scholarly journals Allele Substitution of the Streptokinase Gene Reduces the Nephritogenic Capacity of Group A Streptococcal Strain NZ131

2000 ◽  
Vol 68 (3) ◽  
pp. 1019-1025 ◽  
Author(s):  
Annika Nordstrand ◽  
W. Michael McShan ◽  
Joseph J. Ferretti ◽  
Stig E. Holm ◽  
Mari Norgren
Keyword(s):  
Allelic Variant ◽  
Wild Type ◽  
Poststreptococcal Glomerulonephritis ◽  
Acute Poststreptococcal Glomerulonephritis ◽  
Site Specific Integration ◽  
Allele Substitution ◽  
Group A ◽  
Type Strains ◽  
Streptococcal Strain

ABSTRACT To investigate the role of allelic variants of streptokinase in the pathogenesis of acute poststreptococcal glomerulonephritis (APSGN), site-specific integration plasmids were constructed, which contained either the non-nephritis-associated streptokinase gene (skc5) from the group C streptococcal strainStreptococcus equisimilis H46A or the nephritis-associated streptokinase gene (ska1) from the group A streptococcal nephritogenic strain NZ131. The plasmids were introduced by electroporation and homologous recombination into the chromosome of an isogenic derivative of strain NZ131, in which the streptokinase gene had been deleted and which had thereby lost its nephritogenic capacity in a mouse model of APSGN. The introduction of a non-nephritis-associated allelic variant of streptokinase did not rescue the nephritogenic capacity of the strain. The mutant and the wild-type strains produced equivalent amounts of streptokinase. Complementation of the ska deletion derivative with the original ska allele reconstituted the nephritogenicity of wild-type NZ131. The findings support the hypothesis that the role of streptokinase in the pathogenesis of APSGN is related to the allelic variant of the protein.

scholarly journals Purification and partial characterization of the nephritis strain-associated protein from Streptococcus pyogenes, group A.

1986 ◽  
Vol 163 (3) ◽  
pp. 697-712 ◽  
Author(s):  
K H Johnston ◽  
J B Zabriskie
Keyword(s):  
Biochemical Analysis ◽  
Poststreptococcal Glomerulonephritis ◽  
Isolation And Purification ◽  
Amino Terminal ◽  
Acute Poststreptococcal Glomerulonephritis ◽  
Group A ◽  
Terminal Amino

We report the isolation and purification of the nephritis strain-associated protein (NSAP) first described by Villareal et al. (8). Amino acid analysis, and determination of the first 21 amino-terminal amino acids indicated that this 46 kD protein is a streptokinase. Biochemical analysis confirmed that NSAP could act as a plasminogen activator; immunological investigations indicated that NSAP is antigenically different from streptokinase from group C streptococcus, and possibly represents a unique streptokinase. It is this uniqueness that may contribute to the role of NSAP in the pathogenesis of acute poststreptococcal glomerulonephritis.

scholarly journals Role of Group A Streptococcal Virulence Factors in Adherence to Keratinocytes

2000 ◽  
Vol 68 (3) ◽  
pp. 1215-1221 ◽  
Author(s):  
Gary L. Darmstadt ◽  
Laurel Mentele ◽  
Andreas Podbielski ◽  
Craig E. Rubens
Keyword(s):  
Hyaluronic Acid ◽  
Mutant Strain ◽  
Virulence Factors ◽  
M Protein ◽  
Skin Infections ◽  
Wild Type ◽  
Group A ◽  
Hyaluronic Acid Capsule ◽  
Type Strains

