Biology subjects

Khan, M. M.

Publications and source records attributed to Khan, M. M..

At least 19 recordsLinked to original sources

bioRxiv2026

Cross Study Regulator Activity Analysis Reveals Convergent Programs in alpha Synuclein PFF Models of Parkinson Disease

Alpha-synuclein preformed fibril (PFF) models reproduce features of Parkinson disease (PD), but differences in brain region, cell composition, and experimental design complicate comparisons across studies. We reconstructed study-specific regulatory networks using ARACNe (Algorithm for the Reconstruction of Accurate Cellular Networks) and inferred transcription factor (TF) and transcription cofactor activity using VIPER (Virtual Inference of Protein-activity by Enriched Regulon analysis). Five transcriptomic comparisons from four mouse PFF datasets were analyzed, with human PD single-nucleus RNA-sequencing data used to assess cross-species agreement. In the dopamine-neuron-enriched dataset GSE252967, PFF exposure was associated with increased activity of stress- and inflammation-related TFs, including JUND and RELA, and decreased activity of dopaminergic-identity regulators, including NR4A2, LMX1B, FOXA1, FOXA2, PITX3, and EN1. Several regulators were connected to Th in the inferred network. The substantia nigra bulk dataset GSE296779 showed reduced activity of ATF4- and XBP1-associated protein homeostasis and unfolded protein response programs, supported by regulon-weighted gene-set enrichment. Supporting mouse comparisons showed limited overlap in individual TFs but identified changes in inflammatory, glial, and stress-related programs. Agreement between inferred activity and transcript-level changes ranged from 64% to 100% in these comparisons. Human PD data showed cell-type-dependent agreement with mouse findings. EN1 and PITX3 were shared between the mouse and human networks upstream of Th/TH and showed reduced inferred activity in all seven human cell types, alongside nonsignificant decreases in the mouse dataset. These analyses identified changes in dopaminergic-identity, protein homeostasis, and inflammatory transcriptional programs across mouse PFF models, with partial support in human PD.

neuroscience↗

bioRxiv2026

Genetic Disruption at the CIP2A Locus Modulates T Cell Responses and Attenuates Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) driven by pathogenic T cell-mediated inflammation. Fingolimod (FTY720), an approved therapy for MS, is an established activator of protein phosphatase 2A (PP2A). However the contribution of PP2A in autoimmune neuroinflammation remains incompletely understood. Here, we addressed this question using experimental autoimmune encephalomyelitis (EAE), a murine model of MS, in mice carrying a genetic disruption of the locus encoding cancerous inhibitor of protein phosphatase 2A (CIP2A), an endogenous inhibitor of PP2A. Mice with disruption of the CIP2A locus, the knock out (KO) mice, exhibited attenuated EAE severity compared with wild-type (WT) controls. Histological and flow-cytometric analyses revealed markedly reduced infiltration of mononuclear cells, including CD4 and CD4CXCR6 encephalitogenic T cells, in the CNS of diseased KO mice. Reduced numbers of these T cell populations were also observed in peripheral lymphoid organs of the Cip2a-deficient mice during EAE, while T cell abundance was comparable under steady-state conditions, suggesting impaired activation-induced expansion rather than altered homeostasis or migration. Single-cell RNA sequencing of CNS and lymph node immune cells revealed changes in cell-type abundance and gene expression. Notably, Il17a expression was reduced in CNS CD8+ T cells and showed a similar trend in {gamma}{delta} T cells. Together, our findings reveal that genetic disruption at the CIP2A locus attenuates EAE, possibly by limiting the expansion and accumulation of encephalitogenic T cell populations in CNS. These results identify the CIP2A locus as a previously unrecognized regulator of T cell-driven autoimmune neuroinflammation and provide new insights into mechanisms that restrain pathogenic T cell responses during EAE.

immunology↗

bioRxiv2026

Circulating miR-1285-3p promotes age-associated B cell differentiation through the OXPHOS-IKZF2 axis in SLE

