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.

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 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 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.

Interaction Between the Microbiota, Epithelia, and Immune Cells in the Intestine

2020 ◽  
Vol 38 (1) ◽  
pp. 23-48 ◽  
Author(s):  
Hisako Kayama ◽  
Ryu Okumura ◽  
Kiyoshi Takeda
Keyword(s):  
Epithelial Cells ◽  
Intestinal Microbiota ◽  
Immune Cells ◽  
Oral Tolerance ◽  
Host Cells ◽  
Epithelial Barrier ◽  
Therapeutic Approaches ◽  
Host Health ◽  
Physical Barriers ◽  
Dietary Components

The gastrointestinal tract harbors numerous commensal bacteria, referred to as the microbiota, that benefit host health by digesting dietary components and eliminating pathogens. The intestinal microbiota maintains epithelial barrier integrity and shapes the mucosal immune system, balancing host defense and oral tolerance with microbial metabolites, components, and attachment to host cells. To avoid aberrant immune responses, epithelial cells segregate the intestinal microbiota from immune cells by constructing chemical and physical barriers, leading to the establishment of host-commensal mutualism. Furthermore, intestinal immune cells participate in the maintenance of a healthy microbiota community and reinforce epithelial barrier functions. Perturbations of the microbiota composition are commonly observed in patients with autoimmune diseases and chronic inflammatory disorders. An understanding of the intimate interactions between the intestinal microbiota, epithelial cells, and immune cells that are crucial for the maintenance of intestinal homeostasis might promote advances in diagnostic and therapeutic approaches for various diseases.

scholarly journals In VitroPrevention ofSalmonellaLipopolysaccharide-Induced Damages in Epithelial Barrier Function by VariousLactobacillusStrains

2013 ◽  
Vol 2013 ◽  
pp. 1-6 ◽  
Author(s):  
Chun-Yan Yeung ◽  
Jen-Shiu Chiang Chiau ◽  
Wai-Tao Chan ◽  
Chun-Bin Jiang ◽  
Mei-Lien Cheng ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Tight Junction ◽  
Mononuclear Cells ◽  
Intestinal Epithelial Cells ◽  
Barrier Properties ◽  
Bacterial Invasion ◽  
Epithelial Barrier ◽  
Intestinal Epithelial ◽  
Peripheral Blood Mononuclear

Background.Lactobacillusshows beneficial anti-inflammatory effects onSalmonellainfection. The maintenance of the tight junction (TJ) integrity plays an importance role in avoiding bacterial invasion. WhetherLactobacilluscould be used to regulate the TJ protein expression and distribution in inflamed intestinal epithelial cells was determined.Methods. Using the transwell coculture model,Salmonellalipopolysaccharide (LPS) was apically added to polarized Caco-2 cells cocultured with peripheral blood mononuclear cells in the basolateral compartment. LPS-stimulated Caco-2 cells were incubated with variousLactobacillusstrains. TJ integrity was determined by measuring transepithelial electrical resistance across Caco-2 monolayer. Expression and localization of TJ proteins (zonula-occludens- (ZO-) 1) were determined by Western blot and immunofluorescence microscopy.Results. Various strains ofLactobacilluswere responsible for the different modulations of cell layer integrity. LPS was specifically able to disrupt epithelial barrier and change the location of ZO-1. Our data demonstrate thatLactobacilluscould attenuate the barrier disruption of intestinal epithelial cells caused bySalmonellaLPS administration. We showed thatLactobacillusstrains are associated with the maintenance of the tight junction integrity and appearance.Conclusion. In this study we provide insight that live probiotics could improve epithelial barrier properties and this may explain the potential mechanism behind their beneficial effectin vivo.

scholarly journals Salmonella methylglyoxal detoxification by STM3117-encoded lactoylglutathione lyase affects virulence in coordination with Salmonella pathogenicity island 2 and phagosomal acidification

Microbiology ◽  
2014 ◽  
Vol 160 (9) ◽  
pp. 1999-2017 ◽  
Author(s):  
Sangeeta Chakraborty ◽  
Debalina Chaudhuri ◽  
Arjun Balakrishnan ◽  
Dipshikha Chakravortty
Keyword(s):  
Epithelial Cells ◽  
Virulence Factors ◽  
Oxidative Dna Damage ◽  
Ectopic Expression ◽  
Pathogenicity Island ◽  
Host Cells ◽  
Metabolic Enzyme ◽  
Pathogenicity Islands ◽  
Phagocytic Cells ◽  
Potassium Efflux