ABSTRACT To evaluate the role of putative group A streptococcal virulence factors in the initiation of skin infections, we compared the adherence of a wild-type M49-protein skin-associated strain to that of a series of 16 isogenic mutants created by insertional inactivation of virulence genes. None of the mutants, including the M-protein-deficient (emm mutant) strain, displayed reduced adherence to early-passage cultured human keratinocytes, but adherence of the mutant lacking hyaluronic acid capsule expression (has mutant) was increased 13-fold. In contrast, elimination of capsule expression in M2-, M3-, and M18-protein has mutants increased adherence only slightly (1.3- to 2.3-fold) compared to their respective wild-type strains. A mutant with inactivation of both emm andhas displayed high-level adherence (34.9 ± 4.1%) equal to that of the has mutant strain (40.7 + 8.0%), confirming the lack of involvement of M49 protein in attachment. Moreover, adherence of the M49-protein-deficient (emmmutant) and wild-type strains was increased to the same level (57 and 55%, respectively) following enzymatic digestion of their hyaluronic acid capsule. Adherence of mutants lacking oligopeptide permease (Opp) expression was increased 3.8- to 5.5-fold, in association with decreased cell-associated hyaluronic acid capsule. Finally, soluble CD46 failed to inhibit adherence of M49- and M52-serotype skin strains. We conclude that (i) bacterial M protein and keratinocyte CD46 do not mediate adherence of M49 skin-associated Streptococcus pyogenes to epidermal keratinocytes, (ii) hyaluronic acid capsule impedes the interaction of bacterial adhesins with keratinocyte receptors, (iii) modulation of capsule expression may be important in the pathogenesis of skin infections, and (iv) the molecular interactions in attachment of skin strains of S. pyogenesto keratinocytes are unique and remain unidentified.

Characterization of the Role of the FluG Protein in Asexual Development of Aspergillus nidulans

Genetics ◽  
2001 ◽  
Vol 158 (3) ◽  
pp. 1027-1036 ◽  
Author(s):  
Cletus A D'Souza ◽  
Bee Na Lee ◽  
Thomas H Adams
Keyword(s):  
Aspergillus Nidulans ◽  
Negative Influence ◽  
Asexual Development ◽  
Wild Type ◽  
Significant Similarity ◽  
Developmental Defects ◽  
Asexual Sporulation ◽  
Type Strains

Abstract We showed previously that a ΔfluG mutation results in a block in Aspergillus nidulans asexual sporulation and that overexpression of fluG activates sporulation in liquid-submerged culture, a condition that does not normally support sporulation of wild-type strains. Here we demonstrate that the entire N-terminal region of FluG (∼400 amino acids) can be deleted without affecting sporulation, indicating that FluG activity resides in the C-terminal half of the protein, which bears significant similarity with GSI-type glutamine synthetases. While FluG has no apparent role in glutamine biosynthesis, we propose that it has an enzymatic role in sporulation factor production. We also describe the isolation of dominant suppressors of ΔfluG(dsg) that should identify components acting downstream of FluG and thereby define the function of FluG in sporulation. The dsgA1 mutation also suppresses the developmental defects resulting from ΔflbA and dominant activating fadA mutations, which both cause constitutive induction of the mycelial proliferation pathway. However, dsgA1 does not suppress the negative influence of these mutations on production of the aflatoxin precursor, sterigmatocystin, indicating that dsgA1 is specific for asexual development. Taken together, our studies define dsgA as a novel component of the asexual sporulation pathway.

Immune complex binding Streptococcus pyogenes type M12/emm12 in experimental glomerulonephritis

2013 ◽  
Vol 62 (9) ◽  
pp. 1272-1280 ◽  
Author(s):  
Larissa Burova ◽  
Peter Pigarevsky ◽  
Nadezhda Duplik ◽  
Vlada Snegova ◽  
Alexander Suvorov ◽  
...  
Keyword(s):  
Rabbit Model ◽  
Complement C3 ◽  
Poststreptococcal Glomerulonephritis ◽  
Tnf Α ◽  
Rabbit Igg ◽  
Renal Changes ◽  
Group A ◽  
Study Type ◽  
Binding Component