Age-associated B cells (ABCs) expand in systemic lupus erythematosus (SLE) and contribute to pathogenic humoral immunity, but the mechanisms that restrain their differentiation remain unclear. Here, we identify the transcription factor IKZF2 (Helios) as a regulator that limits ABC differentiation. Transcriptomic and functional analyses showed that suppression of oxidative phosphorylation (OXPHOS) in B cells promoted ABC differentiation and was accompanied by reduced IKZF2 expression. Pharmacologic modulation of mitochondrial metabolism further demonstrated that OXPHOS inhibition promoted, whereas OXPHOS activation restrained, ABC differentiation. Integrative analyses revealed reduced IKZF2 expression in selected B cell subsets from patients with SLE. Functional suppression of IKZF2 enhanced ABC differentiation and attenuated the inhibitory effects of OXPHOS activation, indicating that IKZF2 mediates metabolic control of B cell fate. Mechanistically, IKZF2 restrained early ABC-associated gene programs, including ITGAX and TBX21. Circulating miR-1285-3p in small extracellular vesicles, elevated in SLE, suppressed OXPHOS and recapitulated these effects. Together, these findings identify an OXPHOS-IKZF2 axis that restrains pathogenic B cell differentiation and links extracellular microRNA-mediated metabolic stress to ABC formation in SLE. One-sentence summarySmall EV-associated miR-1285-3p in SLE promotes ABC differentiation by suppressing OXPHOS and relieving IKZF2-mediated restraint.

immunology↗

bioRxiv2026

ATF4 Coordinates Transcriptomic and Structural Adaptations in Aging Muscle

Aging is associated with a progressive loss of skeletal muscle function, known as sarcopenia; however, the molecular mechanisms coordinating cellular stress responses and structural adaptations permissive of sarcopenia remain incompletely understood. In our previous studies, we found aging differentially impacted mitochondrial networks by muscle, suggesting unique stress thresholds and response activation. Here, we investigate the role of activating transcription factor 4 (ATF4), a master regulator of the integrated stress response (ISR), in aged quadriceps muscle using complementary patient and aging mouse models. Older adults exhibited a marked decrease in aerobic capacity, muscle strength, and endurance when compared with young participants. These results paralleled findings in aged mice, with significant loss of muscle mass across multiple hindlimb muscles. Ultrastructural analysis revealed substantial age-related changes in mitochondrial morphology, including increased volume, surface area, and branching index, as well as a shift toward larger, more complex mitochondria. Our data indicate that ATF4 binds directly to the promoter region of the gene encoding TFAM, suggesting a transcriptional regulatory relationship to support DNA stability. These structural and transcriptional changes likely impair oxidative capacity and drive a feed-forward cycle of mitochondrial dysfunction and ISR activation. Our findings indicate that ATF4 coordinates transcriptomic and structural adaptations in aging muscle, identifying the ISR pathway as a potential therapeutic target for preserving muscle function in older adults.

cell biology↗

bioRxiv2025

DYT1 mutation alters gut microbiome composition and gut-brain axis dynamics in a mouse model

Dystonia is a neurological movement disorder characterized by involuntary, sustained, or intermittent muscle contractions, that result in twisting movements, repetitive motor patterns, or abnormal postures. While genetic mutations such as Tor1a+/{Delta}GAG are known contributors, the environmental and peripheral factors influencing disease onset and progression remain poorly understood. Emerging evidence implicates the gut microbiome in shaping neurodevelopment and host behavioral function, yet its contribution to dystonia pathobiology is largely unexplored. Here, we longitudinally profiled the gut microbiome of Tor1a+/{Delta}GAG mouse model using 16S rRNA gene sequencing and uncovered early emerging, persistent disruptions in microbial diversity and community composition that track with progressive motor impairment. Mutant mice exhibited an alteration of key commensal taxa, and molecular signatures indicative of compromised gut-barrier integrity. Parallel transcriptomic profiling of colonic epithelium reveals coordinated dysregulation of pathways governing epithelial stress responses, endoplasmic reticulum homeostasis, lipid signaling, autophagy, and DNA damage and repair, pointing to a previously unrecognized epithelial stress state in Tor1a+/{Delta}GAG mouse model. Integrative microbial-host interaction correlation analyses uncovered robust associations between specific dysbiotic taxa and host signaling pathways. These peripheral perturbations coincide with longitudinal motor deficits, suggesting a mechanistic gut-brain axis linking intestinal dysfunction to central neuronal vulnerability. Together, our findings provide the first experimental framework connecting microbiome perturbations, gut-barrier disruption, and neuronal vulnerability in a genetic model of dystonia. This work positions the gut microbiome and its regulation of epithelial and neuronal homeostasis as a novel entry point for disease modification in individuals carrying deleterious dystonia-associated variants.

