Mis-regulation of Zn and Mn homeostasis is a key phenotype of Cu stress in Streptococcus pyogenes.

All bacteria possess homeostastic mechanisms that control the availability of micronutrient metals within the cell. Cross-talks between different metal homeostasis pathways within the same bacterial organism have been reported widely. In addition, there have been previous suggestions that some metal uptake transporters can promote adventitious uptake of the wrong metal. This work describes the cross-talk between Cu and the Zn and Mn homeostasis pathways in Group A Streptococcus (GAS). Using a ∆copA mutant strain that lacks the primary Cu efflux pump and thus traps excess Cu in the cytoplasm, we show that growth in the presence of supplemental Cu promotes downregulation of genes that contribute to Zn or Mn uptake. This effect is not associated with changes in cellular Zn or Mn levels. Co-supplementation of the culture medium with Zn or, to a lesser extent, Mn alleviates key Cu stress phenotypes, namely bacterial growth and secretion of the fermentation end-product lactate. However, neither co-supplemental Zn nor Mn influences cellular Cu levels or Cu availability in Cu-stressed cells. In addition, we provide evidence that the Zn or Mn uptake transporters in GAS do not promote Cu uptake. Together, the results from this study strengthen and extend our previous proposal that mis-regulation of Zn and Mn homeostasis is a key phenotype of Cu stress in GAS.

The role of CopA in Streptococcus pyogenes copper homeostasis and virulence.

Maintenance of intracellular metal homeostasis during interaction with host niches is critical to the success of bacterial pathogens. To prevent infection, the mammalian innate immune response employs metal-withholding and metal-intoxication mechanisms to limit bacterial propagation. The first-row transition metal ion copper serves critical roles at the host-pathogen interface and has been associated with antimicrobial activity since antiquity. Despite lacking any known copper-utilizing proteins, streptococci have been reported to accumulate significant levels of copper. Here, we report that loss of CopA, a copper-specific exporter, confers increased sensitivity to copper in Streptococcus pyogenes strain HSC5, with prolonged exposure to physiological levels of copper resulting in reduced viability during stationary phase cultivation. This defect in stationary phase survival was rescued by supplementation with exogeneous amino acids, indicating the pathogen had altered nutritional requirements during exposure to copper stress. Furthermore, S. pyogenes HSC5 ΔcopA was substantially attenuated during murine soft-tissue infection, demonstrating the importance of copper efflux at the host-pathogen interface. Collectively, these data indicate that copper can severely reduce the viability of stationary phase S. pyogenes and that active efflux mechanisms are required to survive copper stress in vitro and during infection.

Therapeutic Effect of Bee Venom and Melittin on Skin Infection Caused by Streptococcus pyogenes.

Streptococcus pyogenes (S. pyogenes) bacteria cause almost all primary skin infections in humans. Bee venom (BV) and melittin (Mel) have multiple effects, including antibacterial and anti-inflammatory activities. This study aims to demonstrate their effects on bacterial mouse skin infection using S. pyogenes. The dorsal skin was tape-stripped, then S. pyogenes was topically applied. BV or Mel were topically applied to the lesion. The tissues were stained with hematoxylin and eosin, while immunohistochemical staining was performed with anti-neutrophil. S. pyogenes-infected skin revealed increased epidermal and dermal layers, but it was reduced in the BV and Mel groups. Finding increased neutrophils in the mice infected with S. pyogenes, but the BV and Mel mice showed decreased expression. These results suggest that BV and Mel treatments could reduce the inflammatory reactions and help improve lesions induced by S. pyogenes skin infection. This study provides additional assessment of the potential therapeutic effects of BV and Mel in managing skin infection caused by S. pyogenes, further suggesting that it could be a candidate for developing novel treatment alternative for streptococcal skin infections.

Effects of artificial honey and epigallocatechin-3-gallate on streptococcus pyogenes.

