Distinct Roles in Autophagy and Importance in Infectivity of the Two ATG4 Cysteine Peptidases of Leishmania major

Roderick A.M Williams,Jeremy C Mottram,Graham H Coombs

doi:10.1074/jbc.m112.415372

Abstract

Macroautophagy in Leishmania, which is important for the cellular remodeling required during differentiation, relies upon the hydrolytic activity of two ATG4 cysteine peptidases (ATG4.1 and ATG4.2). We have investigated the individual contributions of each ATG4 to Leishmania major by generating individual gene deletion mutants (Δatg4.1 and Δatg4.2); double mutants could not be generated, indicating that ATG4 activity is required for parasite viability. Both mutants were viable as promastigotes and infected macrophages in vitro and mice, but Δatg4.2 survived poorly irrespective of infection with promastigotes or amastigotes, whereas this was the case only when promastigotes of Δatg4.1 were used. Promastigotes of Δatg4.2 but not Δatg4.1 were more susceptible than wild type promastigotes to starvation and oxidative stresses, which correlated with increased reactive oxygen species levels and oxidatively damaged proteins in the cells as well as impaired mitochondrial function. The antioxidant N-acetylcysteine reversed this phenotype, reducing both basal and induced autophagy and restoring mitochondrial function, indicating a relationship between reactive oxygen species levels and autophagy. Deletion of ATG4.2 had a more dramatic effect upon autophagy than did deletion of ATG4.1. This phenotype is consistent with a reduced efficiency in the autophagic process in Δatg4.2, possibly due to ATG4.2 having a key role in removal of ATG8 from mature autophagosomes and thus facilitating delivery to the lysosomal network. These findings show that there is a level of functional redundancy between the two ATG4s, and that ATG4.2 appears to be the more important. Moreover, the low infectivity of Δatg4.2 demonstrates that autophagy is important for the virulence of the parasite.

Highlights

ATG4 is a cysteine peptidase crucial for macroautophagy
Prolonged incubation in normal medium containing NAC for up to 48 h maintained reactive oxygen species (ROS) at relatively low levels (Fig. 6C, open bars) and the mitochondrial membrane potential close to but significantly lower than that of WT promastigotes (Fig. 6, compare D with B) and there was a significant decrease in the proportion of cells with a defective mitochondrion. These results suggest that the lack of ATG4.2 and consequent impaired autophagic flux is responsible for the increased levels of ROS and that these lead to impaired mitochondrial homeostasis and function
The main defects observed for ⌬atg4.1 were the fewer promastigotes that contained GFP-ATG8-containing autophagosomes (Fig. 2B) and poorer ability of promastigotes to successfully infect macrophages in vitro (Fig. 3, C and D) and in mice (Fig. 3, E-H), even though they appeared to undergo metacyclogenesis in vitro to, slightly slower than, WT parasites (Fig. 3, A and B)

Summary

Introduction

ATG4 is a cysteine peptidase crucial for macroautophagy. Results: Gene deletion mutants show that the two ATG4s of Leishmania perform distinct roles, there is some redundancy. We have investigated the individual contributions of each ATG4 to Leishmania major by generating individual gene deletion mutants (⌬atg4.1 and ⌬atg4.2); double mutants could not be generated, indicating that ATG4 activity is required for parasite viability. Deletion of ATG4.2 had a more dramatic effect upon autophagy than did deletion of ATG4.1 This phenotype is consistent with a reduced efficiency in the autophagic process in ⌬atg4.2, possibly due to ATG4.2 having a key role in removal of ATG8 from mature autophagosomes and facilitating delivery to the lysosomal network. These findings show that there is a level of functional redundancy between the two ATG4s, and that ATG4.2 appears to be the more important. The low infectivity of ⌬atg4.2 demonstrates that autophagy is important for the virulence of the parasite

Objectives

Results

Conclusion