Biology subjects

Yadav, V.

Publications and source records attributed to Yadav, V..

At least 19 recordsLinked to original sources

bioRxiv2026

Trehalose-6-phosphate synthase promotes thermotolerance by governing glycolytic flux in Cryptococcus deneoformans

Growth at physiologically relevant temperatures is essential for fungal pathogenesis and is controlled by several cellular factors. The evolution of fungal thermotolerance is concerning as warming environments may promote the emergence of new pathogens. Trehalose, a disaccharide absent in mammals, plays a central role in thermotolerance by stabilizing proteins and membranes during heat stress. Trehalose is synthesized from glucose-6-phosphate (G6P) and uridine-diphosphate-glucose (UDPG) in two steps catalyzed by trehalose-6-phosphate synthase (Tps1) and trehalose-6-phosphate phosphatase (Tps2). Here, we investigated genetic suppression of Tps1 function in Cryptococcus deneoformans, a species in the Cryptococcus pathogenic species complex. Tps1 is essential for growth at 37{degrees}C in C. deneoformans and spontaneous suppressor mutations restored the growth of tps1{Delta} mutants at 37{degrees}C. Whole-genome sequencing followed by variant calling analysis primarily identified loss-of-function mutations in the gene encoding hexokinase 1 (Hxk1). Targeted gene deletion mutants further showed that loss of either HXK1 or HXK2 can bypass tps1{Delta} in a carbon source-dependent manner. The tps1{Delta} mutant exhibited elevated hexokinase activity, accumulation of G6P and glycogen, and ATP depletion after heat shock. Deletion of HXK1 or HXK2 restored hexokinase activity and partially restored G6P and ATP levels in the tps1{Delta} mutant, while glycogen remained elevated, indicating that excess glycolytic flux underlies the tps1{Delta} high-temperature growth defect. Overall, our study uncovers a previously unappreciated mechanism of Tps1-mediated heat adaptation in C. deneoformans, by revealing that Tps1 functions as a critical metabolic gatekeeper that safeguards glycolytic flux to sustain growth at elevated temperatures. Article summaryTrehalose is crucial for fungal thermal adaptation and mutants lacking trehalose are inviable at 37{degrees}C. This study examined how genetic suppressors restore viability at 37{degrees}C in mutants lacking TPS1, which encodes trehalose-6-phosphate synthase. Through whole-genome sequencing of spontaneous suppressor isolates and variant calling analysis, mutations were identified in HXK1. Gene deletion mutants and biochemical assays show these mutations alter glycolytic flux. We show that tps1{Delta} mutants exhibit unbridled glycolysis, and their growth at 37{degrees}C is restored by hxk1{Delta} mutations that reduce glycolytic flux. This study highlights the interdependence between Tps1 and Hxk1, which may have broader relevance across organisms.

genetics↗

bioRxiv2026

Integration of Morphometric and Machine Learning Approaches Strengthens Yield Prediction and Genetic Divergence Assessment in Annona reticulata under Semi-Arid Conditions.

This study integrated morphometric characterization and machine-learning modelling to identify key predictors of yield in Annona reticulata under semi-arid conditions. Thirty-one canopy, fruit, seed, and biochemical traits were evaluated across 62 genotypes, revealing substantial phenotypic diversity, particularly in structural attributes such as tree growth nature and branch angle. Principal Component Analysis and hierarchical clustering differentiated genotypes into three ideotypes representing high-yielding, structurally stable, and quality-oriented groups. Random Forest modelling and SHapley Additive exPlanations (SHAP) interpretation consistently highlighted leaf breadth, leaf length, fruit shape, and pulp-associated traits as dominant yield predictors, underscoring the coordinated influence of source-sink balance. Integration of SHAP importances with trait stability (CV%) further revealed that moderately variable traits provide reliable selection indices. These findings demonstrate that yield performance is governed by multivariate trait networks rather than isolated descriptors. The proposed framework provides a robust basis for precision phenotyping and strategic parent selection to develop high-yielding, nutritionally enriched, and climate-resilient custard apple cultivars.