In a rabbit model, we have previously reported evidence for a pathogenic role of streptococcal IgG Fc-binding proteins (IgGFcBP) in poststreptococcal glomerulonephritis (PSGN). These proteins, of the M protein family, were shown to trigger anti-IgG production and enhance renal deposition of IgG and/or immune complexes (ICs), with resulting activation of complement and cytokine cascades. In the present study, type M12/emm12, group A streptococci (GAS) were found often to bind artificial ICs, viz. peroxidase–anti-peroxidase rabbit IgG (PAP) or tetanus toxoid–anti-tetanus human IgG (TAT), rather than monomeric IgG. Animals injected with each of four IC binding clinical isolates (from patients with scarlet fever or PSGN) showed pronounced inflammatory and degenerative glomerular changes, morphologically similar to human PSGN, with membrane thickening and IgG and complement C3 deposition, as well as secretion of IL-6 and TNF-α by mesangial and endothelial cells. In contrast, non-binding strains (two from asymptomatic carriers and one from a PSGN case) failed to trigger any renal changes. Only the IC binding strains induced elevated titres of anti-IgG. Though the streptococcal binding component(s) has not been demonstrated, the selective binding of ICs by type M12/emm12 strains appears important for the well-known, marked nephritogenic potential of this GAS type.

scholarly journals Manganese Homeostasis in Group A Streptococcus Is Critical for Resistance to Oxidative Stress and Virulence

mBio ◽  
2015 ◽  
Vol 6 (2) ◽  
Author(s):  
Andrew G. Turner ◽  
Cheryl-lynn Y. Ong ◽  
Christine M. Gillen ◽  
Mark R. Davies ◽  
Nicholas P. West ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Hydrogen Peroxide ◽  
Transition Metal ◽  
Metal Ion ◽  
Double Mutant ◽  
Efflux Pump ◽  
Group A Streptococcus ◽  
Wild Type ◽  
Group A

ABSTRACT Streptococcus pyogenes (group A Streptococcus [GAS]) is an obligate human pathogen responsible for a spectrum of human disease states. Metallobiology of human pathogens is revealing the fundamental role of metals in both nutritional immunity leading to pathogen starvation and metal poisoning of pathogens by innate immune cells. Spy0980 (MntE) is a paralog of the GAS zinc efflux pump CzcD. Through use of an isogenic mntE deletion mutant in the GAS serotype M1T1 strain 5448, we have elucidated that MntE is a manganese-specific efflux pump required for GAS virulence. The 5448ΔmntE mutant had significantly lower survival following infection of human neutrophils than did the 5448 wild type and the complemented mutant (5448ΔmntE::mntE). Manganese homeostasis may provide protection against oxidative stress, explaining the observed ex vivo reduction in virulence. In the presence of manganese and hydrogen peroxide, 5448ΔmntE mutant exhibits significantly lower survival than wild-type 5448 and the complemented mutant. We hypothesize that MntE, by maintaining homeostatic control of cytoplasmic manganese, ensures that the peroxide response repressor PerR is optimally poised to respond to hydrogen peroxide stress. Creation of a 5448ΔmntE-ΔperR double mutant rescued the oxidative stress resistance of the double mutant to wild-type levels in the presence of manganese and hydrogen peroxide. This work elucidates the mechanism for manganese toxicity within GAS and the crucial role of manganese homeostasis in maintaining GAS virulence. IMPORTANCE Manganese is traditionally viewed as a beneficial metal ion to bacteria, and it is also established that most bacteria can tolerate high concentrations of this transition metal. In this work, we show that in group A Streptococcus, mutation of the mntE locus, which encodes a transport protein of the cation diffusion facilitator (CDF) family, results in accumulation of manganese and sensitivity to this transition metal ion. The toxicity of manganese is indirect and is the result of a failure of the PerR regulator to respond to oxidative stress in the presence of high intracellular manganese concentrations. These results highlight the importance of MntE in manganese homeostasis and maintenance of an optimal manganese/iron ratio in GAS and the impact of manganese on resistance to oxidative stress and virulence.

scholarly journals M+ Group A Streptococci Are Phagocytized and Killed in Whole Blood by C5a-Activated Polymorphonuclear Leukocytes

2002 ◽  
Vol 70 (1) ◽  
pp. 350-359 ◽  
Author(s):  
Eric DeMaster ◽  
Norbert Schnitzler ◽  
Qi Cheng ◽  
Patrick Cleary
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Whole Blood ◽  
Polymorphonuclear Leukocytes ◽  
Group A Streptococci ◽  
Wild Type ◽  
Specific Antibodies ◽  
Before And After ◽  
Group A ◽  
Type Strains

