Biology subjects

Bhattacharya, A.

Publications and source records attributed to Bhattacharya, A..

At least 37 records · Page 2Linked to original sources

bioRxiv2026

Strain-specific response to pharmacological cold mimicking in aging mice

BackgroundAging impairs thermogenic and metabolic flexibility, increasing metabolic disease risk. We examined six aging male mouse strains for responses to chronic topical menthol, a pharmacological cold mimetic. Material and methodsMale (C57BL/6J, A/J, BALB/c, C3H/hej, DBA/2J, and FVB/NJ) mice were treated with 4g/kg of 10% menthol weight per volume cream once per day or vehicle as control using finger application for 15 days. After last application, core body temperature measurement, BAT thermography, Nesting behaviour, and metabolic tolerance test were performed, and gene expression was analysed in BAT. ResultsMenthol improved core temperature recovery via thermogenesis and enhanced insulin sensitivity across all strains. However, effects on BAT mass, nesting behaviour, and gluconeogenesis were strain-dependent. ConclusionCollectively, these findings highlight that genetic background modulates metabolic responses to cold mimetics, informing future precision strategies for age-related metabolic disorders.

pharmacology and toxicology↗

bioRxiv2025

M1C is a druggable target for NSCLC KRAS G12C mutant tumors resistant to KRAS inhibitors

Treatment of NSCLC KRAS G12C mutant tumors with the allele-selective sotorasib inhibitor is invariably associated with acquired resistance. The MUC1-encoded oncogenic M1C protein is necessary for self-renewal of NSCLC KRAS mutant cells. We report that treatment of NSCLC KRAS G12C cells with sotorasib induces M1C expression by a STAT1-dependent pathway. In turn, M1C drives sotorasib resistance by NF-{kappa}B-mediated induction of the epithelial-mesenchymal transition (EMT). Targeting M1C(R)NF-{kappa}B signaling (i) suppresses EMT, and (ii) reverses sotorasib resistance. Of translational relevance, treatment with a M1C antibody-drug conjugate (ADC) is effective against sotorasib-resistant NSCLC KRAS G12C cell line and patient-derived tumor xenografts. Clinically, targeted treatment of patients with NSCLC KRAS G12C tumors overexpressing MUC1 associates with decreases in overall survival. These findings identify M1C as a key effector of sotorasib resistance and as a target for treatment of patients with refractory NSCLC KRAS G12C mutant tumors.

cancer biology↗

bioRxiv2025

Chromatin priming and Hunchback recruitment integrate spatial and temporal cues in Drosophila neuroblasts

Neural stem cells generate diverse cell types by integrating spatial and temporal cues to activate neuron-specific terminal selector (TS) genes. In Drosophila neuroblasts (NBs), spatial patterning sets lineage identity, while a temporal transcription factor (TTF) cascade sets birth order. Two proposed mechanisms could integrate these inputs. In direct regulation, spatial transcription factors (STFs) and TTFs co-occupy and regulate TS enhancers within NBs. In epigenetic regulation, STFs first prime NB-specific chromatin, creating selected enhancers that can later recruit TTFs. We tested these models in the NB5-6 and NB7-4 lineages using their candidate STFs, Gooseberry (Gsb) and Engrailed (En), together with the first TTF, Hunchback (Hb). We find that En preferentially occupies pre-accessible chromatin in the NB7-4 lineage, including highly accessible En-Hb co-bound sites. This is consistent with En acting within an already established chromatin landscape whose formation likely depends on additional NB7-4 factors. In contrast, Gsb binds both accessible and less-accessible chromatin in the NB5-6 lineage and can remodel accessibility bidirectionally when ectopically expressed in NB7-4 lineage, with corresponding changes in Hb occupancy. However, Gsb binding alone does not determine which sites become accessible or recruit Hb, indicating that productive Gsb-Hb regulatory states require additional NB5-6-specific inputs. Thus, direct and epigenetic regulation are not alternative mechanisms, but distinct steps within the integration process. We therefore propose a third possibility: distributed STF code model in which these steps -- chromatin priming and direct STF-Hb engagement -- are distributed across the members of each NB-specific STF code. The STF code therefore shapes the enhancer landscape available to Hb and enables productive Hb engagement at lineage-appropriate enhancers.

