Intracellular Group A Streptococcus Induces Golgi Fragmentation To Impair Host Defenses through Streptolysin O and NAD-Glycohydrolase.

Group A Streptococcus (GAS; Streptococcus pyogenes) is a major human pathogen that causes streptococcal pharyngitis, skin and soft tissue infections, and life-threatening conditions such as streptococcal toxic-shock syndrome. During infection, GAS not only invades diverse host cells but also injects effector proteins such as NAD-glycohydrolase (Nga) into the host cells through a streptolysin O (SLO)-dependent mechanism without invading the cells; Nga and SLO are two major virulence factors that are associated with increased bacterial virulence. Here, we have shown that the invading GAS induces fragmentation of the Golgi complex and inhibits anterograde transport in the infected host cells through the secreted toxins SLO and Nga. GAS infection-induced Golgi fragmentation required both bacterial invasion and SLO-mediated Nga translocation into the host cytosol. The cellular Golgi network is critical for the sorting of surface molecules and is thus essential for the integrity of the epithelial barrier and for the immune response of macrophages to pathogens. In epithelial cells, inhibition of anterograde trafficking by invading GAS and Nga resulted in the redistribution of E-cadherin to the cytosol and an increase in bacterial translocation across the epithelial barrier. Moreover, in macrophages, interleukin-8 secretion in response to GAS infection was found to be suppressed by intracellular GAS and Nga. Our findings reveal a previously undescribed bacterial invasion-dependent function of Nga as well as a previously unrecognized GAS-host interaction that is associated with GAS pathogenesis.IMPORTANCE Two prominent virulence factors of group A Streptococcus (GAS), streptolysin O (SLO) and NAD-glycohydrolase (Nga), are linked to enhanced pathogenicity of the prevalent GAS strains. Recent advances show that SLO and Nga are important for intracellular survival of GAS in epithelial cells and macrophages. Here, we found that invading GAS disrupts the Golgi complex in host cells through SLO and Nga. We show that GAS-induced Golgi fragmentation requires bacterial invasion into host cells, SLO pore formation activity, and Nga NADase activity. GAS-induced Golgi fragmentation results in the impairment of the epithelial barrier and chemokine secretion in macrophages. This immune inhibition property of SLO and Nga by intracellular GAS indicates that the invasion of GAS is associated with virulence exerted by SLO and Nga.

Group A Streptococcus modulates RAB1- and PIK3C3 complex-dependent autophagy.

Autophagy selectively targets invading bacteria to defend cells, whereas bacterial pathogens counteract autophagy to survive in cells. The initiation of canonical autophagy involves the PIK3C3 complex, but autophagy targeting Group A Streptococcus (GAS) is PIK3C3-independent. We report that GAS infection elicits both PIK3C3-dependent and -independent autophagy, and that the GAS effector NAD-glycohydrolase (Nga) selectively modulates PIK3C3-dependent autophagy. GAS regulates starvation-induced (canonical) PIK3C3-dependent autophagy by secreting streptolysin O and Nga, and Nga also suppresses PIK3C3-dependent GAS-targeting-autophagosome formation during early infection and facilitates intracellular proliferation. This Nga-sensitive autophagosome formation involves the ATG14-containing PIK3C3 complex and RAB1 GTPase, which are both dispensable for Nga-insensitive RAB9A/RAB17-positive autophagosome formation. Furthermore, although MTOR inhibition and subsequent activation of ULK1, BECN1, and ATG14 occur during GAS infection, ATG14 recruitment to GAS is impaired, suggesting that Nga inhibits the recruitment of ATG14-containing PIK3C3 complexes to autophagosome-formation sites. Our findings reveal not only a previously unrecognized GAS-host interaction that modulates canonical autophagy, but also the existence of multiple autophagy pathways, using distinct regulators, targeting bacterial infection.Abbreviations: ATG5: autophagy related 5; ATG14: autophagy related 14; ATG16L1: autophagy related 16 like 1; BECN1: beclin 1; CALCOCO2: calcium binding and coiled-coil domain 2; GAS: group A streptococcus; GcAV: GAS-containing autophagosome-like vacuole; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; Nga: NAD-glycohydrolase; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns4P: phosphatidylinositol-4-phosphate; RAB: RAB, member RAS oncogene GTPases; RAB1A: RAB1A, member RAS oncogene family; RAB11A: RAB11A, member RAS oncogene family; RAB17: RAB17, member RAS oncogene family; RAB24: RAB24, member RAS oncogene family; RPS6KB1: ribosomal protein S6 kinase B1; SLO: streptolysin O; SQSTM1: sequestosome 1; ULK1: unc-51 like autophagy activating kinase 1; WIPI2: WD repeat domain, phosphoinositide interacting 2.