plant biology↗

bioRxiv2026

Microbial Signal Recognition & Neuronal Mimicry (SRNM) axis in IBD

Inflammatory bowel disease (IBD), encompassing Crohns disease (CD) and ulcerative colitis (UC), is a chronic inflammatory disorder whose pathogenesis involves intricate host-microbiome interactions. Molecular mimicry (the structural or functional resemblance between microbial and host proteins) represents a plausible mechanism by which gut microbiota may trigger or perpetuate autoimmune responses. Here we present a comprehensive, multi-layered molecular mimicry in silico pipeline (MMIP) analysis of 39 baseline shotgun metagenomic samples from Human Microbiome Project 2 (HMP2/IBDMDB). Using DIAMOND-based homology search against the SwissProt followed by UniProt Retrieve/ID Mapping (URIM), we characterized microbial protein functional space through three complementary frameworks: (i) normalized GO term frequency comparison across diagnostic groups, (ii) protein family (PFAM) domain enrichment analysis, and (iii) sequence-level mimicry analysis identifying microbial proteins with direct homology to human proteins. Taxonomic profiling using MetaPhlAn3 pre-computed abundance profiles provided further biological context. CD microbiomes exhibited greater enrichment of immune-relevant biological processes and a higher per-sample sequence-level mimicry rate than UC. CD and UC also showed distinct pathobiont profiles, with CD enriched for oral-origin taxa including Haemophilus parainfluenzae and UC enriched for Fusobacterium nucleatum and Prevotella species. Notably, healthy gut microbiome maintains coordinated mimicry of host neuronal, signal-recognition-particle-associated, and antimicrobial peptide machinery, a repertoire dismantled in IBD and replaced by disease-subtype-specific signatures, alongside a candidate mimicry link between CD-enriched bacteria and NOD2. These results represent the first metagenome-wide characterization of molecular mimicry across IBD subtypes using shotgun metagenomic data, offering new mechanistic insight into how microbial dysbiosis may contribute to immune dysregulation in IBD.

bioinformatics↗

bioRxiv2026

Exploring differential interactional preferences of enzyme-bearing dockarins for cohesin domains in the Clostridium thermocellum cellulosome

Using its 9 sequentially-placed cohesin (Coh) domains as bait, each CipA scaffoldin chain in a Clostridium thermocellum cellulosome binds to (and displays) some combination of 9 out of over [~]70 available dockerin (Doc) domain-bearing lignocellulose-degrading enzymes, at any time. Domains numbered Coh3 through Coh8 show over 94 % pairwise sequence identity with each other, but only [~] 61-76 % identity with Coh1, Coh2, and Coh9. Such identities are much lower ([~] 40-60 %) amongst Doc domains; however, amongst both Coh and Doc domains, polypeptide backbone folds are highly conserved, suggesting that loading preferences of enzyme-bearing Doc domains upon Coh domains must depend upon their relative abundances, and pairwise affinities. To explore this further, we used microscale thermophoresis (MST), size exclusion chromatography (SEC), native polyacrylamide gel electrophoresis (NPGE), mass spectrometry (MS) and bioinformatics-based approaches (BIBA), to examine 28 Coh-Doc pairwise interactions involving recombinant Coh [Coh1, Coh2, Coh3, Coh9] and enzyme-bearing Doc [Cel8A, Cel9F, Man26/5H, Cel9R, Xyn10C, Xyn11D, Xyn10Z] domains. Interactions were found to occur with varying affinities, suggesting that Coh1 prefers Xyn11D; Coh2 prefers Xyn10C; Coh3 prefers Cel9R; Coh9 prefers Cel9R; Coh1/Coh2 prefer Xyn partners; Coh3/Coh9 prefer Cel partners. Dual modes of binding are shown by Coh1 with Xyn10C and Xyn11D; Coh2 with Xyn10Z, Cel8A, and Cel9R; Coh3 with Cel8A, and Cel9R; and Coh9 with Xyn10C, suggesting that Doc domains use either of their two homologous helices (1 and 3) to bind to Coh domains, as earlier proposed. ImportanceBacteria such as Clostridium thermocellum use extracellular enzyme complexes called cellulosomes to degrade and use cellulose. Each complex uses a linear chain of nine cohesin (Coh) domains called a scaffoldin to bind to (and display) any nine of over seventy available xylan or cellulose-degrading enzymes that bear dockerin (Doc) domains. Understanding interactions between Coh and Doc domains facilitates an appreciation of how cellulosomes are assembled and supports the building of protein-engineered constructs that utilize such interactions for many conceivable enzymatic and other applications. The significance of the presented research lies in its demonstration of the differential modes and pairwise affinities of different Coh-Doc interactions, using recombinant protein constructs and a combination of quantitative, semi-quantitative and qualitative analytical methods.