neuroscience↗

bioRxiv2025

Arsenic Trioxide Underpins Delayed Neuroinflammation and Impaired Synaptic Integrity involving Integrative Stress Response Signaling

Arsenic trioxide (ATO), an industrial and environmental chemical with potential for accidental or intentional exposure, exerts profound neurotoxic effects. Its single acute exposure has been linked to delayed neurological and neurodegenerative outcomes. However, the molecular and cellular pathways driving these long-term manifestations remain poorly defined. In this study, combining human iPSC-derived neuronal and in vivo mouse model, we uncovered that the acute ATO exposure activates cellular stress pathway, which drives long-term neurotoxic and associated functional outcomes. We found that ATO induces integrated stress response (ISR) signaling in human iPSC-derived neurons. To model the impact of accidental ATO exposure in vivo, we administered a single high dose of ATO to C57BL/6J wild type mice. Four weeks post-treatment, ATO-exposed mice displayed neuropsychological symptoms and cognitive deficits. Brain analyses of ATO-challenged mice revealed elevated ISR activity marked by the increased phosphorylation of PERK and eIF2 and upregulation of the transcription factors, CHOP and ATF4. Transcriptomic profiling using bulk RNAseq revealed activation of pathways associated with stress and neuroinflammatory responses. Consistently, increased DNA damage and dysregulation of the STING-mediated innate immune response were also found in the brain of ATO-challenged mice. Pharmacological inhibition of the ISR with a small-molecule inhibitor, ISRIB mitigated ISR activation and preserved synaptic integrity in mouse hippocampal cells. In conclusion, our data identify ISR activation and DNA damage-driven immune dysregulation as key pathogenic drivers of ATO-induced delayed neurotoxicity and cognitive deficits and highlight ISR inhibitors as promising therapeutics to mitigate these effects.

neuroscience↗

bioRxiv2025

Harnessing cGAS-STING signaling to counteract the genotoxic-immune nexus in tauopathy

Tauopathies are progressive neurodegenerative disorders characterized by aberrant tau aggregation, cognitive decline, and persistent neuroinflammation, yet the mechanisms driving neuroinflammation and disease progression remain incompletely understood. Here, utilizing human postmortem AD brains and a mouse model of tauopathy, we report that genotoxic stress-induced cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) immune pathway form a self-amplifying loop that fuels neuropathology and cognitive deficits. Targeted disruption of this cycle through either genetic deletion of cGAS or pharmacological inhibition of STING restores immune homeostasis and attenuates tau pathology and cognitive deficits. Our results showed a significant accumulation of DNA double-strand breaks (DDSBs) and impaired DNA repair function, alongside elevated cGAS-STING signaling and type I interferon (IFN-I) responses in human AD brains compared to non-AD. In the PS19 transgenic (PS19Tg) mouse model of tauopathy, we found significantly elevated levels of DDSBs and altered expression of DNA repair proteins during early stages of disease, which preceded the dysregulation of cGAS-STING signaling and emergence of significant neuropathology in the later stage. Interestingly, genetic deletion of cGAS shifted microglial polarization from a pro-inflammatory M1 phenotype toward an anti-inflammatory M2 state, accompanied by a reduction in IFN-I signaling and improved cognitive performance in PS19Tg mice. Pharmacological STING inhibition reshaped the transcriptomic landscape, revealing selective regulation of pathways governing synaptic plasticity, and immune responses. This transcriptional reprogramming was accompanied by suppression of inflammatory responses, reduction in synaptic pathology, and attenuation of tau pathology in PS19Tg mice, underscoring STING as a therapeutic target for tauopathy. In conclusion, our findings reveal that genotoxic-immune crosstalk drives neuroinflammation and tau pathology and identify a conserved, druggable cGAS-STING axis that can be targeted to impede or slow disease progression in tauopathies. O_FIG O_LINKSMALLFIG WIDTH=184 HEIGHT=200 SRC="FIGDIR/small/678980v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@1339c89org.highwire.dtl.DTLVardef@16a1d5borg.highwire.dtl.DTLVardef@1ad4631org.highwire.dtl.DTLVardef@db4615_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2025

