Biology subjects

Webb, D.-L.

Publications and source records attributed to Webb, D.-L..

4 recordsLinked to original sources

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↗

bioRxiv2024

Determinants of the Divergent Salmonella and Shigella Epithelial Colonization Strategies Resolved in Human Enteroids and Colonoids

Despite close relatedness, the major enteropathogens Salmonella and Shigella differ in infectious dose, pathogenesis, and disease kinetics. The prototype strains Salmonella enterica serovar Typhimurium (Salmonella) and Shigella flexneri (Shigella) use Type-3-secretion-systems (T3SSs) to colonize intestinal epithelial cells (IECs), but have evolved partially unique sets of T3SS effectors and accessory virulence factors. A synthesis of how these differences impact the temporal progression of infection in non-transformed human epithelia is missing. Here, we followed Salmonella and Shigella infections of human enteroids and colonoids by time-lapse imaging to pinpoint virulence factor modules that shape the divergent epithelial colonization strategies. By an apical targeting module that integrates flagella and the SPI-4-encoded adhesin system with T3SS, Salmonella accomplishes appreciable numbers of apical invasion events, promptly terminated by IEC death, and thus fostering a polyclonal iterative epithelial colonization strategy. The lack of a corresponding module in Shigella makes this pathogen reliant on external factors such as preexisting damage for rare apical access to the intraepithelial environment. However, Shigella compensates for this ineptness by an intraepithelial expansion module, where tight coupling of OspC3-dependent temporal delay of cell death and IcsA-mediated lateral spread enables intraepithelial Shigella to outrun the IEC death response, fostering an essentially monoclonal colonization strategy.

microbiology↗

bioRxiv2021

High Definition DIC Imaging Uncovers Transient Stages of Pathogen Infection Cycles on the Surface of Human Adult Stem Cell-Derived Intestinal Epithelium

Interactions between individual pathogenic microbes and host tissues involve fast and dynamic processes that ultimately impact the outcome of infection. Using live-cell microscopy, these dynamics can be visualized to study e.g. microbe motility, binding and invasion of host cells, and intra-host-cell survival. Such methodology typically employs confocal imaging of fluorescent tags in tumor-derived cell line infections on glass. This allows high-definition imaging, but poorly reflects the host tissues physiological architecture and may result in artifacts. We developed a method for live-cell imaging of microbial infection dynamics on human adult stem cell-derived intestinal epithelial cell (IEC) layers. These IEC monolayers are grown in alumina membrane chambers, optimized for physiological cell arrangement and fast, but gentle, differential interference contrast (DIC) imaging. This allows sub-second visualization of both microbial and epithelial surface ultrastructure at high resolution without using fluorescent reporters. We employed this technology to probe the behavior of two model pathogens, Salmonella enterica Typhimurium (Salmonella) and Giardia intestinalis (Giardia), at the intestinal epithelial surface. Our results reveal pathogen-specific swimming patterns on the epithelium, showing that Salmonella adheres to the IEC surface for prolonged periods before host-cell invasion, while Giardia uses circular swimming with intermittent attachments to scout for stable adhesion sites. This method even permits tracking of individual Giardia flagella, demonstrating that active flagellar beating and attachment to the IEC surface are not mutually exclusive. Thereby, this work describes a powerful, generalizable, and relatively inexpensive approach to study dynamic pathogen interactions with IEC surfaces at high resolution and under near-native conditions. ImportanceKnowledge of dynamic niche-specific interactions between single microbes and host cells is essential to understand infectious disease progression. However, advances in this field have been hampered by the inherent conflict between the technical requirements for high resolution live-cell imaging on one hand, and conditions that best mimic physiological infection niche parameters on the other. Towards bridging this divide, we present methodology for differential interference contrast (DIC) imaging of pathogen interactions at the apical surface of enteroid-derived intestinal epithelia, providing both high spatial and temporal resolution. This alleviates the need for fluorescent reporters in live-cell imaging and provides dynamic information about microbe interactions with a non-transformed, confluent, polarized and microvilliated human gut epithelium. Using this methodology, we uncover previously unrecognized stages of Salmonella and Giardia infection cycles at the epithelial surface.

microbiology↗