Biology subjects

Khan, I.

Publications and source records attributed to Khan, I..

At least 19 recordsLinked to original sources

bioRxiv2026

TRACKING THE COMPOSITION AND STABILITY OF MICROBIOME ACROSS INDIAN SOCIAL HONEYBEES FORAGING IN A HOMOGENOUS RESOURCE LANDSCAPE

Microbial communities are essential for host health and ecosystem stability. However, whether host identity or shared foraging resources shapes microbiome structure among co-occurring species remains poorly understood. We studied bacterial and fungal communities of four Indian honeybee species in a mustard monoculture resource condition, integrating behavioural observation-based pollinator data with microbial co-occurrence networks derived from metabarcoding. Microbiome composition was linked to host identity rather than foraging behaviour, bee abundance, or landscape use. While core bacterial taxa were shared, relationships among bacterial cobionts, unlike those among fungal genera, remained species-specific. Microbial diversity, along with community structure and function, influenced network stability, with a highly modular microbial network of Apis cerana exhibiting more resilience to simulated perturbations. In summary, host-specific filtering shaped the microbiome more than resource homogenisation, with closely related species facing unique risks of microbial collapse, with broader implications for vulnerability to microbiome imbalance, environmental stress, and emerging infections.

ecology↗

bioRxiv2026

FUS and TAF15 safeguard the critical functions of the ribonucleoprotein network formed by EWSR1 and newly synthesized RNA

The FET family of RNA-binding proteins, FUS, EWSR1, and TAF15, contribute to transcriptional regulation and RNA maturation, but their core functions remain unclear. Chromosomal rearrangements involving FUS, EWSR1, or TAF15 drive multiple cancers, and mutations in the genes encoding the FET proteins are associated with neurodegenerative disease. Here, using nanoscale imaging, we show that endogenous EWSR1 and newly synthesized RNA exhibit a network-like organization with EWSR1 foci forming the nodes of this ribonucleoprotein network. Acute depletion of EWSR1 causes a rapid but transient reduction in nascent RNA levels and cellular metabolic activity without affecting active transcription. Notably, loss of EWSR1 induces a compensatory mechanism involving the reorganization of FUS and TAF15 to closely resemble that of EWSR1, including enhanced clustering with newly synthesized RNA. Together, our findings reveal functional redundancy within the FET protein family that is critical for the homeostatic regulation of nascent RNA levels. In briefSundara Rajan et al. show that endogenous EWSR1 and nascent RNA form a ribonucleoprotein network. EWSR1 depletion transiently reduces nascent RNA and metabolic activity without impairment of transcriptional elongation. Loss of EWSR1 induces compensatory reorganization of FUS and TAF15, revealing a protein family mechanism required for the homeostatic regulation of nascent RNA levels. HighlightsO_LIEWSR1 and nascent RNA form a ribonucleoprotein network C_LIO_LIEWSR1 loss transiently reduces nascent RNA and metabolic activity C_LIO_LIFUS and TAF15 undergo compensatory nuclear reorganization upon EWSR1 loss C_LIO_LIFUS and TAF15 functionally replace EWSR1 C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/713985v1_ufig1.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@b66671org.highwire.dtl.DTLVardef@ff8d49org.highwire.dtl.DTLVardef@194cfd4org.highwire.dtl.DTLVardef@d889f5_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2026

Enriched-GF: A Reproducible High-Yield Autologous Blood-Derived Growth Factor Method for Regenerative Medicine