biochemistry↗

bioRxiv2026

Spatial Variation in Cortex Glia Cell Cycle Supports Central Nervous System Organization in Drosophila

Cortex glia (CG) in the ventral nerve cord (VNC) utilize mechanisms such as endocycling and acytokinetic mitosis to increase their nuclear count and support neighboring neural cells. However, the regulation of their cell cycle is not well understood. Our study examines larval development and reveals that CG nuclei in the thoracic region (tVNC) undergo successive rounds of replication and division, exponentially increasing their number to form a syncytial network around neural cells. In contrast, nuclei in the abdominal region (aVNC) only enhance their DNA content by endocycling. Key regulators, including cyclins and cyclin-dependent kinases, are vital for managing replication and division in CG. Notably, the M-phase regulator String (stg) is crucial for maintaining diploidy in tVNC nuclei and facilitating syncytial network formation. We demonstrate that ectopic stg expression can induce nuclear division in hyperploid aVNC CG. This study highlights regulatory mechanisms in CG, emphasizing the importance of region-specific cell cycle regulation for nervous system organization.

developmental biology↗

bioRxiv2025

The protein phosphatase 2C domain contributes to the pathobiological function of adenylyl cyclase in Cryptococcus neoformans

Adenylyl cyclase produces cyclic adenosine monophosphate, a signalling molecule that controls fungal development and virulence. Fungal adenylyl cyclases are distinguished by the presence of a conserved protein phosphatase 2C domain, whose function remains unknown. Here we show that the protein phosphatase 2C domain of Cac1, the adenylyl cyclase of the human pathogen Cryptococcus neoformans, has an unusual structure but functions as a metal-dependent serine/threonine phosphatase. Deletion of this domain or the adenylyl cyclase catalytic domain demonstrated that the protein phosphatase 2C domain is required for full Cac1 activity, influencing melanin and capsule synthesis, sexual differentiation, titan cell formation, and cell wall integrity. Notably, loss of the protein phosphatase 2C domain induces type 2 helper T-cell-biased immunity and extensive pulmonary damage yet does not cause mortality in mice. Integrated transcriptomic and phosphoproteomic analyses further revealed shared and domain-specific signalling outputs. Together, these findings define a pivotal role for the adenylyl cyclase-linked protein phosphatase 2C domain in C. neoformans pathobiology.

microbiology↗

bioRxiv2025

Dissecting the homeodomain MAT locus and engineering novel tripolar and bipolar mating systems in Cryptococcus amylolentus

Sex in fungi is governed by the mating-type (MAT) locus, which exists as bipolar, pseudobipolar, or tetrapolar systems. The significance and impact of MAT on sexual reproduction, however, remain understudied. Furthermore, the evolution of fungal MAT loci shares features with the evolution of sex chromosomes in plants and animals. Pathogenic Cryptococcus species harbor a bipolar system with a large contiguous MAT locus, whereas closely related species, such as the non-pathogen C. amylolentus possess a tetrapolar system with unlinked P/R and HD loci. The HD locus encodes homeobox domain containing proteins that play an important and evolutionarily conserved role in sexual reproduction. Here, we explored the roles of HD genes in sexual reproduction and determined the implications of a tetrapolar to bipolar MAT transition. With a CRISPR-Cas9 system we developed for C. amylolentus, we generated gene deletion mutants and demonstrated that a single compatible Sxi1-Sxi2 pair is necessary and sufficient for mating. By relocating the HD genes to the P/R locus, we found that the artificially generated bipolar configuration led to defective sexual development, which could be partially restored through additional rounds of sexual reproduction. Transcriptomic profiling further revealed that a Sxi1-Sxi2 heterodimeric complex drives expression of genes required for DNA replication and ergosterol biosynthesis during sexual reproduction. These findings provide the first experimental demonstration of a tetrapolar-to-bipolar transition in a tetrapolar mating species, illuminating MAT locus evolution and homeodomain protein functions in Cryptococcus. ImportanceSexual reproduction is critical for fungal survival and adaptation, yet the mechanisms driving transitions between mating systems remain unclear. With Cryptococcus amylolentus, we provide the first experimental validation of a mating system transition from its original tetrapolar to an intermediate tripolar to a derived bipolar in a tetrapolar species. We show that HD heterodimers phenotypically govern dikaryotic filamentation and also transcriptionally modulate DNA replication. These findings establish a mechanistic basis for how MAT locus reorganization drives bipolar evolution from an ancestral tetrapolar state and reinforce that fertility depends on the coordinated control of MAT locus architecture and regulatory functions.

