<i>Mycobacterium avium</i> subspecies <i>paratuberculosis</i> targets M cells in enteroid-derived monolayers through interactions with β1 integrins

Abstract Paratuberculosis is a global infectious disease caused by the bacterium, Mycobacterium avium subspecies paratuberculosis (MAP). MAP infection of ruminants triggers progressive wasting disease characterized by granulomatous lymphadenitis, enteritis, and severe intestinal pathology that often requires early culling of the animal. The resulting economic burden is significant and MAP exposure in the workplace constitutes a significant zoonotic risk. While it has been established the MAP propagates within resident intestinal immune cells, including macrophages and dendritic cells, significantly less is known about how it attaches, enters and traverses the epithelium. The current paradigm suggests MAP infects the small intestinal epithelium by targeting both enterocytes and M cells, with a potential tropism for the latter. In the current study, we employed emerging enteroid technology to identify the target cells for MAP’s entry into the small intestinal epithelium. We generated mouse enteroid-derived monolayers with functional M cells capable of transcytosis. Upon exposure to MAP, the bacteria were detected within both enterocytes and M cells. Following quantification, it was apparent that MAP exhibited tropism for M cells. Complementary studies using the Caco-2/Raji-B co-culture system provided similar results, wherein MAP was found primarily in cells expressing functional M cell markers. Since other mycobacteria have been shown to initiate cell attachment and entry by using a fibronectin-bridging process, we tested whether these interactions were involved in MAP’s targeting of M cells. We found that MAP’s M cell tropism was significantly enhanced in the presence of fibronectin and that this effect was abolished when monolayers were pretreated with an integrin-blocking peptide. Taken together, our data indicate the MAP preferentially targets M cells and that this process involves a fibronectin-bridging process. Furthermore, our data suggest that targeting M cell-associated integrins could provide a mechanism to reduce MAP infection and transmission within livestock herds. Author Summary In the current study, we sought to determine the target cell for Mycobacterium avium subspecies paratuberculosis (MAP), which is the causative agent of Johne’s disease (JD, also termed paratuberculosis) in ruminants. While MAP primarily infects domestic ruminants including cattle, sheep, goats, and deer, it has also been shown to infect wildlife throughout the world, including cats, rabbits, badgers, and wood mice. Given the significant economic burden of MAP infections in livestock, its role in the pathogenesis of JD has been the focus of much research. However, the broad diversity of MAP-susceptible hosts and reservoirs observed calls into question the true scope of MAP infection and transmission and the true number of susceptible hosts. Furthermore, MAP constitutes a zoonotic threat that some have linked to intestinal pathologies, including Crohn’s disease. To date, it is still not known exactly how MAP attaches, enters and traverses the small intestinal epithelium to eventually propagate within resident macrophages and dendritic cells to cause eventual disease. To address this question, we developed a model of the small intestinal epithelium, from mouse enteroids, that contained functional M cells. We found that MAP selectivity enters M cells and that this involves fibronectin-bridging process that targets M cell-associated β1-integrins.

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