M. tuberculosis PknG manipulates host autophagy flux to promote pathogen intracellular survival

Autophagy. 2022 Mar;18(3):576-594. doi: 10.1080/15548627.2021.1938912. Epub 2021 Jul 7.

Abstract

The eukaryotic-type protein kinase G (PknG), one of the eleven eukaryotic type serine-threonine protein kinase (STPK) in Mycobacterium tuberculosis (Mtb), is involved in mycobacterial survival within macrophages, presumably by suppressing phagosome and autophagosome maturation, which makes PknG an attractive drug target. However, the exact mechanism by which PknG inhibits pathogen clearance during mycobacterial infection remains largely unknown. Here, we show that PknG promotes macroautophagy/autophagy induction but inhibits autophagosome maturation, causing an overall effect of blocked autophagy flux and enhanced pathogen intracellular survival. PknG prevents the activation of AKT (AKT serine/threonine kinase) via competitively binding to its pleckstrin homology (PH) domain, leading to autophagy induction. Remarkably, PknG could also inhibit autophagosome maturation to block autophagy flux via targeting host small GTPase RAB14. Specifically, PknG directly interacts with RAB14 to block RAB14-GTP hydrolysis. Furthermore, PknG phosphorylates TBC1D4/AS160 (TBC1 domain family member 4) to suppress its GTPase-activating protein (GAP) activity toward RAB14. In macrophages and in vivo, PknG promotes Mtb intracellular survival through blocking autophagy flux, which is dependent on RAB14. Taken together, our data unveil a dual-functional bacterial effector that tightly regulates host autophagy flux to benefit pathogen intracellular survival.Abbreviations: AKT: AKT serine/threonine kinase; ATG5: autophagy related 5; BMDMs: bone marrow-derived macrophages; DTT: dithiothreitol; FBS: fetal calf serum; GAP: GTPase-activating protein; MOI: multiplicity of infection; Mtb: Mycobacterium tuberculosis; MTOR: mechanistic target of rapamycin kinase; OADC: oleic acid-albumin-dextrose-catalase; PC, phosphatidylcholine; PH: pleckstrin homology; PI3K: phosphoinositide 3-kinase; PknG: protein kinase G; PtdIns(3,4,5)P3: phosphatidylinositol(3,4,5)-trisphosphate; SQSTM1: sequestosome 1; STPK: serine-threonine protein kinase; TB: tuberculosis; TBC1D4: TBC1 domain family member 4; TPR: tetratricopeptide repeat; ULK1: unc-51 like autophagy activating kinase 1; WT: wild-type.

Keywords: AKT; Mycobacterium tuberculosis; RAB14; autophagy flux; protein kinase G.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Autophagy / physiology
  • Cyclic GMP-Dependent Protein Kinases / metabolism
  • Cyclic GMP-Dependent Protein Kinases / pharmacology
  • GTPase-Activating Proteins / metabolism
  • Humans
  • Mycobacterium tuberculosis* / metabolism
  • Phosphatidylinositol 3-Kinases / metabolism
  • Protein Serine-Threonine Kinases
  • Proto-Oncogene Proteins c-akt / metabolism
  • Serine
  • Tuberculosis* / microbiology
  • rab GTP-Binding Proteins / metabolism

Substances

  • GTPase-Activating Proteins
  • Serine
  • Protein Serine-Threonine Kinases
  • Proto-Oncogene Proteins c-akt
  • Cyclic GMP-Dependent Protein Kinases
  • Rab14 protein, human
  • rab GTP-Binding Proteins

Grants and funding

This work was supported by the National Key Research and Development Program of China (2017YFD0500300 and 2017YFA0505900), the National Natural Science Foundation of China (31830003, 81825014 and 82022041), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB29020000), the National Science and Technology Major Project (2018ZX10101004), Youth Innovation Promotion Association CAS (2018118), and the Open Project Program of the State Key Laboratory of Proteomics (SKLPO202003).