Geographical variation drives adaptive equilibrium of the P. falciparum sickle-associated mutations

The recent discovery of genetic mutations in Plasmodium falciparum--the most lethal malaria parasite--that enable it to overcome the protective effects of sickle cell trait, raises fundamental questions about the underlying biological and evolutionary interactions. Here we develop a geostatistical model to compare sickle haemoglobin genotype frequencies to the Plasmodium falciparum sickle-associated alleles across global populations, and find a robust association at multiple geographical scales, implying that sickle drives positive selection for these parasite mutations. A model of parasite evolution and an analysis of local haplotype patterns suggest that key features of these mutations - that they are polymorphic in all African populations and are mutually correlated despite lying in different genome regions - are caused by geographical variation in selection pressure, and that the alleles may have been maintained by balancing selection over timescales comparable to the age of the sickle mutation itself. The predicted impact of this host-parasite interaction on disease outcomes varies widely across populations, and functional data are needed to discover the biological mechanisms involved.

genetics↗

bioRxiv2025

Interaction of yeast V-ATPase with TLDc protein Rtc5p

The eukaryotic vacuolar H+-ATPase (V-ATPase) is regulated by reversible disassembly into autoinhibited V1-ATPase and Vo proton channel subcomplexes, a mode of regulation conserved from yeast to humans. While signals that govern V-ATPase assembly have been studied in the cellular context, the molecular mechanisms of the process at the level of the enzyme remain poorly understood. We recently discovered that Oxr1p, one of the two TLDc domain proteins in yeast, is essential for rapid V-ATPase disassembly in vivo. How the second TLDc protein, Rtc5p, functions in reversible disassembly, however, is less clear. Here we find that Rtc5p promotes assembly of functional holo V-ATPase from purified V1 and Vo subcomplexes in vitro. CryoEM structures show that Rtc5ps TLDc domain binds the C-terminal domain of the V1-B subunit, with Rtc5ps C-terminal a-helix inserting into the catalytic hexamer, thereby opening a second catalytic site distal to its binding site. Unlike Oxr1p, however, which when deleted produces a distinct phenotype, Rtc5p does not appear to be essential for glucose driven enzyme (re)assembly, hinting at the presence of multiple assembly pathways in vivo.

biochemistry↗

bioRxiv2025

DNA polymerase beta expression in head & neck cancer modulates the poly(ADP-ribose)-mediated replication checkpoint

