Mycobacterium aviumsubspeciesparatuberculosistargets 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.