BackgroundPlatelet-derived Growth factors play key roles in tissue repair and regeneration, yet conventional platelet-rich plasma (PRP) formulations release these mediators inconsistently in vivo due to variability in platelet yield and activation dynamics. To overcome this limitation, direct administration of concentrated platelet-derived growth factor preparations has gained interest, though current manufacturing approaches for human platelet lysate (hPL), growth factor concentrates (GFC), and conditioned serum remain constrained by batch variability, incomplete platelet degranulation, and reliance on anticoagulants. Here, we examine alternative platelet activation workflows to establish a standardized, efficient, and reproducible method for high-yield growth factor recovery suitable for translational and clinical applications. MethodsNine GFC production protocols were compared, employing different combinations of freeze-thaw (FT) cycling, glass bead (GB) agitation, calcium (Ca2) activation, and a novel Enriched Growth Factor (Enriched-GF) method. The objective was to identify a protocol capable of maximizing growth factor yield within a three-hour workflow. Optimal Ca2 concentrations and GB conditions were determined from prior optimization studies and integrated into the Enriched-GF processing scheme. Platelet concentrates (n = 10 per protocol) were processed under each condition, and growth factor levels were quantified using ELISA. ResultsGrowth factor yields differed significantly across protocols. The greatest and most consistent increases in growth factor release were observed with the Enriched-GF method combining GB activation, FT cycling, and Ca2 stimulation. This approach resulted in markedly elevated concentrations of key regenerative mediators, including enhanced EGF release, a 4.5-fold increase in PDGF, maximal TGF-{beta} liberation, and a four-fold increase in FGF2 relative to conventional platelet lysate or conditioned serum preparations. These results were reproducible across independent donor pools, demonstrating robustness and batch-to-batch consistency. ConclusionWe describe a rapid and reproducible method for producing highly concentrated platelet-derived growth factors using a combined GB-FT-Ca2 activation strategy. The Enriched-GF protocol consistently outperformed existing platelet lysate, conditioned serum, and conventional GFC preparation methods, yielding a standardized product with enhanced growth factor content. This Enriched-GF approach offers a clinically practicable solution for applications in regenerative medicine requiring reliable and high-yield growth factor delivery. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/712883v2_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@ac60c3org.highwire.dtl.DTLVardef@104d8eborg.highwire.dtl.DTLVardef@13d5afcorg.highwire.dtl.DTLVardef@120645c_HPS_FORMAT_FIGEXP M_FIG C_FIG Schematic overview of platelet concentrate preparation from whole blood and the generation of different platelet lysates and growth factor-enriched serum using freeze-thaw, calcium gluconate, and glass bead activation methods.

biochemistry↗

bioRxiv2026

Nav1.8 mediates peripheral-to-central nociceptive transmission independently of central presynaptic mechanisms in human DRG-spinal cord circuits

Suzetrigine, a selective Nav1.8 blocker, despite its significant role as a non-opioid analgesic, exhibits modest efficacy. The potential enhancement of Nav1.8 blocker efficacy through engagement of Nav1.8 at central terminals remains uncertain. We established a donor-derived human organotypic nociceptive circuit to determine where and how clinically relevant analgesics suppress pain signaling. Using ELISA and immunohistochemistry, we quantified calcitonin gene-related peptide (CGRP) release from acute explants of adult human spinal cord, dorsal root ganglia (DRG), and an intact DRG-spinal cord preparation preserving primary afferent anatomy and directional signaling. In isolated spinal cords, capsaicin evoked concentration-dependent spinal CGRP release without compromising viability, and clinically used analgesics inhibited it, validating the assay. In the intact circuit, the application of capsaicin to the DRG cell bodies triggered CGRP release exclusively in the spinal cord, consistent with compartmentalized neuropeptide release at central terminals. Selective Nav1.8 inhibition with suzetrigine reduced spinal CGRP release only when applied to the DRG or nerve root, not the spinal cord, indicating that Nav1.8 regulates peripheral action potential propagation rather than presynaptic transmitter release. These findings establish the first intact human pain circuit-based assay to study analgesics and demonstrate that the analgesic efficacy of Nav1.8 inhibitors is unlikely to increase with improved CNS penetration.

