scholarly journals Group A Streptococcus Induces Apoptosis in Human Epithelial Cells

1999 ◽  
Vol 67 (9) ◽  
pp. 4334-4339 ◽  
Author(s):  
Pei-Jane Tsai ◽  
Yee-Shin Lin ◽  
Chih-Feng Kuo ◽  
Huan-Yao Lei ◽  
Jiunn-Jong Wu
Keyword(s):  
Cell Death ◽  
Epithelial Cells ◽  
Group A Streptococcus ◽  
Wild Type ◽  
Group A ◽  
Streptococcal Pyrogenic Exotoxin B ◽  
Induced Apoptosis ◽  
Isogenic Mutants ◽  
Type Strains ◽  
Spe B

ABSTRACT Internalization of group A streptococcus (GAS) by epithelial cells may have a role in causing invasive diseases. The purpose of this study was to examine the fate of GAS-infected epithelial cells. GAS has the ability to invade A-549 and HEp-2 cells. Both A-549 and HEp-2 cells were killed by infection with GAS. Epithelial cell death mediated by GAS was at least in part through apoptosis, as shown by changes in cellular morphology, DNA fragmentation laddering, and propidium iodide staining for hypodiploid cells. A total of 20% of A-549 cells and 11 to 13% of HEp-2 cells underwent apoptosis after 20 h of GAS infection, whereas only 1 to 2% of these cells exhibited spontaneous apoptosis. We further examined whether streptococcal pyrogenic exotoxin B (SPE B), a cysteine protease produced by GAS, was involved in the apoptosis of epithelial cells. The speB isogenic mutants had less ability to induce cell death than wild-type strains. When A-549 cells were cocultured with the mutant and SPE B for 2 h, the percentage of apoptotic cells did not increase although the number of intracellular bacteria increased to the level of wild-type strains. In addition, apoptosis was blocked by cytochalasin D treatment, which interfered with cytoskeleton function. The caspase inhibitors Z-VAD.FMK, Ac-YVAD.CMK, and Ac-DEVD.FMK inhibited GAS-induced apoptosis. These results demonstrate for the first time that GAS induces apoptosis of epithelial cells and internalization is required for apoptosis. The caspase pathway is involved in GAS-induced apoptosis, and the expression of SPE B in the cells enhances apoptosis.

scholarly journals Moraxella catarrhalis Strain O35E Expresses Two Filamentous Hemagglutinin-Like Proteins That Mediate Adherence to Human Epithelial Cells

2007 ◽  
Vol 75 (6) ◽  
pp. 2765-2775 ◽  
Author(s):  
Rachel Balder ◽  
Jonathan Hassel ◽  
Serena Lipski ◽  
Eric R. Lafontaine
Keyword(s):  
Epithelial Cells ◽  
Moraxella Catarrhalis ◽  
Gram Negative Bacteria ◽  
Gene Products ◽  
Wild Type ◽  
Recombinant Bacteria ◽  
Human Epithelial Cells ◽  
Bacterial Outer Membrane ◽  
Isogenic Mutants ◽  
Type Strains

ABSTRACT Two-partner secretion (TPS) systems are a family of proteins being rapidly identified and characterized in a growing number of gram-negative bacteria. TPS systems mediate the secretion of proteins, many involved in virulence traits such as hemolysis, adherence to epithelial cells, inhibition of bacterial growth, and immunomodulation of the host. A TPS system typically consists of a transporter located in the bacterial outer membrane (OM) which is responsible for the recognition and secretion of at least one large exoprotein. Two of the better-characterized TPS systems specify the Bordetella pertussis FHA and Haemophilus influenzae HMW1/HMW2 proteins. We identified three gene products of Moraxella catarrhalis strain O35E that resemble TPS proteins and designated them MhaC (transporter), MhaB1 (exoprotein), and MhaB2 (exoprotein). Western blot analysis using anti-MhaC, or antibodies reacting to both MhaB1 and MhaB2 (MhaB-reactive), revealed that these antigens are expressed in the OM of 63% of isolates tested. Mutations in the mhaC gene specifying the putative transporter of the M. catarrhalis wild-type strains O35E, O12E, and McGHS1 resulted in the absence of MhaB1/MhaB2 in the OM of mutants. These results are therefore consistent with the Mha proteins functioning as a TPS system. Furthermore, we discovered that these mhaC mutants exhibit markedly decreased binding to human epithelial cells relevant to pathogenesis by M. catarrhalis (Chang, HEp2, A549, and/or 16HBE14o−). Expression of O12E MhaC and MhaB1 in a nonadherent strain of Escherichia coli was found to increase the adherence of recombinant bacteria to HEp2 monolayers by sevenfold, thereby demonstrating that this M. catarrhalis TPS system directly mediates binding to human epithelial cells. The construction of isogenic mutants in the mhaB1 and mhaB2 genes of strain O35E also suggests that the MhaB proteins play distinct roles in M. catarrhalis adherence.

