Biology subjects

Ceylan, M.

Publications and source records attributed to Ceylan, M..

4 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

Human jejunal enteroids for studies of epithelial drug transport and metabolism

Intestinal enteroids are stem cell-based "mini-guts" that mimic many aspects of the corresponding epithelial barrier in vivo. Here, we established and characterized differentiated apical-out (AO) and basal-out (BO) jejunal enteroids in suspension and followed their differentiation by quantitative global proteomics and different microscopic techniques. The barrier integrity and function and subcellular location of nutrient and clinically important drug transporters were investigated in the matured enteroids using live-cell microscopy. The presystemic metabolism of two drugs by CYP3A4 was determined and the results were used to predict the pharmacokinetics after oral administration by a PBPK population model. The differentiated AO enteroids displayed a protein profile that overlapped both qualitatively and quantitatively with that of freshly isolated jejunal enterocytes and tissue. They exhibited a morphology that recapitulates the mature villus enterocyte in vivo, formed an intact barrier with a well-developed glycocalyx and are impermeable to the hydrophilic low molecular weight compound lucifer yellow and transported a medium chain fatty acid derivative by FATP4 into lipid deposits. The clinically important ABC-transporters Pgp and BCRP were expressed at near in vivo levels, had the correct subcellular localization and effluxed their substrates. Terfenadine and midazolam were metabolized by CYP3A4 and the results were used to predict the clinical pharmacokinetics of the drugs after oral administration with good accuracy. We conclude that suspended 3D AO enteroids provide a physiologically relevant model for studies of intestinal function that offers convenient access to the apical surface and is easy to dispense in multi-wells formats for large scale experimentation.

cell biology↗

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↗

bioRxiv2023

Illuminating the Function of the Orphan Transporter, SLC22A10 in Humans and Other Primates

SLC22A10 is classified as an orphan transporter with unknown substrates and function. Here we describe the discovery of the substrate specificity and functional characteristics of SLC22A10. The human SLC22A10 tagged with green fluorescent protein was found to be absent from the plasma membrane, in contrast to the SLC22A10 orthologs found in great apes. Estradiol-17{beta}-glucuronide accumulated in cells expressing great ape SLC22A10 orthologs (over 4-fold, p<0.001). In contrast, human SLC22A10 displayed no uptake function. Sequence alignments revealed two amino acid differences including a proline at position 220 of the human SLC22A10 and a leucine at the same position of great ape orthologs. Site-directed mutagenesis yielding the human SLC22A10-P220L produced a protein with excellent plasma membrane localization and associated uptake function. Neanderthal and Denisovan genomes show human-like sequences at proline 220 position, corroborating that SLC22A10 were rendered nonfunctional during hominin evolution after the divergence from the pan lineage (chimpanzees and bonobos). These findings demonstrate that human SLC22A10 is a unitary pseudogene and was inactivated by a missense mutation that is fixed in humans, whereas orthologs in great apes transport sex steroid conjugates.

pharmacology and toxicology↗