BACKGROUND: Streptococcus pyogenes is an important global human pathogen that causes pharyngitis, and antibacterial therapy has become an important part of the overall therapy for pharyngitis. As natural derivatives, honey and green tea are often recommended for patients with pharyngitis in traditional Chinese medicine without experimental theoretical basis on wether the combined effect of honey and green tea on pharyngitis is better than they alone. The aims of this study were to explore the effects of artificial honey (AH) and epigallocatechin-3-gallate (EGCG) on S. pyogenes and elucidate the possible mechanisms, which were investigated using MIC (the minimum inhibitory concentration), FIC (fractional inhibitory concentration) index, growth pattern, biofilm formation and RT-qPCR. RESULTS: The MIC of AH on S. pyogenes was 12.5% (v/v) and the MIC of EGCG was 1250 µg/ml. The FIC index of AH and EGCG was 0.5. The planktonic cell growth, growth pattern and biofilm formation assays showed that AH and EGCG mixture had stronger inhibitory effect on S. pyogenes than they alone. RT-qPCR confirmed that the expression of hasA and luxS gene were inhibited by AH and EGCG mixture. CONCLUSIONS: AH and EGCG mixture can inhibit the planktonic cell growth, biofilm formation and some virulence genes expression of S. pyogenes, better than they alone. The combination of honey and green tea have the potential to treat pharyngitis as natural derivatives, avoiding drug resistance and double infection.

Investigation of Morchella esculenta and Morchella conica for their antibacterial potential against methicillin-susceptible Staphylococcus aureus, methicillin-resistant Staphylococcus aureus and Streptococcus pyogenes.

Antimicrobial resistance is an alarming problem, especially due to emergence of methicillin-resistance Staphylococcus aureus (MRSA). World Health Organization (WHO) has already listed MRSA as a top priority pathogen for the development of novel antibacterial agents. Presently, different therapeutic approaches against bacterial infections are in practice which includes targeting bacterial virulence factors, bacteriophage therapy, and manipulation of the microbiome. Natural products have been efficiently used for centuries to combat bacterial infections. Morchella is a natural fungal product which has been reported to possess broad-spectrum biological activities against bacterial infections. Hence, this study was aimed to evaluate the antibacterial efficacy of two macro-fungi against S. aureus, MRSA, and Streptococcus pyogenes (S. pyogenes). The antibacterial potential of both fungal extracts (Morchella esculenta and Morchella conica) was evaluated using disk diffusion and standard broth microdilution methods. The chemical compounds of both fungi were investigated using ultra-performance liquid chromatography mass spectroscopy (UPLC-MS) analysis. All fungal extracts inhibited growth of tested bacteria with inhibitory zone ranging from 10.66 ± 0.3 to 21.00 ± 1.5 mm. The minimum inhibitory concentration (MIC) of tested bacterial growth ranged from 03.33 to 16.0 mg/ml. It was noteworthy that Morchella extracts prevented S. aureus growth in a bactericidal manner with minimal bactericidal concentration (MBC) of 8-16 mg/ml. The extracts were also more effective against MRSA than currently available antibiotics. In conclusion, the growth inhibition of tested bacteria by fungal extracts revealed their potential as antibacterial agents and their compounds may be used as drug candidates.

Synthesis of Cinnamum zeylanicum and Acacia nilotica Extracts and Their Antibacterial Activity against Staphylococcus aureus and Streptococcus pyogenes.

Different plants are used medically and thofese therapeutic plants have great importance for healing contagious wounds. This herbal treatment is actually also a substitute of different antibiotics and having less side effects on intestinal systems of animals. The foremost concern of this study was to observe the antibacterial activity of Cinnamum zeylanicum and Acacia nilotica. Pathogenic bacteria obtained from wound samples and later identified by biochemical and molecular characterization. Methanol (an organic solvent) was used to extract Cinnamum zeylanicum and Acacia nilotica to check their antimicrobial exertion by using agar diffusion method. Different antibiotics such as, ampicillin, oflaxocin, ticarcillin and cefexime, showed their susceptibility toward antibiotics. The zone of inhibitions for antibiotic and plant extracts' antibacterial activity were measured. Pathogenic bacteria were identified as Staphylococcus aureus and Streptococcus pyogenesby molecular characterization. These bacteria showed susceptibility to antibiotics and also the plant extracts. Antibiotic oflaxocin showed maximum activity against these two pathogens (12.25 ± 0.44 and 12.375 ± 0.47) while antibiotic cefixime showed minimum effect (1.25 ± 0.28 and 0.625 ± 0.25). Plant extracts showed significant antibacterial activity with maximum activity (14 ± 0.9 by Acacia nilotica and 12 ± 0.5 by Cinnamum zeylanicum) in 100% solution. It can be concluded thatmethanolic extract of traditional therapeutic plants proved to be a promising source of antimicrobial agents against antibiotic resistant bacteria. Cinnamum zeylanicum and Acacia nilotica were observed to be competent as antibacterial tool against pathogenic bacterial strains.