scholarly journals Role of CsrR, Hyaluronic Acid, and SpeB in the Internalization of Streptococcus pyogenes M Type 3 Strain by Epithelial Cells

2002 ◽  
Vol 70 (2) ◽  
pp. 462-469 ◽  
Author(s):  
Jeries Jadoun ◽  
Osnat Eyal ◽  
Shlomo Sela
Keyword(s):  
Hyaluronic Acid ◽  
Epithelial Cells ◽  
Group A Streptococcus ◽  
Acid Synthesis ◽  
Complex Interactions ◽  
Bacterial Uptake ◽  
Group A ◽  
Isogenic Mutants ◽  
Hyaluronic Acid Capsule ◽  
Type 3

ABSTRACT Internalization of group A streptococcus by human epithelial cells has been extensively studied during the past 6 years. It is now clear that multiple mechanisms are involved in this process. We have previously demonstrated that the CsrR global regulator controls the internalization of an invasive M type 3 strain through regulation of the has (hyaluronic acid synthesis) operon, as well as another, unknown gene(s). Recently, it was reported that the CsrR-regulated cysteine protease (SpeB) is also involved in bacterial uptake. In this study we have examined the roles of CsrR, hyaluronic acid capsule, and SpeB in streptococcal internalization. We have constructed isogenic mutants of the M3 serotype deficient in the csrR, hasA, and speB genes and tested their ability to be internalized by HEp-2 epithelial cells. Inactivation of csrR abolished internalization, while inactivation of either hasA or speB increased the internalization efficiency. Mutation in csrR derepressed hasA transcription and lowered the activity of SpeB, while no effect on speB transcription was observed. The speB mutant expressed smaller amounts of capsule, while the hasA mutant transcribed more csrR and speB mRNAs. Thus, it seems that complex interactions between CsrR, SpeB, and capsule are involved in modulation of group A streptococcus internalization.

scholarly journals Effect of Group A Streptococcal Cysteine Protease on Invasion of Epithelial Cells

1998 ◽  
Vol 66 (4) ◽  
pp. 1460-1466 ◽  
Author(s):  
Pei-Jane Tsai ◽  
Chih-Feng Kuo ◽  
Kuei-Yuan Lin ◽  
Yee-Shin Lin ◽  
Huan-Yao Lei ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cysteine Protease ◽  
Protease Activity ◽  
Extracellular Proteins ◽  
Wild Type ◽  
Respiratory Epithelial Cells ◽  
Group A ◽  
Human Respiratory Epithelial Cells ◽  
Respiratory Epithelial ◽  
Type Strains

ABSTRACT Cysteine protease of group A streptococci (GAS) is considered an important virulence factor. However, its role in invasiveness of GAS has not been investigated. We demonstrated in this study that two strains of protease-producing GAS had the ability to invade A-549 human respiratory epithelial cells. Isogenic protease mutants were constructed by using integrational plasmids to disrupt thespeB gene and confirmed by Southern hybridization and Western immunoblot analyses. No extracellular protease activity was produced by the mutants. The mutants had growth rates similar to those of the wild-type strains and produced normal levels of other extracellular proteins. When invading A-549 cells, the mutants had a two- to threefold decrease in activity compared to that of the wild-type strains. The invasion activity increased when the A-549 cells were incubated with purified cysteine protease and the mutant. However, blockage of the cysteine protease with a specific cysteine protease inhibitor, E-64, decreased the invasion activity of GAS. Intracellular growth of GAS was not found in A-549 cells. The presence or absence of protease activity did not affect the adhesive ability of GAS. These results suggested that streptococcal cysteine protease can enhance the invasion ability of GAS in human respiratory epithelial cells.

scholarly journals Degradation of Complement 3 by Streptococcal Pyrogenic Exotoxin B Inhibits Complement Activation and Neutrophil Opsonophagocytosis