Head and Neck Squamous Cell Carcinoma (HNSCC) imposes a significant health burden, necessitating innovative therapeutic strategies to enhance treatment efficacy. Current treatments such as surgery, radiation, and chemotherapy have limited effectiveness and yield severe side effects, emphasizing the need for targeted therapies. We have focused on DNA polymerase beta (Pol{beta}) and its roles in replication stress, cellular responses to DNA damaging therapies, and DNA damage response modifiers. Our investigations reveal a regulatory role for base excision repair (BER) proteins, including Pol{beta}, in the cellular response to inhibitors of poly(ADP-ribose) glycohydrolase (PARG), an enzyme involved in poly(ADP-ribose) (PAR) degradation. The inhibition of PARG, in HNSCC cells, elicits replication stress. Further, this activates the PAR-induced S-phase/ATR checkpoint, leading to a block to replication, cell cycle arrest, and the onset of apoptosis. However, Pol{beta} overexpression mitigates this response, reducing replication-stress induced PAR foci formation, suggesting a modulation of replication checkpoint activation. We found that PARG inhibitor treatment is ineffective on HNSCC cells that overexpress Pol{beta}, implying that the PARG inhibitor-induced PAR and apoptotic response is dependent on the level of Pol{beta}. Further, our in vitro experiments demonstrate that combining PARG and ATR/CHK1 inhibitors overcomes Pol{beta}-mediated treatment resistance in HNSCC cells, producing enhanced effects as compared to the individual treatment conditions. Our findings suggest a possible treatment paradigm for HNSCC, employing ATR or CHK1 inhibitors in combination with PARG inhibitors. This strategy offers a promising path for more effective HNSCC treatments, potentially overcoming Pol{beta}-related resistance.

cancer biology↗

bioRxiv2025

Monoclonal Nanobodies Alter the Activity and Assembly of the Yeast Vacuolar H+-ATPase

The vacuolar ATPase (V-ATPase; V1Vo) is a multi-subunit rotary nanomotor proton pump that acidifies organelles in virtually all eukaryotic cells, and extracellular spaces in some specialized tissues of higher organisms. Evidence suggests that metastatic breast cancers mislocalize V-ATPase to the plasma membrane to promote cell survival and facilitate metastasis, making the V-ATPase a potential drug target. We have generated a library of camelid single-domain antibodies (Nanobodies; Nbs) against lipid-nanodisc reconstituted yeast V-ATPase Vo proton channel subcomplex. Here, we present an in-depth characterization of three anti-Vo Nbs using biochemical and biophysical in vitro experiments. We find that the Nbs bind Vo with high affinity, with one Nb inhibiting holoenzyme activity and another one preventing enzyme assembly. Using cryoEM, we find that two of the Nbs bind the c subunit ring of the Vo on the lumen side of the complex. Additionally, we show that one of the Nbs raised against yeast Vo can pull down human V-ATPase (HsV1Vo). Our research demonstrates Nb versatility to target and modulate the activity of the V-ATPase, and highlights the potential for future therapeutic Nb development.

biochemistry↗

bioRxiv2024

The inflammasome sensor NLRP3 interacts with REV7 to maintain genome integrity through homologous recombination

DNA double strand break (DSB) is a highly toxic lesion that can generate genome instability, a major source of tumorigenesis. DSBs are mainly repaired by non-homologous end joining (NHEJ) or homologous recombination (HR). The selection of the DSB repair pathway primarily depends on the DNA resection of the DSB ends. Indeed, HR is initiated by resection at the DSB generating 3 single stranded extension. The shieldin complex prevents resection fostering DSB repair toward NHEJ. Here, we reveal that the inflammasome sensor NLRP3 facilitates DNA end resection to promote the HR pathway in an inflammasome-independent manner. Strikingly, NLRP3 silencing decreases HR efficiency, as evidenced by RAD51 foci and functional HR assays. Mechanistically, we describe that NLRP3 interacts with REV7, a subunit of the shieldin complex, and its depletion increases REV7 recruitment to IR-induced DSBs. Similar to cancer cells harboring HR mutated genes, we find that NLRP3 deficient cells are sensitive to PARP inhibitors (PARPi) and exhibit an epistatic relationship with BRCA1 deficiency. Remarkably, loss of REV7 in NLRP3-depleted cells induces PARPi resistance by restoring HR. This study unravels the crucial role of the innate immune receptor NLRP3 in regulating the selection of DSB repair pathways to maintain genome integrity.

molecular biology↗

bioRxiv2024

Dynamic multi-omics and mechanistic modeling approach uncovers novel mechanisms of kidney fibrosis progression