Group A Streptococcus NAD-Glycohydrolase Inhibits Caveolin 1-Mediated Internalization Into Human Epithelial Cells.

Group A Streptococcus (GAS) invades epithelial cells causing persistent infection. GAS has a variety of effector proteins that modulate host systems to affect their survival in host environments. The main effector proteins of GAS are NAD-glycohydrolase (Nga) and streptolysin O (SLO). Although Nga has NADase activity and shows SLO-dependent cytotoxicity, some clinical isolates harbor NADase-inactive subtypes of Nga, and the function of NADase-inactive Nga is still unclear. In this study, we found that deletion of nga enhanced the internalization of GAS into HeLa and Ca9-22 cells. Amino acid substitution of Nga R289K/G330D (NADase-inactive) does not enhance GAS invasion, suggesting that Nga may inhibit the internalization of GAS into host cells in an NADase-independent manner. Moreover, double deletion of slo and nga showed similar invasion percentages compared with wild-type GAS, indicating the important role of SLO in the inhibition of GAS invasion by Nga. Furthermore, enhanced internalization of the nga deletion mutant was not observed in Cav1-knockout HeLa cells. Altogether, these findings demonstrate an unrecognized NADase-independent function of Nga as a negative regulator of CAV1-mediated internalization into epithelial cells.

RAB30 regulates PI4KB (phosphatidylinositol 4-kinase beta)-dependent autophagy against group A Streptococcus.

Macroautophagy/autophagy plays an important role in the immune response to invasion by intracellular pathogens such as group A Streptococcus (GAS; Streptococcus pyogenes). We previously identified RAB30, a Golgi-resident GTPase, as a novel anti-bacterial autophagic regulator in the formation of GAS-containing autophagosome-like vacuoles (GcAVs); however, the precise mechanism underlying this process remains elusive. Here, we elucidate a novel property of RAB30: the ability to recruit PI4KB (phosphatidylinositol 4-kinase beta) to the Golgi apparatus and GcAVs. We found that trans-Golgi network (TGN) vesicles were incorporated into GcAVs via RAB30 to promote GcAV formation. Moreover, depletion of phosphatidylinositol-4-phosphate (PtdIns4P), a phosphatidylinositol enriched in the TGN, by wortmannin and phenylarsine oxide, followed by subsequent repletion with exogenous PtdIns4P revealed that PtdIns4P is crucial for GcAV formation. Furthermore, we identify an interaction between RAB30 and PI4KB, in which the knockdown of RAB30 decreased the localization of PI4KB to the TGN and GcAVs. Finally, PI4KB knockout suppressed autophagy by inhibiting GcAV formation, resulting in the increased survival of GAS. Our results demonstrate a novel autophagosomal formation mechanism involving coordinative functions of RAB30 and PI4KB distinct from those utilized in canonical autophagy. Abbreviations: GAS: group A Streptococcus; GcAVs: GAS-containing autophagosome-like vacuoles; PI4KB: phosphatidylinositol 4-kinase beta; PtdIns: phosphatidylinositol; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns4P: phosphatidylinositol-4-phosphate; PtdIns5P: phosphatidylinositol-5-phosphate; SLO: streptolysin O; TGN: trans-Golgi network; TGOLN2: trans-golgi network protein 2; PH: plekstrin homology; OSBP: oxysterol binding protein.

LAMTOR2/LAMTOR1 complex is required for TAX1BP1-mediated xenophagy.