developmental biology↗

bioRxiv2025

Comparative Evaluation of Assumption Lean Community Detection Methods for Human Connectome Networks

Community detection on resting-state functional connectivity provides a principled lens on mesoscale organization in functional brain networks. Currently, the choice of community count K, lacks a standardized or principled guideline. We conducted a systematic benchmark of three assumption-lean approaches on weighted functional connectivity matrices: the Weighted Stochastic Block Model, Spectral Clustering, and K-means Clustering. Performance was assessed on synthetic networks with known ground truth and on three neuroimaging cohorts which included adult and infant datasets. We compared several strategies for selecting K, including the silhouette index and other approaches commonly used in the existing literature. These were evaluated alongside a likelihood-based criterion for the weighted stochastic block model that employs bootstrap confidence intervals for differences in log-likelihood between successive values of K. For the synthetic networks, WSBM and Spectral Clustering correctly identified the true number of communities, whereas K-means Clustering did not. In adult datasets, most indices did not yield a unique optimum, whereas the likelihood-based criterion selected K = 11, which is consistent with established sensory and association systems. In infants and toddlers, the same procedure supports a larger K around 15 and reveals developmentally distinct mesoscale architecture, including anterior and posterior subdivisions within default mode and fronto parietal systems.

neuroscience↗

bioRxiv2025

Pleckstrin homology domain-containing serine/threonine kinase plays a crucial role in the survival and phagocytosis of Entamoeba histolytica.

Kinases are critical regulators of parasite survival, cytoskeletal dynamics, and endocytic processes. Entamoeba histolytica encodes 372 kinases, many of which remain functionally uncharacterized. This study characterizes EhPHDK, an AGC-family kinase essential for pseudopod formation, phagocytosis, and pinocytosis. Structural modelling suggests an autoregulatory mechanism via its PH domain, potentially modulating kinase activity. Bio-layer interferometry identified a crucial amino acid sequence facilitating interaction with EhCaBP1, implicating EhPHDK in endocytic regulation. Live-cell imaging and immunostaining confirmed its dynamic recruitment to the plasma membrane, colocalizing with EhCaBP1 during phagocytosis initiation and progression. Gene silencing severely impaired parasite viability, reducing growth by 90%, while RBC uptake assays demonstrated its indispensable role in phagocytosis. The regulatory interplay between EhPHDK and cytoskeletal components underscores its function in actin-driven processes. Given its divergence from host kinases, EhPHDK presents a promising therapeutic target for amebiasis. This study provides novel insights into kinase-mediated cytoskeletal remodelling, advancing our understanding of E. histolytica pathogenesis and offering potential avenues for intervention in parasitic survival mechanisms. HighlightsO_LIEhPHDK kinase regulates pseudopod formation, phagocytosis, and pinocytosis. C_LIO_LIStructural modelling suggests PH domain autoregulation of kinase activity. C_LIO_LIEhPHDK interacts with EhCaBP1, influencing endocytosis and cytoskeletal dynamics. C_LIO_LIGene silencing reduces parasite survival by 90%, impairing phagocytosis. C_LIO_LIEhPHDK is a potential therapeutic target, diverging from host kinases. C_LI

cell biology↗

bioRxiv2025

Evolutionary history and recurrent host adaptation in ancient Salmonella enterica

