI-anchored heparan sulfate proteoglycan (HSPG). In a Drosophila amyloidosis model, glial GPC4 expression exacerbates motor deficits and reduces lifespan, indicating that glial GPC4 contributes to a toxic cellular program during neurodegeneration. In cell culture, GPC4 enhances microglia phagocytosis of tau aggregates, and shed GPC4 can act in trans to facilitate tau aggregate uptake and seeding in neurons. Additionally, our data demonstrate that GPC4-mediated effects are amplified in the presence of APOE. These studies offer a mechanistic framework linking A{beta} and tau pathology through microglial HSPGs and APOE.

neuroscience↗

bioRxiv2025

Consequences of training data composition for deep learning models in single-cell biology

Foundation models for single-cell transcriptomics have the potential to augment (or replace) purpose-built tools for a variety of common analyses, especially when data are sparse. Recent work with large language models has shown that training data composition greatly shapes performance; however, to date, single-cell foundation models have ignored this aspect, opting instead to train on the largest possible corpus. We systematically investigate the consequences of training dataset composition on the behavior of deep learning models of single-cell transcriptomics, focusing on human hematopoiesis as a tractable model system and including cells from adult and developing tissues, disease states, and perturbation atlases. We find that (1) these models generalize poorly to unseen cell types, (2) adding malignant cells to a healthy cell training corpus does not necessarily improve modeling of unseen malignant cells, and (3) including an embryonic stem cell differentiation atlas during training improves performance on out-of-distribution tasks. Our results emphasize the importance of diverse training data and suggest strategies to optimize future single-cell foundation models.

genomics↗

bioRxiv2025

Antagonistic kinesin-14s within a single chromosomal drive haplotype

In maize, there are two meiotic drive systems that operate on large tandem repeat arrays called knobs that are found on chromosome arms. One meiotic drive haplotype, Abnormal chromosome 10 (Ab10), encodes two kinesin proteins that interact with two distinct tandem repeat arrays in a sequence-specific manner to confer meiotic drive. The kinesin KINDR associates with knob180 repeats while the kinesin TRKIN associates with TR-1 repeats. Prior data show that meiotic drive is conferred primarily by the KINDR/knob180 system, with the TRKIN/TR-1 system having little or no role. The second meiotic drive haplotype, K10L2, shows low levels of meiotic drive and only encodes the TRKIN/TR-1 system. Here we used long-read sequencing to assemble the K10L2 haplotype and showed that it has strong homology to an internal portion of the Ab10 haplotype. We also carried out CRISPR mutagenesis of Trkin to test the role of Trkin on Ab10 and K10L2. The data indicate that the Trkin gene on Ab10 does not improve drive or fitness but instead has a weak deleterious effect when paired with a normal chromosome 10. The deleterious effect is more severe when Ab10 is paired with K10L2: in this context functional Trkin on either chromosome nearly abolishes Ab10 drive. We modeled the effect of Trkin on Ab10 and found it should not persist in the population. We conclude that Trkin either confers an advantage to Ab10 in untested circumstances or that it is in the process of being purged from the Ab10 population. ARTICLE SUMMARYMendels first law states that paired chromosomes are transmitted through meiosis at equal frequencies. Some chromosome variants, however, are transmitted at higher frequencies in a process called meiotic drive. We wanted to know the function of a motor protein called TRKIN that is encoded on a maize meiotic drive chromosome. Surprisingly, we found that TRKIN provides no advantage to the meiotic driver and instead seems to be deleterious, suggesting it had a function in a wild ancestor but is now being purged from the population. The results illustrate how genomes are shaped by often-conflicting forces of selection and selfish genetic elements.

evolutionary biology↗

bioRxiv2025

Orthologous transcription factor replacement reveals that stable TFIIIC complexes are required for proper mitotic chromosome segregation