ABSTRACT Historically, resistance to phagocytosis has been determined by incubating group A streptococci in human blood and comparing the numbers of CFU before and after incubation. Utilizing a flow cytometry-based technique, we have investigated the phagocytosis of M+ group A streptococci by polymorphonuclear leukocytes (PMNs) in heparinized human peripheral whole blood. Intracellular labeling of streptococci with a nontoxic fluorescent dye allowed us to quantify the association and phagocytosis of M+ streptococci by PMNs in whole blood in the presence or absence of C5a, a physiologically important chemotactic activator of PMNs. We found that wild-type strains of group A streptococci that are resistant to phagocytosis (determined by the classical Lancefield method) readily associate with C5a-activated whole-blood PMNs. In the absence of opsonizing M-type-specific antibodies, the M+ streptococci associated with PMNs are phagocytized and killed. In addition, blockade of the β2 integrin, CD11b/CD18, with anti-human CD11b monoclonal antibody inhibited association between M+ streptococci and C5a-activated PMNs. These findings establish a new relationship between M+ streptococci and PMNs, in which C5a-activated PMNs have the capacity to kill M+ streptococci in whole blood through a receptor-mediated phagocytic mechanism.

scholarly journals The Role of Ethylene Production in Virulence of Pseudomonas syringae pvs. glycinea and phaseolicola

2001 ◽  
Vol 91 (5) ◽  
pp. 511-518 ◽  
Author(s):  
Helge Weingart ◽  
Henriette Ullrich ◽  
Klaus Geider ◽  
Beate Völksch
Keyword(s):  
Pseudomonas Syringae ◽  
Ethylene Production ◽  
Type Strain ◽  
Wild Type Strain ◽  
Wild Type ◽  
In Planta ◽  
Population Sizes ◽  
Soybean Plants ◽  
Type Strains

The importance of ethylene production for virulence of Pseudomonas syringae pvs. glycinea and phaseolicola was assayed by comparing bacterial multiplication and symptom development in bean and soybean plants inoculated with ethylene-negative (efe) mutants and wild-type strains. The efe mutants of Pseudomonas syringae pv. glycinea were significantly reduced in their ability to grow in planta. However, the degree of reduction was strain-dependent. Population sizes of efe mutant 16/83-E1 that did not produce the phototoxin coronatine were 10- and 15-fold lower than those of the wild-type strain on soybean and on bean, and 16/83-E1 produced very weak symptoms compared with the wild-type strain. The coronatine-producing efe mutant 7a/90-E1 reached fourfold and twofold lower population sizes compared with the wild-type strain on soybean and bean, respectively, and caused disease symptoms typical of the wild-type strain. Experiments with ethylene-insensitive soybeans confirmed these results. The virulence of the wild-type strains was reduced to the same extent in ethylene-insensitive soybean plants as the virulence of the efe mutants in ethylene-susceptible soybeans. In contrast, the virulence of Pseudomonas syringae pv. phaseolicola was not affected by disruption of the efe gene.

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 Extracellular Cysteine Protease Produced by Streptococcus pyogenes Participates in the Pathogenesis of Invasive Skin Infection and Dissemination in Mice

1999 ◽  
Vol 67 (4) ◽  
pp. 1779-1788 ◽  
Author(s):  
Slawomir Lukomski ◽  
Charles A. Montgomery ◽  
Jacqueline Rurangirwa ◽  
Robert S. Geske ◽  
James P. Barrish ◽  
...  
Keyword(s):  
Mutant Strain ◽  
Cysteine Protease ◽  
Skin Infection ◽  
Immunogold Electron Microscopy ◽  
Wild Type ◽  
Systemic Dissemination ◽  
Group A ◽  
Protease Expression ◽  
Striking Contrast