neuroscience↗

bioRxiv2026

TRACKING EVOLUTIONARY COSTS OF IMMUNE ADAPTATION AGAINST SINGLE VERSUS COINFECTING PATHOGENS

Immunity against pathogens is costly and can adversely affect fitness through resource allocation or physiological trade-offs. Infection by multiple pathogens may further worsen the effects if hosts require the activation of multiple immune responses, which in turn can elevate the overall immunological costs. Poor body condition and stressful environments can also exacerbate these trade-offs. In this study, we experimentally tested these possibilities using Tribolium castaneum populations that were evolving against either a single or a set of coinfecting bacterial pathogens. Contrary to our expectations, none of these evolved beetles showed any measurable trade-offs between pathogen resistance vs major fitness traits, such as reproduction or lifespan. Instead, they appeared to increase reproductive success and resistance to starvation, suggesting improved body condition that could mask underlying fitness costs. However, evolved beetles showed reduced quinone production, an externally secreted antimicrobial defence, indicating trade-offs between internal vs external immunity. Finally, we identified reproductive costs only under limited resource availability, but not under suboptimal resource quality, suggesting that trade-offs can be highly condition-dependent. Overall, this study provides a unique comparison across pathogens and infection types, highlighting the importance of analysing variation in life-history traits within relevant ecological contexts to understand fitness costs of evolving immunity.

evolutionary biology↗

bioRxiv2025

Correlative evaluation of anticancer effects of a modified methioninase MGL-KPV using 2D and 3D cell models, human cancer xenografts in zebrafish embryos and Balb/c nude mice

PurposeThe emergence of new models of tumor growth requires a comparison of the results of the evaluation of the anticancer effect of single drug candidate for the selection of a relevant model. MethodsWe assessed the anticancer activity of the modified enzyme MGL-KPV using four models: 2D cultures, 3D spheroids, zebrafish embryo xenografts, and Balb/c nude mice. Cytotoxicity was tested in cancer cell lines and fibroblasts; efficacy was evaluated in zebrafish and mice. The effect of MGL does not require direct contact with cancer cells, making it unique drug candidate for in vitro and in vivo comparison. ResultsMGL-KPV showed selective cytotoxicity with IC50 values of 0.23-2.21 U/mL (2D) and 1.43-4.82 U/mL (3D), sparing fibroblasts. HCT-116 and Panc-1 were most sensitive. In zebrafish xenografts, tumor size reduced by 70% (HCT-116) and 43% (Panc-1); in mice -- by 45% and 34%, respectively. ConclusionsMGL-KPV demonstrated significant anticancer activity across all models. These results support step-by-step non-clinical proof-of-concept studies approach for amino-acid cleaving enzymes with anticancer properties.

pharmacology and toxicology↗

bioRxiv2025

Paternal over- and under-nutrition program fetal and placental development in a sex-specific manner in mice

The association between sub-optimal paternal diet and offspring well-being is becoming established. However, the underlying mechanisms are yet to be fully defined. The aim of this study was to establish the impact of over- and under-nutrition, with or without macronutrient supplementation, on male reproductive fitness and post-fertilisation development. Male C57/BL6J mice were fed either control diet (CD), isocaloric low protein diet (LPD), high fat/sugar Western diet (WD) or LPD or WD supplemented with methyl-donors and carriers (MD-LPD or MD-WD respectively) for 8 weeks before mating with virgin C57/BL6J females. Placental tissue was collected at embryonic day (E)8.5, to assess early placental (ectoplacental cone) morphology and metabolism and E17.5 for sex-specific transcriptomic profiling. Post-mating, stud male tissues were harvested for assessment of testicular morphology and gene expression, gut microbiota composition and metabolic status. WD and MD-WD males displayed increased adiposity, hepatic cholesterol and free fatty acids and gut microbiota dysbiosis when compared to CD fed males. In the testes, WD and MD-WD perturbed the expression of genes associated with metabolism and transcription regulation. Additionally, we observed differential expression of multiple genes within the Wnt signalling pathway, central in the regulation of cellular proliferation, migration, survival, and cell fate determination during development. Despite no impact on fundamental male fertility, significant changes in ectoplacental cone metabolism, fetal growth, and placental gene expression were observed in response to specific dietary regimens. Interestingly, while CD male and female placentas displayed 301 genome-wide, sexually-dimorphic genes, LPD, MD-LPD, WD and MD-WD male and female placentas possessed only 13, 0, 14 and 15 sexually-dimorphic genes respectively. Our data show that while sub-optimal paternal diet has minimal impact on male fertility, fetal and placental development are perturbed in a sex-specific manner.

