Streptolysin O and its Co-Toxin NAD-glycohydrolase Protect Group A Streptococcus from Xenophagic Killing

ABSTRACT A search for homologs of the Bacillus subtilis PhoP response regulator in the group A streptococcus (GAS) genome revealed three good candidates. Inactivation of one of these, recently identified as csrR (J. C. Levin and M. R. Wessels, Mol. Microbiol. 30:209–219, 1998), caused the strain to produce mucoid colonies and to increase transcription ofhasA, the first gene in the operon for capsule synthesis. We report here that a nonpolar insertion in this gene also increased transcription of ska (encoding streptokinase),sagA (streptolysin S), and speMF (mitogenic factor) but did not affect transcription of slo(streptolysin O), mga (multiple gene regulator of GAS),emm (M protein), scpA (complement C5a peptidase), or speB or speC (pyrogenic exotoxins B and C). The amounts of streptokinase, streptolysin S, and capsule paralleled the levels of transcription of their genes in all cases. Because CsrR represses genes unrelated to those for capsule synthesis, and because CsrA-CsrB is a global regulatory system inEscherichia coli whose mechanism is unrelated to that of these genes in GAS, the locus has been renamed covR, for “control of virulence genes” in GAS. Transcription of thecovR operon was also increased in the nonpolar insertion mutant, indicating that CovR represses its own synthesis as well. All phenotypes of the covR nonpolar insertion mutant were complemented by the covR gene on a plasmid. CovR acts on operons expressed both in exponential and in stationary phase, demonstrating that the CovR-CovS pathway is separate from growth phase-dependent regulation in GAS. Therefore, CovR is the first multiple-gene repressor of virulence factors described for this important human pathogen.

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

mBio ◽

10.1128/mbio.01974-20 ◽

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.

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NAD-Glycohydrolase Production and speA and speC Distribution in Group A Streptococcus (GAS) Isolates Do Not Correlate with Severe GAS Diseases in the Australian Population

Journal of Clinical Microbiology ◽

10.1128/jcm.40.7.2642-2644.2002 ◽

2002 ◽

Vol 40 (7) ◽

pp. 2642-2644 ◽

Cited By ~ 11

Author(s):

A. DelVecchio ◽

M. Maley ◽

B. J. Currie ◽

K. S. Sriprakash

Keyword(s):

Group A Streptococcus ◽

Australian Population ◽

Nad Glycohydrolase ◽

Group A

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

Journal of Biological Chemistry ◽

10.1074/jbc.m511674200 ◽

2006 ◽

Vol 281 (12) ◽

pp. 8216-8223 ◽

Cited By ~ 44

Author(s):

Athanasios Michos ◽

Ioannis Gryllos ◽

Anders Håkansson ◽

Amit Srivastava ◽

Efi Kokkotou ◽

...

Keyword(s):

Cytotoxic Effects ◽

Nad Glycohydrolase ◽

Streptolysin O ◽

Group A

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

Open Forum Infectious Diseases ◽

10.1093/ofid/ofz360.2268 ◽

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.

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Streptolysin O Rapidly Impairs Neutrophil Oxidative Burst and Antibacterial Responses to Group A Streptococcus

Frontiers in Immunology ◽

10.3389/fimmu.2015.00581 ◽

2015 ◽

Vol 6 ◽

Cited By ~ 37

Author(s):

Satoshi Uchiyama ◽

Simon Döhrmann ◽

Anjuli M. Timmer ◽

Neha Dixit ◽

Mariam Ghochani ◽

...

Keyword(s):

Oxidative Burst ◽

Group A Streptococcus ◽

Streptolysin O ◽

Group A ◽

Neutrophil Oxidative Burst

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

10.1101/2020.07.16.207894 ◽

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.

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Streptolysin O Inhibits Clathrin-Dependent Internalization of Group A Streptococcus

mBio ◽

10.1128/mbio.00332-10 ◽

2011 ◽

Vol 2 (1) ◽

Cited By ~ 19

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.

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