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The free-living amoeba Acanthamoeba castellanii is an ecologically, clinically, and evolutionarily important microorganism. A. castellanii amoebae can be directly pathogenic to humans and serve as reservoirs for bacterial pathogens (e.g., Legionella pneumophila), but they also regulate the proliferation of other microorganisms in the soil. Despite their importance, no reliable genetic system has been developed, hampering the use of A. castellanii and related species as model organisms. Transfecting A. castellanii with plasmids is possible with commercial kits, but it is expensive, inefficient, and vulnerable to product discontinuation. In this contribution, we present a method for efficient transfection of A. castellanii with readily available and inexpensive polyethylenimines. We systematically explore the method’s parameters, achieving up to 100-fold higher efficiency compared to currently used protocols. This method provides a robust step toward a complete genetic toolbox for A. castellanii, thereby expanding its utility as a model organism.

Porous membranes are frequently used as supports of cell monolayers in functional studies of epithelial and endothelial barriers. However, commonly used conventional polymer-based membranes such as those made of polycarbonate (transwells) do not mimic the structural and biochemical properties of native basement membranes, which may limit cellular differentiation and function. Here, we use a nanofibrillar membrane that mimics the microenvironment of the basement membrane and is made of recombinant spider silk functionalized with the integrin-binding RGD motif of fibronectin and coated with human kidney-specific laminin-521 (FN-silk). The FN-silk membranes were evaluated as culture substrates for renal epithelial cells (RPTEC/TERT1), a cell type notoriously difficult to differentiate in culture. FN-silk membrane structure, cellular morphology, mRNA expression, barrier properties and transporter activity were assessed using scanning and transmission electron microscopy, RNA-sequencing, permeability and transport assays. Both FN-silk and conventional membranes supported barrier integrity and tight junction expression. In contrast to cultures on conventional membranes, the RPTEC monolayers on FN-silk exhibited a differentiated morphology, low expression of cell death markers, and, directional anion and cation transport. Further, the commonly used conventional membranes released endocrine-disrupting bisphenols that activated estrogen-mediated signaling. In summary, these findings indicate that FN-silk membranes with kidney-specific basement membrane laminin 521 reduces cellular stress, supports and maintains cellular differentiation and preserves important cellular functions. Our work establishes FN-silk membranes as a next-generation biomaterial that enables the differentiation of renal epithelial monolayers into a physiologically relevant in vitro model.

Studying the pathogenesis of human-adapted microorganisms is challenging, since small animal models often fail to recapitulate human physiology. Hence, the comprehensive genetic and regulatory circuits driving the infection process of principal human pathogens such as Shigella flexneri remain to be defined. We combined large-scale Shigella infections of enteroids and colonoids with transposon-directed insertion sequencing and Bayesian statistical modeling to address infection bottlenecks, thereby establishing the comprehensive genome-wide map of Shigella genes required to infect human intestinal epithelium. This revealed the Shigella virulence effectors essential for epithelial cell colonization across geometries and intestinal segments, identified over 100 chromosomal genes involved in the process and uncovered a post-transcriptional mechanism whereby tRNA-modification enzymes and differential codon usage exert global control of a bacterial virulence program. Our findings provide a broadly applicable framework for combining advanced organotypic tissue culture with functional genomics and computational tools to map human–microorganism interactions at scale.

Despite their relatedness, Salmonella and Shigella enteropathogens 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). However, they have evolved 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-1, Salmonella accomplishes appreciable numbers of apical IEC invasion events. These are promptly counteracted by IEC death, thus fostering a polyclonal iterative epithelial colonization strategy. The lack of a corresponding apical targeting module in Shigella makes this pathogen reliant on external factors, for example, preexisting damage, for rare apical access to the intraepithelial environment. However, Shigella compensates for this ineptness by an intraepithelial expansion module reliant on the tight coupling of OspC3-dependent temporal delay of cell death and IcsA-mediated mobility. This module enables intraepithelial Shigella to evade the IEC death response just long enough to expand laterally, thus fostering an essentially monoclonal colonization strategy. Taken together, the study reveals how a small set of virulence factors shapes divergent epithelial colonization strategies by related enterobacteria.

Mast cells (MCs) lodge within barrier tissues and respond to infectious microbes. Recent work demonstrated that MCs differentiate their cytokine response to extracellular versus invasive Gram-negative enterobacteria by a two-step activation mechanism that integrates Toll-like-receptor (TLR) sensing with signals elicited by type-III-secretion-system (TTSS) effectors during bacterial invasion. However, multiple MC subtypes exist, and it remains unclear how their phenotypic heterogeneity impacts microbial interactions. We find that murine MCs maintained in IL-3, or differentiated toward a connective-tissue phenotype (CT-MCs), respond potently to the enteropathogen Salmonella enterica Typhimurium (S.Tm) through two-step activation, with the TLR component explained by functional TLR4 and TLR2. By contrast, murine mucosal mast cells (M-MCs) express insignificant levels of these TLRs, therefore being unresponsive to extracellular S.Tm, but still mounting a response to invasive bacteria. Following invasion, MC granule maintenance by serglycin restricts S.Tm vacuolar and cytosolic colonization. Notably, this has no impact on the cytokine release from infected MCs, thus uncoupling S.Tm<acute accent>s intracellular life-cycle from the MC cytokine response. Finally, human LUVA MCs employ a variant of two-step activation where TLR2/6 signaling combines with the TTSS-elicited signals. Together, this study explains how MC subtypes can respond differently to S.Tm-infection depending on their TLR expression and granule features.