2008 ◽  
Vol 76 (3) ◽  
pp. 1163-1169 ◽  
Author(s):  
Chih-Feng Kuo ◽  
Yee-Shin Lin ◽  
Woei-Jer Chuang ◽  
Jiunn-Jong Wu ◽  
Nina Tsao
Keyword(s):  
Complement Activation ◽  
Group A Streptococcus ◽  
Normal Serum ◽  
Enzyme Linked Immunosorbent Assay ◽  
Alternative Complement ◽  
Isogenic Mutant Strain ◽  
Group A ◽  
Streptococcal Pyrogenic Exotoxin B ◽  
Important Virulence Factor ◽  
Spe B

ABSTRACT Streptococcal pyrogenic exotoxin B (SPE B), a cysteine protease, is an important virulence factor in group A streptococcus (GAS) infection. The inhibition of phagocytic activity by SPE B may help prevent bacteria from being ingested. In this study, we examined the mechanism SPE B uses to enable bacteria to resist opsonophagocytosis. Using an enzyme-linked immunosorbent assay, we found that SPE B-treated serum impaired the activation of the classical, the lectin, and the alternative complement pathways. In contrast, C192S, a SPE B mutant lacking protease activity, had no effect on complement activation. Further study showed that cleavage of serum C3 by SPE B, but not C192S, blocked zymosan-induced production of reactive oxygen species in neutrophils as a result of decreased deposition of C3 fragments on the zymosan surface. Reconstitution of C3 into SPE B-treated serum unblocked zymosan-mediated neutrophil activation dose dependently. SPE B-treated, but not C192S-treated, serum also impaired opsonization of C3 fragments on the surface of GAS strain A20. Moreover, the amount of C3 fragments on the A20 cell surface, a SPE B-producing strain, was less than that on its isogenic mutant strain, SW507, after opsonization with normal serum. A20 opsonized with SPE B-treated serum was more resistant to neutrophil killing than A20 opsonized with normal serum, and SPE B-mediated resistance was C3 dependent. These results suggest a novel SPE B mechanism, one which degrades serum C3 and enables GAS to resist complement damage and opsonophagocytosis.

scholarly journals The Streptococcus pyogenes Capsule Is Required for Adhesion of Bacteria to Virus-Infected Alveolar Epithelial Cells and Lethal Bacterial-Viral Superinfection

2004 ◽  
Vol 72 (10) ◽  
pp. 6068-6075 ◽  
Author(s):  
Shigefumi Okamoto ◽  
Shigetada Kawabata ◽  
Yutaka Terao ◽  
Hideaki Fujitaka ◽  
Yoshinobu Okuno ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Influenza A ◽  
Alveolar Epithelial Cells ◽  
Invasive Group ◽  
Alveolar Epithelial ◽  
Streptolysin S ◽  
Streptolysin O ◽  
Group A ◽  
Streptococcal Pyrogenic Exotoxin B ◽  
Isogenic Mutants

ABSTRACT An apparent worldwide resurgence of invasive group A Streptococcus (GAS) infections remains unexplained. However, we recently demonstrated in mice that when an otherwise nonlethal intranasal GAS infection is preceded by a nonlethal influenza A virus (IAV) infection, induction of lethal invasive GAS infections is often the result. In the present study, we established several isogenic mutants from a GAS isolate and evaluated several virulence factors as candidates responsible for the induction of invasive GAS infections. Disruption of the synthesis of the capsule, Mga, streptolysin O, streptolysin S, or streptococcal pyrogenic exotoxin B of GAS significantly reduced mortality among mice superinfected with IAV and a mutant. In addition, the number of GAS organisms adhering to IAV-infected alveolar epithelial cells was markedly reduced with the capsule-depleted mutant, although this was not the case with the other mutants. Wild-type GAS was found to bind directly to IAV particles, whereas the nonencapsulated mutant showed much less ability to bind. These results suggest that the capsule plays a key role in the invasion of host tissues by GAS following superinfection with IAV and GAS.

scholarly journals Biofilm formation by group A Streptococcus: a role for the streptococcal regulator of virulence (Srv) and streptococcal cysteine protease (SpeB)

