scholarly journals 2590. Streptolysin O Enhances Binding of the Group A Streptococcal NAD+-Glycohydrolase Toxin to Oropharyngeal Keratinocytes

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S900-S900
Author(s):  
Jorge J Velarde ◽  
Nicola Lynskey ◽  
Alessandro Piai ◽  
James Chou ◽  
Michael Wessels
Keyword(s):  
Flow Cytometry ◽  
Cell Surface ◽  
Solution Structure ◽  
Host Cells ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Nad Glycohydrolase ◽  
Streptolysin O ◽  
Group A ◽  
Translocation Domain

Abstract Background Streptolysin O (SLO) and the NAD+-glycohydrolase (NADase) are co-toxins secreted by group A Streptococcus (GAS) that play a significant role in virulence. NADase requires SLO for translocation into the host cell cytoplasm, a process termed cytolysin-mediated translocation (CMT). Recently, we noted that interaction of the two toxins mutually increased their stability. Although NADase is predicted to bind to the host cell surface, this interaction is incompletely understood. Here, we investigate potential mechanisms by which NADase binds to oropharyngeal keratinocytes. Methods The amino terminal region of NADase has been implicated in CMT, but the structure of the putative translocation domain has not been characterized. We determined the solution structure of this domain by NMR spectroscopy. We used flow-cytometry and confocal microscopy to investigate whether NADase could interact directly with oropharyngeal keratinocytes. Finally, since we expect that NADase and SLO are co-expressed from the same operon, are secreted in a coordinated fashion, and interact in solution, we tested whether SLO affects NADase binding to host cells. Results The solution structure of the NADase translocation domain revealed a β-sandwich fold with an elongated N-terminal intrinsically disordered region. Structural homology searches (DALI) identified a potential carbohydrate binding module, suggesting the translocation domain could play a role in glycan binding. We also demonstrated by flow-cytometry that purified recombinant NADase toxin is able to independently interact with the cell surface of oropharyngeal keratinocytes. Importantly, interaction with SLO significantly enhanced the association of NADase with the cell surface, resulting in a 5-fold increase of the geometric mean fluorescence intensity. Conclusion The structure of the NADase translocation domain reveals a potential carbohydrate binding module, which may mediate binding of the toxin to a cell-surface glycan. Binding of NADase to host cells is markedly enhanced by its interaction with SLO. We conclude that interaction of the two toxins contributes to the CMT process by functionally increasing the local concentration of NADase at the cell surface. Disclosures All authors: No reported disclosures.

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

mBio ◽  
2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Takashi Nozawa ◽  
Junpei Iibushi ◽  
Hirotaka Toh ◽  
Atsuko Minowa-Nozawa ◽  
Kazunori Murase ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Virulence Factors ◽  
Golgi Complex ◽  
Host Cells ◽  
Bacterial Invasion ◽  
Epithelial Barrier ◽  
Nad Glycohydrolase ◽  
Streptolysin O ◽  
Group A ◽  
Golgi Fragmentation

ABSTRACT 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.

scholarly journals Intracellular Group A Streptococcus induces Golgi fragmentation to impair host defenses through Streptolysin O and NAD-glycohydrolase

2020 ◽  
Author(s):  
Takashi Nozawa ◽  
Junpei Iibushi ◽  
Hirotaka Toh ◽  
Atsuko Minowa-Nozawa ◽  
Kazunori Murase ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Virulence Factors ◽  
Golgi Complex ◽  
Host Cells ◽  
Bacterial Invasion ◽  
Epithelial Barrier ◽  
Nad Glycohydrolase ◽  
Streptolysin O ◽  
Group A ◽  
Golgi Fragmentation

AbstractGroup 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 thus is essential for epithelial barrier integrity and 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 GAS pathogenesis.ImportanceTwo prominent virulence factors of GAS, SLO and Nga, have been established to be linked to enhanced pathogenicity of 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 disrupt the Golgi complex in host cells by SLO and Nga. We showed that GAS-induced Golgi fragmentation requires bacterial invasion into host cells, SLO pore-formation activity, and Nga NADase activity. GAS-induced Golgi fragmentation resulted in the impairment of 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.

