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 The Majority of 9,729 Group A Streptococcus Strains Causing Disease Secrete SpeB Cysteine Protease: Pathogenesis Implications

2015 ◽  
Vol 83 (12) ◽  
pp. 4750-4758 ◽  
Author(s):  
Randall J. Olsen ◽  
Anjali Raghuram ◽  
Concepcion Cantu ◽  
Meredith H. Hartman ◽  
Francisco E. Jimenez ◽  
...  
Keyword(s):  
Necrotizing Fasciitis ◽  
Cysteine Protease ◽  
Protease Activity ◽  
Sequence Data ◽  
Group A Streptococcus ◽  
Whole Genome Sequence ◽  
Wild Type ◽  
Tissue Destruction ◽  
Lethal Challenge ◽  
Group A

Group A streptococcus (GAS), the causative agent of pharyngitis and necrotizing fasciitis, secretes the potent cysteine protease SpeB. Several lines of evidence suggest that SpeB is an important virulence factor. SpeB is expressed in human infections, protects mice from lethal challenge when used as a vaccine, and contributes significantly to tissue destruction and dissemination in animal models. However, recent descriptions of mutations in genes implicated in SpeB production have led to the idea that GAS may be under selective pressure to decrease secreted SpeB protease activity during infection. Thus, two divergent hypotheses have been proposed. One postulates that SpeB is a key contributor to pathogenesis; the other, that GAS is under selection to decrease SpeB during infection. In order to distinguish between these alternative hypotheses, we performed casein hydrolysis assays to measure the SpeB protease activity secreted by 6,775 GAS strains recovered from infected humans. The results demonstrated that 84.3% of the strains have a wild-type SpeB protease phenotype. The availability of whole-genome sequence data allowed us to determine the relative frequencies of mutations in genes implicated in SpeB production. The most abundantly mutated genes were direct transcription regulators. We also sequenced the genomes of 2,954 GAS isolates recovered from nonhuman primates with experimental necrotizing fasciitis. No mutations that would result in a SpeB-deficient phenotype were identified. Taken together, these data unambiguously demonstrate that the great majority of GAS strains recovered from infected humans secrete wild-type levels of SpeB protease activity. Our data confirm the important role of SpeB in GAS pathogenesis and help end a long-standing controversy.

scholarly journals Replacement of Histidine 340 with Alanine Inactivates the Group A Streptococcus Extracellular Cysteine Protease Virulence Factor

2000 ◽  
Vol 68 (6) ◽  
pp. 3716-3719 ◽  
Author(s):  
Siddeswar Gubba ◽  
Vincent Cipriano ◽  
James M. Musser
Keyword(s):  
Virulence Factor ◽  
Cysteine Protease ◽  
Structural Integrity ◽  
Group A Streptococcus ◽  
Invasive Disease ◽  
Site Directed Mutagenesis ◽  
Enzyme Active Site ◽  
Group A ◽  
Human Fibronectin ◽  
Insight Into

ABSTRACT Streptococcus pyogenes expresses a highly conserved extracellular cysteine protease that is a virulence factor for invasive disease, including soft tissue infection. Site-directed mutagenesis was used to generate a His340Ala recombinant mutant protein that was made as a stable 40-kDa zymogen by Escherichia coli. Purified His340Ala protein was proteolytically inactive when bovine casein and human fibronectin were used as substrates. Wild-type 28-kDa streptococcal protease purified from S. pyogenes processed the 40-kDa mutant zymogen to a 28-kDa mature form, a result suggesting that the derivative protein retained structural integrity. The data are consistent with the hypothesis that His340 is an enzyme active site residue, an idea confirmed by recent solution of the zymogen crystal structure (T. F. Kagawa, J. C. Cooney, H. M. Baker, S. McSweeney, M. Liu, S. Gubba, J. M. Musser, and E. N. Baker, Proc. Natl. Acad. Sci. USA 97:2235–2240, 2000). The data provide additional insight into structure-function relationships in thisS. pyogenes virulence factor.

scholarly journals The most common causative bacteria in maternal sepsis-related deaths in Japan were group A Streptococcus: A nationwide survey

2019 ◽  
Vol 25 (1) ◽  
pp. 41-44 ◽  
Author(s):  
Hiroaki Tanaka ◽  
Shinji Katsuragi ◽  
Junichi Hasegawa ◽  
Kayo Tanaka ◽  
Kazuhiro Osato ◽  
...  
Keyword(s):  
Group A Streptococcus ◽  
Nationwide Survey ◽  
Group A ◽  
Maternal Sepsis ◽  
Streptococcus A

scholarly journals Quorum Sensing Influences Burkholderia thailandensis Biofilm Development and Matrix Production