Salmonella enterica subsp. enterica is an extremely diverse bacterial pathogen causing frequent infections and foodborne disease among human populations. More than 1500 different bacterial strains (serovars) have been described, many with a wide host range. A small number of serovars are adapted to infect specific hosts: of these, serovars Typhi and Paratyphi A, B, and C cause primate-specific systemic infections (typhoid and paratyphoid fever). Although Paratyphi C is one of the rarest human-specific serovars today, it was once widespread, and all ancient Salmonella genomes published to date belong to or are ancestral to this lineage. Here, we present 53 new ancient Salmonella genomes spanning Eurasia and dating between 3500 BCE and 1300 CE. This rich genomic dataset allows us to reconstruct the evolutionary history of this pathogen in unprecedented detail. We identify multiple extinct prehistoric lineages that caused infections throughout Eurasia. Multiple lineage replacement events are observed throughout prehistoric and historic times, and Bayesian phylogenetic analysis is used to date and identify host adaptation events within this lineage. We find that host-adapted sublineages Paratyphi C, Choleraesuis, and Typhisuis continued to evolve host specificity independently from each other. We reconstruct signals of convergent host adaptation in the studied lineages and other host-adapted strains by analysing shared pseudogenes and recurrent gene gain and loss events. This analysis demonstrates a role for host interactions as a particular target of selection, highlighting the gradual adaptation of this S. enterica lineage to humans that coincides with the intensification of animal husbandry in pastoralist and sedentary farming societies.

genetics↗

bioRxiv2025

Molecular Configuration, Regulation and Function of Heterochannel Electrical Synapses

Neuron-specific expression of particular gap junction channel components defines the configuration and functional properties of electrical synapses. However, how a neuron utilises multiple, simultaneously expressed channel proteins - connexins or innexins -to make meaningful connections with distinct synaptic partners remains largely unknown. Using the posterior mechanosensory circuit in C. elegans, we discovered that individual electrical synapses can be formed by clustering together molecularly distinct gap junction channel-types made of three different innexin proteins, INX-1, UNC-7, and UNC-9. In this previously unknown configuration, which we term as heterochannel synapses, molecularly distinct gap junction channel types functionally collaborate to regulate posterior touch sensory behaviour, enhancing functional robustness. We show that the synaptic trafficking of the molecularly different channel types within a heterochannel synapse is independently regulated by discrete and conserved kinesin motor proteins, while distinct molecular pathways involving channel-specific retrograde kinesins regulate their turnover. These independent, channel-specific regulations also make individual synapse-level alterations in the composition of heterochannel synapses possible under altered environmental conditions, providing a novel mechanism for electrical synapse plasticity. Finally, we present evidence of heterochannel electrical synapses in C. elegans locomotory circuits and in the cerebellar Purkinje neurons of zebrafish larvae. Altogether, we demonstrate a novel heterochannel organization of electrical synapses, their regulation, and functional importance, which may be a conserved feature of metazoan nervous system.

neuroscience↗

bioRxiv2025

Comparative Evaluation of Solid-Phase and Membrane Mimetic Strategies in Membrane Proteome Coverage and Disease-State Analysis

Membrane proteins (MPs) are vital to cellular signaling, metabolism, and disease pathology, yet remain underrepresented in proteomics. To address this, several independent workflows have been developed to enable the profiling of the membrane proteome, however the relative advantages and limitations of each method remain poorly defined. Here, we systematically compare four classical solid-phase membrane proteomic workflows (SP3, SP4, FASP, S-Trap) and three membrane mimetic strategies (Peptidisc, nanodisc, and SMALP copolymer) for mass spectrometry-based membrane proteome profiling, using healthy (LFD) and obese (HFD) mouse liver tissue. We found that the solid-phase methods yield higher total protein identifications, while the membrane mimetic systems enrich MPs. SMALP copolymer displays intermediate characteristics between the solid-phase and membrane mimetic workflows. Peptidisc and nanodisc stand out for their enrichment of MPs, although Peptidisc shows better enrichment of plasma membrane integral MPs, particularly those with 11+ transmembrane segments. In the context of HFD-induced liver proteome remodeling, the Peptidisc workflow outperformed the other six methods by capturing the highest number of differentially expressed MPs and demonstrating the greatest accuracy in detecting MP-level dysregulation. Collectively, this comparative analysis highlights the trade-offs between depth of proteome coverage and MP enrichment across workflows, underscoring the importance of method selection based on total protein counts, MP enrichment, and the accurate detection of MP-level dysregulation. HighlightsO_LISystematic comparison of seven workflows for membrane proteomics C_LIO_LISolid-phase methods enrich soluble proteins; mimetics enrich membrane proteins C_LIO_LISMALP displays intermediate performance between other workflows C_LIO_LIPeptidisc captures the most dysregulated membrane proteins in diseased liver C_LIO_LIPeptidisc most accurate in detecting membrane protein dysregulation C_LI In Brief StatementThis study presents a systematic comparison of seven proteomic workflows for membrane protein profiling. Solid-phase methods yield higher total protein identifications, whereas membrane mimetics enrich more membrane proteins. Among tested methods on the diseased mouse liver, Peptidisc captures more differentially expressed membrane proteins and demonstrates superior accuracy in detecting membrane protein-level dysregulation. These findings provide a practical framework for selecting proteomic strategies tailored to membrane protein enrichment and biological insight. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=131 SRC="FIGDIR/small/672181v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1217dc7org.highwire.dtl.DTLVardef@1c8c188org.highwire.dtl.DTLVardef@119db19org.highwire.dtl.DTLVardef@7f5642_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

