Legionella longbeachae effector protein RavZ inhibits autophagy and regulates phagosome ubiquitination during infection

Abstract

Legionella organisms are ubiquitous environmental bacteria that are responsible for human Legionnaires’ disease, a fatal form of severe pneumonia. These bacteria replicate intracellularly in a wide spectrum of host cells within a distinct compartment termed the Legionella-containing vacuole (LCV). Effector proteins translocated by the Dot/Icm apparatus extensively modulate host cellular functions to aid in the biogenesis of the LCV and intracellular proliferation. RavZ is an L. pneumophila effector that functions as a cysteine protease to hydrolyze lipidated LC3, thereby compromising the host autophagic response to bacterial infection. In this study, we characterized the RavZ (RavZLP) ortholog in L. longbeachae (RavZLLO), the second leading cause of Legionella infections in the world. RavZLLO and RavZLP share approximately 60% sequence identity and a conserved His-Asp-Cys catalytic triad. RavZLLO is recognized by the Dot/Icm systems of both L. pneumophila and L. longbeachae. Upon translocation into the host, it suppresses autophagy signaling in cells challenged with both species, indicating the functional redundancy of RavZLLO and RavZLP. Additionally, ectopic expression of RavZLLO but not RavZLP in mammalian cells reduces the levels of cellular polyubiquitinated and polyneddylated proteins. Consistent with this process, RavZLLO regulates the accumulation of polyubiquitinated species on the LCV during L. longbeachae infection.

Ubiquitin‐targeted bacterial effectors: rule breakers of the ubiquitin system

“…RavZ binds LC3 via an N‐terminal LC3‐interacting region (LIR) motif, which facilitates extraction of LC3 from the membrane and removal of the PE moiety (Yang et al , 2017a). Recent studies have also implicated RavZ in clearing ubiquitin from the bacteria‐containing vacuole (Kubori et al , 2017; Shi et al , 2023b), although direct DUB activity remains to be shown. The strategy RavZ uses to simultaneously remove and inactivate LC3 is akin to how the foot‐and‐mouth disease viral protease Lb pro cleaves ISG15 (Swatek et al , 2018), but whether this strategy has been adopted by other pathogen‐encoded Ub/Ub‐like proteases is an open question.…”

The emerging role and therapeutic implications of bacterial and parasitic deubiquitinating enzymes

“…The effector RavZ was identified as a functional and important DUB in various Legionella species and was one of the first examples of bacterial effector proteins that manipulate host autophagy (30)(31)(32). RavZ harbors cysteine protease activity and hydrolyzes lipidconjugated LC3, a Ub-like protein incorporated in autophagic processes on autophagosome membranes (58).…”

“…This hydrolysis reduces the interaction of LC3 with other autophagosome proteins, thereby downregulating autophagy (30). Moreover, further studies revealed the interference of Ub recruitment to the replication vacuoles during Legionella infection upon RavZ activity (31,32). This raises the question of whether RavZ harbors DUB-like activity of RavZ in addition to its general cysteine protease activity towards lipid-conjugated substrates.…”