Cranberry and Sumac Extracts Exhibit Antibacterial and Anti-Adhesive Effects Against Streptococcus pyogenes.

Group A Streptococci (GAS) or Streptococcus pyogenes is responsible for acute bacterial pharyngitis in children as well as adults. Streptococcal pharyngitis is initiated by successful attachment and colonization of the bacteria, followed by the establishment of the biofilm in various environments. In this study, we examined the antibacterial activities of in-house prepared aqueous and ethanolic extracts of 10 Atlantic Canada fruits in the context of minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), time-kill kinetics, and adhesion inhibition properties against S. pyogenes. Per our findings, MIC and MBC for all the tested extracts ranged from 0.25 to 8 mg/mL and from 4 to 64 mg/mL, respectively. Accordingly, at 1/2 × MBC, cranberry and sumac extracts also lowered the attachment of GAS to the uncoated and fibronectin-coated substratum. Particularly, cranberry and sumac aqueous extracts were more effective against the adhesion of S. pyogenes ATCC 19615 to the fibronectin-coated surface than a clinical strain. In conclusion, ethanolic and aqueous extracts of cranberry and sumac could potentially be incorporated into natural health products designed for the amelioration of strep throat, yet a detailed understanding of its mode of action (e.g., biofilm inhibition and eradication) could pave its path to the field of antibacterial natural health product discovery, design, and development.

Antibacterial, antifungal and enzyme inhibitory effects of selected plants from Turkey.

In this study, antibacterial, antifungal, antihyaluronidase, anticollagenase and antielastase activity of Hypericum bithynicum, Malva neglecta, Morus alba, Rubus discolor, Sambucus ebulus and Smilax excelsa were investigated. Methanol extracts of M. neglecta and R. discolor and all extracts of H. bithynicum were more active against Staphylococcus epidermidis. Similarly, water extracts of M. alba and S. ebulus were more active against Streptococcus pneumonia. Additionally, S. ebulus and S. excelsa had prominent antifungal activity on Candida albicans. Besides, methanol extract of M. neglecta and n-hexane extract of H. bithynicum were determined to have significant antihyaluronidase activity. Only R. discolor showed significant antielastase effect.

Hybrid donor-acceptor polymer nanoparticles and combination antibiotic for mitigation of pathogenic bacteria and biofilms.

Biofilms pose a significant clinical problem in skin and soft tissue infections. Their resistance to antibiotics has spurred investigations into alternative treatments, such as nanoparticle-mediated photothermal ablation. Non-toxic Hybrid Donor- Acceptor (DA) Polymer nanoParticles (H-DAPPs) were developed for fluorescence imaging (using poly(3-hexylthiophene-2,5 diyl) (P3HT)) and rapid, near-infrared photothermal ablation (NIR- PTA) (using poly[4,4-bis(2-ethylhexyl)-cyclopenta[2,1-b;3,4-b']dithiophene-2,6-diyl-alt-2,1,3-benzoselenadiazole-4,7-diyl] (PCPDTBSe)). H-DAPPs were evaluated alone, and in combination with antibiotics, against planktonic S. aureus and S. pyogenes, and S. aureus biofilms. H-DAPPs NIR-PTA (15-700 µg/ mL) can generate rapid temperature changes of 27.6-73.1 °C, which can eradicate planktonic bacterial populations and reduce biofilm bacterial viability by more than 4- log (> 99.99%) with exposure to 60 s of 800 nm light. Reductions were confirmed via confocal analysis, which suggested that H-DAPPs PTA caused bacterial inactivation within the biofilms, but did not significantly reduce biofilm polysaccharides. SEM imaging revealed structural changes in biofilms after H-DAPPs PTA. S. aureus biofilms challenged with 100 µg/mL of H-DAPPs (H-DAPPs-100) to induce an average temperature of 55.1 °C, and the minimum biofilm eradication concentration (MBEC) of clindamycin, resulted in up to ~3- log decrease in bacterial viability compared to untreated biofilms and those administered H-DAPPs-100 PTA only, and up to ~2- log compared to biofilms administered only clindamycin. This study demonstrates that polymer nanoparticle PTA can mitigate biofilm infection and may improve antimicrobial efficacy.