Matrix-M^® adjuvant, containing saponins, delivers a potent adjuvant effect and good safety profile. Given that Matrix-M is composed of Matrix-A and Matrix-C particles, comprising different saponin fractions, understanding their distinct roles can provide deeper insight into the mechanism of action of Matrix-M and guide future applications. Here, we demonstrate that the antigen and Matrix-M, Matrix-A, or Matrix-C colocalize in lysosomes following uptake by bone marrow–derived dendritic cells. Matrix-M, Matrix-A, and Matrix-C induce lysosomal membrane permeabilization (LMP), but Matrix-C shows the highest LMP potential. LMP is required for interleukin (IL)-1β and IL-18 secretion in vitro. In vivo, a robust adjuvant effect of Matrix-M, Matrix-A, and Matrix-C is observed, both in the presence and absence of the NLRP3 inflammasome. LMP induced by Matrix-M, as well as Matrix-A and Matrix-C, also enables antigen cross-presentation. Thus, Matrix-induced LMP explains the capability of Matrix-M–adjuvanted protein vaccines to induce CD8⁺ T-cell responses.

Pathogen invasion of intestinal epithelial cells (IECs) is a key gut infection event. The dynamics of this process in intact epithelia and the factors governing IEC susceptibility remain underexplored. We present a resource for live-cell infection imaging in human enteroid-/colonoid-derived IEC layers across maturation states and in the presence or absence of soluble mucus production. Utilization of this resource shows how human IEC maturation fortifies the apical surface against invasion by Salmonella Typhimurium (S.Tm). Immature IEC layers appear permissive to S.Tm invasion, but maturation toward an enterocyte/colonocyte phenotype reduces the susceptibility by up to 10-fold. This shift couples to downregulated expression of actin regulatory proteins exploited by the pathogen, an increased dependence on the S.Tm effector SipA, and the build-up of the apical IEC glycocalyx linked to surface-attached mucins like MUC13 and nullifiable by StcE-enzyme treatment. This underscores how the maturation state of human IECs governs susceptibility to bacterial invasion.

Inflammasomes are central components of mammalian innate immunity activated upon the detection of danger signals such as pathogen ligands. This activation generally leads to pyroptosis and interleukin release, but specific responses vary between cell types. Here, we study nucleotide-binding, leucine-rich repeat (NLR) family apoptosis inhibitory protein (NAIP)/NLR CARD-containing protein 4 (NLRC4) inflammasome dynamics of intestinal epithelial cells of murine enteroids at the single-cell level. Using an apoptosis-associated speck-like protein containing a CARD (ASC)::GFP reporter and a cell-membrane-permeable NAIP/NLRC4 agonist, we observe pyroptosis with or without ASC polymerization. We use fluidic force microscopy to inject NAIP ligands into cells, revealing a dose-dependent response. High doses promote ASC polymerization, while lower doses lead to survival or NLRC4-dependent cell death in the absence of ASC polymerization. Using a fluorescence resonance energy transfer (FRET) reporter as a proxy for interleukin maturation, we reveal that single epithelial cell caspase-1 dynamics are affected by the differentiation state of cells and NAIP ligand dosing. Our results indicate an inflammasome response that is tuned at multiple levels in intestinal epithelial cells.

Development of oral drug delivery systems that penetrate the colonic mucus remains challenging. Artificial models of porcine colonic mucus have been developed that mimic the rheology and viscosity of the native mucus and its contents of mucins, protein, and lipids. However, they are less representative with regard to the zeta potential, a factor of importance for charged molecules and particles. This study therefore aimed to improve the existing porcine artificial colonic mucus model by exchanging the polymer backbone (used for viscosity) to more closely mimic the charge of porcine native colonic mucus. Polymers studied were poly(acrylic acid), hydroxyethylcellulose, sodium hyaluronate, sodium alginate, and pectin. The resulting porcine artificial colonic mucus was assayed for apparent viscosity, storage modulus, pH, water content, zeta potential, and pore size. The two best-performing polymers (poly(acrylic acid) and hydroxyethylcellulose) were then assayed with diffusion of FITC-dextran, particle tracking of nanoparticles, and binding of FITC-dextran and contrasted to data generated in porcine native colonic mucus (PNCM). Of the two polymers, PACM based on HEC generated zeta potential and binding kinetics similar to those of PNCM. We conclude that the choice of polymer in PACMs is critical for improving their use in drug development. The extensive characterization of the PACMs further points toward the importance of complementary techniques to determine rheological characteristics, mesh, and pore size.

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°C, while it 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 upholds pINV replication to a level favourable 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 adequate pINV level, sustained by pcnB, underpins the successful execution of Shigella´s infection cycle.