Kidney fibrosis, characterized by excessive extracellular matrix deposition, is a progressive disease that, despite affecting 10% of the population, lacks specific treatments and suitable biomarkers. This study presents a comprehensive, time-resolved multi-omics analysis of kidney fibrosis using an in vitro model system based on human kidney PDGFR{beta}+ mesenchymal cells aimed at unraveling disease mechanisms. Using transcriptomics, proteomics, phosphoproteomics, and secretomics we quantified over 14,000 biomolecules across seven time points following TGF-{beta} stimulation. This revealed distinct temporal patterns in the expression and activity of known and potential kidney fibrosis markers and modulators. Data integration resulted in time-resolved multi-omic network models which allowed us to propose mechanisms related to fibrosis progression through early transcriptional reprogramming. Using siRNA knockdowns and phenotypic assays, we validated predictions and regulatory mechanisms underlying kidney fibrosis. In particular, we show that several early-activated transcription factors, including FLI1 and E2F1, act as negative regulators of collagen deposition and propose underlying molecular mechanisms. This work advances our understanding of the pathogenesis of kidney fibrosis and provides a resource to be further leveraged by the community. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=71 SRC="FIGDIR/small/618507v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@d2bfaborg.highwire.dtl.DTLVardef@257d34org.highwire.dtl.DTLVardef@13f06f3org.highwire.dtl.DTLVardef@e16e6c_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG

systems biology↗

bioRxiv2024

Exploring the Antifibrotic Potential of the heparan sulfate mimetic OTR4120: Insights from Preclinical Models

RationaleIdiopathic pulmonary fibrosis (IPF) is a debilitating lung disease characterized by excessive deposition of extracellular matrix (ECM), resulting in lung function impairment. Heparan sulfate mimetics (HSm) have been suggested to have potential antifibrotic effects by regulating ECM. This study aims to investigate the impact of a specific HSm named OTR4120 on fibrotic processes in ex vivo, in vitro and in vivo models. MethodsHuman Precision Cut Lung Slices (hPCLS) treated with a fibrotic cocktail alone or with OTR4120 were evaluated using second-harmonic imaging microscopy (SHIM) to assess collagen deposition. Human embryonic fibroblast WI-38 cell line and primary fibroblasts obtained from human donors were differentiated into myofibroblasts (MYF) using TGF-{beta}1 and treated with OTR4120 or a control vehicle. Gene expression analysis for MYF markers was performed using quantitative PCR (qPCR). Protein expression of MYF markers was evaluated using immunofluorescence techniques. Bulk-RNA sequencing analysis on WI-38 cells cultured under different experimental conditions was conducted. Finally, the therapeutic effects of OTR4120 on a bleomycin-induced fibrosis mouse model were investigated. ResultsSHIM analysis on OTR4120-treated hPCLS showed a decrease in collagen deposition. OTR4120 treatment of primary fibroblasts and WI-38 cells exposed to TGF-{beta}1 significantly reduced the expression of MYF markers. Bulk-RNA sequencing analysis on OTR4120-treated WI-38 cells showed significant impacts on fibrosis-related processes. Therapeutic application of OTR4120 in vivo to bleomycin-induced fibrosis mice resulted in enhanced fibrosis resolution. ConclusionOTR4120 has potential therapeutic benefits as an antifibrotic agent in the context of lung fibrosis. Further investigations are necessary to understand the precise mechanism through which OTR4120 exerts its antifibrotic effects.

cell biology↗

bioRxiv2024

Identification of Antiviral Drug Candidates Against Monkeypox DNA Polymerase and Profilin-like Protein A42R Utilizing an In-Silico Approach