ARP2/3 complex mediates the neuropathology of PTEN-deficient human neural cells downstream of mTORC1 and mTORC2 hyperactivation

Mutations in the phosphatase and tensin homolog (PTEN) gene are linked to severe neurodevelopmental disorders. Loss of PTEN causes hyperactivation of both mechanistic target of rapamycin (mTOR) complexes, mTORC1 and mTORC2. Recent studies have shown that this dual hyperactivation is required for the neuropathology observed in PTEN-deficient human stem cell-derived neural cells. However, the molecular effectors that integrate these synergistic signals remain unknown. Here, we identify the actin-regulating ARP2/3 complex as a critical point of convergence downstream of mTORC1 and mTORC2. We show that concurrent hyperactivation of both complexes drives increased filamentous actin and elevated levels of the ARP2/3 complex subunits in PTEN-deficient human neural precursors and neurons. Pharmacological or genetic inhibition of ARP2/3 is sufficient to rescue multiple disease-relevant phenotypes, including neural precursor hyperproliferation, neuronal hypertrophy, and electrical hyperactivity, without affecting the upstream mTORC1 or mTORC2 hyperactivation. Together, these findings reveal the PTEN-mTOR-ARP2/3 signaling axis as a core mechanism of neuropathology and highlight ARP2/3 inhibition as a potential therapeutic strategy for PTEN-related neurodevelopmental disorders.

neuroscience↗

bioRxiv2025

Quantification method affects replicability of eQTL analysis, colocalization, and TWAS

eQTL mapping and TWAS are widely used to contextualize GWAS, yet the impact of RNA-seq processing choices remains unexplored. We find that RNA-seq quantification method and transcriptomic reference substantially affect eQTL detection and gene expression prediction with significant downstream impact on colocalization and TWAS results. Our findings demonstrate that seemingly minor methodological decisions substantially affect these common analyses, highlighting the need for standardized practices to ensure reproducible genetic association studies.

genomics↗

bioRxiv2025

The landscape of tertiary lymphoid structures in endometrial cancer revealed through harmonized multi-level transcriptomics

Tertiary lymphoid structures (TLSs) are ectopic lymphoid tissues that form within tumors. TLSs are associated with improved prognosis and better responses to immune checkpoint inhibitors (ICI) in cancer. However, the contribution of genetic, immune, and (micro)environmental factors to TLS formation and function remain incompletely understood. Using harmonized bulk and single-cell RNA-sequencing coupled with spatial transcriptomics, we present a comprehensive characterization of tumor-associated TLSs in endometrial cancer (EC). Our integrated datasets reveal the cellular architecture of TLSs, their organizing principles--including chemokine-receptor gradients--and TLS activity compared to paired tumor-draining lymph nodes, as well as spatial preferences in IgA/IgG plasma cell and T cell distribution. We identified a consensus set of transcriptional components that independently predict the presence of TLS in EC. Furthermore, we demonstrate that clinical neoadjuvant ICI in EC patients shifts the cellular balance in favor of immunogenic CXCL13+ PDCD1+ T cells, providing a mechanistic insight into ICI-induced TLSs formation. Our data offers a systems-level understanding of TLSs and a roadmap for their therapeutic exploitation.

