Human Intestinal Organoids as a Model to Study Intestinal Infection by the Foodborne Pathogen Salmonella enterica

Abstract

Salmonella enterica is a prominent human pathogen causing over 100 million infections every year. Serovars of S. enterica are very closely related, but due to differences in infection progression in animal models, host responses that drive human infection outcomes are not well understood. Nontyphoidal serovars, like S. enterica serovar Typhimurium (STM) and Enteritidis (SE), cause an inflammatory infection in the gut and recruit neutrophils to clear the infection. In contrast, typhoidal serovars, including serovar Typhi (ST), infect via the same route yet clinically go largely undetected prior to spreading systemically to cause severe disease. Both groups utilize Type-three secretion systems (T3SSs) to infect. T3SS-1 secretes proteins to promote invasion into non-phagocytic cells. T3SS-2 in contrast, is active once bacteria are intracellular and modulate host cell dynamics to create a niche that is favorable for bacterial replication. In this dissertation, I summarize work using a human intestinal organoid (HIO) model, a 3-dimensional tissue culture model consisting of primary human intestinal epithelial cells and mesenchyme, to study host responses to three major serovars relevant to human health, STM, SE, and ST. First, we used a transcriptomics approach to define which epithelial responses to infection were dependent on either T3SS-1 or T3SS-2. Surprisingly, we found that T3SS-1 mutant (T3SS-1mut) bacteria, previously reported not to induce a robust inflammatory response in cell lines, elicited a similar response to WT bacteria when cultured in the luminal space of the HIO. Additionally, we found that WT STM suppressed cell cycle progression, which was dependent on T3SS-1 and T3SS-2. Next, we infected HIOs with STM, SE, and ST and measured serovar-specific HIO transcriptional responses and evaluated differences in bacterial invasion, replication and host cellular functions. We found that HIOs responded uniquely to all three serovars including distinct responses between the two non-typhoidal serovars. Each serovar varied in its ability to invade and replicate within HIOs and to trigger HIO stress pathways, leading to different outcomes in host cell death, replication, and reactive oxygen species formation. Although the HIOs allow us to study human intestinal epithelial responses to Salmonella, there are critical host components absent from HIOs that influence infection outcome. Neutrophils are dominant early responders to non-typhoidal serovar infections, which are characterized by gastroenteritis. To investigate the contribution of neutrophils in host defense against non-typhoidal serovars, we co-cultured neutrophils with HIOs and characterized the infection. We found that instead of killing bacteria, neutrophils enhanced epithelial intrinsic defenses. This included increased production of pro-inflammatory markers and enhanced extrusion of epithelial cells into the HIO lumen. Enhanced cell shedding decreased intracellular bacterial burden in the epithelial monolayer, suggesting that neutrophils can protect against Salmonella infection by promoting expulsion of infected cells from the intestine. Overall, this dissertation provides insights into human epithelial responses specific to individual Salmonella enterica serovars and emphasizes intriguing differences between closely related serovars that deserve further study. We also found a new and unexpected role of luminal bacteria in inducing host inflammatory responses, an aspect of intestinal infection biology that has been challenging to study using standard cell culture models. Lastly, by co-culturing HIOs with neutrophils we revealed an underappreciated contribution of innate immune cells in controlling epithelial defense mechanisms during infection by Salmonella.