Biology subjects

Pelaseyed, T.

Publications and source records attributed to Pelaseyed, T..

10 recordsLinked to original sources

bioRxiv2026

A bare host cell membrane with minimal glycocalyx is an optimal surface for targeting by virulence-primed Salmonella Typhimurium

Gut pathogens such as Salmonella enterica serovar Typhimurium target intestinal epithelial cells for adhesion and type-3-secretion-system-dependent invasion, while also invading multiple other cell types as the infection progresses. Mechanistic studies have uncovered virulence factors involved in this process, but host cell determinants affecting Salmonella cell surface targeting remain less deeply explored. Furthermore, cell surface protein expression and glycosylation patterns differ dramatically between epithelial and blood-derived cell types, and even across maturation states of the same cell type. Here, we explored bottom-up reconstruction of the host cell surface, using simplistic suspension-growing K562 cells, to determine the contribution of individual cell surface constituents during Salmonella targeting. Combined with flow cytometry and a stringently tunable gene expression system, this model enabled high-throughput analysis and combined genetic manipulations in both pathogen and host cells. Transcriptomic and proteomic data along with lectin characterization revealed minimal K562 surface glycosylation at baseline. Chemical manipulations substantiated the role of cell membrane cholesterol in promoting Salmonella targeting, whereas ectopic expression of glycoproteins such as transmembrane mucins introduced a size-dependent steric barrier towards invading bacteria. Strikingly, even established glycoprotein receptors for Salmonella adhesins hampered rather than promoted invasion, suggesting that adhesins are required to overcome cellular glycocalyces in vivo, while a bare host cell membrane would be the pathogens preferred interaction surface.

microbiology↗

bioRxiv2025

Cryo-EM structure of CLCA1 identifies CLCA1 as a founding member of a novel metzincin family.

Calcium-activated chloride channel regulator 1 (CLCA1) is implicated in several diseases, especially mucus-associated airway diseases, but its molecular function and regulation have remained unclear. By determining the structure of CLCA1 by negative stain electron microscopy and cryo-EM, we could confirm that CLCA1 forms large oligomeric complexes which adopts a compact domain organization comprising a metallohydrolase (MH), von Willebrand type A (VWA), {beta}-sheet-rich (BSR), inhibitory (ID), and fibronectin type III-like (FnIII-l) domains. The unusually large MH domain bears hallmarks of metzincins but is distinguished by several unique features including an atypical active site zinc-coordination environment and a second Zn2+ -coordination site. Unlike classical metzincins, CLCA1 lacks a pro-domain; instead, a C-terminal inhibitory loop occludes the MH active site, providing an alternative mechanism of autoinhibition. The adjacent VWA domain, resolved in its closed state, is poised for conformational change upon ligand binding, suggesting a route for allosteric regulation of protease activity. Structural and functional assays support a role for CLCA1 in cleaving glycosylated substrates, leading to alterations in mucin architecture consistent with a regulated function in mucus remodeling. Together, these data establish CLCA1 as the founding member of a new eukaryotic metzincin family, here termed CLCAsins, with unique regulatory mechanisms.

molecular biology↗

bioRxiv2025

Proteomic analysis of bronchoalveolar lavage fluid after lung transplantation associates stable allograft function with less lung damage at 12 months

IntroductionFreedom from chronic lung allograft dysfunction (CLAD) is a key objective after lung transplantation, yet predicting its onset remains challenging. This study investigated whether early proteomic changes in bronchoalveolar lavage fluid (BALF) can differentiate between patients maintaining stable graft function at 36 months and those developing CLAD within the first year. Additionally, findings were compared to proteomic data from non-transplanted individuals. MethodsBALF samples were collected at one and twelve months post-transplant from 43 lung transplant recipients together with clinical parameters. Proteomic analysis was performed using mass spectrometry with label-free quantification for global protein profiling and heavy-labelled peptides for absolute quantification of mucins and related proteins. Differentially expressed proteins were identified and analyzed through pathway enrichment to explore biological mechanisms associated with CLAD. ResultsNo significant proteomic differences were detected at one month. By twelve months, 63 proteins were differentially expressed between patients who developed early CLAD and those with stable function. Mucin levels declined in stable patients but remained elevated in both groups compared to healthy controls. Cartilage acidic protein 1 was significantly higher in stable patients at twelve months and correlated with better pulmonary function. Pathway analysis linked several altered proteins in CLAD patients to networks associated with lung injury and remodelling. ConclusionProtein profiles in BALF that resemble those of healthy lungs are associated with sustained graft function, while persistent expression of lung injury markers is associated with early CLAD. This suggests an adaptive process is needed for long-term post-transplant success.

molecular biology↗

bioRxiv2025

Developmental factors drive the compartmentalised and discontinuous maturation of the small intestinal epithelium during the early postnatal period