immunology↗

bioRxiv2025

Lifestyle transition from rural to urban setting changes the gut bacterial profile in an ethnic community of northeast India

The human gut microbiome undergoes transformation due to various exogenous and endogenous influences. Lifestyle has been one of the major contributors of gut microbiome composition. Our study investigated the effect of migration on the gut microbiome of the Mishing ethnic community in India associated with urban migration from their pastural lifestyle. Gut bacterial diversity was profiled using 16S rRNA amplicon sequencing. We observed notable alterations in microbial diversity and composition, including changes in the core microbiome. While Bifidobacterium, Lactococcus and Streptococcus significantly increased (p < 0.01) among the migrant population there was depletion in bacteria such as Succinivibrio and Prevotella. This change in core microbiome was further reflected on the functional profile of the bacterial community harbored by the urban and rural dwellers. While the rural population was enriched in fatty acid biosynthesis pathways, amino acid and Vitamin B2 biosynthesis pathways were more prominent in the microbiome of the urban dwellers. We further observed increase in diversity and richness of the microbiome following migration. Co-analysis with ASVs from food samples consumed by the Mishing rural population revealed that 51 ASVs among the rural dwellers were from a widely consumed beverage, Apong which were completely absent among the migrants. Our analyses highlight the dietary preferences, particularly abstinence from specific foods and beverages, may modulate the alterations in gut microbial profiles. While the cross-sectional design and amplicon sequencing limited our ability to assess temporal and functional changes, our findings highlight a measurable impact of lifestyle and dietary transitions on the gut microbiome.

microbiology↗

bioRxiv2025

Long-read transcriptome assembly reveals vast isoform diversity in the placenta associated with metabolic and endocrine function

The placenta is critical for fetal development and mediates the effects of pregnancy complications on offspring metabolic health, yet it is often poorly characterized in genomic studies. Existing transcriptomic analyses rely on adult tissue-based references, which overlook developmentally important isoform diversity. We used largest-in-class long-read RNA-seq (N=72) to create a comprehensive placental transcriptome reference, identifying 37,661 high-confidence isoforms (14,985 novel) across 12,302 genes (2,759 novel). Contrary to characterizations of the placenta as a "transcriptomic void," we found transcriptional breadth and complexity comparable to adult tissues, with extraordinary splicing diversity in genes controlling obesity, lactogen production and growth, including 108 distinct CSH1 (placental lactogen) isoforms. This improved reference offers two advantages: First, it reduced inferential uncertainty in isoform quantification by 30% and increased the yield of high-confidence transcripts. Applying this reference to short-read RNA-seq datasets (N=344) of gestational diabetes mellitus (GDM), we found that placental transcription mediated 36% of GDM effects on birth weight, with novel CSH1 isoforms identified as key mediators. We further uncovered ancestry-specific effects, with distinct CSH1 isoforms mediating larger effects in European (24.4%) than Asian (13.4%) populations. Our results establish that utilizing long-read-based, tissue-specific transcriptomic annotations is critical, enabling isoform-resolved analyses that provide greater sensitivity than conventional gene-level approaches for understanding placental function and context-specific variation across diverse biobanks.

genomics↗

bioRxiv2025

Interactome Analysis of the CC2D1A Scaffold Reveals Novel Neuronal Interactions and a Postsynaptic Role