developmental biology↗

bioRxiv2025

DIET IMPACTS THE EFFECTS OF THERMAL STRESS ON INFECTION OUTCOMES

BackgroundClimate change can lead to warmer environments, facilitating higher pathogen growth and increasing disease burden. Climate change can also reduce the nutritional value of food resources, causing malnutrition, which can lead to impaired immunity and disease vulnerability. However, despite these associations, no experiments have tested how dietary manipulations and temperature variations interact to drive the effects of pathogenic infections. In this work, we conducted laboratory experiments with the model insect Drosophila melanogaster to test these effects. ResultsVirgin flies raised on low-carbohydrate diets exhibited improved post-infection survival rates and more resilience to pathogen burden compared to those reared on standard or high-carbohydrate diets. However, such a survival advantage of the low-carbohydrate diet was lost in mated individuals. Notably, temperature stress decreased survival rates in flies only when they were reared on a standard diet. This effect was not observed in flies raised on high- or low-carbohydrate diets, indicating that the impact of temperature on infection responses might depend on diet. Besides, flies developing on a low-carbohydrate diet showed greater fertility at ambient temperatures than those on other diets, indicating an apparent lack of fitness costs associated with improved post-infection response. Also, high-carbohydrate diets conferred greater resistance to starvation, suggesting differences in resource allocation among somatic maintenance tasks that could increase vulnerability to pathogens. ConclusionOverall, these findings imply a complex interaction between diet, temperature, and fitness traits that influences variation in infection responses. As many organisms around the world face environmental warming and poor diets, examining these patterns and processes can provide a deeper understanding of variations in health and disease responses amid a changing climate.

ecology↗

bioRxiv2025

CNNM1 is Involved in Spermatogonial Stem Cell Maintenance in the Mouse

Male reproductive development starts early during embryogenesis with the formation of spermatogonial stem cells (SSCs) from their neonatal precursors, the gonocytes. Previously, we reported high levels of CNNM1 expression in the testicular germ cells of neonatal mice, which were maintained in the SSCs of pubertal and adult mice. CNNM1 exhibited a progressive decline in expression levels as the SSCs progressed through the spermatogenic differentiation sequence. However, the specific functional role of CNNM1 during spermatogenesis remains elusive. Here, we studied the expression pattern of Cnnm1 during gonocyte to spermatogonia transition (GST), and expression levels were found to be higher at the initial establishment phase of SSCs marked with a higher rate of cell proliferation. GDNF-mediated self-renewal/proliferation of the C18-4 spermatogonial cell line enhanced the expression of CNNM1. Overexpression of CNNM1 in C18-4 spermatogonial cell lines resulted in the upregulation of genes involved in cell proliferation, nucleic acid metabolism, male germ cell development and various pathways involved in cell cycle regulation, whereas CNNM1 knockdown altered the cell cycle progression. Based on the expression analysis and proteome profiling, we conclude that CNNM1 regulates and maintains the self-renewal, proliferation, and survival of mouse spermatogonial cells. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=196 HEIGHT=200 SRC="FIGDIR/small/683346v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@7aa116org.highwire.dtl.DTLVardef@e5c74borg.highwire.dtl.DTLVardef@17f23f4org.highwire.dtl.DTLVardef@11795a_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2025

