IL-33/ST2 Axis Plays a Protective Effect in Streptococcus pyogenes Infection through Strengthening of the Innate Immunity.

Group A Streptococcus (GAS) causes invasive human diseases with the cytokine storm. Interleukin-33 (IL-33)/suppression of tumorigenicity 2 (ST2) axis is known to drive TH2 response, while its effect on GAS infection is unclear. We used an air pouch model to examine the effect of the IL-33/ST2 axis on GAS-induced necrotizing fasciitis. GAS infection induced IL-33 expression in wild-type (WT) C57BL/6 mice, whereas the IL-33- and ST2-knockout mice had higher mortality rates, more severe skin lesions and higher bacterial loads in the air pouches than those of WT mice after infection. Surveys of infiltrating cells in the air pouch of GAS-infected mice at the early stage found that the number and cell viability of infiltrating cells in both gene knockout mice were lower than those of WT mice. The predominant effector cells in GAS-infected air pouches were neutrophils. Absence of the IL-33/ST2 axis enhanced the expression of inflammatory cytokines, but not TH1 or TH2 cytokines, in the air pouch after infection. Using in vitro assays, we found that the IL-33/ST2 axis not only enhanced neutrophil migration but also strengthened the bactericidal activity of both sera and neutrophils. These results suggest that the IL-33/ST2 axis provided the protective effect on GAS infection through enhancing the innate immunity.

AR-12 Has a Bactericidal Activity and a Synergistic Effect with Gentamicin against Group A Streptococcus.

Streptococcus pyogenes (group A Streptococcus (GAS) is an important human pathogen that can cause severe invasive infection, such as necrotizing fasciitis and streptococcal toxic shock syndrome. The mortality rate of streptococcal toxic shock syndrome ranges from 20% to 50% in spite of antibiotics administration. AR-12, a pyrazole derivative, has been reported to inhibit the infection of viruses, intracellular bacteria, and fungi. In this report, we evaluated the bactericidal activities and mechanisms of AR-12 on GAS infection. Our in vitro results showed that AR-12 dose-dependently reduced the GAS growth, and 2.5 µg/mL of AR-12 significantly killed GAS within 2 h. AR-12 caused a remarkable reduction in nucleic acid and protein content of GAS. The expression of heat shock protein DnaK and streptococcal exotoxins was also inhibited by AR-12. Surveys of the GAS architecture by scanning electron microscopy revealed that AR-12-treated GAS displayed incomplete septa and micro-spherical structures protruding out of cell walls. Moreover, the combination of AR-12 and gentamicin had a synergistic antibacterial activity against GAS replication for both in vitro and in vivo infection. Taken together, these novel findings obtained in this study may provide a new therapeutic strategy for invasive GAS infection.

Dextromethorphan Attenuates NADPH Oxidase-Regulated Glycogen Synthase Kinase 3ß and NF-κB Activation and Reduces Nitric Oxide Production in Group A Streptococcal Infection.

Group A Streptococcus (GAS) is an important human pathogen that causes a wide spectrum of diseases, including necrotizing fasciitis and streptococcal toxic shock syndrome. Dextromethorphan (DM), an antitussive drug, has been demonstrated to efficiently reduce inflammatory responses, thereby contributing to an increased survival rate of GAS-infected mice. However, the anti-inflammatory mechanisms underlying DM treatment in GAS infection remain unclear. DM is known to exert neuroprotective effects through an NADPH oxidase-dependent regulated process. In the present study, membrane translocation of NADPH oxidase subunit p47phox and subsequent reactive oxygen species (ROS) generation induced by GAS infection were significantly inhibited via DM treatment in RAW264.7 murine macrophage cells. Further determination of proinflammatory mediators revealed that DM effectively suppressed inducible nitric oxide synthase (iNOS) expression and NO, tumor necrosis factor alpha, and interleukin-6 generation in GAS-infected RAW264.7 cells as well as in air-pouch-infiltrating cells from GAS/DM-treated mice. GAS infection caused AKT dephosphorylation, glycogen synthase kinase-3ß (GSK-3ß) activation, and subsequent NF-κB nuclear translocation, which were also markedly inhibited by treatment with DM and an NADPH oxidase inhibitor, diphenylene iodonium. These results suggest that DM attenuates GAS infection-induced overactive inflammation by inhibiting NADPH oxidase-mediated ROS production that leads to downregulation of the GSK-3ß/NF-κB/NO signaling pathway.