The temporal, spatial and cellular diversity of the small intestinal epithelium during the postnatal period, a critical time window that accompanies the transition from placental energy supply to enteral feeding, the establishment of the enteric microbiota and postnatal immune maturation, has not been systematically investigated. Here, we used laser capture microdissection and bulk RNA-Seq, proteomics, and single cell RNA-Seq to analyse the total, organ site, crypt- and villus-specific intestinal epithelium of specific pathogen-free, germ-free and Salmonella-infected mice during the postnatal period. We identified key temporal and organ-site specific expression patterns that revealed a weak effect of the microbiota but strong influence of developmental regulators during early life. We also determined age-dependent signalling pathway and transcription factor activity and characterised age- and cell type-specific developmental trajectories revealing a distinct compartmentalised maturation process along the proximal-to-distal length and crypt-villus axis and a discontinuous appearance of goblet/Paneth cell and absorptive enterocyte transcriptional profiles. Finally, we described the cell type-specific response to neonatal enteric infection. Taken together, our findings identify the epithelium as an integral element in the maturation of postnatal mucosal tissues and in the establishment of host-microbe homeostasis.

immunology↗

bioRxiv2024

Maturation of Human Intestinal Epithelial Cell Layers Fortifies the Apical Surface against Salmonella Attack

The active invasion of intestinal epithelial cells (IECs) represents a key event in the infection cycle of many gut pathogens. Studies of how Salmonella enterica Typhimurium (S.Tm) bacteria enter transformed cell lines have shaped the paradigm for swift type-three-secretion-system-1 (TTSS-1)-driven IEC invasion, fueled by expansive membrane ruffles. However, comparative studies suggest that non-transformed IECs in the intact gut context comprise a much more challenging target for the attack. The molecular and cellular features that explain these discrepancies remain undefined. By live-cell imaging in human enteroid-and colonoid-derived IEC layers, we demonstrate that the maturation state of gut epithelia dramatically impacts permissiveness to S.Tm invasion. IEC layers kept under immature progenitor-cell-promoting conditions are permissive to the bacterial invasion, whereas maturation towards an enterocyte/colonocyte fate reduces the frequency of S.Tm-induced epithelial entry structures, and lowers the invasion efficiency by up to tenfold. This phenotypic shift during IEC maturation couples to an altered expression of actin regulatory proteins implicated in the invasion process, and an increased dependence on the S.Tm TTSS-1 effector SipA for successful entry. In addition, IEC maturation involves upregulation of cell surface mucins, e.g. MUC13, and shifts in glycocalyx composition, as revealed by multiple lectin stainings. Enzymatic treatment of the apical surface with the StcE mucinase converts maturing IEC layers back to the S.Tm-invasion-permissive state of their immature counterparts. Taken together, these results showcase how the maturation state of human IECs dictates the susceptibility to invasion by a prototype enterobacterium.

cell biology↗

bioRxiv2024

Neonatal microbiota colonization drives maturation of primary and secondary goblet cell mediated protection in the pre-weaning colon

In the distal colon, mucus secreting goblet cells primarily confer protection from luminal microorganisms via generation of a sterile inner mucus layer barrier structure. Bacteria-sensing sentinel goblet cells provide a secondary defensive mechanism that orchestrates mucus secretion in response to microbes that breach the mucus barrier. Previous reports have identified mucus barrier deficiencies in adult germ-free mice, thus implicating a fundamental role for the microbiota in programming mucus barrier generation. In this study, we have investigated the natural neonatal development of the mucus barrier and sentinel goblet cell-dependent secretory responses upon postnatal colonization. Combined in vivo and ex vivo analyses of pre- and post-weaning colonic mucus barrier and sentinel goblet cell maturation demonstrated a sequential microbiota-dependent development of these primary and secondary goblet cell-intrinsic protective functions, with dynamic changes in mucus processing dependent on innate immune signalling via MyD88, and development of functional sentinel goblet cells dependent on the NADPH/Dual oxidase family member Duox2. Our findings therefore identify new mechanisms of microbiota-goblet cell regulatory interaction and highlight the critical importance of the pre-weaning period for the normal development of colonic barrier function.

immunology↗

bioRxiv2024

BPP43_05035 is a Brachyspira pilosicoli cell surface adhesin that weakens the integrity of the epithelial barrier during infection

