Mycobacterium avium subspecies paratuberculosis targets M cells in enteroid-derived monolayers through interactions with β1 integrins.

Paratuberculosis is an 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 immune cells, less is known about how it traverses the epithelium. It's currently thought that 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 developed and validated an enteroid-based in vitro assay containing functional M cells to identify the target cells for MAP's entry. Upon exposure to MAP, the bacteria were detected within both enterocytes and M cells, however quantitative image analysis revealed significant tropism for the latter. Complementary studies using the Caco-2/Raji-B co-culture system provided similar results. 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 enhanced by fibronectin and that this effect was abolished when monolayers were pretreated with an integrin blocking peptide. Our data demonstrate that MAP preferentially targets M cells and that this involves a fibronectin-bridging process. Furthermore, our study supports the utility of M cell containing enteroids to study host-pathogen interaction at the intestinal epithelium.