Protection against Klebsiella pneumoniae using lithium chloride in an intragastric infection model.

Intragastric Klebsiella pneumoniae infections of mice can cause liver abscesses, necrosis of liver tissues, and bacteremia. Lithium chloride, a widely prescribed drug for bipolar mood disorder, has been reported to possess anti-inflammatory properties. Using an intragastric infection model, the effects of LiCl on K. pneumoniae infections were examined. Providing mice with drinking water containing LiCl immediately after infection protected them from K. pneumoniae-induced death and liver injuries, such as necrosis of liver tissues, as well as increasing blood levels of aspartate aminotransferase and alanine aminotransferase, in a dose-dependent manner. LiCl administered as late as 24 h postinfection still provided protection. Monitoring of the LiCl concentrations in the sera of K. pneumoniae-infected mice showed that approximately 0.33 mM LiCl was the most effective dose for protecting mice against infections, which is lower than the clinically toxic dose of LiCl. Surveys of bacterial counts and cytokine expression levels in LiCl-treated mice revealed that both were effectively inhibited in blood and liver tissues. Using in vitro assays, we found that LiCl (5 µM to 1 mM) did not directly interfere with the growth of K. pneumoniae but made K. pneumoniae cells lose the mucoid phenotype and become more susceptible to macrophage killing. Furthermore, low doses of LiCl also partially enhanced the bactericidal activity of macrophages. Taken together, these data suggest that LiCl is an alternative therapeutic agent for K. pneumoniae-induced liver infections.

Kallistatin modulates immune cells and confers anti-inflammatory response to protect mice from group A streptococcal infection.

Group A streptococcus (GAS) infection may cause severe life-threatening diseases, including necrotizing fasciitis and streptococcal toxic shock syndrome. Despite the availability of effective antimicrobial agents, there has been a worldwide increase in the incidence of invasive GAS infection. Kallistatin (KS), originally found to be a tissue kallikrein-binding protein, has recently been shown to possess anti-inflammatory properties. However, its efficacy in microbial infection has not been explored. In this study, we transiently expressed the human KS gene by hydrodynamic injection and investigated its anti-inflammatory and protective effects in mice via air pouch inoculation of GAS. The results showed that KS significantly increased the survival rate of GAS-infected mice. KS treatment reduced local skin damage and bacterial counts compared with those in mice infected with GAS and treated with a control plasmid or saline. While there was a decrease in immune cell infiltration of the local infection site, cell viability and antimicrobial factors such as reactive oxygen species actually increased after KS treatment. The efficiency of intracellular bacterial killing in neutrophils was directly enhanced by KS administration. Several inflammatory cytokines, including tumor necrosis factor alpha, interleukin 1ß, and interleukin 6, in local infection sites were reduced by KS. In addition, KS treatment reduced vessel leakage, bacteremia, and liver damage after local infection. Therefore, our study demonstrates that KS provides protection in GAS-infected mice by enhancing bacterial clearance, as well as reducing inflammatory responses and organ damage.

Regulatory role of GSK-3 ß on NF- κ B, nitric oxide, and TNF- α in group A streptococcal infection.

Group A streptococcus (GAS) imposes a great burden on humans. Efforts to minimize the associated morbidity and mortality represent a critical issue. Glycogen synthase kinase-3 ß (GSK-3 ß) is known to regulate inflammatory response in infectious diseases. However, the regulation of GSK-3 ß in GAS infection is still unknown. The present study investigates the interaction between GSK-3 ß , NF- κ B, and possible related inflammatory mediators in vitro and in a mouse model. The results revealed that GAS could activate NF- κ B, followed by an increased expression of inducible nitric oxide synthase (iNOS) and NO production in a murine macrophage cell line. Activation of GSK-3 ß occurred after GAS infection, and inhibition of GSK-3 ß reduced iNOS expression and NO production. Furthermore, GSK-3 ß inhibitors reduced NF- κ B activation and subsequent TNF- α production, which indicates that GSK-3 ß acts upstream of NF- κ B in GAS-infected macrophages. Similar to the in vitro findings, administration of GSK-3 ß inhibitor in an air pouch GAS infection mouse model significantly reduced the level of serum TNF- α and improved the survival rate. The inhibition of GSK-3 ß to moderate the inflammatory effect might be an alternative therapeutic strategy against GAS infection.

