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

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.

SUBMITTER: Ge P

PROVIDER: S-EPMC9037497 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis (Mtb). In the lungs, macrophages and neutrophils are the first immune cells that have contact with the infecting mycobacteria. Neutrophils are phagocytic cells that kill microorganisms through several mechanisms, which include the lytic enzymes and antimicrobial peptides that are found in their lysosomes, and the production of reactive oxygen species. Neutrophils also release extracellular vesicles (EVs) (100-1,000?nm in diameter) to the extracellular milieu; these EVs consist of a lipid bilayer surrounding a hydrophilic core and participate in intercellular communication. We previously demonstrated that human neutrophils infected in vitro with Mtb H37Rv release EVs (EV-TB), but the effect of these EVs on other cells relevant for the control of Mtb infection, such as macrophages, has not been completely analyzed. In this study, we characterized the EVs produced by non-stimulated human neutrophils (EV-NS), and the EVs produced by neutrophils stimulated with an activator (PMA), a peptide derived from bacterial proteins (fMLF) or Mtb, and observed that the four EVs differed in their size. Ligands for toll-like receptor (TLR) 2/6 were detected in EV-TB, and these EVs favored a modest increase in the expression of the co-stimulatory molecules CD80, a higher expression of CD86, and the production of higher amounts of TNF-? and IL-6, and of lower amounts of TGF-?, in autologous human macrophages, compared with the other EVs. EV-TB reduced the amount of intracellular Mtb in macrophages, and increased superoxide anion production in these cells. TLR2/6 ligation and superoxide anion production are known inducers of autophagy; accordingly, we found that EV-TB induced higher expression of the autophagy-related marker LC3-II in macrophages, and the co-localization of LC3-II with Mtb inside infected macrophages. The intracellular mycobacterial load increased when autophagy was inhibited with wortmannin in these cells. In conclusion, our results demonstrate that neutrophils produce different EVs in response to diverse activators, and that EV-TB activate macrophages and promote the clearance of intracellular Mtb through early superoxide anion production and autophagy induction, which is a novel role for neutrophil-derived EVs in the immune response to Mtb.

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

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