microbiology↗

bioRxiv2025

Phylogenetic Analysis of Beta-Lactamases Reveals Distinct Evolutionary Patterns of Chromosomal and Plasmid-Encoded BLs and the Mosaic Role of VIM Linking NDM and IMP

Beta-lactamases (BLs) are a major driver of antibiotic resistance in pathogens like Acinetobacter baumannii, Enterobacteriaceae and Pseudomonas aeruginosa. This study explores the structural stability and functional divergence of metallo-beta-lactamases (MBLs) and AmpC through study of protein-protein interaction (PPI) network analysis, phylogenetics and identification of conserved domains and motifs. The study also involves analysis of co-evolutionary dynamics of BLs with related genes. PPI analysis identified NagZ as central interacting partner for BLs in P. aeruginosa and Enterobacteriaceae, suggesting its pivotal role in BL expression, and highlighting its probable role as a potential drug target. In contrast, A. baumannii exhibited such a high diversity in protein interactions, that considering a single protein as topmost interacting partner was difficult. Phylogenetic analysis revealed strong co-evolutionary trends between functionally-related proteins. A. baumannii and Enterobacteriaceae were found to be closely related in case of not just the chromosomally-encoded MBL-fold containing proteins, but also the true MBLs which are plasmid-encoded. On the other hand, similar evolutionary patterns for chromosomally-encoded BLs like AmpC and related genes was found in A. baumannii and P. aeruginosa. Analysis of PFAM domains showed that catalytic and substrate binding domains are more conserved than accessory ones. Several motifs, such as, ASNGLI were found to be conserved across all MBLs and thus carry the potential to be significant drug targets. Finally, inter-BL analysis revealed that VIM acts as an evolutionary link by acting like a mosaic between IMP and NDM as supported by intermediate GC content, positioning in phylogenetic trees and shared sequence features.

evolutionary biology↗

bioRxiv2025

Thermoregulation network governing virulence of a critical human fungal pathogen

Cryptococcus neoformans is an environmental fungal pathogen that causes meningoencephalitis in humans, requiring thermal adaptation to human body temperature. We employed several orthogonal complementary approaches to elucidate molecular mechanisms of calcineurin signaling, which is essential for thermotolerance of many fungal species, and in so doing, delineated a thermoregulatory network. First, genetic suppressors for loss of calcineurin activity identified mutations in a kinase, Yak1, as the primary suppression mechanisms. Second, the development and utilization of the proximity labeling tool TurboID identified novel subcellular interactions of calcineurin during thermal stress. Third, investigations employing phosphoproteome, RNA-sequencing, and Ribo-sequencing revealed a major role for calcineurin in controlling translation initiation machinery during thermal stress adaptation. Fourth, truncation alleles revealed domain-specific roles of calcineurin catalytic A subunit in thermotolerance, meiosis, and virulence. Combined, this study presents a comprehensive analysis of thermotolerance-governing networks and mechanisms in a fungal pathogen of global impact.

microbiology↗

bioRxiv2025

Inhibitors of trehalose-6-phosphate synthase activity in fungal pathogens compromise thermal tolerance pathways