Synergistic effects of streptolysin S and streptococcal pyrogenic exotoxin B on the mouse model of group A streptococcal infection.

Streptococcus pyogenes is a group A streptococcus (GAS) and an important human pathogen that causes a variety of diseases. Streptococcal pyrogenic exotoxin B (SPE B) and streptolysin S (SLS) are important virulence factors involved in GAS infection, but it is not clear which one is more virulent. Using an air pouch infection model, the wild-type strain NZ131, its isogenic mutants, and complementary mutants were used to examine the effects of SPE B and SLS on GAS infection. The results of the skin lesion and mouse mortality assays showed that although SPE B and SLS had a synergistic effect on GAS infection, SPE B played a more important role in local tissue damage while SLS had a more prominent effect on mouse mortality. Surveys of the exudates from the air pouch revealed that the expression of inflammatory cytokines was significantly inhibited in the sagB/speB-double-mutant JM4-infected mice. Furthermore, in vivo and in vitro studies showed that the isogenic mutant strains were more susceptible to the immune cell killing than the wild-type strain and that the sagB/speB-double-mutant JM4 was the most sensitive among these strains. Moreover, infection with the sagB/speB-double-mutant JM4 strain caused the least amount of macrophage apoptosis compared to infection with the wild-type NZ131 and the other complementary strains, which express only SPE B or SLS or both. Taken together, these results indicate that both SPE B and SLS contributed to GAS evasion from immune cell killing, local tissue damage, and mouse mortality.

Experimental phage therapy in treating Klebsiella pneumoniae-mediated liver abscesses and bacteremia in mice.

Intragastric inoculation of mice with Klebsiella pneumoniae can cause liver abscesses, necrosis of liver tissues, and bacteremia. A newly isolated phage (φNK5) with lytic activity for K. pneumoniae was used to treat K. pneumoniae infection in an intragastric model. Both intraperitoneal and intragastric administration of a single dose of φNK5 lower than 2 × 10(8) PFU at 30 min after K. pneumoniae infection was able to protect mice from death in a dose-dependent manner, but the efficacy achieved with a low dose of φNK5 by intragastric treatment provided the more significant protection. Phage φNK5 administered as late as 24 h after K. pneumoniae inoculation was still protective, while intraperitoneal treatment with phage was more efficient than intragastric treatment as a result of the dissemination of bacteria into the circulation at 24 h postinfection. Surveys of bacterial counts for mice treated with φNK5 by the intraperitoneal route revealed that the bacteria were eliminated effectively from both blood and liver tissue. K. pneumoniae-induced liver injury, such as liver necrosis, as well as blood levels of aspartate aminotransferase and alanine aminotransferase and inflammatory cytokine production, was significantly inhibited by φNK5 treatment. These data suggest that a low dose of φNK5 is a potential therapeutic agent for K. pneumoniae-induced liver infection.

Dextromethorphan efficiently increases bactericidal activity, attenuates inflammatory responses, and prevents group a streptococcal sepsis.

Group A streptococcus (GAS) is an important human pathogen that causes a wide spectrum of diseases, ranging from mild throat and skin infections to severe invasive diseases such as necrotizing fasciitis and streptococcal toxic shock syndrome. Dextromethorphan (DM), a dextrorotatory morphinan and a widely used antitussive drug, has recently been reported to possess anti-inflammatory properties. In this study, we investigated the potential protective effect of DM in GAS infection using an air pouch infection mouse model. Our results showed that DM treatment increased the survival rate of GAS-infected mice. Bacterial numbers in the air pouch were lower in mice treated with DM than in those infected with GAS alone. The bacterial elimination efficacy was associated with increased cell viability and bactericidal activity of air-pouch-infiltrating cells. Moreover, DM treatment prevented bacterial dissemination in the blood and reduced serum levels of the proinflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), and IL-1ß and the chemokines monocyte chemotactic protein 1 (MCP-1), macrophage inflammatory protein 2 (MIP-2), and RANTES. In addition, GAS-induced mouse liver injury was reduced by DM treatment. Taken together, DM can increase bacterial killing and reduce inflammatory responses to prevent sepsis in GAS infection. The consideration of DM as an adjunct treatment in combination with antibiotics against bacterial infection warrants further study.