The Solution Structure of the CBM4-2 Carbohydrate Binding Module from a ThermostableRhodothermus marinusXylanase†

Biochemistry ◽  
2002 ◽  
Vol 41 (18) ◽  
pp. 5712-5719 ◽  
Author(s):  
Peter J. Simpson ◽  
Stuart J. Jamieson ◽  
Maher Abou-Hachem ◽  
Eva Nordberg Karlsson ◽  
Harry J. Gilbert ◽  
...  
Keyword(s):  
Solution Structure ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module

scholarly journals Enhancement of Streptolysin O Activity and Intrinsic Cytotoxic Effects of the Group A Streptococcal Toxin, NAD-Glycohydrolase

2006 ◽  
Vol 281 (12) ◽  
pp. 8216-8223 ◽  
Author(s):  
Athanasios Michos ◽  
Ioannis Gryllos ◽  
Anders Håkansson ◽  
Amit Srivastava ◽  
Efi Kokkotou ◽  
...  
Keyword(s):  
Cytotoxic Effects ◽  
Nad Glycohydrolase ◽  
Streptolysin O ◽  
Group A

scholarly journals Streptolysin O Inhibits Clathrin-Dependent Internalization of Group A Streptococcus

mBio ◽  
2011 ◽  
Vol 2 (1) ◽  
Author(s):  
Lauren K. Logsdon ◽  
Anders P. Håkansson ◽  
Guadalupe Cortés ◽  
Michael R. Wessels
Keyword(s):  
Epithelial Cells ◽  
Group A Streptococcus ◽  
Bacterial Species ◽  
Expression System ◽  
Human Keratinocytes ◽  
Host Cells ◽  
Local Perturbation ◽  
Lysosomal Degradation ◽  
Streptolysin O ◽  
Group A

ABSTRACTGroup AStreptococcus(GAS) can be internalized by epithelial cells, including keratinocytes from human skin or pharyngeal epithelium. Internalization of GAS by epithelial cells has been postulated both to play a role in host defense and to provide a sanctuary site for GAS survival. The cholesterol-binding cytolysin streptolysin O (SLO) appears to enhance virulence in part by inhibiting GAS internalization by human keratinocytes and by disrupting the lysosomal degradation of internalized GAS. We now report that low-level production of SLO by an inducible expression system reduced GAS internalization by keratinocytes. Induced SLO expression also prevented lysosomal colocalization with intracellular bacteria and acidification of GAS-containing vacuoles. Exogenous recombinant SLO mimicked the inhibitory effect of SLO secretion on GAS entry but not that on colocalization with the lysosomal marker LAMP-1, implying that disruption of lysosomal degradation requires intracellular secretion of SLO. The internalization of SLO-negative GAS was blocked by the depletion of host cell cholesterol and by the inhibition or knocking down of the expression of clathrin or dynamin. SLO also inhibited the cellular uptake of other cargos that are internalized by clathrin-mediated uptake or by macropinocytosis. We conclude that SLO interferes with the internalization of GAS through local perturbation of the keratinocyte cell membrane and disruption of a clathrin-dependent uptake pathway.IMPORTANCEStreptolysin O (SLO) is a member of a family of pore-forming toxins, the cholesterol-dependent cytolysins, that are produced by many Gram-positive bacterial pathogens. While SLO can lyse host cells at high doses, much smaller amounts appear to contribute to pathogenesis by inhibiting the internalization of group AStreptococcus(GAS) by pharyngeal keratinocytes and by preventing efficient intracellular killing by lysosomal fusion. This study provides evidence that SLO blocks a clathrin-dependent pathway for the internalization of GAS through effects on the cell surface, whereas inhibition of lysosomal fusion depends on the intracellular production of SLO. These observations may have broader implications for understanding the pathogenesis of multiple bacterial species that produce cholesterol-dependent cytolysins.

scholarly journals The NADase-Negative Variant of the Streptococcus pyogenes Toxin NAD + Glycohydrolase Induces JNK1-Mediated Programmed Cellular Necrosis

mBio ◽  
2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Sukantha Chandrasekaran ◽  
Michael G. Caparon
Keyword(s):  
Cell Death ◽  
Epithelial Cells ◽  
Host Cell ◽  
Streptococcus Pyogenes ◽  
Host Cells ◽  
Programmed Necrosis ◽  
Nad Glycohydrolase ◽  
Streptolysin O ◽  
Different Types ◽  
Infected Cells

