The selective autophagy receptors Optineurin and p62 are both required for zebrafish host resistance to mycobacterial infection

Mycobacterial pathogens are the causative agents of chronic infectious diseases like tuberculosis and leprosy. Autophagy has recently emerged as an innate mechanism for defense against these intracellular pathogens. In vitro studies have shown that mycobacteria escaping from phagosomes into the cytosol are ubiquitinated and targeted by selective autophagy receptors. However, there is currently no in vivo evidence for the role of selective autophagy receptors in defense against mycobacteria, and the importance of autophagy in control of mycobacterial diseases remains controversial. Here we have used Mycobacterium marinum (Mm), which causes a tuberculosis-like disease in zebrafish, to investigate the function of two selective autophagy receptors, Optineurin (Optn) and SQSTM1 (p62), in host defense against a mycobacterial pathogen. To visualize the autophagy response to Mm in vivo, optn and p62 zebrafish mutant lines were generated in the background of a GFP-Lc3 autophagy reporter line. We found that loss-of-function mutation of optn or p62 reduces autophagic targeting of Mm, and increases susceptibility of the zebrafish host to Mm infection. Transient knockdown studies confirmed the requirement of both selective autophagy receptors for host resistance against Mm infection. For gain-of-function analysis, we overexpressed optn or p62 by mRNA injection and found this to increase the levels of GFP-Lc3 puncta in association with Mm and to reduce the Mm infection burden. Taken together, our results demonstrate that both Optn and p62 are required for autophagic host defense against mycobacterial infection and support that protection against tuberculosis disease may be achieved by therapeutic strategies that enhance selective autophagy. Author summary Tuberculosis is a serious infectious disease that claims over a million lives annually. Vaccination provides insufficient protection and the causative bacterial pathogen, Mycobacterium tuberculosis, is becoming increasingly resistant to antibiotic therapy. Therefore, there is an urgent need for novel therapeutic strategies. Besides searches for new antibiotics, considerable efforts are made to identify drugs that improve the immune defenses of the infected host. One host defense pathway under investigation for therapeutic targeting is autophagy, a cellular housekeeping mechanism that can direct intracellular bacteria to degradation. However, evidence for the anti-mycobacterial function of autophagy is largely based on studies in cultured cells. Therefore, we set out to investigate anti-mycobacterial autophagy using zebrafish embryos, which develop hallmarks of tuberculosis following infection with Mycobacterium marinum. Using red-fluorescent mycobacteria and a green-fluorescent zebrafish autophagy reporter we could visualize the anti-mycobacterial autophagy response in a living host. We generated mutant and knockdown zebrafish for two selective autophagy receptors, Optineurin and p62, and found that these have reduced anti-bacterial autophagy and are more susceptible to infection. Moreover, we found that increased expression of these receptors enhances anti-bacterial autophagy and protects against infection. These results provide new evidence for the host-protective function of selective autophagy in mycobacterial disease.