Mutations in antimicrobial peptides differently affect sleep and plasticity

Molecules implicated in host defense can, independent of their roles as immune mediators, regulate sleep. In mammals, cytokines such as Tumor Necrosis Factor (TNF ) and Interleukin 1{beta} (IL1{beta}) promote sleep. In Drosophila, infection stimulates the systemic release of a battery of anti-microbial peptides (AMPs). These fly immune effectors have been understudied as sleep modulators. We obtained mutants in different AMPs and characterized their functions in sleep and sleep dependent outputs. AMP mutants sleep less with a longer sleep latency, and lowered arousal thresholds. Group C mutant flies, doubly mutant for the anti-fungal peptides Metchinikowin (Mtk) and Drosomycin (Drs), showed the greatest impairments. These Group C mutants also showed an exaggerated sleep rebound. Sleep rebound was unaltered in other mutants. Different sets of AMP mutants exhibited specific disruptions in socialization and rocking induced sleep. These data are a detailed characterization of sleep regulation in AMP mutants. We also evaluated sleep functions. Group C mutants uniquely exhibited normal learning and memory, and lower synapse abundance, despite sleeping the least. Group C mutants are thus able to carry out some sleep functions without sleeping very much. Glial knockdown of Metchnikowin and Drosomycin mimicked the sleep phenotypes of the null mutants, these genes thus act from glia. Sleep and memory defects in AMP mutants was reversible - enhancing sleep of Bomanin mutants pharmacologically or behaviorally improved learning and memory. Together, these data suggest that AMPs are potent sleep modulators and that different classes of AMPs differently affect sleep and sleep dependent outcomes.

neuroscience↗

bioRxiv2025

ADAPTATION AGAINST LONG-TERM PROTEIN DEPRIVATION TRADES OFF WITH IMMUNOCOMPETENCE AND THE ABILITY TO SURVIVE PATHOGENIC INFECTIONS

Insufficient protein intake, leading to malnutrition, is a major global health concern that compromises the immune system and increases susceptibility to diseases. In scenarios where protein availability is constrained, organisms may experience strong selection to efficiently allocate their resources between immunity vs other energy-demanding processes, such as reproduction, resulting in evolutionary trade-offs. Additionally, in many species, protein deficiency has a more significant impact on female reproduction than on males, potentially leading to pronounced sexually dimorphic trade-offs involving immunity and infection outcomes. However, there are no experiments to test these possibilities. In this work, we demonstrate that in Drosophila melanogaster populations selected for increased early-life reproduction under protein limitations, evolved virgin females indeed suffered a greater reduction in their resistance to the pathogen Providencia rettgeri and showed lower survival following infection than males, corroborating our expectations. However, mating resulted in a loss of sexually dimorphic infection outcomes, causing both sexes to exhibit nearly identical infection costs and reduced infection tolerance compared to their ancestral, unselected populations. Moreover, several immune components, including the Toll- and IMD-mediated inducible immune pathways, were either less upregulated or more downregulated in the selected flies, which may contribute to their heightened susceptibility to pathogens. Downregulation in key metabolic pathways and genes related to phagocytosis, melanisation and ROS-mediated defence after infection in selected flies can also be associated with their increased pathogen vulnerability. Taken together, our work thus reveals the reproductive status- and sex-specific plasticity of immune investments and post-infection health in response to evolutionary constraints under chronic protein deprivation.

evolutionary biology↗

bioRxiv2025

Clonal Expansion of Alveolar Fibroblast Progeny Drives Pulmonary Fibrosis

Pulmonary fibrosis has been called a fibroproliferative disease but the functional importance of proliferating fibroblasts to pulmonary fibrosis has not been systematically examined. In response to alveolar injury, resting alveolar fibroblasts differentiate into fibrotic fibroblasts that express high levels of collagens. However, what role, if any, proliferation plays in the accumulation of fibrotic fibroblasts remains unclear. Through EdU incorporation, genetic lineage tracing, and single cell RNA sequencing, we resolve the proliferation dynamics of lung fibroblasts during post-injury fibrogenesis. Our data show substantial DNA replication in progeny of alveolar fibroblasts in two models of pulmonary fibrosis. By genetically labeling individual cells, we observe clonal expansion of alveolar fibroblast descendants principally in regions of fibrotic remodeling. The transcriptome of proliferating fibroblasts closely resembles that of fibrotic fibroblasts, suggesting that fibroblasts can first differentiate into fibrotic fibroblasts and then proliferate. Genetic ablation of proliferating fibroblasts and selective inhibition of cytokinesis in alveolar fibroblast descendants significantly mitigates pulmonary fibrosis and rescues lung function. Furthermore, fibroblasts in precision-cut lung slices from human fibrotic lungs exhibit higher proliferation rates than those in non-diseased lungs. This work establishes fibroblast proliferation as a critical driver of pulmonary fibrosis and suggests that specifically targeting fibroblast proliferation could be a new therapeutic strategy for fibrotic diseases.