Microbiology ◽  
2009 ◽  
Vol 155 (1) ◽  
pp. 46-52 ◽  
Author(s):  
Christopher D. Doern ◽  
Amity L. Roberts ◽  
Wenzhou Hong ◽  
Jessica Nelson ◽  
Slawomir Lukomski ◽  
...  
Keyword(s):  
Cysteine Protease ◽  
Biofilm Formation ◽  
Group A Streptococcus ◽  
Immunoblot Analysis ◽  
Wild Type ◽  
Flow Conditions ◽  
Group A ◽  
Streptococcus A ◽  
Insight Into ◽  
Western Immunoblot Analysis

Recently, biofilms have become a topic of interest in the study of the human pathogen group A Streptococcus (GAS). In this study, we sought to learn more about the make-up of these structures and gain insight into biofilm regulation. Enzymic studies indicated that biofilm formation by GAS strain MGAS5005 required an extracellular protein and DNA component(s). Previous results indicated that inactivation of the transcriptional regulator Srv in MGAS5005 resulted in a significant decrease in virulence. Here, inactivation of Srv also resulted in a significant decrease in biofilm formation under both static and flow conditions. Given that production of the extracellular cysteine protease SpeB is increased in the srv mutant, we tested the hypothesis that increased levels of active SpeB may be responsible for the reduction in biofilm formation. Western immunoblot analysis indicated that SpeB was absent from MGAS5005 biofilms. Complementation of MGAS5005Δsrv restored the biofilm phenotype and eliminated the overproduction of active SpeB. Inhibition of SpeB with E64 also restored the MGAS5005Δsrv biofilm to wild-type levels.

scholarly journals Insufficient Acidification of Autophagosomes Facilitates Group A Streptococcus Survival and Growth in Endothelial Cells

mBio ◽  
2015 ◽  
Vol 6 (5) ◽  
Author(s):  
Shiou-Ling Lu ◽  
Chih-Feng Kuo ◽  
Hao-Wen Chen ◽  
Yi-Shuan Yang ◽  
Ching-Chuan Liu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Epithelial Cells ◽  
Bacterial Growth ◽  
Group A Streptococcus ◽  
Multiorgan Failure ◽  
Specific Inhibitor ◽  
Low Ph ◽  
Bafilomycin A1 ◽  
Group A

ABSTRACTGroup A streptococcus (GAS) is an important human pathogen, and its invasion via blood vessels is critically important in serious events such as bacteremia or multiorgan failure. Although GAS was identified as an extracellular bacterium, the internalization of GAS into nonphagocytic cells may provide a strategy to escape from immune surveillance and antibiotic killing. However, GAS has also been reported to induce autophagy and is efficiently killed within lysosome-fused autophagosomes in epithelial cells. In this study, we show that GAS can replicate in endothelial cells and that streptolysin O is required for GAS growth. Bacterial replication can be suppressed by altering GAS gene expression in an acidic medium before internalization into endothelial cells. The inhibitory effect on GAS replication can be reversed by treatment with bafilomycin A1, a specific inhibitor of vacuolar-type H+-ATPase. Compared with epithelial cells in which acidification causes autophagy-mediated clearance of GAS, there was a defect in acidification of GAS-containing vesicles in endothelial cells. Consequently, endothelial cells fail to maintain low pH in GAS-containing autophagosomes, thereby permitting GAS replication inside LAMP-1- and LC3-positive vesicles. Furthermore, treatment of epithelial cells with bafilomycin A1 resulted in defective GAS clearance by autophagy, with subsequent bacterial growth intracellularly. Therefore, low pH is a key factor for autophagy-mediated suppression of GAS growth inside epithelial cells, while defective acidification of GAS-containing vesicles results in bacterial growth in endothelial cells.IMPORTANCEPrevious reports showed that GAS can induce autophagy and is efficiently killed within lysosome-fused autophagosomes in epithelial cells. In endothelial cells, in contrast, induction of autophagy is not sufficient for GAS killing. In this study, we provide the first evidence that low pH is required to prevent intracellular growth of GAS in epithelial cells and that this mechanism is defective in endothelial cells. Treatment of GAS with low pH altered GAS growth rate and gene expression of virulence factors and resulted in enhanced susceptibility of GAS to intracellular lysosomal killing. Our findings reveal the existence of different mechanisms of host defense against GAS invasion between epithelial and endothelial cells.

