Phg1/TM9 Proteins Control Intracellular Killing of Bacteria by Determining Cellular Levels of the Kil1 Sulfotransferase in Dictyostelium

Abstract

Dictyostelium discoideum has largely been used to study phagocytosis and intracellular killing of bacteria. Previous studies have shown that Phg1A, Kil1 and Kil2 proteins are necessary for efficient intracellular killing of Klebsiella bacteria. Here we show that in phg1a KO cells, cellular levels of lysosomal glycosidases and lysozyme are decreased, and lysosomal pH is increased. Surprisingly, overexpression of Kil1 restores efficient killing in phg1a KO cells without correcting these lysosomal anomalies. Conversely, kil1 KO cells are defective for killing, but their enzymatic content and lysosomal pH are indistinguishable from WT cells. The killing defect of phg1a KO cells can be accounted for by the observation that in these cells the stability and the cellular amount of Kil1 are markedly reduced. Since Kil1 is the only sulfotransferase characterized in Dictyostelium, an (unidentified) sulfated factor, defective in both phg1a and kil1 KO cells, may play a key role in intracellular killing of Klebsiella bacteria. In addition, Phg1B plays a redundant role with Phg1A in controlling cellular amounts of Kil1 and intracellular killing. Finally, cellular levels of Kil1 are unaffected in kil2 KO cells, and Kil1 overexpression does not correct the killing defect of kil2 KO cells, suggesting that Kil2 plays a distinct role in intracellular killing.

Dictyostelium EHD associates with Dynamin and participates in phagosome maturation

“…Glycosidase activity, phagosomal pH and proteolysis activity, and phagosomes purification Glycosidase activities in cell pellets and extracellular fluid were assessed as previously described (Froquet et al, 2008;Le Coadic et al, 2013). For phagosomal pH measurement, 3-µm carboxylated silica beads coupled to the pH-sensitive fluorophore FITC and to the pH-insensitive fluorophore Alexa-Fluor-594 were used.…”

The Nonaspanins TM9SF2 and TM9SF4 Regulate the Plasma Membrane Localization and Signalling Activity of the Peptidoglycan Recognition Protein PGRP-LC in <b><i>Drosophila</i></b>

“…They are found in the endosomal compartments of yeast, Dictyostelium and human cells where they possibly contribute to cell migration, vesicular transport, endocytic trafficking and autophagy [11,12,17,18,19,20,21]. In Dictyostelium, TM9SF4/Phg1A is required for the phagocytosis and killing of bacteria [16,22,23]. Moreover, the two Dictyostelium TM9 proteins Phg1A and Phg1B synergistically contribute to the expression and/or localization of transmembrane proteins [14,24].…”

Defective lysosome maturation and Legionella pneumophila replication in Dictyostelium ArfGAP ACAP-A mutant cells

“…Interestingly, only the mature form of cathepsin D was detected in WT and acapA 2 cells (data not shown), suggesting that there was normal maturation of lysosomal enzymes in the absence of ACAP-A. Next, we measured the proteolytic activity of phagosomes in living cells using beads coupled to Alexa Fluor 594 and to BSA labeled with DQ-Green at a self-quenching concentration, which become dequenched upon proteolysis of BSA Le Coadic et al, 2013). We observed that protease activity was substantially increased in acapA 2 cells (Fig.…”

“…After 4 h at 22˚C, migration distances were calculated by measuring displacement of cell front. Glycosidase activities (N-acetyl bglucosaminidase and a-mannosidase) and cathepsin D content in cell pellet and culture medium after 3 days of culture were assessed as previously described (Froquet et al, 2008;Le Coadic et al, 2013). For enzymatic activities and western immunoblot analysis, equal amounts of protein were analyzed.…”

“…For enzymatic activities and western immunoblot analysis, equal amounts of protein were analyzed. Phagosomal proteolysis of DQ-BSA-labeled beads was determined as reported previously Le Coadic et al, 2013). After bead uptake, the cell suspension was analyzed by flow cytometry to determine the intensity of the DQ-green fluorescent marker in cells containing phagocytosed beads (Le Coadic et al, 2013).…”