Transcription factors are speculated to play crucial roles in adaptive evolution. Using ortholog replacement of essential transcription factors (eTFs) from other yeast species, we investigated how eTFs can change. Several orthologs could not fully complement Saccharomyces cerevisiae mutants, indicating that functions or interactions of these eTFs have changed, rendering them incompatible. We further characterized TFIIIC, a fast-evolving protein complex that assists RNA polymerase III-mediated transcription, which exhibited complete or partial incompatibility in several subunits. In the orthologous Tfc7-replacement line, binding of TFIIIC to tRNA genes was reduced, yet tRNA abundance was not severely affected. However, the chromosomes of Tfc7-replacement cells were often mis-segregated during mitosis and their fitness was further reduced in a spindle checkpoint mutant. Our chromatin-immunoprecipitation experiments uncovered that unstable TFIIIC binding results in defective cohesion loading, leading to chromosome mis-segregation. Swapping the highly divergent C-terminal domain of Tfc7 orthologs rescued its interaction with Tfc1 and cell fitness, supporting that incompatibility is caused by altered interactions between complex subunits. Our results reveal distinct essential functions of a well-studied protein complex.

evolutionary biology↗

bioRxiv2025

Erythropoietin decreases apoptosis and promotes Schwann cell repair and phagocytosis following nerve crush injury in mice

After peripheral nerve trauma, insufficient clearance of phagocytic debris significantly hinders nerve regeneration. Without sufficient myelin debris clearance, Schwann cells (SCs) undergo increased apoptosis, impairing functional recovery. There is no treatment for peripheral nerve crush injury (PNCI). Erythropoietin (EPO) is an FDA-approved drug for anemia, which may help in the treatment of PNCI by transdifferentiating resident SCs into repair SCs (rSCs) and enhancing phagocytosis to facilitate the removal of cellular debris. For the first time, we conducted bulk RNA sequencing on mice with calibrated sciatic nerve crush injuries (SNCIs) on days 3, 5, and 7 post-SNCI to uncover transcriptomic changes with and without EPO treatment. We found EPO altered several biological pathways and associated genes, particularly those involved in cell apoptosis, differentiation, proliferation, phagocytosis, myelination, and neurogenesis. We validated the effects of EPO on SNCI on early (days 3/5) and intermediate (day 7) post-SNCI, and found EPO treatment reduced apoptosis (TUNEL), and enhanced SC repair (c-Jun and p75-NTR), proliferation (Ki67), and the phagocytosis of myelin debris by rSCs at crush injury sites. This improvement corresponded with an enhanced sciatic functional index (SFI). We also confirmed these findings in-vitro. EPO significantly enhanced SC repair during early de-differentiation, marked by high c-Jun and p75-NTR protein levels, and later re-differentiation with high EGR2 and low c-Jun and p75-NTR levels. These changes occurred under lipopolysaccharide (LPS) stress at 24 and 72h, respectively, compared to LPS treatment alone. Under LPS stress, EPO also significantly increased rSCs proliferation and phagocytosis of myelin or dead SCs. In conclusion, our findings support EPO may enhance the function of rSCs in debris clearance as a basis for its possible use in treating nerve trauma.

neuroscience↗

bioRxiv2025

Invasive lobular carcinoma integrated multi-omics analysis reveals silencing of Arginosuccinate synthase and upregulation of nucleotide biosynthesis in tamoxifen resistance