cell biology↗

bioRxiv2025

β2-integrins as biomarkers in urothelial cancer

{beta}2-integrins are a family of adhesion proteins expressed in immune cells that play multiple roles in anti-tumor immunity. {beta}2-integrins regulate tumor infiltration of anti-tumorigenic immune cells such as cytotoxic CD8+ T cells and NK cells. However, they also regulate the activity of myeloid cells, such as macrophages, which can have both anti- and pro-tumorigenic properties. The role of {beta}2-integrins in urothelial cancer remains poorly understood. Here, we have investigated the role of different {beta}2-integrins, and their cytoplasmic regulators, in urothelial cancer, by utilizing RNA expression data. We found that ITGAL (encoding for CD11a) and FERMT3 (encoding for the integrin regulator kindlin-3) have a positive correlation with patient survival. EcoTyper analysis revealed increased infiltration of CD8+ T cells and NK cells in ITGAL high samples, but ITGAL or FERMT3 expression did not correlate with response to immunotherapy. In contrast, ITGAM and ITGAX (which encode for myeloid markers CD11b and CD11c) and FLNA (encoding for the integrin regulator filamin A) correlated with poor survival and reduced responsiveness to immunotherapy and critically regulate the tumor myeloid immune landscape (M1/M2 macrophages, cDC1 dendritic cells). Therefore, different {beta}2-integrins may be used as biomarkers to differentiate urothelial cancer patients with different immune landscapes, responding differently to therapy.

cancer biology↗

bioRxiv2025

Proximate Composition and Mineral Analysis of Housefly Maggots and Mealworms Compared to Fish and Soybean Meals

The nutritional profiles of dried yellow mealworms and housefly maggots were analyzed and compared with conventional protein sources in poultry diets such as fish meal (FM) and soybean meal (SBM). Nutrient composition was determined through proximate analysis, while amino acid and mineral contents were analyzed using HPLC-based amino acid analyzer and atomic absorption spectrophotometer, respectively. Metabolizable energy (ME) was calculated to estimate energy content per kilogram of feed. The crude protein content of housefly maggots (57.8%) was comparable to that of fish meal (58.5%), which was the highest among the evaluated feeds, while mealworms had a slightly lower protein content (51.02%). The highest ether extract values were recorded for mealworms (27.3%) and maggots (26.9%), surpassing those in FM and SBM. The highest ME was calculated for housefly maggots (4,074 kcal/kg), followed by mealworms (3,988 kcal/kg) and FM (3,824 kcal/kg). Mineral profiling showed the highest calcium content in mealworms (5.23 g/kg), followed by maggots (5.13 g/kg). Phosphorus and zinc were recorded highest in housefly maggots, at 16.8 g/kg and 98.5 mg/kg, respectively. The limiting amino acids lysine, leucine, and isoleucine were highest in housefly maggots, followed by FM and mealworms, showing comparable values between the insect meals and FM. Methionine content was recorded highest in mealworms (2.3 g/100g), closely followed by housefly maggots (2.27 g/100g). The study demonstrated that housefly maggots and mealworms have a nutritive profile superior to soybean meal and comparable to fish meal.

zoology↗

bioRxiv2025

High-resolution Staphylococcus profiling reveals intra-species diversity in a single skin niche

