The PGRS Domain of Mycobacterium tuberculosis PE_PGRS Protein Rv0297 Is Involved in Endoplasmic Reticulum Stress-Mediated Apoptosis through Toll-Like Receptor 4.

Sonam Grover,Seyed E Hasnain,Manjunath P,Aditi Singh,Lothar H Wieler,Yadvir Singh,Torsten Semmler,Karsten Tedin,Tarina Sharma,Nasreen Z Ehtesham,Sakshi Kohli,Anuradha Chowdhary

doi:10.1128/mbio.01017-18

Abstract

ABSTRACTThe genome of Mycobacterium tuberculosis, the causal organism of tuberculosis (TB), encodes a unique protein family known as the PE/PPE/PGRS family, present exclusively in the genus Mycobacterium and nowhere else in the living kingdom, with largely unexplored functions. We describe the functional significance of the PGRS domain of Rv0297, a member of this family. In silico analyses revealed the presence of intrinsically disordered stretches and putative endoplasmic reticulum (ER) localization signals in the PGRS domain of Rv0297 (Rv0297PGRS). The PGRS domain aids in ER localization, which was shown by infecting macrophage cells with M. tuberculosis and by overexpressing the protein by transfection in macrophage cells followed by activation of the unfolded protein response, as evident from increased expression of GRP78/GRP94 and CHOP/ATF4, leading to disruption of intracellular Ca2+ homeostasis and increased nitric oxide (NO) and reactive oxygen species (ROS) production. The consequent activation of the effector caspase-8 resulted in apoptosis of macrophages, which was Toll-like receptor 4 (TLR4) dependent. Administration of recombinant Rv0297PGRS (rRv0297PGRS) also exhibited similar effects. These results implicate a hitherto-unknown role of the PGRS domain of the PE_PGRS protein family in ER stress-mediated cell death through TLR4. Since this protein is already known to be present at later stages of infection in human granulomas it points to the possibility of it being employed by M. tuberculosis for its dissemination via an apoptotic mechanism.

Highlights

The genome of Mycobacterium tuberculosis, the causal organism of tuberculosis (TB), encodes a unique protein family known as the PE/PPE/polymorphic guanine-cytosine-rich sequences (PGRSs) family, present exclusively in the genus Mycobacterium and nowhere else in the living kingdom, with largely unexplored functions
Our initial experiments revealed that Rv0297PGRS alone or in fusion with the PE domain resulted in cell death (Fig. S3)
When HEK293T cells were transfected with various constructs and stained with nuclear stain Hoechst 33342 (30 h and 48 h postinfection), those cells transfected with constructs harboring Rv0297PGRS showed rounding-off phenomena after 30 h (Fig. S3A)

Summary

Introduction

The genome of Mycobacterium tuberculosis, the causal organism of tuberculosis (TB), encodes a unique protein family known as the PE/PPE/PGRS family, present exclusively in the genus Mycobacterium and nowhere else in the living kingdom, with largely unexplored functions. Administration of recombinant Rv0297PGRS (rRv0297PGRS) exhibited similar effects These results implicate a hitherto-unknown role of the PGRS domain of the PE_PGRS protein family in ER stress-mediated cell death through TLR4. Since this protein is already known to be present at later stages of infection in human granulomas it points to the possibility of it being employed by M. tuberculosis for its dissemination via an apoptotic mechanism. 10% of the coding capacity of the M. tuberculosis genome is dedicated to the PE and PPE gene family members, so termed due to the occurrence of PE and PPE domains close to the N-terminal region [3,4,5] This family is present exclusively in the genus Mycobacterium and nowhere else in the living kingdom [4]. Localization of Rv1818c to mitochondria resulted in induction of cell death [18] and was shown to be involved in enhanced survival of Mycobacterium smegmatis in macrophages [11]

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