Infections caused by fungal pathogens such as Candida and Cryptococcus are associated with high mortality rates, partly due to limitations in the current antifungal arsenal. This highlights the need for antifungal drug targets with novel mechanisms of action. The trehalose biosynthesis pathway is a promising antifungal drug target because trehalose biosynthesis is essential for virulence in Cryptococcus neoformans and Candida albicans and is also a mediator of fungal stress responses, such as thermotolerance. To exploit its untapped antifungal potentials, we screened the St. Jude 3-point pharmacophore library to identify small molecule inhibitors of the first enzyme in the trehalose biosynthesis pathway, trehalose-6-phosphate synthase (Tps1). Structure-guided optimization of a potent hit, SJ6675, yielded a water-soluble inhibitor named 4456dh. Employing biochemical, structural and cell-based assays, we demonstrate that 4456dh inhibits Tps1 enzymatic activity, suppresses trehalose synthesis and exerts a fungicidal effect. Notably, the structure of Tps1 in complex with 4456 reveals that 4456 occupies the substrate binding pocket. Importantly, 4456dh renders normally thermotolerant fungal pathogens unable to survive at elevated temperatures, which is critical as we investigate the emergence of fungi from the environment due to a warming climate. Overall, this work develops the water-soluble 4456dh as an early-stage antifungal drug that has a distinct mechanism of action compared to existing clinical antifungals. ImportanceThe rise of fungal infections in recent years is alarming due to an increase in the vulnerable immunocompromised population, global temperature increase and limited antifungal treatment options. One of the major hurdles in developing new drugs is the identification of fungal-specific antifungal drug targets due to highly conserved cellular machinery between fungi and humans. Here, we describe a small molecule inhibitor, 4456dh, of the trehalose biosynthesis pathway. This pathway is present in fungi but not humans. Trehalose plays a critical role in stress responses such as thermotolerance in fungal pathogens and is essential for their virulence. We show that treatment with 4456dh blocks production of trehalose and renders fungal cells inviable. Thus far, 4456dh is active against two fungal pathogens of critical importance suggesting a broad-spectrum activity.

microbiology↗

bioRxiv2025

Calcineurin controls the cytokinesis machinery during thermal stress in Cryptococcus deneoformans

Calcineurin is a highly conserved phosphatase that plays a central role in sensing calcium and governing transcriptional, post-transcriptional, and post-translational signaling networks. Calcineurin is a heterodimer consisting of a catalytic A subunit and a regulatory B subunit. Through downstream effectors, calcineurin signaling drives myriad responses in different organisms. In the fungal pathogenic Cryptococcus species complex that infects humans, calcineurin governs thermotolerance and is essential for growth at high temperature and pathogenesis. In Cryptococcus deneoformans, the underlying molecular functions of this critical signaling cascade are not well understood. In this study, we conducted a genetic screen and identified genetic changes that suppress the requirement for calcineurin during high-temperature growth. Our results identified two mechanisms that bypass the requirement for calcineurin function. The first mechanism involves segmental aneuploidy via both amplification as well as loss of chromosome fragments. The second mechanism involves dominant amino acid substitution mutations in the genes encoding three proteins, Chs6, Imp2, and Cts1, orthologs of components of the Ingression Progression Complex required for septation and budding in Saccharomyces cerevisiae. Loss of calcineurin activity causes chitin and chitosan accumulation and severe budding defects, whereas suppressor mutations largely restore growth and cytokinesis in the absence of calcineurin. These findings reveal the calcineurin signaling cascade controls a conserved cytokinesis machinery at the mitotic exit network during thermal stress. Significance StatementCellular responses to external stress conditions involve activation of signaling cascades, which act via effectors to govern key cellular processes. One such signaling cascade involves the phosphatase calcineurin, the conserved target of cyclosporin A and FK506 that governs myriad cellular functions through its substrates. In human fungal pathogens, calcineurin is critical for pathogenicity. Here, we employed a genetic screen to map the functional interactions of this signaling cascade and repeatedly identified mutations in components of a single protein complex, suggesting a focused downstream response. With genetic and biochemical approaches, we established that these proteins play a crucial role in cell division at high temperature enabling cell division and revealing new avenues of research that could identify potential antifungal drug targets.

genetics↗

bioRxiv2025

Bioenergetic reprogramming of macrophages reduces drug tolerance in Mycobacterium tuberculosis