The skin microbiome is dominated by a few key genera, among which Staphylococcus is one of the most well-characterised. Recent studies have examined the roles of various Staphylococcus species such as S. epidermidis and S. hominis within broader skin microbial communities. However, these investigations often rely on isolates from multiple individuals and hence, limiting their ability to capture intra-community interactions. In this study, we focused on the axillary microbiome of a single healthy individual to characterise the genetic and functional diversity of resident Staphylococcus isolates. Using a low-cost, high-throughput DNA extraction and long-read whole genome sequencing pipeline, we generated complete genomes for 93 isolates spanning seven genetically distinct lineages across three major skin species. These comprised of one dominant with three additional lineages of S. epidermidis, two of S. hominis and one of S. capitis. Functional and metabolic analyses revealed species- and strain-specific features, suggesting potential metabolic cross-feeding and specialisation within this community, including within strains of S. epidermidis. These findings highlight the metabolic complexity and potential interdependence of staphylococci inhabiting a single skin site and the need for strain-level resolution of the community. The strains form part of the York Skin Microbiome (YSM) collection, a growing open biobank of genetically diverse skin isolates from matched individuals.

microbiology↗

bioRxiv2025

Metagenomic exploration of the bacteriome reveals natural Wolbachia infections in yellow fever mosquito Aedes aegypti and Asian tiger mosquito Aedes albopictus

BackgroundDengue and associated complications are spreading to non-endemic regions of Pakistan. Vector control, the foremost and widely adopted strategy for managing dengue has been implemented through various measures in Pakistan. Biological control through the use of Wolbachia, a bacterium naturally present in various insect genera, including Aedes, has demonstrated promising results globally. In this study we collected Aedes species and investigated their microbiomes with a particular focus on identifying the endosymbiont Wolbachia. MethodsMosquitoes were collected via Gravitraps in the Peshawar region of northwest Pakistan. The identity of the mosquitoes was initially confirmed through morphological characters followed by molecular identification using species-specific Cytochrome oxidase I (COI) primers. The DNA from female Ae. aegypti and Ae. albopictus was further subjected to 16S rRNA sequencing. The hypervariable regions V3/V4 of 16S rRNA were used for sequencing using the paired-end Illumina MiSeq platform. ResultsThe phylogenetic analysis of the COI gene in our samples demonstrated similarity to Aedes species previously documented in Pakistan. In comparative analysis of their microbiomes, Ae. albopictus was found to harbor 921 bacterial species, while Ae. aegypti only had 239 species. The metagenomic analysis revealed Wolbachia pipientis infection in Ae. aegypti while co-infection of Wolbachia pipientis and Wolbachia bourtzisii was detected in Ae. albopictus microbiota. ConclusionBoth Ae. aegypti and Ae. albopictus are present in Peshawar region of Khyber Pakhtoonkhwa province of Pakistan. Comparative analysis of the bacteriome showed higher bacterial diversity for Ae. albopictus as compared to Ae. aegypti. The investigation revealed Wolbachia infection in both Aedes mosquitoes species.

molecular biology↗

bioRxiv2025

Murine Leukemia Virus GlycoGag Antagonizes SERINC5 via ER-phagy Receptor RETREG1

Serine incorporator 5 (SERINC5) is a host restriction factor that targets certain enveloped viruses, including human immunodeficiency virus type 1 (HIV-1) and murine leukemia virus (MLV). It integrates into the viral envelope from the cell surface, inhibiting viral entry. SERINC5 is transported to the cell surface via polyubiquitination, while a single K130R mutation retains it in the cytoplasm. Both HIV-1 Nef and MLV glycoGag proteins antagonize SERINC5 by reducing its expression in producer cells. Here, we report that MLV glycoGag employs selective autophagy to downregulate SERINC5, demonstrating a more potent mechanism for decreasing its cell surface expression. Although glycoGag is a type II integral membrane protein, it primarily localizes to the cytoplasm and undergoes rapid proteasomal degradation. Employing the K130R mutant, we show that Nef, primarily associated with the plasma membrane, downregulates SERINC5 only after it has trafficked to the cell surface, whereas glycoGag can reduce its expression before reaching the plasma membrane while still in the cytoplasm. Nonetheless, an interaction with SERINC5 stabilizes and recruits glycoGag to the plasma membrane, enabling it to downregulate SERINC5 from the cell surface. Through affinity-purified mass spectrometry analysis combined with CRISPR/Cas9 knockouts, we find that glycoGags activity depends on reticulophagy regulator 1 (RETREG1), an ER-phagy receptor. Further knockout experiments of critical autophagy genes demonstrate that glycoGag downregulates cytoplasmic SERINC5 via micro-ER-phagy. These findings provide crucial new insights into the ongoing arms race between retroviruses and SERINC5 during infection. AUTHOR SUMMARYHIV-1 Nef and MLV glycoGag are unrelated viral proteins, yet both counteract the same host restriction factor, SERINC5, to facilitate productive infection. In this study, we report a novel pathway through which glycoGag downregulates SERINC5. We demonstrate that while Nef downregulates SERINC5 only after it has trafficked to the cell surface, glycoGag can directly downregulate SERINC5 in the cytoplasm before it reaches the plasma membrane. Furthermore, we show that this pathway is mediated by the ER-phagy receptor RETREG1, which targets SERINC5 for degradation via micro-ER-phagy. This mechanism provides a more effective means of blocking SERINC5 antiviral activity. These findings reveal that retroviruses have evolved different strategies to antagonize SERINC5, highlighting the critical role of SERINC5 in restricting retroviral infections.