Predictive modeling of blood pressure during hemodialysis: a comparison of linear model, random forest, support vector regression, XGBoost, LASSO regression and ensemble method.

BACKGROUND: Intradialytic hypotension (IDH) is commonly occurred and links to higher mortality among patients undergoing hemodialysis (HD). Its early prediction and prevention will dramatically improve the quality of life. However, predicting the occurrence of IDH clinically is not simple. The aims of this study are to develop an intelligent system with capability of predicting blood pressure (BP) during HD, and to further compare different machine learning algorithms for next systolic BP (SBP) prediction. METHODS: This study presented comprehensive comparisons among linear regression model, least absolute shrinkage and selection operator (LASSO), tree-based ensemble machine learning models (random forest [RF] and extreme gradient boosting [XGBoost]), and support vector regression to predict the BP during HD treatment based on 200 and 48 maintenance HD patients containing a total of 7,180 and 2,065 BP records for the training and test dataset, respectively. Ensemble method also was computed to obtain better predictive performance. We compared the developed models based on R2, root mean square error (RMSE) and mean absolute error (MAE). RESULTS: We found that RF (R2=0.95, RMSE=6.64, MAE=4.90) and XGBoost (R2=1.00, RMSE=1.83, MAE=1.29) had comparable predictive performance on the training dataset. However, RF (R2=0.49, RMSE=16.24, MAE=12.14) had more accurate than XGBoost (R2=0.41, RMSE=17.65, MAE=13.47) on testing dataset. Among these models, the ensemble method (R2=0.50, RMSE=16.01, MAE=11.97) had the best performance on testing dataset for next SBP prediction. CONCLUSIONS: We compared five machine learning and an ensemble method for next SBP prediction. Among all studied algorithms, the RF and the ensemble method have the better predictive performance. The prediction models using ensemble method for intradialytic BP profiling may be able to assist the HD staff or physicians in individualized care and prompt intervention for patients' safety and improve care of HD patients.

Streptolysin S induces mitochondrial damage and macrophage death through inhibiting degradation of glycogen synthase kinase-3ß in Streptococcus pyogenes infection.

Group A Streptococcus (GAS) infection is associated with a variety of human diseases. Previous studies indicate GAS infection leads to RAW264.7 cell death, but the mechanism is unclear. Here, analyzing the timing of reactive oxygen species (ROS) production and using mitochondrial ROS scavenger, we found the wild type GAS-induced RAW264.7 cell death was associated with mitochondrial ROS. The wild type GAS infection could activate glycogen synthase kinase-3ß (GSK-3ß). Inhibition of GSK-3ß activity by lithium chloride or decreasing GSK-3ß expression by lentivirus-mediated short hairpin RNA for GSK-3ß could not only decrease the wild type GAS-induced mitochondrial ROS generation, mitochondria damage and cell death, but also reduced GAS intracellular replication. Streptolysin S (SLS), a GAS toxin, played the important role on GAS-induced macrophage death. Compared to the wild type GAS with its isogenic sagB mutant (SLS mutant)-infected macrophages, we found sagB mutant infection caused less mitochondrial ROS generation and cell death than those of the wild type GAS-infected ones. Furthermore, the sagB mutant, but not the wild type or the sagB-complementary mutant, could induce GSK-3ß degradation via a proteasome-dependent pathway. These results suggest that a new mechanism of SLS-induced macrophage death was through inhibiting GSK-3ß degradation and further enhancing mitochondrial damage.

HECT E3 Ubiquitin Ligase-Regulated Txnip Degradation Facilitates TLR2-Mediated Inflammation During Group A Streptococcal Infection.