Autophagy is associated with apoptosis in cisplatin injury to renal tubular epithelial cells

2008 ◽  
Vol 294 (4) ◽  
pp. F777-F787 ◽  
Author(s):  
Cheng Yang ◽  
Varsha Kaushal ◽  
Sudhir V. Shah ◽  
Gur P. Kaushal
Keyword(s):  
Cell Death ◽  
Epithelial Cells ◽  
Beclin 1 ◽  
Lag Phase ◽  
Tubular Epithelial Cells ◽  
Renal Tubular Epithelial Cells ◽  
Normal Cells ◽  
Life And Death ◽  
Renal Tubular ◽  
Induced Apoptosis

Autophagy has emerged as another major “programmed” mechanism to control life and death much like “programmed cell death” is for apoptosis in eukaryotes. We examined the expression of autophagic proteins and formation of autophagosomes during progression of cisplatin injury to renal tubular epithelial cells (RTEC). Autophagy was detected as early as 2–4 h after cisplatin exposure as indicated by induction of LC3-I, conversion of LC3-I to LC3-II protein, and upregulation of Beclin 1 and Atg5, essential markers of autophagy. The appearance of cisplatin-induced punctated staining of autophagosome-associated LC3-II upon GFP-LC3 transfection in RTEC provided further evidence for autophagy. The autophagy inhibitor 3-methyladenine blocked punctated staining of autophagosomes. The staining of normal cells with acridine orange displayed green fluorescence with cytoplasmic and nuclear components in normal cells but displayed considerable red fluorescence in cisplatin-treated cells, suggesting formation of numerous acidic autophagolysosomal vacuoles. Autophagy inhibitors LY294002 or 3-methyladenine or wortmannin inhibited the formation of autophagosomes but induced apoptosis after 2–4 h of cisplatin treatment as indicated by caspase-3/7 and -6 activation, nuclear fragmentation, and cell death. This switch from autophagy to apoptosis by autophagic inhibitors further suggests that the preapoptotic lag phase after treatment with cisplatin is mediated by autophagy. At later stages of cisplatin injury, apoptosis was also found to be associated with autophagy, as autophagic inhibitors and inactivation of autophagy proteins Beclin 1 and Atg5 enhanced activation of caspases and apoptosis. Our results demonstrate that induction of autophagy mounts an adaptive response, suppresses cisplatin-induced apoptosis, and prolongs survival of RTEC.

scholarly journals M1 Protein Triggers a Phosphoinositide Cascade for Group A Streptococcus Invasion of Epithelial Cells

2003 ◽  
Vol 71 (10) ◽  
pp. 5823-5830 ◽  
Author(s):  
Sai Sudha Purushothaman ◽  
Beinan Wang ◽  
P. Patrick Cleary
Keyword(s):  
Epithelial Cells ◽  
Group A Streptococcus ◽  
Dominant Negative ◽  
M Protein ◽  
Lipid Kinase ◽  
Cytoskeletal Rearrangement ◽  
M1 Protein ◽  
Serovar Typhimurium ◽  
Group A

ABSTRACT Invasion of nonphagocytic cells by bacteria provides a favorable niche for persistence and evasion of host defenses and antibiotics. M protein is a major virulence factor because it promotes high-frequency invasion of epithelial cells by group A Streptococcus (GAS) and also renders the bacterium resistant to phagocytosis. In this study, we investigated the role of M1 protein from serotype M1 strain 90-226 in regulating mammalian signal transduction and cytoskeletal rearrangement for bacterial entry. LY294002 and wortmannin, which are inhibitors of phosphatidylinositol 3-kinase (PI 3-K) blocked invasion of epithelial cells by GAS by 75 and 80%, respectively, but failed to inhibit invasion by Salmonella enterica serovar Typhimurium. Also, epithelial cells transiently transfected with dominant negative p85 and p110 genes, the regulatory and catalytic subunits of PI 3-K, respectively, were less able to be invaded by GAS. To separate the influence of other streptococcal virulence factors from M protein, Lactococcus lactis was engineered to express M1 protein on its surface. L. lactis(pLM1) invaded epithelial cells efficiently in vitro, and PI 3-K inhibitors blocked 90% of this invasion. Purified soluble M1 protein stimulated the formation of stress fibers and actin tuffs on epithelial cells. LY294002 and wortmannin inhibited these cellular changes. A phosphoinositide analogue also inhibited the invasion of epithelial cells by GAS. Therefore, M1 protein, either directly or via bound fibronectin, initiates signals that depend on the lipid kinase PI 3-K pathway, which paves the way for cytoskeletal rearrangement that internalize the bacterium.

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