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Group A Streptococcus Prevents the Acidification of Phagolysosomes in Macrophages
Author(s) -
Franscioni Henry,
Snipper Joshua,
No Hyewon,
Okumura Cheryl
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.925.6
Subject(s) - phagolysosome , streptolysin , lysosome , phagosome , bafilomycin , phagocytosis , atpase , endosome , microbiology and biotechnology , chemistry , biology , biochemistry , enzyme , intracellular , autophagy , bacterial protein , gene , apoptosis
Group A Streptococcus (GAS) is a gram‐positive bacterium that can cause a variety of ailments ranging from pharyngitis to necrosis fasciitis. Phagocytes are important first responders to GAS infection, and typically clear the pathogen by phagocytosis and subsequent degradation by fusing the phagosome with the lysosome. Critical to this process is the acidification of the lysosome by the multi‐subunit vacuolar ATPase (V‐ATPase), which pumps hydrogen ions into the lysosomal compartment that activates the degradative enzymes of the lysosome. Studies in our lab have shown that GAS is able to prevent the acidification of the phagolysosome, possibly by using the pore‐forming toxin streptolysin O (SLO), which would allow leakage of protons. SLO binds to cholesterol, a component found in lipid raft structures that facilitate protein assembly, including the V‐ATPase complex. Here, we examined whether SLO prevents phagolysosome acidification by inhibiting the recruitment and assembly of V‐ATPase on the phagolysosome. We used immunofluorescence to probe macrophages infected with both the GAS wild type (M1WT) and an isogenic SLO mutant strain (M1ΔSLO) with an antibody for one of the V‐ATPase subunits. Cells infected with either strain appeared to have no differences in recruitment of V‐ATPase. We also fractionated cells to isolate phagolysosomes in order to determine whether the entire V‐ATPase complex is present in phagolysosomes by mass spectrometry. Purity of the cell fractions was verified by Western blotting using antibodies for EEA‐1 (early endosome/phagosome marker) and LAMP‐2 (lysosome marker). We further probed the phagolysosome fraction with antibodies for 2 V‐ATPase subunits and concluded that SLO does not appear to affect the recruitment of either of these subunits. Preliminary mass spectrometry data indicate that the remaining V‐ATPase subunits appear to be present in phagolysosomes containing wild type GAS. These results indicate that V‐ATPase recruitment is not significantly altered in GAS‐infected phagosomes. Testing the activity of the V‐ATPase is ongoing. These results should shed light on the mechanism by which GAS prevents phagolysosomal acidification.