Loss of the protein scaffold Coiled-coil and C2 domain containing 1A (CC2D1A) leads to intellectual disability (ID), autism spectrum disorder (ASD), and other neurodevelopmental presentations in humans. CC2D1A interactions have been studied in different cell lines proposing diverse roles in endolysosomal maturation and intracellular signaling, but the composition and functional mechanisms of the CC2D1A interactome remain poorly understood, especially in the brain. We performed comprehensive proteomic analyses to characterize CC2D1A binding partners, first comparing immunoprecipitations with three different anti-CC2D1A antibodies in HEK293 cells and then probing the mouse hippocampus. In HEK cells, Gene Ontology (GO) analysis revealed broad interaction networks in the nucleus, mitochondrion, and cytoplasmic vesicles sharing functions in organelle organization, vesicle mediated transport, and protein metabolism. These are unified by the best characterized CC2D1A interactor, the ESCRT III component CHMP4B, and define a pleiotropic role for CC2D1A in membrane trafficking and protein homeostasis. In the hippocampus, using stringent criteria and additional controls, including a Cc2d1a hypomorph mouse line, we identified 10 high-confidence interactors in addition to CHMP4B (TNIK, G3BP2, CEP135, MAPKAP1, SHFL, PPT1, PNKD, VAMP5, and PPP6R2) revealing roles for RNA regulation and synaptic function. The HEK studies had also pointed to CC2D1B, the only paralog of CC2D1A, as an interactor. We confirmed that not only the two proteins can bind in the brain, but also localize in different synaptic compartments, showing that CC2D1A is uniquely enriched in the post-synapse. This supports a unique function of CC2D1A in regulation of synaptic transmission that could explain the more severe cognitive deficits in humans and mice upon its loss. To our knowledge these findings provide the most comprehensive characterization of the CC2D1A interactome to date, elucidating novel, multifaceted, and dynamic cellular functions, providing potential implications for its role in neurodevelop-mental disorders.

molecular biology↗

bioRxiv2025

Evaluation of Leishmania Homologue of Activated C Kinase (LACK) of Leishmania donovani in comparison to glycoprotein 63 as vaccine candidate against visceral leishmaniasis

Leishmaniasis, caused by Leishmania protozoa transmitted via sand fly bites, affects over 12 million people annually, manifesting as self-limiting cutaneous lesions or fatal visceral leishmaniasis (VL). The diseases immune response involves a Th1/Th2 paradigm, with Th1 promoting resistance and Th2 linked to susceptibility. Despite no available human vaccine, the Leishmania homologue of activated C kinase (LACK) protein has shown promise as a candidate due to its conservation across species. In this study, we purified native 34-kDa LACK protein from Leishmania donovani promastigotes and compared its efficacy with gp63 in cationic DSPC liposomes. While gp63 exhibited protective efficacy, LACK failed to protect BALB/c mice. Recombinant LACK, which was cloned, expressed, and purified, also did not confer protection. Immunological assays revealed a Th2-biased immune response characterized by a high IgG1/IgG2a ratio, elevated Th2 cytokines, and an unaltered delayed-type hypersensitivity (DTH) response, highlighting its limited potential. ImportanceThis study underscores the limitations of LACK as a standalone vaccine candidate against VL. Both native and recombinant LACK forms, despite liposomal encapsulation, elicited a Th2- dominated immune response, inadequate for protection. These findings suggest that LACKs intrinsic immunogenic properties may not support the robust Th1 response crucial for combating Leishmania donovani infections. Future research should prioritize alternative antigens or combination formulations to promote Th1-driven immunity. This work contributes to the growing body of knowledge guiding vaccine development for VL, emphasizing the need for immunogens that effectively target protective immune pathways.

immunology↗

bioRxiv2025

Prediction of physical characteristics of disordered proteins using molecular simulation and physics-informed multiple machine learning strategies

