Wild-type Group A Streptococcus Research Articles

FBXO2/SCF ubiquitin ligase complex directs xenophagy through recognizing bacterial surface glycan.

Xenophagy, also known as antibacterial selective autophagy, degrades invading bacterial pathogens such as group A Streptococcus (GAS) to defend cells. Although invading bacteria are known to be marked with ubiquitin and selectively targeted by xenophagy, how ubiquitin ligases recognize invading bacteria is poorly understood. Here, we show that FBXO2, a glycoprotein-specific receptor for substrate in the SKP1/CUL1/F-box protein (SCF) ubiquitin ligase complex, mediates recognition of GlcNAc side chains of the GAS surface carbohydrate structure and promotes ubiquitin-mediated xenophagy against GAS. FBXO2 targets cytosolic GAS through its sugar-binding motif and GlcNAc expression on the GAS surface. FBXO2 knockout resulted in decreased ubiquitin accumulation on intracellular GAS and xenophagic degradation of bacteria. Furthermore, SCF components such as SKP1, CUL1, and ROC1 are required for ubiquitin-mediated xenophagy against GAS. Thus, SCFFBXO2 recognizes GlcNAc residues of GAS surface carbohydrates and functions in ubiquitination during xenophagy.

M Protein of Group a Streptococcus Plays an Essential Role in Inducing High Expression of A20 in Macrophages Resulting in the Downregulation of Inflammatory Response in Lung Tissue.

Group A streptococcus (GAS), a common pathogen, is able to escape host immune attack and thus survive for longer periods of time. One of the mechanisms used by GAS is the upregulated expression of immunosuppressive molecules, which leads to a reduction in the production of inflammatory cytokines in immune cells. In the present study, we found that macrophages produced lower levels of proinflammatory cytokines (IL-1β, TNF-α, IL-6) when challenged with GAS than they did when challenged with Escherichia coli (E. coli). Simultaneously, in a mouse model of lung infection, GAS appeared to induce a weaker inflammatory response compared to E. coli. Our data also indicated that the expression of the A20 transcriptional regulator was higher in GAS-infected macrophages than that in macrophages infected with E. coli, and that high expression of A20 correlated with a reduction in the production of TRAF6. SiRNA targeting of A20 led to the increased production of TRAF6, IL-1β, TNF-α, and IL-6, suggesting that A20 inhibits synthesis of these key proinflammatory cytokines. We also investigated the pathway underlying A20 production and found that the synthesis of A20 depends on My88, and to a lower extent on TNFR1. Finally, we showed a significant reduction in the expression of A20 in macrophages stimulated by M protein-mutant GAS, however, a speB-GAS mutant, which is unable to degrade M protein, induced a greater level of A20 production than wild type GAS. Collectively, our data suggested that M protein of GAS was responsible for inducing A20 expression in macrophages, which in turn down-regulates the inflammatory cytokine response in order to facilitate GAS in evading immune surveillance and thus prolong survival in the host.

Group A Streptococcus Prevents the Acidification of Phagolysosomes in Macrophages

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.