Monkeypox virus (MPXV) is emerging as a major concern in the field of infectious diseases. Current treatments are limited, highlighting the need for new therapeutic options. The use of computational methods, such as molecular docking and molecular dynamic (MD) simulations, is a valuable approach in identifying potential compounds that can target specific proteins of the virus, like the DNA polymerase and profilin-like protein A42R in this case, with the aim of controlling the disease. Our study focused on screening various libraries of compounds for predicted binding to MPXV DPol and A42R proteins, with the top-performing molecules identified based on their docking scores. Among these, Dorsilurin K and Mangostin in complex with DPol, whereas [2-oxo-2-[3-(3,4,5,6-tetrahydro-2H-azepin-7-ylsulfamoyl)anilino]ethyl] 3,5-dimethylbenzoate and N-[4-[2-[4-(4-methylphenyl)sulfonylpiperazin-1-yl]-2-oxoethoxy]phenyl]furan-2-carboxamide in complex with A42R stand out with notably high docking scores, suggesting they may have a good affinity for binding to the DPol and A42R proteins of MPXV respectively. MD simulations confirmed the stability of these ligand-protein complexes followed by evaluation of the ADMET and oral bioavailability analysis. However, it is important that computational methods can suggest promising candidates, in vitro and eventually in vivo studies are essential to validate these therapeutic candidates. Further studies on these compounds will provide insights into their efficacy, safety, and potential side effects. In conclusion, this study offers promising avenues for developing potential treatments for Monkeypox. If the identified compounds prove effective in further studies, it could be a significant breakthrough in managing this zoonotic disease.

pharmacology and toxicology↗

bioRxiv2024

Sex Dimorphism in Expression of Immune Response Genes in Drosophila

Sex dimorphism in immunity is commonly observed in a wide variety of taxa and is thought to arise from fundamental life history differences between females and males. In Drosophila melanogaster, infection with different pathogens typically results in different modes of immune sex dimorphism, with male-or female-bias, likely due to specific disease-causing mechanisms of pathogens and host-pathogen interactions. Studies showed that some pathways, such as IMD and Toll, can explain these sex-dimorphic immune responses in Drosophila. However, it is unclear if sex differences in the immune response observed in D. melanogaster are conserved, even in closely related species. One window into identifying conserved and evolving sex differences in the immune response is to examine the sex-differential expression of immunity-related genes. Here, we aim to understand whether two closely related species, D. melanogaster and D. simulans, show conserved sex dimorphism in innate immunity, focusing on associated changes in gene expression in response to infection with a gram-negative bacterium, Providencia rettgeri. Survival, bacterial load, and bacterial load upon death (BLUD) were investigated to assess overall sex differences. D. melanogaster females and males differed significantly in survival, whereas D. simulans did not. Enrichment analyses revealed that both sexes and species upregulate genes involved in similar immune-related biological processes, but downregulated groups differed. We identified conserved sex differential gene expression of genes in the bacterial infection response pathways IMD, Toll, Jak/STAT, their regulators, and other immune-related gene classes (e.g., BOMs), as well as sex and species differences. In D. melanogaster, the effector antimicrobial peptides (AMPs) regulated by IMD were more highly upregulated relative to D. simulans in both sexes. Moreover, D. melanogaster females uniquely initiated high levels of gene expression that were involved in negative feedback mechanisms that controlled the overstimulation of IMD. Genes in the Toll pathway were also sex-differentially expressed with a higher level of upregulation in D. melanogaster. Remarkably, comparing expression across species, we find that D. simulans likely employs both the conventional peptidoglycan recognition-driven PRR-SPE-Spz pathway and the microbial protease recognition-based Psh-dependent activation of Toll; in contrast, D. melanogaster appears to solely rely on the PRR-SPE-Spz pathway in this context. In summary, our findings indicate that sex differences are conserved in both species for the majority of upregulated genes, while downregulation patterns and specific gene subsets show notable differences between sexes or species.

genetics↗

bioRxiv2024

Regulation of sex differences in innate immunity by sex-determining gene transformer in Drosophila melanogaster