Eradication of Mycobacterium tuberculosis (Mtb) requires strategies targeting bacteria inside the host. Mtb exhibits heterogeneity in redox metabolism inside macrophages to evade killing by anti-TB drugs. If and how macrophage physiology correlates with bacterial redox heterogeneity and drug tolerance remains unclear. Using a fluorescent reporter of mycobacterial redox potential, flow sorting, and RNA sequencing of infected macrophages, we characterized transcriptional and metabolic responses of macrophages harboring redox-diverse Mtb populations. We found that macrophages with suppressed glycolysis and elevated oxidative phosphorylation (OXPHOS) correlated with Mtb populations exhibiting reductive stress and drug tolerance. Conversely, macrophages with elevated glycolysis and suppressed OXPHOS displayed higher mitochondrial reactive oxygen species through reverse electron transport, resulting in oxidative stress in Mtb and enhancing drug efficacy. Computational and genetic approaches identified Nrf2 as a key regulator of macrophage bioenergetics driving redox heterogeneity and drug tolerance in Mtb. Redirecting macrophage metabolism from OXPHOS to glycolysis using an FDA- approved antiemetic drug, meclizine, subverted redox heterogeneity and diminished drug tolerance in macrophages and mice. The pharmacological profile of meclizine (Cmax and AUClast) indicated no adverse interactions with first-line anti-TB drugs in mice. Our data demonstrate the feasibility of reprogramming macrophage metabolism to reduce drug tolerance in Mtb infection.

microbiology↗

bioRxiv2025

NF-κB mediates transactivation of HNRNPD, resulting in PTEN destabilization and constitutive activation of the PI3K-AKT pathway in oral cancer cells

Heterogeneous Ribonucleoprotein D (hnRNPD), an RNA binding protein transcriptionally upregulated by NF-{kappa}B transcription factor, is associated with poor outcome of Oral Squamous Cell Carcinoma (OSCC). However, the role of hnRNPD in OSCC remains elusive. This study reveals that hnRNPD positively affects the proliferation, migration, invasion, and survival of OSCC cells. Transcriptome profiling in hnRNPD knockout cells identified significant upregulation of PTEN and inhibition of the PI3K/AKT/mTOR axis. HnRNPD mediates the destabilization of PTEN mRNA by binding to the class II AU-Rich Element (ARE) in 3UTR of PTEN. The expression of hnRNPD and PTEN are strongly negatively correlated in OSCC tissue specimens, further corroborating hnRNPD-mediated PTEN destabilization. The hnRNPD knockout inhibited autophagy, evident by an accumulation of autophagic vesicles and decreased autophagic flux. Mechanistically, the hnRNPD knockout reduced the expression of NF-{kappa}B, eventually downregulating its transcriptional target LC3b, a key mediator of autophagy. SA-{beta}-Galactosidase staining in hnRNPD KO cells conclusively demonstrated the onset of cellular senescence. The present study demonstrates hnRNPD-driven positive modulation of autophagy via NF-{kappa}B, independent of the PI3K/AKT/mTOR axis, highlighting it as a novel therapeutic target for treating oral cancer. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/631360v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@11f6844org.highwire.dtl.DTLVardef@f65124org.highwire.dtl.DTLVardef@1492571org.highwire.dtl.DTLVardef@13931dd_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIHnRNPD mediates oral cancer cell proliferation, migration, invasion, and survival. C_LIO_LIHnRNPD acts as a novel regulator of the PI3K/AKT/mTOR axis by destabilization of PTEN. C_LIO_LINF-{kappa}B/RelA downregulated on knockout of HNRNPD, inhibiting autophagy through downregulating its transcriptional target LCB-II. C_LIO_LIHnRNPD mediates cellular senescence in oral cancer cells. C_LI

cancer biology↗

bioRxiv2024

Distinct evolutionary trajectories following loss of RNA interference in Cryptococcus neoformans