We introduce a novel hybrid machine learning (ML) framework to predict the radius of gyration and other conformational properties of intrinsically disordered proteins (IDPs). Our model integrates sequence information with physical features derived from a coarse-grained (CG) model validated by experimental data. Specifically, we combine hidden states from sequence-based models with 23 physical features projected into a shared latent space, and apply an attention mechanism that assigns weights to each residue to highlight the most informative regions of the sequence. This attention-guided fusion significantly improves predictive accuracy across multiple metrics, including MAPE and MSE, while also enhancing confidence in the predictions. We trained and evaluated our models on Brownian dynamics (BD) simulation results for approximately 7,000 IDPs from the MobiDB database (each with > 99% disorder score). We find that sequence-based models consistently outperform feature-only models, with the GRU achieving the best performance among sequence-only approaches. Moreover, combining sequence and feature information further improves accuracy across all architectures, with the hybrid biGRU model delivering the best overall predictive performance. SHAP analysis reveals the relative importance of physical features, offering model explainability and guiding feature selection. Notably, using a small number of top features often reduces model complexity and improves generalization. Furthermore an integrated gradient analysis reveals that apart from the length of the IDPs, the three parameters (SCD, SHD, and f *) play key role in ML predictions. Our framework provides a fast, interpretable, and scalable tool for predicting IDP behavior, enabling efficient initial screening prior to costly molecular simulations.

biophysics↗

bioRxiv2025

The intracellular vacuolar pathogen Coxiella burnetii promotes exocytosis in a type IVB secretion system dependent manner

Coxiella burnetii is an obligate intracellular bacterial pathogen that causes a zoonotic disease known as Q fever. Upon internalization into host cells, Coxiella extensively remodels host vesicle trafficking and replicates within acidic, lysosome-derived, spacious vacuoles by secreting a suite of effectors through the type IVB secretion system (T4BSS), and hijacking the host cell machinery. However, how fundamental lysosomal functions, such as exocytosis, are subverted during Coxiella infection has not been well understood. In this study, we aimed to investigate the regulation and role of exocytosis in the context of Coxiella infection. Biochemical and fluorescence-based imaging approaches indicated that C. burnetii infection promotes release of extracellular vesicles (EVs). Increase in extracellular levels of endolysosomal proteins (LAMP1 and cathepsin D) and surface LAMP1 expression were identified in both phagocytic and non-phagocytic cells, during later stages of infection. Interestingly, infection-induced exocytosis was dependent on the activity of the bacterial T4BSS. Modulating the activity of TRPML1, a host calcium channel that induces exocytosis by regulating the release of calcium from lysosomes into the cytosol, using synthetic agonist/antagonist, led to a decrease in intracellular C. burnetii replication, suggesting a complex, tightly regulated role of TRPML1 during infection. Together, this study demonstrates that infection by the lysosome-adapted pathogen Coxiella burnetii activates exocytosis in a temporal, host and bacterial factors-dependent manner.

cell biology↗

bioRxiv2025

Phosphoribosyl ubiquitination of SNARE proteins regulate autophagy in Legionella infection

Legionella pneumophila is an intracellular pathogen that causes Legionnaires disease. The bacteria releases effector proteins some of which remodel the host autophagic-lysosomal pathways. One effector is RavZ, which delipidates ATG8 proteins, making Legionella-infected cells deficient in autophagy. Here we show that SidE effectors mediate the phosphoribosyl ubiquitination (PR-Ub) of autophagic SNARE proteins STX17 and SNAP29. STX17 modification induces recruitment of STX17+ membranes from the endoplasmic reticulum to Legionella-containing phagosomes to form replicative vacuoles. Using proximity labeling, biochemistry and Legionella infection studies, we have discovered a mechanism by which autophagy is hijacked by bacteria to recruit ER membranes to the bacterial vacuole which has autophagy markers but do not fuse with lysosomes. Mass spectrometric identification of PR-Ub sites and mutational studies show that PR-Ub modification of STX17 alters its interaction with ATG14L, which drives the recruitment of ER membranes to the bacterial vacuole in a PI3K dependent manner. On the other hand, PR-Ub of SNAP29 inhibits the formation of the autophagosomal SNARE complex (STX17-SNAP29-VAMP8) by steric hindrance, thereby preventing the fusion of bacterial vacuoles with lysosomes.

cell biology↗