Application of Green Algal Planktochlorella nurekis Biomasses to Modulate Growth of Selected Microbial Species.

As microalgae are producers of proteins, lipids, polysaccharides, pigments, vitamins and unique secondary metabolites, microalgal biotechnology has gained attention in recent decades. Microalgae can be used for biomass production and to obtain biotechnologically important products. Here, we present the application of a method of producing a natural, biologically active composite obtained from unicellular microalgae of the genus Planktochlorella sp. as a modulator of the growth of microorganisms that can be used in the cosmetics and pharmaceutical industries by exploiting the phenomenon of photo-reprogramming of metabolism. The combination of red and blue light allows the collection of biomass with unique biochemical profiles, especially fatty acid composition (Patent Application P.429620). The ethanolic and water extracts of algae biomass inhibited the growth of a number of pathogenic bacteria, namely Enterococcus faecalis, Staphylococcus aureus PCM 458, Streptococcus pyogenes PCM 2318, Pseudomonas aeruginosa, Escherichia coli PCM 2209 and Candida albicans ATCC 14053. The algal biocomposite obtained according to our procedure can be used also as a prebiotic supplement. The presented technology may allow the limitation of the use of antibiotics and environmentally harmful chemicals commonly used in preparations against Enterococcus faecalis, Staphylococcus aureus, Streptococcus pyogenes, Pseudomonas aeruginosa, Escherichia coli or Candida spp.

Characterization of the antibacterial activity from ethanolic extracts of the botanical, Larrea tridentata.

BACKGROUND: ß-lactam antibiotics are a class of broad-spectrum antibiotics consisting of all antibiotic agents that contain a ß-lactam ring in their molecular structures. ß-lactam antibiotics are only known to be isolated from fungi (e.g. Acremonium chrysogenum, Penicillium chrysogenum and Aspergillus nidulans) and bacteria (e.g. Streptomyces clavuligerus). We have shown that botanical extracts prepared from Larrea tridentata have strong antimicrobial activity against several bacteria, including members of Staphylococcus and Streptococcus genera. METHODS: Through resistance studies, inhibitor assays, and ELISA testing, we demonstrated L. tridentata extracts may contain a ß-lactam type antibiotic activity. RESULTS: Based on the estimated ß-lactam concentration within the extract, the antimicrobial activity of the L. tridentata extract was approximately 2000-8000-fold greater against Staphylococcus as compared to other ß-lactams, penicillin or ampicillin. In the L. tridentata extract, this increased activity was found to be associated with the likely presence of a cofactor leading to increased potentiation of the ß-lactam activity. This potentiation activity was also observed to enhance the activity of exogenously added natural penicillin antibiotics. CONCLUSIONS: Although constituents were not isolated in this study, the results obtained strongly support the presence of ß-lactam type antibiotic activity and antibiotic potentiation activity present in ethanolic extracts prepared from L. tridentata.

In-vitro and in-vivo evaluation of the antibacterial potential of Typha elephantina.

The present study was aimed to evaluate the in-vitro and in-vivo antibacterial effects of the Typha elephantina aqueous extract (TE.AQ), ethanolic extract (TE.ET) and T. elephantina methanolic extract (TE.ME) against eight selected clinical pathogens. The test samples were tested for in-vitro analysis (by disc diffusion method) at different concentrations of 5, 15, 25, 50 and 100 mg/dL against both gram positive and gram-negative strains. The highest potential was observed in TE.ME at a concentration of 100 mg/dL against Pseudomonas aeruginosa exhibiting 19.67 ± 0.577 mm zone of inhibition (ZOI). The same fraction also showed good activity against Staphylococus aureus with ZOI of 17.50 ± 0.70 mm. The TE.ET was found most active against P. aeruginosa and Streptococcus pyogenes having ZOI of 18.53 ± 0.503 and 16.2 ± 1.55 mm respectively at a concentration of 100 mg/dL. The most sensitive bacteria P. aeruginosa was selected for in-vivo study (using poultry chicks) for induction of infection in chicks. The effects of TE.AQ, TE.ET and TE.ME were determined at concentrations of 300 mg/kg body weight based on hematological parameters, liver enzymes and gross pathological findings of lungs and livers. The findings of the in-vivo study in chick's model showed that treatment of experimental animals with TE.ME significantly restored the hematological parameters, liver enzymes and architecture of lungs and livers. Based on scientific evidence, the current study suggests that TE.ME may serve as a best and new natural antibacterial agent and can be used against infections caused by P. aeruginosa.