While increased mutation rates typically have negative consequences in multicellular organisms, hypermutation can be advantageous for microbes adapting to the environment. Previously, we identified two hypermutator Cryptococcus neoformans clinical isolates that rapidly develop drug resistance due to transposition of a retrotransposon, Cnl1. Cnl1-mediated hypermutation is caused by a nonsense mutation in the gene encoding a novel RNAi component, Znf3, combined with a tremendous transposon burden. To elucidate adaptative mechanisms following RNAi loss, two bioinformatic pipelines were developed to identify RNAi loss-of-function mutations in a collection of 387 sequenced C. neoformans isolates. Remarkably, several RNAi-loss isolates were identified that are not hypermutators and have not accumulated transposons. To test if these RNAi loss-of-function mutations can cause hypermutation, the mutations were introduced into a non-hypermutator strain with a high transposon burden, which resulted in a hypermutator phenotype. To further investigate if RNAi-loss isolates can become hypermutators, in vitro passaging was performed. Although no hypermutators were found in two C. neoformans RNAi-loss strains after short-term passage, hypermutation was observed in a passaged C. deneoformans strain with increased transposon burden. Consistent with a two-step evolution, when an RNAi-loss isolate was crossed with an isolate containing a high Cnl1 burden, F1 hypermutator progeny inheriting a high transposon burden were identified. In addition to Cnl1 transpositions, insertions of a novel gigantic DNA transposon KDZ1 ([~]11 kb), contributed to hypermutation in the progeny. Our results suggest that RNAi loss is relatively common (7/387, [~]1.8%) and enables distinct evolutionary trajectories: hypermutation following transposon accumulation or survival without hypermutation. Significance StatementThere is a dearth of antifungal drugs available to treat Cryptococcus neoformans, a human fungal pathogen of global impact. We previously identified natural hypermutators with a loss-of-function mutation in the RNAi machinery and transposon expansion. Here, we identified several novel natural isolates with RNAi defects, none of which are hypermutators or have undergone transposon expansion. Furthermore, we demonstrate that these isolates can lie on a pathway to hypermutation following introduction of a transposon burden. In addition, a novel DNA transposon class was discovered that contributes to antifungal drug resistance. These findings highlight the importance of transposons in driving rapid adaptation in the absence of RNAi and reveal distinct evolutionary trajectories following RNAi loss, a relatively common event in C. neoformans.

genetics↗

bioRxiv2024

A Mem-dELISA platform for dual color and ultrasensitive digital detection of colocalized proteins on extracellular vesicles

Accurate, multiplex, and ultrasensitive measurement of different colocalized protein markers on individual tumor-derived extracellular vesicles (EVs) and dimerized proteins with multiple epitopes could provide insights into cancer heterogeneity, therapy management and early diagnostics that cannot be extracted from bulk methods. However, current digital protein assays lack certain features to enable robust colocalization, including multi-color detection capability, large dynamic range, and selectivity against background proteins. Here, we report a lithography-free, inexpensive (< $0.1) and ultrasensitive dual-color Membrane Digital ELISA (Mem-dELISA) platform by using track-etched polycarbonate (PCTE) membranes to overcome these shortcomings. Their through-pores remove air bubbles through wicking before they are sealed on one side by adhesion to form microwells. Immunomagnetic bead-analyte complexes and substrate solution are then loaded into the microwells from the opposite side, with >80% loading efficiency, before sealing with oil. This enables duplex digital protein colorimetric assay with beta galactosidase and alkaline phosphatase enzymes. The platform achieves 5 logs of dynamic range with a limit of detection of 10 aM for both Biotinylated {beta}-galactosidase (B-{beta}G) and Biotin Alkaline Phosphatase Conjugated (B-ALP) proteins. We demonstrate its potential by showing that a higher dosage of paclitaxel suppresses EpCAM-positive EVs but not GPC-1 positive EVs from breast cancer cells, a decline in chemo-resistance that cannot be detected with Western blot analysis of cell lysate. The Mem-dELISA is poised to empower researchers to conduct ultrasensitive, high throughput protein colocalization studies for disease diagnostics, treatment monitoring and biomarker discovery.