ABSTRACT The role of an extracellular cysteine protease encoded by thespeB gene in group A Streptococcus (GAS) skin infection was studied with a mouse model. Mice were injected subcutaneously with a wild-type GAS serotype M3 strain or a cysteine protease-inactivated isogenic derivative grown to stationary phase. The mortality rate of mice injected with the M3 speB mutant strain was significantly decreased (P < 0.0008) compared to that of animals injected with the wild-type parental organism. The abscesses formed in animals infected with the cysteine protease mutant strain were significantly smaller (P< 0.0001) than those caused by the wild-type organism and slowly regressed over 3 to 4 weeks. In striking contrast, infection with the wild-type GAS isolate generated necrotic lesions, and in some animals the GAS disseminated widely from the injection site and produced extensive cutaneous damage. All of these animals developed bacteremia and died. GAS dissemination was accompanied by severe tissue and blood vessel necrosis. Cysteine protease expression in the infected tissue was identified by immunogold electron microscopy. These data demonstrate that cysteine protease expression contributes to soft tissue pathology, including necrosis, and is required for efficient systemic dissemination of the organism from the initial site of skin inoculation.

scholarly journals One-step multiplex real-time RT-PCR assay for detecting and genotyping wild-type group A rotavirus strains and vaccine strains (Rotarix® and RotaTeq®) in stool samples

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e1560 ◽  
Author(s):  
Rashi Gautam ◽  
Slavica Mijatovic-Rustempasic ◽  
Mathew D. Esona ◽  
Ka Ian Tam ◽  
Osbourne Quaye ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Wild Type ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Qrt Pcr ◽  
Group A ◽  
One Step ◽  
Stool Samples ◽  
Pcr Assays ◽  
Type Strains

Background.Group A rotavirus (RVA) infection is the major cause of acute gastroenteritis (AGE) in young children worldwide. Introduction of two live-attenuated rotavirus vaccines, RotaTeq® and Rotarix®, has dramatically reduced RVA associated AGE and mortality in developed as well as in many developing countries. High-throughput methods are needed to genotype rotavirus wild-type strains and to identify vaccine strains in stool samples. Quantitative RT-PCR assays (qRT-PCR) offer several advantages including increased sensitivity, higher throughput, and faster turnaround time.Methods.In this study, a one-step multiplex qRT-PCR assay was developed to detect and genotype wild-type strains and vaccine (Rotarix® and RotaTeq®) rotavirus strains along with an internal processing control (Xeno or MS2 RNA). Real-time RT-PCR assays were designed for VP7 (G1, G2, G3, G4, G9, G12) and VP4 (P[4], P[6] and P[8]) genotypes. The multiplex qRT-PCR assay also included previously published NSP3 qRT-PCR for rotavirus detection and Rotarix® NSP2 and RotaTeq® VP6 qRT-PCRs for detection of Rotarix® and RotaTeq® vaccine strains respectively. The multiplex qRT-PCR assay was validated using 853 sequence confirmed stool samples and 24 lab cultured strains of different rotavirus genotypes. By using thermostablerTthpolymerase enzyme, dsRNA denaturation, reverse transcription (RT) and amplification (PCR) steps were performed in single tube by uninterrupted thermocycling profile to reduce chances of sample cross contamination and for rapid generation of results. For quantification, standard curves were generated using dsRNA transcripts derived from RVA gene segments.Results.The VP7 qRT-PCRs exhibited 98.8–100% sensitivity, 99.7–100% specificity, 85–95% efficiency and a limit of detection of 4–60 copies per singleplex reaction. The VP7 qRT-PCRs exhibited 81–92% efficiency and limit of detection of 150–600 copies in multiplex reactions. The VP4 qRT-PCRs exhibited 98.8–100% sensitivity, 100% specificity, 86–89% efficiency and a limit of detection of 12–400 copies per singleplex reactions. The VP4 qRT-PCRs exhibited 82–90% efficiency and limit of detection of 120–4000 copies in multiplex reaction.Discussion.The one-step multiplex qRT-PCR assay will facilitate high-throughput rotavirus genotype characterization for monitoring circulating rotavirus wild-type strains causing rotavirus infections, determining the frequency of Rotarix® and RotaTeq® vaccine strains and vaccine-derived reassortants associated with AGE, and help to identify novel rotavirus strains derived by reassortment between vaccine and wild-type strains.

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