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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.

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.

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.

Mast cells localize to mucosal tissues and contribute to innate immune defense against infection. How mast cells sense, differentiate between, and respond to bacterial pathogens remains a topic of ongoing debate. Using the prototype enteropathogen Salmonella Typhimurium (S.Tm) and other related enterobacteria, here we show that mast cells can regulate their cytokine secretion response to distinguish between extracellular and invasive bacterial infection. Tissue-invasive S.Tm and mast cells colocalize in the mouse gut during acute Salmonella infection. Toll-like Receptor 4 (TLR4) sensing of extracellular S.Tm, or pure lipopolysaccharide, causes a modest induction of 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 mast cells 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 drive the full-blown mast cell response. Supernatants from S.Tm-infected mast cells boost macrophage survival and maturation from bone-marrow progenitors. Taken together, this study shows that mast cells can differentiate between extracellular and host-cell invasive enterobacteria via a two-step activation mechanism and tune their inflammatory output accordingly.

Bacterial host cell invasion has routinely been investigated by gentamicin protection assays, which are laborsome and suffer from pronounced experimental noise. This chapter describes an internally controlled, medium- to high-throughput method that resolves the capacity of multiple Salmonella virulence factor mutant strains to bind and invade host cells. The method, widely applicable to also other pathogens, is based on the combination of consortia of genetically tagged isogenic bacterial strains and a modified gentamicin protection assay. These protocols provide a flexible tool box to stringently quantify host cell binding and invasive properties of different mutants. Moreover, the method can be applied to both infections of cultured host cells and in vivo animal models, providing a comparable genetic readout, which greatly facilitates comparisons across experimental models.

Sulfur is an essential element of life. Thiol-containing metabolites in all organisms are involved in the regulation of diverse biological processes. Especially, the microbiome produces bioactive metabolites or biological intermediates of this compound class. The analysis of thiol-containing metabolites is challenging due to the lack of specific tools, making these compounds difficult to investigate selectively. We have now developed a new methodology comprising bicyclobutane for chemoselective and irreversible capturing of this metabolite class. We utilized this new chemical biology tool immobilized onto magnetic beads for the investigation of human plasma, fecal samples, and bacterial cultures. Our mass spectrometric investigation detected a broad range of human, dietary and bacterial thiol-containing metabolites and we even captured the reactive sulfur species cysteine persulfide in both fecal and bacterial samples. The described comprehensive methodology represents a new mass spectrometric strategy for the discovery of bioactive thiol-containing metabolites in humans and the microbiome.

The behaviors of infectious bacteria are commonly studied in bulk. This is effective to define the general properties of a given isolate, but insufficient to resolve subpopulations and unique single-microbe behaviors within the bacterial pool. We here employ microscopy to study single-bacterium characteristics among Salmonella enterica serovar Typhimurium (S.Tm), as they prepare for and launch invasion of epithelial host cells. We find that during the bacterial growth cycle, S.Tm populations switch gradually from fast planktonic growth to a host cell-invasive phenotype, characterized by flagellar motility and expression of the Type-three-secretion-system-1. The indistinct nature of this shift leads to the establishment of a transient subpopulation of S.Tm "doublets"-waist-bearing bacteria anticipating cell division-which simultaneously express host cell invasion machinery. In epithelial cell culture infections, these S.Tm doublets outperform their "singlet" brethren and represent a hyperinvasive subpopulation. Atop both glass and enteroid-derived monolayers, doublets swim along markedly straighter trajectories than singlets, thereby diversifying search patterns and improving the surface exploration capacity of the total bacterial population. The straighter swimming, combined with an enhanced cell-adhesion propensity, suffices to account for the hyperinvasive doublet phenotype. This work highlights bacterial cell length heterogeneity as a key determinant of target search patterns atop epithelia.

The ratio of the two virulence-regulatory proteins VirF(21):VirF(30) increases at low temperature and can dictate the invasive properties of Shigella flexneri. Shigella spp, the etiological agents of bacillary dysentery in humans, have evolved an intricate regulatory strategy to ensure fine-tuned expression of virulence genes in response to environmental stimuli. A key component in this regulation is VirF, an AraC-like transcription factor, which at the host temperature (37 degrees C) triggers, directly or indirectly, the expression of > 30 virulence genes important for invasion of the intestinal epithelium. Previous work identified two different forms of VirF with distinct functions: VirF(30) activates virulence gene expression, while VirF(21) appears to negatively regulate virF itself. Moreover, VirF(21) originates from either differential translation of the virF mRNA or from a shorter leaderless mRNA (llmRNA). Here we report that both expression of the virF(21) llmRNA and the VirF(21):VirF(30) protein ratio are higher at 30 degrees C than at 37 degrees C, suggesting a possible involvement of VirF(21) in minimizing virulence gene expression outside the host (30 degrees C). Ectopic elevation of VirF(21) levels at 37 degrees C indeed suppresses Shigella ' s ability to infect epithelial cells. Finally, we find that the VirF(21) C-terminal portion, predicted to contain a Helix-Turn-Helix motif (HTH2), is required for the functionality of this negative virulence regulator.

The gut epithelium serves to maximize the surface for nutrient and fluid uptake, but at the same time must provide a tight barrier to pathogens and remove damaged intestinal epithelial cells (IECs) without jeopardizing barrier integrity. How the epithelium coordinates these tasks remains a question of significant interest. We used imaging and an optical flow analysis pipeline to study the dynamicity of untransformed murine and human intestinal epithelia, cultured atop flexible hydrogel supports. Infection with the pathogen Salmonella Typhimurium (S.Tm) within minutes elicited focal contractions with inward movements of up to similar to 1,000 IECs. Genetics approaches and chimeric epithelial monolayers revealed contractions to be triggered by the NAIP/NLRC4 inflammasome, which sensed type-III secretion system and flagellar ligands upon bacterial invasion, converting the local tissue into a contraction epicenter. Execution of the response required swift sublytic Gasdermin D pore formation, ion fluxes, and the propagation of a myosin contraction pulse across the tissue. Importantly, focal contractions preceded, and could be uncoupled from, the death and expulsion of infected IECs. In both two-dimensional monolayers and three-dimensional enteroids, multiple infection-elicited contractions coalesced to produce shrinkage of the epithelium as a whole. Monolayers deficient for Caspase-1(-11) or Gasdermin D failed to elicit focal contractions but were still capable of infected IEC death and expulsion. Strikingly, these monolayers lost their integrity to a markedly higher extent than wild-type counterparts. We propose that prompt NAIP/NLRC4/Caspase-1/Gasdermin D/myosin-dependent contractions allow the epithelium to densify its cell packing in infected regions, thereby preventing tissue disintegration due to the subsequent IEC death and expulsion process.