Thioredoxin-interacting protein (Txnip) inhibits the activity of thioredoxin (Trx) to modulate inflammatory responses. The burden of inflammation caused by microbial infection is strongly associated with disease severity; however, the role of Txnip in bacterial infection remains unclear. In Group A Streptococcus (GAS)-infected macrophages, Txnip was degraded independent of glucose consumption and streptococcal cysteine protease expression. Treatment with proteasome inhibitors reversed GAS-induced Txnip degradation. The activation of Toll-like receptor 2 (TLR2) initiated Txnip degradation, while no further Txnip degradation was observed in TLR2-deficient bone marrow-derived macrophages. NADPH oxidase-regulated NF-κB activation and pro-inflammatory activation were induced and accompanied by Txnip degradation during GAS infection. Silencing Txnip prompted TLR2-mediated inducible nitric oxide synthase (iNOS)/NO, TNF-α, and IL-6 production whereas the blockage of Txnip degradation by pharmacologically inhibiting the HECT E3 ubiquitin ligase with heclin and AMP-dependent protein kinase with dorsomorphin effectively reduced such effects. Our findings reveal that TLR2/NADPH oxidase-mediated Txnip proteasomal degradation facilitates pro-inflammatory cytokine production during GAS infection.

Group A Streptococcus Induces LAPosomes via SLO/ß1 Integrin/NOX2/ROS Pathway in Endothelial Cells That Are Ineffective in Bacterial Killing and Suppress Xenophagy.

Group A streptococcus (GAS) is an important human pathogen which can cause fatal diseases after invasion into the bloodstream. Although antibiotics and immune surveillance are the main defenses against GAS infection, GAS utilizes internalization into cells as a major immune evasion strategy. Our previous findings revealed that light chain 3 (LC3)-associated single membrane GAS-containing vacuoles in endothelial cells are compromised for bacterial clearance due to insufficient acidification after fusion with lysosomes. However, the characteristics and the activation mechanisms of these LC3-positive compartments are still largely unknown. In the present study, we demonstrated that the LC3-positive GAS is surrounded by single membrane and colocalizes with NADPH oxidase 2 (NOX2) complex but without ULK1, which are characteristics of LC3-associated phagocytosis (LAP). Inhibition of NOX2 or reactive oxygen species (ROS) significantly reduces GAS multiplication and enhances autolysosome acidification in endothelial cells through converting LAP to conventional xenophagy, which is revealed by enhancement of ULK1 recruitment, attenuation of p70s6k phosphorylation, and formation of the isolation membrane. We also clarify that the inactivation of mTORC1, which is the initiation signal of autophagy, is inhibited by NOX2- and ROS-activated phosphatidylinositol 3-kinase (PI3K)/AKT and MEK/extracellular signal-regulated kinase (ERK) pathways. In addition, streptolysin O (SLO) of GAS is identified as a crucial inducer of ROS for ß1 integrin-mediated LAP induction. After downregulation of ß1 integrin, GAS multiplication is reduced, accompanied with LAP inhibition and xenophagy induction. These results demonstrate that GAS infection preferentially induces ineffective LAP to evade xenophagic killing in endothelial cells through the SLO/ß1 integrin/NOX2/ROS pathway.IMPORTANCE Our previous reports showed that the LC3-associated GAS-containing single membrane vacuoles are inefficient for bacterial clearance in endothelial cells, which may result in bacteremia. However, the characteristics and the induction mechanisms of these LC3-positive vacuoles are still largely unknown. Here we provide the first evidence that these LC3-positive GAS-containing single membrane compartments appear to be LAPosomes, which are induced by NOX2 and ROS. Through NOX2- and ROS-mediated signaling, GAS preferentially induces LAP and inhibits bacteriostatic xenophagy in endothelial cells. We also provide the first demonstration that ß1 integrin acts as the receptor for LAP induction through GAS-produced SLO stimulation in endothelial cells. Our findings reveal the underlying mechanisms of LAP induction and autophagy evasion for GAS multiplication in endothelial cells.

Degradation of complement 3 by streptococcal pyrogenic exotoxin B inhibits complement activation and neutrophil opsonophagocytosis.

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.

Glossogin, a novel phenylpropanoid from Glossogyne tenuifolia, induced apoptosis in A549 lung cancer cells.