Xenophagy, also known as antibacterial autophagy, plays a role in host defence against invading pathogens such as Group A Streptococcus (GAS) and Salmonella. In xenophagy, autophagy receptors are used in the recognition of invading pathogens and in autophagosome maturation and autolysosome formation. However, the mechanism by which autophagy receptors are regulated during bacterial infection remains poorly elucidated. In this study, we identified LAMTOR2 and LAMTOR1, also named p14 and p18, respectively, as previously unrecognised xenophagy regulators that modulate the autophagy receptor TAX1BP1 in response to GAS and Salmonella invasion. LAMTOR1 was localized to bacterium-containing endosomes, and LAMTOR2 was recruited to bacterium-containing damaged endosomes in a LAMTOR1-dependent manner. LAMTOR2 was dispensable for the formation of autophagosomes targeting damaged membrane debris surrounding cytosolic bacteria, but it was critical for autolysosome formation, and LAMTOR2 interacted with the autophagy receptors NBR1, TAX1BP1, and p62 and was necessary for TAX1BP1 recruitment to pathogen-containing autophagosomes. Notably, knockout of TAX1BP1 caused a reduction in autolysosome formation and subsequent bacterial degradation. Collectively, our findings demonstrated that the LAMTOR1/2 complex is required for recruiting TAX1BP1 to autophagosomes and thereby facilitating autolysosome formation during bacterial infection.

NLRX1 Negatively Regulates Group A Streptococcus Invasion and Autophagy Induction by Interacting With the Beclin 1-UVRAG Complex.

Group A Streptococcus (GAS) can invade epithelial cells; however, these bacteria are targeted and eventually destroyed by autophagy. Members of the Nod-like receptor (NLR) family are thought to be critical for the autophagic response to invasive bacteria. However, the intracellular sensors within host cells that are responsible for bacterial invasion and the induction of autophagy are largely unknown. Thus, our aim was to examine the role of one such NLR, namely NLRX1, in invasion and autophagy during GAS infection. We found that GAS invasion was markedly increased in NLRX1 knockout cells. This led to the potentiation of autophagic processes such as autophagosome and autolysosome formation. NLRX1 was found to interact with Beclin 1 and UVRAG, members of Beclin1 complex, and knockout of these proteins inhibited invasion and autophagy upon GAS infection. Especially, NLRX1 interacted with Beclin 1 via its NACHT domain and this interaction was responsible for the NLRX1-mediated inhibition of invasion and autophagic processes including autophagosome and autolysosome formation during GAS infection. These findings demonstrate that NLRX1 functions as a negative regulator to inactivate the Beclin 1-UVRAG complex, which regulates invasion and autophagy during GAS infection. Thus, our study expands our knowledge of the role of NLRX1 during bacterial invasion and autophagy and could lead to further investigations to understand pathogen-host cell interactions, facilitating novel targeted therapeutics.

Bcl-xL Affects Group A Streptococcus-Induced Autophagy Directly, by Inhibiting Fusion between Autophagosomes and Lysosomes, and Indirectly, by Inhibiting Bacterial Internalization via Interaction with Beclin 1-UVRAG.

Anti-apoptotic Bcl-2 and Bcl-xL are proposed to regulate starvation-induced autophagy by directly interacting with Beclin 1. Beclin 1 is also thought to be involved in multiple vesicle trafficking pathways such as endocytosis by binding to Atg14L and UVRAG. However, how the interaction of Bcl-2 family proteins and Beclin 1 regulates anti-bacterial autophagy (xenophagy) is still unclear. In this study, we analyzed these interactions using Group A Streptococcus (GAS; Streptococcus pyogenes) infection as a model. GAS is internalized into epithelial cells through endocytosis, while the intracellular fate of GAS is degradation by autophagy. Here, we found that Bcl-xL but not Bcl-2 regulates GAS-induced autophagy. Autophagosome-lysosome fusion and the internalization process during GAS infection were promoted in Bcl-xL knockout cells. In addition, knockout of Beclin 1 phenocopied the internalization defect of GAS. Furthermore, UVRAG interacts not only with Beclin 1 but also with Bcl-xL, and overexpression of UVRAG partially rescued the internalization defect of Beclin 1 knockout cells during GAS infection. Thus, our results indicate that Bcl-xL inhibits GAS-induced autophagy directly by suppressing autophagosome-lysosome fusion and indirectly by suppressing GAS internalization via interaction with Beclin 1-UVRAG.