Streptococcus pyogenes upregulates arginine catabolism to exert its pathogenesis on the skin surface.

The arginine deiminase (ADI) pathway has been found in many kinds of bacteria and functions to supplement energy production and provide protection against acid stress. The Streptococcus pyogenes ADI pathway is upregulated upon exposure to various environmental stresses, including glucose starvation. However, there are several unclear points about the advantages to the organism for upregulating arginine catabolism. We show that the ADI pathway contributes to bacterial viability and pathogenesis under low-glucose conditions. S. pyogenes changes global gene expression, including upregulation of virulence genes, by catabolizing arginine. In a murine model of epicutaneous infection, S. pyogenes uses the ADI pathway to augment its pathogenicity by increasing the expression of virulence genes, including those encoding the exotoxins. We also find that arginine from stratum-corneum-derived filaggrin is a key substrate for the ADI pathway. In summary, arginine is a nutrient source that promotes the pathogenicity of S. pyogenes on the skin.

Antibacterial, anti-inflammatory and antioxidant activities of Mahanintangtong and its constituent herbs, a formula used in Thai traditional medicine for treating pharyngitis.

BACKGROUND: Mahanintangtong is listed in the Thailand's National List of Essential Medicines (NLEM). It is used to treat non-specific fevers and illnesses such as pharyngitis and chickenpox. In this study, we investigated the biological activities of the different medicinal plants used in the Mahanintangtong formula. METHODS: The plant materials were extracted by maceration and decoction. Antimicrobial activity, assessed by disc diffusion method, the minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) were compared with commercially available standard antibiotics. To elucidate the anti-inflammatory mechanisms, inhibition of nitric oxide (NO), tumor necrosis factor α (TNF-α), and interleukin 6 (IL-6) production was tested by Griess and ELISA techniques. Antioxidant activity was measured by ABTS and DPPH scavenging assays. RESULTS: The extracts with the best antimicrobial activities were carbonized Tectona grandis showing against Streptococcus pyogenes, Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa. The ethanol extract of Dracaena loureiroi wood exhibited the highest NO and IL-6 inhibitory activity with IC50 values of 9.42 ± 1.81 and 12.02 ± 0.30 µg/mL, respectively. The ethanol extract of Pogostemon cablin had the highest TNF-α inhibitory with IC50 values of 10.68 ± 0.02 µg/mL. In anti-free radical testing, the ethanol extract of D. loureiroi displayed high antioxidant activity by both ABTS and DPPH assays. CONCLUSION: The ethanol extracts from carbonized T. grandis and Mahanintangtong showed good antimicrobial activity, especially against S. pyogenes, and good anti-inflammatory activity. These findings are relevant to the pathogenesis of pharyngitis and justify additional studies to see if Mahanintangtong could have clinical utility.

Do Antimicrobial Resistance Patterns Matter? An Algorithm for the Treatment of Patients With Impetigo.

BACKGROUND: Impetigo, a highly contagious bacterial skin infection commonly occurring in young children, but adults may also be affected. The superficial skin infection is mainly caused by Staphylococcus aureus (S. aureus) and less frequently by Streptococcus pyogenes (S. pyogenes). Antimicrobial resistance has become a worldwide concern and needs to be addressed when selecting treatment for impetigo patients. An evidence-based impetigo treatment algorithm was developed to address the treatment of impetigo for pediatric and adult populations. METHODS: An international panel of pediatric dermatologists, dermatologists, pediatricians, and pediatric infectious disease specialists employed a modified Delphi technique to develop the impetigo treatment algorithm. Treatment recommendations were evidence-based, taking into account antimicrobial stewardship and the increasing resistance to oral and topical antibiotics. RESULTS: The algorithm includes education and prevention of impetigo, diagnosis and classification, treatment measures, and follow-up and distinguishes between localized and widespread or epidemic outbreaks of impetigo. The panel adopted the definition of localized impetigo of fewer than ten lesions and smaller than 36 cm2 area affected in patients of two months and up with no compromised immune status. Resistance to oral and topical antibiotics prescribed for the treatment of impetigo such as mupirocin, retapamulin, fusidic acid, have been widely reported. CONCLUSIONS: When prescribing antibiotics, it is essential to know the local trends in antibiotic resistance. Ozenoxacin cream 1% is highly effective against S. pyogenes and S. aureus, including methycyllin-susceptible and resistant strains (MRSA), and may be a suitable option for localized impetigo.J Drugs Dermatol. 2021;20(2):134-142. doi:10.36849/JDD.5475 THIS ARTICLE HAD BEEN MADE AVAILABLE FREE OF CHARGE. PLEASE SCROLL DOWN TO ACCESS THE FULL TEXT OF THIS ARTICLE WITHOUT LOGGING IN. NO PURCHASE NECESSARY. PLEASE CONTACT THE PUBLISHER WITH ANY QUESTIONS.