Intracellular pathogens such as Salmonella enterica serovar Typhimurium (S. Typhimurium) manipulate their host cells through the interplay of various virulence factors. A multitude of such virulence factors are encoded on the genome of S. Typhimurium and are usually organized in pathogenicity islands. The virulence-associated genomic stretch of STM3117–3120 has structural features of pathogenicity islands and is present exclusively in non-typhoidal serovars of Salmonella. It encodes metabolic enzymes predicted to be involved in methylglyoxal metabolism. STM3117-encoded lactoylglutathione lyase significantly impacts the proliferation of intracellular Salmonella. The deletion mutant of STM3117 (Δlgl) fails to grow in epithelial cells but hyper-replicates in macrophages. This difference in proliferation outcome was the consequence of failure to detoxify methylglyoxal by Δlgl, which was also reflected in the form of oxidative DNA damage and upregulation of kefB in the mutant. Within macrophages, the toxicity of methylglyoxal adducts elicits the potassium efflux channel (KefB) in the mutant which subsequently modulates the acidification of mutant-containing vacuoles (MCVs). The perturbation in the pH of the MCV milieu and bacterial cytosol enhances the Salmonella pathogenicity island 2 translocation in Δlgl, increasing its net growth within macrophages. In epithelial cells, however, the maturation of Δlgl-containing vacuoles were affected as these non-phagocytic cells maintain less acidic vacuoles compared to those in macrophages. Remarkably, ectopic expression of Toll-like receptors 2 and 4 on epithelial cells partially restored the survival of Δlgl. This study identified a novel metabolic enzyme in S. Typhimurium whose activity during intracellular infection within a given host cell type differentially affected the virulence of the bacteria.

scholarly journals Emergence of a New Highly Successful Acapsular Group A Streptococcus Clade of Genotype emm89 in the United Kingdom

mBio ◽  
2015 ◽  
Vol 6 (4) ◽  
Author(s):  
Claire E. Turner ◽  
James Abbott ◽  
Theresa Lamagni ◽  
Matthew T. G. Holden ◽  
Sophia David ◽  
...  
Keyword(s):  
United Kingdom ◽  
Hyaluronic Acid ◽  
Genome Sequencing ◽  
Virulence Factors ◽  
Invasive Disease ◽  
Whole Genome ◽  
The United Kingdom ◽  
Streptolysin O ◽  
Group A ◽  
Hyaluronic Acid Capsule

ABSTRACTGroup AStreptococcus(GAS) genotypeemm89 is increasingly recognized as a leading cause of disease worldwide, yet factors that underlie the success of thisemmtype are unknown. Surveillance identified a sustained nationwide increase inemm89 invasive GAS disease in the United Kingdom, prompting longitudinal investigation of this genotype. Whole-genome sequencing revealed a recent dramatic shift in theemm89 population with the emergence of a new clade that increased to dominance over previousemm89 variants. Temporal analysis indicated that the clade arose in the early 1990s but abruptly increased in prevalence in 2008, coinciding with an increased incidence ofemm89 infections. Although standard variable typing regions (emmsubtype,teetype,softype, and multilocus sequence typing [MLST]) remained unchanged, uniquely the emergent clade had undergone six distinct regions of homologous recombination across the genome compared to the rest of the sequencedemm89 population. Two of these regions affected known virulence factors, the hyaluronic acid capsule and the toxins NADase and streptolysin O. Unexpectedly, and in contrast to the rest of the sequencedemm89 population, the emergent clade-associated strains were genetically acapsular, rendering them unable to produce the hyaluronic acid capsule. The emergent clade-associated strains had also acquired an NADase/streptolysin O locus nearly identical to that found inemm12 and modernemm1 strains but different from the rest of the sequencedemm89 population. The emergent clade-associated strains had enhanced expression of NADase and streptolysin O. The genome remodeling in the new clade variant and the resultant altered phenotype appear to have conferred a selective advantage over otheremm89 variants and may explain the changes observed inemm89 GAS epidemiology.IMPORTANCESudden upsurges or epidemic waves are common features of group A streptococcal disease. Although the mechanisms behind such changes are largely unknown, they are often associated with an expansion of a single genotype within the population. Using whole-genome sequencing, we investigated a nationwide increase in invasive disease caused by the genotypeemm89 in the United Kingdom. We identified a new clade variant that had recently emerged in theemm89 population after having undergone several core genomic recombination-related changes, two of which affected known virulence factors. An unusual finding of the new variant was the loss of the hyaluronic acid capsule, previously thought to be essential for causing invasive disease. A further genomic adaptation in the NADase/streptolysin O locus resulted in enhanced production of these toxins. Recombination-related genome remodeling is clearly an important mechanism in group AStreptococcusthat can give rise to more successful and potentially more pathogenic variants.

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