Invasive Lobular Carcinoma (ILC), a distinct subtype of breast cancer is hallmarked by E-Cadherin loss, slow proliferation, and strong hormone receptor positivity. ILC faces significant challenges in clinical management due to advanced stage at diagnosis, late recurrence, and development of resistance to endocrine therapy - a cornerstone of ILC treatment. To elucidate the mechanisms underlying endocrine resistance in ILC, ILC cell lines (MDA-MB-134-VI, SUM44PE) were generated to be resistant to tamoxifen, a selective estrogen receptor modulator. The tamoxifen-resistant (TAMR) cells exhibit a 2-fold increase tamoxifen IC50 relative to parental cells. Metabolomics and RNA-sequencing revealed deregulation of alanine, aspartate, and glutamate metabolism, purine metabolism, and arginine and proline metabolism in TAMR cells. Among the fifteen commonly dysregulated genes in these pathways, low ASS1 expression was identified in the TAMR cells and was significantly correlated with poor outcome in ILC patients, specifically in the context of endocrine therapy. Our study reveals methylation mediated silencing of ASS1 in TAMR cells as a likely mechanism of downregulation. Demethylation restored ASS1 expression and correspondingly reduced tamoxifen IC50 toward parental levels. Nucleic acid biosynthesis is augmented in TAMR cells, evidenced by increase in nucleotide intermediates. Both TAMR cell lines demonstrated increased expression of several nucleic acid biosynthesis enzymes, including PAICS, PRPS1, ADSS2, CAD, and DHODH. Furthermore, CAD, the key multifunctional protein of de novo pyrimidine biosynthesis pathway is differentially activated in TAMR cells. Treating TAMR cell with Decitabine, a demethylating agent, or Farudodstat, a pyrimidine biosynthesis inhibitor, markedly augmented efficacy of tamoxifen. Collectively, our study unveils ASS1 downregulation as a novel mechanism underlying acquired tamoxifen resistance in ILC and establishes a metabolic link between ASS1 and nucleic acid biosynthesis. Restoring ASS1 expression or inhibiting pyrimidine biosynthesis restored tamoxifen sensitivity. ASS1 could be a potential biomarker and therapeutic target in tamoxifen resistant ILC patients, warranting further investigation.

cancer biology↗

bioRxiv2025

Combinatorial phenotypic landscape enables bacterial resistance to phage infection

AbstractSuccess of phage therapies is limited by bacterial defenses against phages. While a large variety of anti- phage defense mechanisms has been characterized, how expression of these systems is distributed across individual cells and how their combined activities translate into protection from phages has not been studied. Using bacterial single-cell RNA sequencing, we profiled the transcriptomes of [~]50,000 cells from cultures of a human pathobiont, Bacteroides fragilis, infected with a lytic bacteriophage. We quantified the asynchronous progression of phage infection in single bacterial cells and reconstructed the infection timeline, characterizing both host and phage transcriptomic changes as infection unfolded. We discovered a subpopulation of bacteria that remained uninfected and determined the heterogeneously expressed host factors associated with protection. Each cells vulnerability to phage infection was defined by combinatorial phase-variable expression of multiple genetic loci, including capsular polysaccharide (CPS) biosynthesis pathways, restriction-modification systems (RM), and a previously uncharacterized operon likely encoding fimbrial genes. By acting together, these heterogeneously expressed phase-variable systems and anti-phage defense mechanisms create a phenotypic landscape where distinct protective combinations enable the survival and re-growth of bacteria expressing these phenotypes without acquiring additional mutations. The emerging model of complementary action of multiple protective mechanisms heterogeneously expressed across an isogenic bacterial population showcases the potent role of phase variation and stochasticity in bacterial anti-phage defenses. One Sentence SummaryCombinatorial phenotypic states with differential vulnerability to phage infection across a Bacteroides fragilis population enable a small number of super-resistant bacterial cells to evade the phage without the need for acquiring mutations.

systems biology↗

bioRxiv2025

Pseudomonas superinfection drives Pf phage transmission within airway infections in patients with cystic fibrosis

Pf bacteriophages, lysogenic viruses that infect Pseudomonas aeruginosa (Pa), are implicated in the pathogenesis of chronic Pa infections; phage-infected (Pf+) strains are known to predominate in people with cystic fibrosis (pwCF) who are older and have more severe disease. However, the transmission patterns of Pf underlying the progressive dominance of Pf+ strains are unclear. In particular, it is unknown whether phage transmission commonly occurs horizontally between bacteria within the airway via viral particles or if Pf+ bacteria are mostly acquired via new Pseudomonas infections. Here, we have studied Pa genomic sequences from 3 patient cohorts totaling 663 clinical isolates from 105 pwCF. We identify Pf+ isolates and analyze transmission patterns of Pf within patients between genetically similar groups of bacteria called "clone types". We find that Pf is predominantly passed down vertically within Pa lineages and rarely via horizontal transfer between clone types within the airway. Conversely, we find extensive evidence of Pa superinfection by a new, genetically distinct Pa that is Pf+. Finally, we find that clinical isolates show reduced activity of the type IV pilus and reduced susceptibility to Pf in vitro. These results cast new light on the transmission of virulence-associated phages in the clinical setting.

microbiology↗