Glossogyne tenuifolia has been shown to exhibit good antioxidant and anticancer activity. In this study, a new phenylpropanoid compound, glossogin (1'-acetoxy-4-O-isovalyryleugenol), was isolated from ethyl acetate extract of G. tenuifolia by using column chromatography and HPLC. Its chemical structure was determined by (1)H and (13)C NMR, MS and IR spectroscopic evidence. This compound showed the cytotoxicity against A549 human lung cancer cell line and it induced the progressing apoptosis on A549 cells. This apoptosis was verified as A549 cells were arrested at the sub-G(1) phase. The apoptosis was accompanied by release of cytochrome C and activation of caspase-9 and -3. It was also associated with the decrease in Bcl-2 and Bcl-xL protein levels, and the increase in Bad protein expression. Data analysis suggests glossogin exerted significant apoptotic effect on A549 cells through the mitochondrial pathway. Hence, our findings showed that glossogin exhibited potential anticancer activity against lung cancer through proliferating inhibition and apoptosis induction of cancer cells.

Efficacy of φkm18p phage therapy in a murine model of extensively drug-resistant Acinetobacter baumannii infection.

PURPOSE: Few effective antibiotics are available for treating extensively drug-resistant Acinetobacter baumannii (XDRAB) sepsis. Phage therapy may show potential in treating XDRAB infections. MATERIALS AND METHODS: We studied φkm18p phage therapy in BALB/c and C57BL/6 mice models of XDRAB bacteremia. RESULTS: We observed survival rates of nearly 100% in groups given phage therapy concurrent with XDRAB at different multiplicities of infection. In mice that received phage therapy after a 1-hour delay, the survival rate decreased to about 50%. The bacterial load in the blood decreased from 108 to 102 and 103 colony-forming units (CFU)/mL in the concurrent treatment group. In the phage therapy group, the levels of the cytokines, such as tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6), were low at 3 hours after infection. Although some phage-resistant mutants were isolated after phage therapy, a cytotoxicity study showed that they had reduced fitness. CONCLUSION: Phage therapy in XDRAB bacteremia increased the animal survival rates, decreased the bacteremia loads, and decreased the levels of inflammatory markers TNF-α and IL-6. However, the reduced therapeutic effect with delayed administrations may be a concern in developing a successful phage therapy for treating acute infections of multidrug-resistant pathogens.

Abrogation of streptococcal pyrogenic exotoxin B-mediated suppression of phagocytosis in U937 cells by Cordyceps sinensis mycelium via production of cytokines.

Streptococcal pyrogenic exotoxin B (SPE B) is a virulent factor in group A streptococcal infection. We previously showed that SPE B reduced phagocytosis in human monocytic U937 cells. Here we show that the mycelium extract of Cordyceps sinensis (CS), a Chinese immunomodulatory herbal medicine, increased phagocytosis in U937 cells. Neither heat nor trypsin pretreatment prevented CS extract from causing this increase. Further studies indicated that SPE B-mediated suppression of U937 cell phagocytic activity was abrogated by CS extract. Factors in the conditioned medium from CS-extract-treated U937 cells were responsible for blocking the SPE B-mediated suppression of phagocytosis. Heating the conditioned medium eliminated the increase, which suggested that the U937-cell protein products augmented phagocytosis. Analyzing cytokine mRNA expression of U937 cells revealed increases in interferon-gamma (IFN-gamma), interleukin (IL)-12 p35 and p40, and tumor necrosis factor-alpha (TNF-alpha), but not in IL-1beta, IL-6, or IL-8. Treating U937 cells with anti-IFN-gamma, IL-12, and TNF-alpha antibodies also eliminated the conditioned medium-induced increase in phagocytosis. Taken together, SPE B inhibited phagocytosis, but CS mycelium extract abrogated this inhibition by causing cytokine production.

Immunization with a streptococcal multiple-epitope recombinant protein protects mice against invasive group A streptococcal infection.

Streptococcus pyogenes (group A Streptococcus; GAS) causes clinical diseases, including pharyngitis, scarlet fever, impetigo, necrotizing fasciitis and streptococcal toxic shock syndrome. A number of group A streptococcus vaccine candidates have been developed, but only one 26-valent recombinant M protein vaccine has entered clinical trials. Differing from the design of a 26-valent recombinant M protein vaccine, we provide here a vaccination using the polyvalence epitope recombinant FSBM protein (rFSBM), which contains four different epitopes, including the fibronectin-binding repeats domain of streptococcal fibronectin binding protein Sfb1, the C-terminal immunogenic segment of streptolysin S, the C3-binding motif of streptococcal pyrogenic exotoxin B, and the C-terminal conserved segment of M protein. Vaccination with the rFSBM protein successfully prevented mortality and skin lesions caused by several emm strains of GAS infection. Anti-FSBM antibodies collected from the rFSBM-immunized mice were able to opsonize at least six emm strains and can neutralize the hemolytic activity of streptolysin S. Furthermore, the internalization of GAS into nonphagocytic cells is also reduced by anti-FSBM serum. These findings suggest that rFSBM can be applied as a vaccine candidate to prevent different emm strains of GAS infection.

