Targeting of autophagy by Yersinia enterocolitica in epithelial cells

Abstract

Autophagy is a cellular pathway that delivers cytoplasmic proteins, lipids and organelles to lysosomes for degradation. It occurs at basal levels to maintain cellular homeostasis and is upregulated in response to various stress conditions. Moreover, autophagy has also emerged as an innate immune response that selectively targets intracellular bacteria in the cytosol or within damaged vacuoles in a selective process, called xenophagy, in order to restrict bacterial growth. Importantly, some bacteria have evolved strategies to combat autophagy or to exploit the autophagy machinery to promote intracellular growth. Until now, little is known about the role of autophagy in Y. enterocolitica infection of epithelial cells. This work provides evidence that, after invading epithelial cells, autophagic processes are triggered which may influence the lifestyle of Y. enterocolitica. We have shown that Yersinia-induced autophagy is mediated by the Yersinia surface protein invasin, which requires viable bacteria. Once within the epithelial cells, a population of Y. enterocolitica resides within autophagosome-like vacuoles that display markers of autophagosomes (LC3) and late endosomes (LAMP-1 but no v-ATPase or cathepsin D). Notably, the ability of these vacuoles to fuse with lysosomes and the concomitant acidification are blocked. Transmission electron microscopy studies revealed that these vacuoles consist in their ultrastructure mainly of double or multiple membranes around the bacteria, characteristic of xenophagy. Furthermore, manipulation of the autophagic responses by using a knockout embryonic mouse fibroblast line deficient in autophagy (Atg5-/- MEFs) suggested that the bacteria may employ the autophagy machinery to create a niche that could support intracellular bacterial survival and replication. This may eventually assist spread of the bacteria from the infected cells. It appears that the Yersinia-containing vacuoles (YCVs) are targeted by ubiquitin, and the autophagy receptors p62 and NDP52 to promote autophagy. Finally, using wild-type mice and mice deficient in intestinal epithelial cell autophagy, pilot experiments were performed to investigate whether autophagy is hijacked in vivo by Y. enterocolitica to promote the dissemination of the bacteria from the intestinal mucosa to deeper tissues. However, in these experiments we did not observe a significant difference in the dissemination of the bacteria in autophagy deficient versus competent mice, which suggests a more complex picture of epithelial autophagy in the pathogenesis of intestinal yersiniosis. In total, our study shows that Y. enterocolitica may manipulate the normal autophagy defense machinery of epithelial cells to promote survival, replication and spread of the bacteria from the infected cells. Whether this is beneficial for the pathogen in vivo remains to be specified.