Biology subjects

Di Martino, M. L.

Publications and source records attributed to Di Martino, M. L..

7 recordsLinked to original sources

bioRxiv2026

Apical-Out Ileal Organoids: A Versatile Platform for Probing Gut-Nanomaterial Interactions

The human ileal epithelium forms a critical interface governing interactions with luminal microorganisms and nanomaterials, yet physiologically relevant in vitro models with accessible apical surfaces remain limited. Here, human apical-out ileoids derived from healthy ileal biopsies are established as a platform that recapitulates the architecture and complexity of the native ileal epithelium. Assessment of donor- and passage-dependent variability identifies a representative ileoid line and a robust culture window. Integrated proteomic, imaging, ultrastructural, and functional analyses demonstrate preservation of key epithelial features, including brush-border organization, barrier integrity, absorptive capacity, and multiple endocytic pathways. Intact ileoids internalize nanoparticles of distinct surface charge, including empty lipid nanoparticles compositionally matched to approved mRNA vaccine formulations, establishing that uptake can be quantified across an unperturbed apical surface. This uptake is restrained by a retained mucin-rich glycocalyx. Cleavage of surface-attached mucins selectively enhances uptake of negatively charged nanoparticles, leaving near-neutral and positively charged nanoparticles unchanged, and increasing entry of Salmonella bacteria. These findings reveal the glycocalyx as a charge-selective regulator of epithelial uptake. In conclusion, our study provides a foundation for nanoparticle-epithelium investigations using human apical-out ileoids, and a versatile, translationally relevant platform for nanomedicine delivery, host-microbe interactions, and epithelial transport.

cell biology↗

bioRxiv2026

Dual Screen for Gut Metabolites Suppressing Enterobacterial Growth and Invasiveness Reveals Structure - Activity Relationships among Anti-Infective Indoles

The antibiotic resistance crisis makes characterization of new anti-infective molecules a pressing matter. Molecules that suppress bacterial growth and survival, virulence, or the combination of these traits, all warrant further exploration. Naturally occurring microbe-host ecosystems, such as the human gut, provide incompletely tapped resources in this regard. We developed a flexible platform to parallelly assess how gut metabolites affect the growth and epithelial cell invasion capacity of the enteropathogens Salmonella enterica Typhimurium (Salmonella) and Shigella flexneri. By screening a gut metabolite library, the assays identified multiple anti-infective compound classes and extended previously reported antibacterial activities for e.g. medium chain fatty acids, bile acids, purine nucleotides, and indole. Importantly, a targeted follow-up screen combined with chemical biology iterations showed how the anti-infective activity of indole is impacted by its derivatization. Specifically, a methyl group at either of the carbons of the indole scaffold potentiated the suppressive effect on type-III-secretion-system-mediated virulence, flagellar motility (for Salmonella), and growth, in a concentration-dependent manner. By contrast, N1-methylation markedly attenuated the activity of indole and its C-derivatized versions. The study, hence, offers assays for dual growth and virulence analysis of invasive enterobacteria exposed to anti-infective candidate molecules, and informs on structure-activity relationships among indole metabolites.

microbiology↗

bioRxiv2025

The pcnB gene sustains Shigella flexneri virulence

The enteropathogen Shigella flexneri employs a Type Three Secretion System (T3SS) to colonize intestinal epithelial cells. Genes encoding the T3SS are located on a large IncFII virulence plasmid, pINV. T3SS expression comes at the expense of slowed Shigella growth and is therefore strictly controlled by both transcriptional and post-transcriptional mechanisms. Following up on a recent genome-wide screen, we here show that the chromosomal gene pcnB, encoding the poly-A polymerase I (PAP-I), slows Shigella growth at 37{degrees}C, while at the same time promotes early colonization of a human epithelial enteroid model. Proteomic profiling revealed that pcnB drives global increase of the Shigella T3SS virulence program. Accordingly, pcnB sustains pINV replication to a level optimal for Shigella virulence. This is achieved through increased degradation of the antisense RNA CopA, involved in plasmid replication control. The pcnB effect on pINV replication was found to also ensure longer-term intraepithelial expansion of Shigella following human intestinal epithelium invasion. Our findings exemplify how an optimal pINV level is necessary for the execution of Shigellas infection cycle. AUTHOR SUMMARYBacterial infections represent a major global threat. Understanding the genetic determinants promoting infections is crucial to overcome this threat. Shigella is an intracellular bacterial pathogen that invades and disseminates in the intestinal epithelium, causing bacillary dysentery in humans. Shigellas ability to cause disease relies on the delivery of effector proteins into the host cells through an injection machinery, with most of the genes involved in this process located on a large virulence plasmid. Here we show that the chromosomal gene pcnB sustains an optimal virulence plasmid level. This is crucial for Shigella to maximize virulence protein expression and thereby efficiently invade, replicate and spread within the intestinal epithelium.

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↗

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↗

bioRxiv2023

A Two-Step Activation Mechanism Enables Mast Cells to Differentiate their Response between Extracellular and Invasive Enterobacterial Infection

Mast cells (MCs) localize to mucosal tissues and contribute to innate immune defenses against infection. How MCs sense, differentiate between, and respond to bacterial pathogens remains a topic of ongoing debate. Using the prototype enteropathogen Salmonella Typhimurium (S.Tm) and other closely related enterobacteria, we here demonstrate that MCs can regulate their cytokine secretion response to distinguish between extracellular and invasive bacterial infection. Tissue-invasive S.Tm and MCs colocalize in the Salmonella-infected mouse gut. Toll-like Receptor 4 (TLR4) sensing of extracellular S.Tm, or pure LPS, causes a slow and modest induction of MC cytokine transcripts and proteins, including IL-6, IL-13, and TNF. By contrast, type-III-secretion-system-1 (TTSS-1)-dependent S.Tm invasion of both mouse and human MCs triggers rapid and potent inflammatory gene expression and >100-fold elevated cytokine secretion. The S.Tm TTSS-1 effectors SopB, SopE, and SopE2 here elicit a second activation signal, including Akt phosphorylation downstream of effector translocation, which combines with TLR activation to promote the full-blown MC response. Supernatants from S.Tm-infected MCs boost macrophage survival and maturation from bone-marrow progenitors. Taken together, this study shows that MCs can differentiate between extracellular and host-cell invasive enterobacteria via a two-step activation mechanism and tune their inflammatory output accordingly.

immunology↗