ABSTRACT Virulence factors are often multifunctional and contribute to pathogenesis through synergistic mechanisms. For the human pathogen Streptococcus pyogenes , two factors that act synergistically are the S. pyogenes NAD + glycohydrolase (SPN) and streptolysin O (SLO). Through distinct mechanisms, SLO forms pores in host cell membranes and translocates SPN into the host cell cytosol. Two natural variants of SPN exist, one that exhibits NADase activity and one that lacks this function, and both versions are translocated and act in concert with SLO to cause an accelerated death response in epithelial cells. While NADase + SPN is known to trigger a metabolic form of necrosis through the depletion of NAD + , the mechanism by which NADase − SPN induces cell death was unknown. In the studies described here, we examined the pathway of NADase − cell death through analysis of activation patterns of mitogen-activated protein kinases (MAPKs). S. pyogenes infection resulted in activation of members of three MAPK subfamilies (p38, ERK, and JNK). However, only JNK was activated in an SLO-specific manner. NADase − SPN induced necrosis in HeLa epithelial cells associated with depolarization of mitochondrial membranes, activation of NF-κB, and the generation of reactive oxygen species. Remarkably, RNA interference (RNAi) silencing of JNK protected cells from NADase − -SPN-mediated necrosis, suggesting that NADase − SPN triggers a form of programmed necrosis dependent on JNK signaling. Taken together, these data demonstrate that SPN acts with SLO to elicit necrosis through two different mechanisms depending on its NADase activity, i.e., metabolic (NADase + ) or programmed (NADase − ), leading to distinct inflammatory profiles. IMPORTANCE Many bacterial pathogens produce toxins that alter how infected host cells interact with the immune system. For Streptococcus pyogenes , two toxins, a NAD + glycohydrolase (SPN) and streptolysin O (SLO), act in combination to cause infected cells to die. However, there are two natural forms of SPN, and these variants cause dying cells to produce different types of signaling molecules. The NADase + form of SPN kills cells by depleting reserves of NAD + and cellular energy. The other form of SPN lacks this activity (NADase − ); thus, the mechanism by which this variant induces toxicity was unknown. Here, we show that infected cells recognize NADase − SPN through a specific signaling molecule called JNK, which causes these cells to undergo a form of cellular suicide known as programmed necrosis. This helps us to understand how different forms of toxins alter host cell signaling to help S. pyogenes cause different types of diseases.

scholarly journals Engineered Lactococcus lactis Secreting IL-23 Receptor-Targeted REX Protein Blockers for Modulation of IL-23/Th17-Mediated Inflammation

2019 ◽  
Vol 7 (5) ◽  
pp. 152 ◽  
Author(s):  
Tina Vida Plavec ◽  
Milan Kuchař ◽  
Anja Benko ◽  
Veronika Lišková ◽  
Jiří Černý ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Surface ◽  
Lactococcus Lactis ◽  
Probiotic Bacterium ◽  
Host Cells ◽  
Bacterial Surface ◽  
Cdna Sequences ◽  
Secretion Signal ◽  
Antibody Conjugate

Lactococcus lactis, a probiotic bacterium of food origin, has recently been demonstrated as a suitable strain for the production and in vivo delivery of therapeutically important proteins into the gut. We aimed to engineer recombinant L. lactis cells producing/secreting REX binding proteins that have been described as IL-23 receptor (IL-23R) blockers and IL-23R antagonists suppressing the secretion of cytokine IL-17A, a pivotal step in the T-helper Th17-mediated pro-inflammatory cascade, as well as in the development of autoimmune diseases, including inflammatory bowel disease (IBD). To reach this goal, we introduced cDNA sequences coding for REX009, REX115, and REX125 proteins into plasmid vectors carrying a Usp45 secretion signal, a FLAG tag sequence consensus, and a LysM-containing cA surface anchor (AcmA), thus allowing cell–surface peptidoglycan anchoring. These plasmids, or their non-FLAG/non-AcmA versions, were introduced into L. lactis host cells, thus generating unique recombinant L. lactis–REX strains. We demonstrate that all three REX proteins are expressed in L. lactis cells and are efficiently displayed on the bacterial surface, as tested by flow cytometry using an anti-FLAG antibody conjugate. Upon 10-fold concentration of the conditioned media, a REX125 secretory variant can be detected by Western blotting. To confirm that the FLAG/non-FLAG REX proteins displayed by L. lactis retain their binding specificity, cell-surface interactions of REX proteins with an IL-23R-IgG chimera were demonstrated by flow cytometry. In addition, statistically significant binding of secreted REX009 and REX115 proteins to bacterially produced, soluble human IL-23R was confirmed by ELISA. We conclude that REX-secreting L. lactis strains were engineered that might serve as IL-23/IL-23R blockers in an experimentally induced mouse model of colitis.

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