2016 ◽  
Vol 198 (19) ◽  
pp. 2643-2650 ◽  
Author(s):  
Boo Shan Tseng ◽  
Charlotte D. Majerczyk ◽  
Daniel Passos da Silva ◽  
Josephine R. Chandler ◽  
E. Peter Greenberg ◽  
...  
Keyword(s):  
Quorum Sensing ◽  
Biofilm Formation ◽  
Bacterial Species ◽  
Matrix Material ◽  
Close Relative ◽  
Wild Type ◽  
Flow Conditions ◽  
Burkholderia Thailandensis ◽  
Content Type ◽  
Biofilm Development

ABSTRACTMembers of the genusBurkholderiaare known to be adept at biofilm formation, which presumably assists in the survival of these organisms in the environment and the host. Biofilm formation has been linked to quorum sensing (QS) in several bacterial species. In this study, we characterizedBurkholderia thailandensisbiofilm development under flow conditions and sought to determine whether QS contributes to this process.B. thailandensisbiofilm formation exhibited an unusual pattern: the cells formed small aggregates and then proceeded to produce mature biofilms characterized by “dome” structures filled with biofilm matrix material. We showed that this process was dependent on QS.B. thailandensishas three acyl-homoserine lactone (AHL) QS systems (QS-1, QS-2, and QS-3). An AHL-negative strain produced biofilms consisting of cell aggregates but lacking the matrix-filled dome structures. This phenotype was rescued via exogenous addition of the three AHL signals. Of the threeB. thailandensisQS systems, we show that QS-1 is required for proper biofilm development, since abtaR1mutant, which is defective in QS-1 regulation, forms biofilms without these dome structures. Furthermore, our data show that the wild-type biofilm biomass, as well as the material inside the domes, stains with a fucose-binding lectin. ThebtaR1mutant biofilms, however, are negative for fucose staining. This suggests that the QS-1 system regulates the production of a fucose-containing exopolysaccharide in wild-type biofilms. Finally, we present data showing that QS ability during biofilm development produces a biofilm that is resistant to dispersion under stress conditions.IMPORTANCEThe saprophyteBurkholderia thailandensisis a close relative of the pathogenic bacteriumBurkholderia pseudomallei, the causative agent of melioidosis, which is contracted from its environmental reservoir. Since most bacteria in the environment reside in biofilms,B. thailandensisis an ideal model organism for investigating questions inBurkholderiaphysiology. In this study, we characterizedB. thailandensisbiofilm development and sought to determine if quorum sensing (QS) contributes to this process. Our work shows thatB. thailandensisproduces biofilms with unusual dome structures under flow conditions. Our findings suggest that these dome structures are filled with a QS-regulated, fucose-containing exopolysaccharide that may be involved in the resilience ofB. thailandensisbiofilms against changes in the nutritional environment.

scholarly journals Assessing the Role of Pharyngeal Cell Surface Glycans in Group A Streptococcus Biofilm Formation

Antibiotics ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 775
Author(s):  
Heema K. N. Vyas ◽  
Anuk D. Indraratna ◽  
Arun Everest-Dass ◽  
Nicolle H. Packer ◽  
David M. P. De Oliveira ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Cell Surface ◽  
Treatment Failure ◽  
Antibiotic Treatment ◽  
Biofilm Formation ◽  
Extracellular Polymeric Substances ◽  
Group A Streptococcus ◽  
Sialic Acid Residue ◽  
Group A ◽  
Terminal Mannose

Group A Streptococcus (GAS) causes 700 million infections and accounts for half a million deaths per year. Antibiotic treatment failure rates of 20–40% have been observed. The role host cell glycans play in GAS biofilm formation in the context of GAS pharyngitis and subsequent antibiotic treatment failure has not been previously investigated. GAS serotype M12 GAS biofilms were assessed for biofilm formation on Detroit 562 pharyngeal cell monolayers following enzymatic removal of all N-linked glycans from pharyngeal cells with PNGase F. Removal of N-linked glycans resulted in an increase in biofilm biomass compared to untreated controls. Further investigation into the removal of terminal mannose and sialic acid residues with α1-6 mannosidase and the broad specificity sialidase (Sialidase A) also found that biofilm biomass increased significantly when compared to untreated controls. Increases in biofilm biomass were associated with increased production of extracellular polymeric substances (EPS). Furthermore, it was found that M12 GAS biofilms grown on untreated pharyngeal monolayers exhibited a 2500-fold increase in penicillin tolerance compared to planktonic GAS. Pre-treatment of monolayers with exoglycosidases resulted in a further doubling of penicillin tolerance in resultant biofilms. Lastly, an additional eight GAS emm-types were assessed for biofilm formation in response to terminal mannose and sialic acid residue removal. As seen for M12, biofilm biomass on monolayers increased following removal of terminal mannose and sialic acid residues. Collectively, these data demonstrate that pharyngeal cell surface glycan structures directly impact GAS biofilm formation in a strain and glycan specific fashion.

