Dok proteins are recruited to the phagosome and degraded in a GP63-dependent manner during Leishmania major infection

Abstract

Three adaptor molecules of the Dok family, Dok-1, Dok-2 and Dok-3 are expressed in macrophages and are involved in the negative regulation of signaling in response to lipopolysaccharide and various cytokines and growth factors. We investigated the role and the fate of these proteins following infection with Leishmania major promastigotes in macrophages. The protozoan parasite L. major causes cutaneous leishmaniasis and is known for its capacity to alter host-cell signaling and function. Dok-1/Dok-2(-/-) bone marrow-derived macrophages displayed normal uptake of L. major promastigotes. Following Leishmania infection, Dok-1 was barely detectable by confocal microscopy. By contrast, phagocytosis of latex beads or zymosan led to the recruitment of Dok-1 to phagosomes. In the absence of the Leishmania pathogenesis-associated metalloprotease GP63, Dok-1 was also, partially, recruited to phagosomes containing L. major promastigotes. Further biochemical analyses revealed that similar to Dok-1, Dok-2 and Dok-3 were targets of GP63. Moreover, we showed that upon infection with wild-type or Δgp63 L. major promastigotes, production of nitric oxide and tumor necrosis factor by interferon-γ-primed Dok-1/Dok-2(-/-) macrophages was reduced compared to WT macrophages. These results suggest that Dok proteins may be important regulators of macrophage responses to Leishmania infection.

Dok-1 and Dok-2 Are Required To Maintain Herpes Simplex Virus 1-Specific CD8+T Cells in a Murine Model of Ocular Infection

“…We have previously shown that in IFN-␥-primed macrophages, nitric oxide (NO) and TNF secretion is reduced in DKO cells (45). Thus, Dok-1/2 deficiency could be expected to result in less efficient clearing of the virus during acute infection; however, we found that viral titers in tear films and the kinetics of viral clearance in the eyes were similar in DKO and WT mice.…”

Leishmania major Promastigotes Evade LC3-Associated Phagocytosis through the Action of GP63

“…The promastigote surface glycolipid lipophosphoglycan (LPG) plays a major role in this inhibition by disrupting phagosomal lipid microdomains [ 8 – 10 ]. Recent evidence indicates that the glycophosphatidylinositol (GPI)-anchored zinc-dependent metalloprotease GP63 also contributes to the Leishmania- induced phagosomal remodeling [ 7 , 11 ]. Hence, during phagocytosis, GP63 is released from the parasite and rapidly gains access to various intracellular compartments where it cleaves a number of regulators of macrophage function [ 7 , 11 – 16 ], including regulators of membrane fusion [ 7 , 15 ].…”

“…Recent evidence indicates that the glycophosphatidylinositol (GPI)-anchored zinc-dependent metalloprotease GP63 also contributes to the Leishmania- induced phagosomal remodeling [ 7 , 11 ]. Hence, during phagocytosis, GP63 is released from the parasite and rapidly gains access to various intracellular compartments where it cleaves a number of regulators of macrophage function [ 7 , 11 – 16 ], including regulators of membrane fusion [ 7 , 15 ]. One such molecule is the soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) Vesicle-associated membrane protein 8 (VAMP8), which we recently showed to control the early recruitment of the NADPH oxidase (NOX2) to phagosomes [ 7 ].…”

The Leishmania-Macrophage Interactions: Role of E-NTPDases and Purinergic Signaling

“…For example, liphosphoglycan (LPG), glycosylinositol phospholipids (GIPLs), proteophosphoglycans (PPGs), secreted acid phosphatases (SAPs) and cysteine protease B have been well-explored in this matter [26][27][28][29][30][31][32][33] . The GP63, a zinc-metalloprotease named glycoprotein 63 (GP63), is another critical virulence factor that has been fully explored [34][35][36][37][38] . However, the E-NTPDases (ecto-nucleoside triphosphate diphosphohydrolases), while less-extensively studied, have been demonstrated and believed to be important infectivity and virulence factors [39][40][41][42][43][44][45][46][47][48] .…”