microbiology↗

bioRxiv2024

Distinct roles for the Staphylococcus aureus POT transporter DtpT in di/tripeptide uptake and glutathione utilisation inside human macrophages

Peptides available in biological niches inhabited by the human pathogen Staphylococcus aureus serve as a rich source of amino acids required for growth and successful host colonisation. Uptake of peptides by S. aureus involves at least two transport systems: the di/tripeptide permease DtpT and the oligopeptide ABC transporter Opp3. Here we study the individual and combined functions of DtpT and Opp3 in enabling utilisation of diverse di-/tripeptides via a high-throughput phenotypic screen. We reveal that DtpT is the primary route of uptake for dipeptides, and although many peptides can be utilised via either of the two transport systems, we demonstrate a clear preference for Asp/Glu-containing peptides among DtpT substrates. To better understand the substrate preferences of DtpT, the protein was purified and reconstituted into proteoliposomes. Active transport of diverse di- and tripeptides was demonstrated, supporting the conclusions of the phenotypic screen. During this in vitro analysis, we discovered that DtpT could transport the biologically prevalent tripeptide glutathione (GSH). Bacterial growth assays demonstrate that dtpT is essential for GSH utilisation in the absence of the known glutathione transporter, Gis, identifying DtpT as the second GSH uptake system of S. aureus. We demonstrate that GSH transport is required by S. aureus for complete fitness during in vitro macrophage infection experiments. Finally, based on analysis of the DtpT structure and identification of key residues needed for GSH binding and transport, we suggest that GSH transport may be conserved in the DtpT orthologue of Listeria monocytogenes. Together, these data reveal important new functions for DtpT in the utilisation of diverse peptides and point toward a novel role for DtpT (and, potentially, other bacterial POT proteins) in glutathione acquisition during intracellular infection. Author summaryThe environments where bacterial pathogens thrive are often rich in proteins and their degradation products, including oligopeptides, which can be taken up by the bacterium and used as nutrients. Understanding how this occurs could help us find ways to tackle the growth of these pathogens during infection. Here we examine how the major human pathogen Staphylococcus aureus takes up oligopeptides. We demonstrate that a membrane transporter protein, DtpT, is the major route of dipeptide uptake and reveal over 100 new di-/tripeptide targets for this protein. Our findings highlight a defined role for DtpT in the accumulation of Aspartate- and Glutamate-containing peptides, which may serve as relevant nitrogen sources during infection. We also provide the first evidence that DtpT transports the prevalent human metabolite reduced glutathione, and demonstrate that DtpT functions alongside the previously identified Gis glutathione transport system to support intracellular survival of this pathogen inside macrophages. Overall, our findings provide a clear example of how substrate selectivity allows DtpT to fulfil specific biological roles in S. aureus, and this functional specialisation may be a common feature of homologous peptide transporters in other bacterial pathogens and across the Tree of Life.

microbiology↗