Methylomic Changes of Autophagy-Related Genes by Legionella Effector Lpg2936 in Infected Macrophages.

Ahmed I Abd El Maksoud,Dalia Elebeedy,Nasser H Abass,Ahmed M Awad,Ghada M Nasr,Tamer Roshdy,Hany Khalil

doi:10.3389/fcell.2019.00390

Abstract

Legionella pneumophila (L. pneumophila) is a Gram-negative bacterium that infects the human respiratory tract causing Legionnaires’ disease, a severe form of pneumonia. Recently, rising evidence indicated the ability of Legionella to regulate host defense via its type 4 secretion system including hundreds of effectors that promote intracellular bacterial replication. The host defense against such invaders includes autophagic machinery that is responsible for degradation events of invading pathogens and recycling of cell components. The interplay between host autophagy and Legionella infection has been reported, indicating the role of bacterial effectors in the regulation of autophagy during intracellular replication. Here, we investigated the potential impact of Legionella effector Lpg2936 in the regulation of host autophagy and its role in bacterial replication using mice-derived macrophages and human lung epithelial cells (A549 cells). First, monitoring of autophagic flux following infection revealed a marked reduction of Atg7 and LC3B expression profile and low accumulation levels of autophagy-related LC3-I, LC3-II, and the Atg12–Atg5 protein complex. A novel methyladenine alteration was observed due to irreversible changes of GATC motif to G(6 mA) TC in the promoter region of Atg7 and LC3B indicated by cleaved genomic-DNA using the N6 methyladenine-sensitive restriction enzyme DpnI. Interestingly, RNA interference (RNAi) of Lpg2936 in infected macrophages showed dramatic inhibition of bacterial replication by restoring the expression of autophagy-related proteins. This is accompanied by low production levels of bacterial-associated pro-inflammatory cytokines. Furthermore, a constructed Lpg2936 segment in the GFP expression vector was translocated in the host nucleus and successfully induced methyladenine changes in Atg7 and LC3B promoter region and subsequently regulated autophagy in A549 cells independent of infection. Finally, treatment with methylation inhibitors 5-AZA and (2)-Epigallocatechin-3-gallate (EGCG) was able to restore autophagy-related gene expression and to disrupt bacterial replication in infected macrophages. This cumulative evidence indicates the methylation effect of Legionella effector Lpg2936 on the host autophagy-related molecules Atg7 and LC3B and subsequent reduction in the expression levels of autophagy effectors during intracellular replication of L. pneumophila.

Highlights

Legionella pneumophila is a Gram-negative bacterium that replicates in macrophages and causes a severe form of pneumonia called Legionnaires’ disease (LD) (Horwitz, 1983)
We demonstrate the direct role of the L. pneumophila effector Lpg2936 in the methyladenine changes of autophagy-related Atg7 enzyme and LC3B-asscoited protein to ensure intracellular bacterial replication
To determine whether Legionella effector Lpg2936 is involved in the regulation of host autophagy, infected mice-derived macrophages were pre-transfected with siRNA that targets Lpg2936

Summary

Introduction

Legionella pneumophila is a Gram-negative bacterium that replicates in macrophages and causes a severe form of pneumonia called Legionnaires’ disease (LD) (Horwitz, 1983). L. pneumophila blazes a variety of intracellular communication by transferring more than 300 effectors through its secretion system, Icm/Dot type-IV, which modulates cellular immune response including cell signal transduction, autophagic machinery, apoptosis, and cytokine secretion (Hempstead and Isberg, 2013; Nevo et al, 2014). Autophagy is a cellular process in which double-membraned vesicles are formed to surround the cytosolic defective contents by recruiting specific autophagy associated proteins (ATGs) and deliver these contents to the lysosomes for degradation (Klionsky et al, 2012). The unlipidated LC3-I is activated by the protease Atg4B through exposing a C-terminal glycine residue Both conjugation reactions required E1-like activating enzyme, Atg, while distinct E2-like enzymes, Atg and Atg, are utilized for lipidated LC3II and the Atg12–Atg complex, respectively (Fujita et al, 2008a)

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