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ABSTRACT 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 intrahost-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 tissue’s 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 layers are grown in apical imaging chambers, optimized for physiological cell arrangement and fast, but gentle, differential interference contrast (DIC) imaging. This allows subsecond 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 serovar Typhimurium and Giardia intestinalis, at the intestinal epithelial surface. Our results reveal pathogen-specific swimming patterns on the epithelium and show that Salmonella lingers on the IEC surface for prolonged periods before host cell invasion, while Giardia uses circular swimming with intermittent attachments to scout for stable adhesion sites. The method even permits tracking of individual Giardia flagella, demonstrating that active flagellar beating and attachment to the IEC surface are not mutually exclusive. This work describes a generalizable and relatively inexpensive approach to resolving dynamic pathogen-IEC layer interactions, applicable even to genetically nontractable microorganisms. IMPORTANCE Knowledge 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 the one hand and conditions that best mimic physiological infection niche parameters on the other. Toward bridging this divide, we present a 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 nontransformed, 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.

Pathogenic gut bacteria are common causes of intestinal disease. Enteroids—cultured three-dimensional replicas of the mammalian gut—offer an emerging model system to study disease mechanisms under conditions that recapitulate key features of the intestinal tract.

ABSTRACT Bacterial host cell invasion mechanisms depend on the bacterium’s virulence factors and the properties of the target cell. The enteropathogen Salmonella enterica serovar Typhimurium (S.Tm) invades epithelial cell types in the gut mucosa and a variety of immune cell types at later infection stages. The molecular mechanism(s) of host cell entry has, however, been studied predominantly in epithelial cell lines. S.Tm uses a type three secretion system (TTSS-1) to translocate effectors into the host cell cytosol, thereby sparking actin ruffle-dependent entry. The ruffles also fuel cooperative invasion by bystander bacteria. In addition, several TTSS-1-independent entry mechanisms exist, involving alternative S.Tm virulence factors, or the passive uptake of bacteria by phagocytosis. However, it remains ill-defined how S.Tm invasion mechanisms vary between host cells. Here, we developed an internally controlled and scalable method to map S.Tm invasion mechanisms across host cell types and conditions. The method relies on host cell infections with consortia of chromosomally tagged wild-type and mutant S.Tm strains, where the abundance of each strain can be quantified by qPCR or amplicon sequencing. Using this methodology, we quantified cooccurring TTSS-1-dependent, cooperative, and TTSS-1-independent invasion events in epithelial, monocyte, and macrophage cells. We found S.Tm invasion of epithelial cells and monocytes to proceed by a similar MOI-dependent mix of TTSS-1-dependent and cooperative mechanisms. TTSS-1-independent entry was more frequent in macrophages. Still, TTSS-1-dependent invasion dominated during the first minutes of interaction also with this cell type. Finally, the combined action of the SopB/SopE/SopE2 effectors was sufficient to explain TTSS-1-dependent invasion across both epithelial and phagocytic cells. IMPORTANCE Salmonella enterica serovar Typhimurium (S.Tm) is a widespread and broad-host-spectrum enteropathogen with the capacity to invade diverse cell types. Still, the molecular basis for the host cell invasion process has largely been inferred from studies of a few selected cell lines. Our work resolves the mechanisms that Salmonellae employ to invade prototypical host cell types, i.e., human epithelial, monocyte, and macrophage cells, at a previously unattainable level of temporal and quantitative precision. This highlights efficient bacterium-driven entry into innate immune cells and uncovers a type III secretion system effector module that dominates active bacterial invasion of not only epithelial cells but also monocytes and macrophages. The results are derived from a generalizable method, where we combine barcoding of the bacterial chromosome with mixed consortium infections of cultured host cells. The application of this methodology across bacterial species and infection models will provide a scalable means to address host-pathogen interactions in diverse contexts.

ABSTRACT FinO domain proteins such as ProQ of the model pathogen Salmonella enterica have emerged as a new class of major RNA-binding proteins in bacteria. ProQ has been shown to target hundreds of transcripts, including mRNAs from many virulence regions, but its role, if any, in bacterial pathogenesis has not been studied. Here, using a Dual RNA-seq approach to profile ProQ-dependent gene expression changes as Salmonella infects human cells, we reveal dysregulation of bacterial motility, chemotaxis, and virulence genes which is accompanied by altered MAPK (mitogen-activated protein kinase) signaling in the host. Comparison with the other major RNA chaperone in Salmonella, Hfq, reinforces the notion that these two global RNA-binding proteins work in parallel to ensure full virulence. Of newly discovered infection-associated ProQ-bound small noncoding RNAs (sRNAs), we show that the 3′UTR-derived sRNA STnc540 is capable of repressing an infection-induced magnesium transporter mRNA in a ProQ-dependent manner. Together, this comprehensive study uncovers the relevance of ProQ for Salmonella pathogenesis and highlights the importance of RNA-binding proteins in regulating bacterial virulence programs. IMPORTANCE The protein ProQ has recently been discovered as the centerpiece of a previously overlooked “third domain” of small RNA-mediated control of gene expression in bacteria. As in vitro work continues to reveal molecular mechanisms, it is also important to understand how ProQ affects the life cycle of bacterial pathogens as these pathogens infect eukaryotic cells. Here, we have determined how ProQ shapes Salmonella virulence and how the activities of this RNA-binding protein compare with those of Hfq, another central protein in RNA-based gene regulation in this and other bacteria. To this end, we apply global transcriptomics of pathogen and host cells during infection. In doing so, we reveal ProQ-dependent transcript changes in key virulence and host immune pathways. Moreover, we differentiate the roles of ProQ from those of Hfq during infection, for both coding and noncoding transcripts, and provide an important resource for those interested in ProQ-dependent small RNAs in enteric bacteria.