Preclinical safety and immunogenicity of Streptococcus pyogenes (Strep A) peptide vaccines.

We have developed two candidate vaccines to protect against multiple strains of Strep A infections. The candidates are combinatorial synthetic peptide vaccines composed of a M protein epitope (J8 or p*17) and a non-M protein epitope (K4S2). To enhance immunogenicity, each peptide is conjugated to the carrier protein CRM197 (CRM) and formulated with aluminium hydroxide adjuvant Alhydrogel (Alum) to make the final vaccines, J8-CRM + K4S2-CRM/Alum and p*17-CRM + K4S2-CRM/Alum. The safety and toxicity of each vaccine was assessed. Sprague Dawley rats were administered three intramuscular doses, over a six-week study with a 4-week recovery period. A control group received CRM only formulated with Alum (CRM/Alum). There was no evidence of systemic toxicity in the rats administered either vaccine. There was an associated increase in white blood cell, lymphocyte and monocyte counts, increased adrenal gland weights, adrenocortical hypertrophy, and increased severity of granulomatous inflammation at the sites of injection and the associated inguinal lymph nodes. These changes were considered non-adverse. All rats administered vaccine developed a robust and sustained immunological response. The absence of clinical toxicity and the development of an immunological response in the rats suggests that the vaccines are safe for use in a phase 1 clinical trial in healthy humans.

Zinc oxide nanoparticles inhibit bacterial biofilm formation via altering cell membrane permeability.

In the current scenario nanoparticles (NPs) have gained a breathtaking impetus due to their multidimensional applications in varied fields. In the present effort, biogenic synthesis of Zinc Oxide nanoparticles (ZnO NPs) was carried out using aqueous extract of dried powder of Emblica officinalis (Amla). Physicochemical characterization of nanoparticles was carried out via UV-Visible (UV-Vis) spectroscopy, Fourier transform infrared spectroscopy (FTIR), X-ray diffractometer (XRD), Scanning electron microscopy (SEM) and Transmission electron microscopy (TEM) wherein the particles were found to be quasi spherical and with a size ranging between 3 and 11 nm. The ZnO nanoparticles exhibited significant antibacterial activity against bacteria as Streptococcus pyogenes MTCC 442, Bacillus cereus MTCC 1272, Escherichia coli MTCC 1687 and Pseudomonas aeruginosa MTCC 4673. The nanoparticles displayed high anti-biofilm activity toward all the bacterial strains, when tested against three different base materials viz. glass, plastic and metal (Aluminum). Further, the nanoparticle treatment of bacterial cells caused changes in their cell membrane permeability, leading to leakage of nucleic acid from the bacterial cells, thereby defining it as the most probable mechanism for their anti-biofilm potential.

Activation of activin/Smad2 and 3 signaling pathway and the potential involvement of endothelial­mesenchymal transition in the valvular damage due to rheumatic heart disease.