AbstractSex dimorphism is one of the key features of dioecious organisms, which shapes almost all the aspects of life history traits from early development to cessation of life. However, the specific regulatory differences between males and females that underlie sex dimorphism are underexplored for most quantitative traits, including immune sex dimorphism. Complex patterns of variation, for example, with respect to numbers and types of contributing alleles, dynamic changes in gene expression, and context dependency, have created challenges in understanding the mechanistic basis of sex differences in immunity. To investigate the regulatory basis of sex dimorphism in immunity, we focused on the sex determination hierarchy master switch, transformer (tra), in Drosophila melanogaster. Two different perturbations of sex determination were examined, pseudomales (tra-mutant) and pseudofemales (tra-overexpression). Changes in gene expression patterns in response to the infection of Providencia rettgeri, an extracellular gram-negative bacterium, were examined for each of these perturbations and for controls with wild-type sex determination phenotypes. To the best of our knowledge, this is the first study to report the immune response to bacterial infection in any insects with mutant or overexpressed tra genotypes. Survival and bacterial load were first characterized to assess the overall impact of tra on sex differences in immunity. To examine the regulatory role of the tra gene, sex differentially regulated genes were identified in comparisons of wound control to bacterial infected flies for males, females, and tra-mutant or tra-overexpression animals. The survival assay showed that in the absence of the female-specific isoform of tra (TraF), tra-mutant XX animals showed significantly lower survival than wild-type females, and their survival rate aligned with males. The overexpression of TraF in the XY animals resulted in a surprising outcome, much higher survival of these animals relative to both males and females. The DEG analysis of genes with a significant interaction between sex and infection status identified 235 and 417 genes regulated upstream and downstream of tra in the sex hierarchy pathway, respectively. Interestingly, the GO enrichment analysis found 49 bacterial infection response-related biological processes enriched among genes regulated downstream of tra, including the Toll signaling pathway, and no enrichment for immune response-related categories among genes regulated upstream of tra. Further analysis of the genes and regulators of the Toll signaling pathway identified a significant regulatory role of tra or its downstream targets in signal detection, transduction, cellular response, and regulators of the pathway. Moreover, several genes regulated downstream of tra can regulate IMD pathways via the transcription factor NF-kB-Relish and some of its regulators, such as Diap2/IAP2 and Charon. Overall, the findings highlighted a strong potential role of tra to establish immune sex dimorphism in Drosophila.

genomics↗

bioRxiv2024

In-host evolution of classic to convergent Klebsiella pneumoniae sequence type 147 isolates and impact of associated capsular changes on different morphotypes

Klebsiella pneumoniae, an important opportunistic pathogen, has long been categorized into two distinct pathotypes: the often multidrug-resistant classic (cKp) and the highly virulent hypervirulent (hvKp). However, a recent global trend has witnessed the emergence of convergent strains, seamlessly combining antimicrobial resistance with hypervirulence. Our study delved into a series of K. pneumoniae isolates sourced from the same patient, all belonging to the international, high-risk clonal lineage of sequence type 147. As reported in a previous study, these isolates exhibited diverse morphotypes on blood agar, ranging from small white to normal-sized white, grey, or grey and dry (g/d) colonies. Through an interplay of omics and phenotypic experiments, we unraveled the intricate mechanisms governing these distinct colony morphologies and their implications on bacterial virulence and resilience. While the earlier isolates demonstrated modest levels of resistance and virulence, their later counterparts showed significantly heightened levels, attributed to the acquisition of additional plasmids. Bioinformatics analysis unveiled a chromosomal insertion of a hybrid plasmid in one isolate, marking an unprecedented in-host microevolution from the classic to the convergent pathotype. All morphotypes exhibited positive insertion sequences around or within the K loci, with the grey or g/d phenotypes arising from impaired K loci. Despite lower serum resistance, these morphotypes demonstrated superior adhesion to human epithelial cells. Interestingly, while capsule-deficient strains are conventionally associated with decreased virulence, our isolates displayed high mortality rates in the Galleria mellonella infection model. In conclusion, our findings not only provide unprecedented insights into in-host microevolution within a patient, transitioning from the classic to the convergent pathotype, but also contribute significantly to the understanding of the diverse morphotypes exhibited by K. pneumoniae.

microbiology↗