Galectin-3 Inhibits Galectin-8/Parkin-Mediated Ubiquitination of Group A Streptococcus.

Group A streptococcus (GAS) is an important human pathogen that causes a wide variety of cutaneous and systemic infections. Although originally thought to be an extracellular bacterium, numerous studies have demonstrated that GAS can trigger internalization into nonimmune cells to escape from immune surveillance or antibiotic-mediated killing. Epithelial cells possess a defense mechanism involving autophagy-mediated targeting and killing of GAS within lysosome-fused autophagosomes. In endothelial cells, in contrast, we previously showed that autophagy is not sufficient for GAS killing. In the present study, we showed higher galectin-3 (Gal-3) expression and lower Gal-8 expression in endothelial cells than in epithelial cells. The recruitment of Gal-3 to GAS is higher and the recruitment of Gal-8 to GAS is lower in endothelial cells than in epithelial cells. We further showed that Gal-3 promotes GAS replication and diminishes the recruitment of Gal-8 and ubiquitin, the latter of which is a critical protein for autophagy sequestration. After knockdown of Gal-3 in endothelial cells, the colocalization of Gal-8, parkin, and ubiquitin-decorated GAS is significantly increased, as is the interaction of Gal-8 and parkin, an E3 ligase. Furthermore, inhibition of Gal-8 in epithelial cells attenuates recruitment of parkin; both Gal-8 and parkin contribute to ubiquitin recruitment and GAS elimination. Animal studies confirmed that Gal-3-knockout mice develop less-severe skin damage and that GAS replication can be detected only in the air pouch and not in organs and endothelial cells. These results demonstrate that Gal-3 inhibits ubiquitin recruitment by blocking Gal-8 and parkin recruitment, resulting in GAS replication in endothelial cells.IMPORTANCE In epithelial cells, GAS can be efficiently killed within the lysosome-fused autophaosome compartment. However, we previously showed that, in spite of LC-3 recruitment, the autophagic machinery is not sufficient for GAS killing in endothelial cells. In this report, we provide the first evidence that Gal-3, highly expressed in endothelial cells, blocks the tagging of ubiquitin to GAS by inhibiting recruitment of Gal-8 and parkin, leading to an enhancement of GAS replication. We also provide the first demonstration that Gal-8 can interact with parkin, the critical E3 ligase, for resistance to intracellular bacteria by facilitating the decoration of bacteria with ubiquitin chains. Our findings reveal that differential levels of Gal-3 and Gal-8 expression and recruitment to GAS between epithelial cells and endothelial cells may contribute to the different outcomes of GAS elimination or survival and growth of GAS in these two types of cells.

Streptococcal pyrogenic exotoxin B inhibits apoptotic cell clearance by macrophages through protein S cleavage.

Clearance of apoptotic cells by macrophages plays an important role in maintaining tissue homeostasis. Previous study indicated that streptococcal pyrogenic exotoxin B (SPE B) reduces phagocytic activity in group A streptococcus (GAS) infection. Here, we demonstrate that SPE B causes an inhibitory effect on protein S-mediated phagocytosis. In the presence of SPE B, serum- and purified protein S-mediated phagocytosis of apoptotic cells were significantly inhibited. The binding abilities of protein S to apoptotic cells were decreased by treatment with SPE B. Bacterial culture supernatants from GAS NZ131 strain also caused a reduction of protein S binding to apoptotic cells, but speB mutant strain did not. SPE B directly cleaved protein S in vitro and in vivo, whereas a lower level of cleavage occurred in mice infected with a speB isogenic mutant strain. SPE B-mediated initial cleavage of protein S caused a disruption of phagocytosis, and also resulted in a loss of binding ability of protein S-associated C4b-binding protein to apoptotic cells. Taken together, these results suggest a novel pathogenic role of SPE B that initiates protein S degradation followed by the inhibition of apoptotic cell clearance by macrophages.