scholarly journals Characterization of Biofilm Formation by Clinically Relevant Serotypes of Group A Streptococci

2006 ◽  
Vol 72 (4) ◽  
pp. 2864-2875 ◽  
Author(s):  
Cordula Lembke ◽  
Andreas Podbielski ◽  
Carlos Hidalgo-Grass ◽  
Ludwig Jonas ◽  
Emanuel Hanski ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Laser Scanning ◽  
Group A Streptococcus ◽  
Three Dimensional ◽  
Positive Control ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Subsequent Formation ◽  
Group A

ABSTRACT Streptococcus pyogenes (group A streptococcus [GAS]) is a frequent cause of purulent infections in humans. As potentially important aspects of its pathogenicity, GAS was recently shown to aggregate, form intratissue microcolonies, and potentially participate in multispecies biofilms. In this study, we show that GAS in fact forms monospecies biofilms in vitro, and we analyze the basic parameters of S. pyogenes in vitro biofilm formation, using Streptococcus epidermidis as a biofilm-positive control. Of nine clinically important serotype strains, M2, M6, M14, and M18 were found to significantly adhere to coated and uncoated polystyrene surfaces. Fibronectin and collagen types I and IV best supported primary adherence of serotype M2 and M18 strains, respectively, whereas serotype M6 and M14 strains strongly bound to uncoated polystyrene surfaces. Absorption measurements of safranin staining, as well as electron scanning and confocal laser scanning microscopy, documented that primary adherence led to subsequent formation of three-dimensional biofilm structures consisting of up to 46 bacterial layers. Of note, GAS isolates belonging to the same serotype were found to be very heterogeneous in their biofilm-forming behavior. Biofilm formation was equally efficient under static and continuous flow conditions and consisted of the classical three steps, including partial disintegration after long-term incubation. Activity of the SilC signaling peptide as a component of a putative quorum-sensing system was found to influence the biofilm structure and density of serotype M14 and M18 strains. Based on the presented methods and results, standardized analyses of GAS biofilms and their impact on GAS pathogenicity are now feasible.

scholarly journals Sodium Dodecyl Sulfate Hypersensitivity of clpP and clpB Mutants of Escherichia coli

2002 ◽  
Vol 68 (8) ◽  
pp. 4117-4121 ◽  
Author(s):  
Soumitra Rajagopal ◽  
Narasimhan Sudarsan ◽  
Kenneth W. Nickerson
Keyword(s):  
Escherichia Coli ◽  
Sodium Dodecyl Sulfate ◽  
Uronic Acid ◽  
Sodium Dodecyl ◽  
Immunoblot Analysis ◽  
Wild Type ◽  
E Coli ◽  
Dodecyl Sulfate ◽  
Shock Proteins ◽  
Western Immunoblot Analysis

ABSTRACT We studied the hypersensitivity of clpP and clpB mutants of Escherichia coli to sodium dodecyl sulfate (SDS). Both wild-type E. coli MC4100 and lon mutants grew in the presence of 10% SDS, whereas isogenic clpP and clpB single mutants could not grow above 0.5% SDS and clpA and clpX single mutants could not grow above 5.0% SDS. For wild-type E. coli, cellular ClpP levels as determined by Western immunoblot analysis increased ca. sixfold as the levels of added SDS increased from 0 to 2%. Capsular colanic acid, measured as uronic acid, increased ca. sixfold as the levels of added SDS increased from 2 to 10%. Based on these findings, 3 of the 19 previously identified SDS shock proteins (M. Adamowicz, P. M. Kelley, and K. W. Nickerson, J. Bacteriol. 173:229-233, 1991) are tentatively identified as ClpP, ClpX, and ClpB.

scholarly journals Crystal structure of the zymogen form of the group A Streptococcus virulence factor SpeB: An integrin-binding cysteine protease

2000 ◽  
Vol 97 (5) ◽  
pp. 2235-2240 ◽  
Author(s):  
T. F. Kagawa ◽  
J. C. Cooney ◽  
H. M. Baker ◽  
S. McSweeney ◽  
M. Liu ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Virulence Factor ◽  
Cysteine Protease ◽  
Group A Streptococcus ◽  
Group A

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.

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