The anaerobic spirochete Brachyspira causes intestinal spirochetosis, characterized by the intimate attachment of bacterial cells to the colonic mucosa, potentially leading to symptoms such as diarrhea, abdominal pain, and weight loss. Despite the clinical significance of Brachyspira infections, the mechanism behind the interaction between Brachyspira and the colonic epithelium is not known. In this study, we characterized the molecular mechanism of B. pilosicoli-epithelium interaction and its impact on the epithelial barrier during infection. Through a proteomics approach, we identified BPP43_05035 as a candidate B. pilosicoli adhesion protein that mediates bacterial attachment to cultured human colonic epithelial cells. The crystal structure of BPP43_05035 revealed a globular lipoprotein with a six-bladed beta-propeller domain. Blocking the native BPP43_05035 on B. pilosicoli, either with a specific antibody or via competitive inhibition, abrogated its binding to epithelial cells. Furthermore, the binding of BPP43_05035 to epithelial cells required surface-exposed host N-glycans. Proximity labeling and interaction assays revealed that BPP43_05035 bound to tight junctions, thereby increasing the permeability of the epithelial monolayer. Extending our investigation to human patients, we identified a downregulation of tight junction and brush border genes in B. pilosicoli-infected patients carrying detectible levels of epithelium-bound BPP43_05035. Collectively, our findings identify BPP43_05035 as a B. pilosicoli adhesin that weakens the colonic epithelial barrier during infection.

microbiology↗

bioRxiv2024

MUC17 is an essential small intestinal glycocalyx component that is disrupted in Crohn's disease

Crohns disease (CD) is the chronic inflammation of the ileum and colon triggered by bacteria, but insights into molecular perturbations at the bacteria-epithelium interface are limited. We report that membrane mucin MUC17 protects small intestinal enterocytes against commensal and pathogenic bacteria. In non-inflamed CD ileum, reduced MUC17 levels correlated with a compromised glycocalyx, allowing bacterial contact with enterocytes. Muc17 deletion in mice rendered the small intestine prone to atypical infection while maintaining resistance to colitis. The loss of Muc17 resulted in spontaneous deterioration of epithelial homeostasis and extra-intestinal translocation of bacteria. Finally, Muc17-deficient mice harbored specific small intestinal bacterial taxa observed in CD. Our findings highlight MUC17 as an essential line of defense in the small intestine with relevance for early epithelial defects in CD. One-sentence summaryMembrane mucin MUC17 protects enterocytes against bacterial attachment and constitutes an early defect in Crohns disease.

physiology↗

bioRxiv2023

The MYO1B and MYO5B motor proteins and the SNX27 sorting nexin regulate membrane mucin MUC17 trafficking in enterocytes

A dense glycocalyx, composed of the megaDalton-sized membrane mucin MUC17, coats the microvilli in the apical brush border of transporting intestinal epithelial cells, called enterocytes. The establishment of the MUC17-based glycocalyx in the mouse small intestine occurs at the critical suckling-weaning transition. The enterocytic glycocalyx extends 1 {micro}m into the intestinal lumen and prevents the gut bacteria from directly attaching to the enterocytes. To date, the mechanism behind apical targeting of MUC17 to the brush border remains unknown. Here, we show that the actin-based motor proteins MYO1B and MYO5B, and the sorting nexin SNX27 regulate the intracellular trafficking of MUC17 in enterocytes. We demonstrate that MUC17 turnover at the brush border is slow and controlled by MYO1B and SNX27. Furthermore, we report that MYO1B regulates MUC17 protein levels in enterocytes, whereas MYO5B specifically governs MUC17 levels at the brush border. Together, our results extend our understanding of the intracellular trafficking of membrane mucins and provide mechanistic insights into how defective trafficking pathways render enterocytes sensitive to bacterial invasion.

cell biology↗

bioRxiv2022

Discovery of a MUC3B gene reconstructs the membrane mucin gene cluster on human chromosome 7

Human tissue surfaces are coated with mucins, a family of macromolecular sugar-laden proteins serving diverse functions from lubrication to formation of selective biochemical barriers against harmful microorganisms and molecules. Membrane mucins are a distinct group of mucins that are attached to epithelial cell surfaces where they create a dense glycocalyx facing the extracellular environment. All mucin proteins carry long stretches of tandemly repeated sequences that undergo extensive O-linked glycosylation to form linear mucin domains. However, the repetitive nature of mucin domains makes them prone to recombination and render their genetic sequences particularly difficult to read with standard sequencing technologies. As a result, human mucin genes suffer from significant sequence gaps that have hampered investigation of gene function in health and disease. Here we leveraged a recent human genome assembly to identify a previously unmapped MUC3B gene located within a cluster of four structurally related membrane mucin genes that we entitle the MUC3 cluster at q22 locus in chromosome 7. We found that MUC3B shares high sequence identity with the known MUC3A gene, and that the two genes are governed by evolutionarily conserved regulatory elements. Furthermore, we show that MUC3A, MUC3B, MUC12 and MUC17 in the human MUC3 cluster are exclusively expressed in intestinal epithelial cells. Our results complete existing genetic gaps in the MUC3 cluster that is a conserved genetic unit during primate evolution. We anticipate our results to be the starting point for detection of new polymorphisms in the MUC3 cluster associated with human diseases. Moreover, our study provides the basis for exploration of intestinal mucin gene function in widely used experimental models such as human intestinal organoids and genetic mouse models.

genetics↗