Rheumatic heart disease (RHD) is an autoimmune disease caused by rheumatic fever following group A hemolytic streptococcal infection and primarily affects the mitral valve. RHD is currently a major global health problem. However, the exact pathological mechanisms associated with RHD­induced cardiac valve damage remain to be elucidated. The endothelial­mesenchymal transition (EndMT) serves a key role in a number of diseases with an important role in cardiac fibrosis and the activin/Smad2 and 3 signaling pathway is involved in regulating the EndMT. Nevertheless, there are no studies to date, to the best of the authors' knowledge, investigating the association between RHD and EndMT. Thus, the aim of the current study was to investigate the potential role of EndMT in cardiac valve damage and assess whether activin/Smad2 and 3 signaling was activated during RHD­induced valvular injury in a rat model of RHD induced by inactivated Group A streptococci and complete Freund's adjuvant. Inflammation and fibrosis were assessed by hematoxylin and eosin and Sirius red staining. Serum cytokine and rheumatoid factor levels were measured using ELISA kits. Expression levels of activin/Smad2 and 3 signaling pathway­related factors [activin A, Smad2, Smad3, phosphorylated (p­)Smad2 and p­Smad3], EndMT­related factors [lymphoid enhancer factor­1 (LEF­1), Snail1, TWIST, zinc finger E­box­binding homeobox (ZEB)1, ZEB2, α smooth muscle actin (α­SMA) and type I collagen α 1 (COL1A1)], apoptosis­related markers (BAX and cleaved caspase­3) and valvular inflammation markers (NF­κB and p­NF­κB) were detected using reverse transcription­quantitative PCR and western blot analyses. Compared with the control group, the degree of valvular inflammation and fibrosis, serum levels of IL­6, IL­17, TNF­α and expression of apoptosis­related markers (BAX and cleaved caspase­3) and valvular inflammation marker (p­NF­κB), activin/Smad2 and 3 signaling pathway­related factors (activin A, p­Smad2 and p­Smad3), EndMT­related factors (LEF­1, Snail1, TWIST, ZEB 1, ZEB2, α­SMA and COL1A1) were significantly increased in the RHD group. These results suggested that the activin/Smad2 and 3 signaling pathway was activated during the development of valvular damage caused by RHD and that the EndMT is involved in RHD­induced cardiac valve damage.

Role of Glutathione in Buffering Excess Intracellular Copper in Streptococcus pyogenes.

Copper (Cu) is an essential metal for bacterial physiology but in excess it is bacteriotoxic. To limit Cu levels in the cytoplasm, most bacteria possess a transcriptionally responsive system for Cu export. In the Gram-positive human pathogen Streptococcus pyogenes (group A Streptococcus [GAS]), this system is encoded by the copYAZ operon. This study demonstrates that although the site of GAS infection represents a Cu-rich environment, inactivation of the copA Cu efflux gene does not reduce virulence in a mouse model of invasive disease. In vitro, Cu treatment leads to multiple observable phenotypes, including defects in growth and viability, decreased fermentation, inhibition of glyceraldehyde-3-phosphate dehydrogenase (GapA) activity, and misregulation of metal homeostasis, likely as a consequence of mismetalation of noncognate metal-binding sites by Cu. Surprisingly, the onset of these effects is delayed by ∼4 h even though expression of copZ is upregulated immediately upon exposure to Cu. Further biochemical investigations show that the onset of all phenotypes coincides with depletion of intracellular glutathione (GSH). Supplementation with extracellular GSH replenishes the intracellular pool of this thiol and suppresses all the observable effects of Cu treatment. These results indicate that GSH buffers excess intracellular Cu when the transcriptionally responsive Cu export system is overwhelmed. Thus, while the copYAZ operon is responsible for Cu homeostasis, GSH has a role in Cu tolerance and allows bacteria to maintain metabolism even in the presence of an excess of this metal ion.IMPORTANCE The control of intracellular metal availability is fundamental to bacterial physiology. In the case of copper (Cu), it has been established that rising intracellular Cu levels eventually fill the metal-sensing site of the endogenous Cu-sensing transcriptional regulator, which in turn induces transcription of a copper export pump. This response caps intracellular Cu availability below a well-defined threshold and prevents Cu toxicity. Glutathione, abundant in many bacteria, is known to bind Cu and has long been assumed to contribute to bacterial Cu handling. However, there is some ambiguity since neither its biosynthesis nor uptake is Cu-regulated. Furthermore, there is little experimental support for this physiological role of glutathione beyond measuring growth of glutathione-deficient mutants in the presence of Cu. Our work with group A Streptococcus provides new evidence that glutathione increases the threshold of intracellular Cu availability that can be tolerated by bacteria and thus advances fundamental understanding of bacterial Cu handling.