cancer biology↗

bioRxiv2024

In-silico evaluation of Mirror repeats in some selected genes ofCandida albicans

All cellular processes in a living cell are controlled by its genetic material. DNA in majority of the domains acts as a regulatory molecule by controlling various vital functions. The genetic makeup of DNA or RNA (in viruses) is unique in all the domains. Their uniqueness is determined by the presence of various types of repetitive patterns of bases. These includes inverted repeats, tandem repeats, VNTRs, palindromes etc. Among many repetitive pattern types, Mirror repeats (MR) found to be dispersed throughout the genes or genomes. These sequences are associated with various functional features like their involvement in H-DNA formation, in replication & transcription, nervous system related diseases development etc. The major focus of this investigation is to identify MR sequences from some selected genes of Candida albicans using a bioinformatics based pipeline. The approach refers to as FPCB which utilized some manual steps to extract out MR sequence from any targeted gene or genome. The current study find out that the identified Mirror repeats found to be dispersed throughout the selected genes along with variable length. Among them the maximum & minimum MR sequences were reported in the gene FAS2 (108) & HIS1 (15) respectively. The present study will be helpful to provide a new insight for molecular as well as computational based studies of Candida albicans as well as other related fungal groups.

bioinformatics↗

bioRxiv2023

A mitochondrial quality control mechanism reverses the phagosome maturation arrest caused by Mycobacterium tuberculosis

Phagosome maturation arrest (PMA) imposed by Mycobacterium tuberculosis (Mtb) is a classic tool that helps Mtb evade macrophage anti-bacterial responses. The exclusion of RAB7, a small GTPase, from Mtb-phagosomes causes PMA. Here, we report an unexpected mechanism that triggers crosstalk between the mitochondrial quality control (MQC) and the phagosome maturation pathways that reverses the PMA. CRISPR-mediated p62/SQSTM1 depletion (p62KD) does not appear to impact mitochondrial quality. The p62KD cells are restrictive to Mtb growth, triggered by an increasingly oxidative environment and increased lysosomal targeting. The lysosomal targeting of Mtb is facilitated by enhanced TOM20+ mitochondria-derived vesicles (MDVs) biogenesis, a key MQC mechanism. In p62KD cells, TOM20+-MDVs biogenesis is MIRO1/MIRO2-dependent and gets delivered to lysosomes for degradation in a RAB7-dependent manner. Upon infection in p62KD cells, TOM20+-MDVs get extensively targeted to Mtb-phagosomes, inadvertently facilitating RAB7 recruitment, PMA reversal and lysosomal targeting of Mtb; the phenotype also replicated in p62/SQSTM1 knockout cells. Triggering MQC collapse in p62KDcells further diminishes Mtb survival, signifying cooperation between redox- and lysosome-mediated mechanisms. The MQC-anti-bacterial pathway crosstalk could be exploited for host-directed anti-tuberculosis therapies.

microbiology↗

bioRxiv2023

SIRT1 and SIRT3 impact host mitochondrial function and host- Salmonella pH balance during infection

Mitochondria are an important organelle regulating energy homeostasis. Mitochondrial health and dynamics are crucial determinants of the outcome of several bacterial infections. SIRT3, a major mitochondrial sirtuin, along with SIRT1 regulates key mitochondrial functions. This led to considerable interest in understanding the role of SIRT1 and SIRT3 in governing mitochondrial functions during Salmonella infection. Here, we show that loss of SIRT1 and SIRT3 function either by shRNA-mediated knockdown or inhibitor treatment led to increased mitochondrial dysfunction with alteration in mitochondrial bioenergetics alongside increased mitochondrial superoxide generation in the Salmonella-infected macrophages. Consistent with dysfunctional mitochondria, mitophagy was induced along with altered mitochondrial fusion-fission dynamics in S. Typhimurium-infected macrophages. Additionally, the mitochondrial bioenergetic alteration promotes acidification of the infected macrophage cytosolic pH. This host cytosolic pH imbalance skewed the intra-phagosomal and intra- bacterial pH in the absence of SIRT1 and SIRT3, resulting in decreased SPI-2 gene expression. Our results suggest a novel role of SIRT1 and SIRT3 in maintaining the intracellular Salmonella niche by modulating the mitochondrial bioenergetics and dynamics in the infected macrophages. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=84 SRC="FIGDIR/small/557159v2_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@486cbaorg.highwire.dtl.DTLVardef@da2fb0org.highwire.dtl.DTLVardef@70cd46org.highwire.dtl.DTLVardef@1b4d0bd_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