Ming T. Lin

Interleukin-1 receptor antagonist attenuates the heat stroke-induced neuronal damage by reducing the cerebral ischemia in rats

The effects of interleukin-1 receptor antagonist (IL-Ira) on both local cerebral blood flow and neuronal damage of the hypothalamus, corpus striatum, cortex or thalamus were assessed in rats with heat stroke. Heat stroke was induced by exposing the urethane-anesthetized rats to a high ambient temperature (42 degrees C). Damage to the hypothalamus, corpus striatum, cortex or thalamus was scored on a scale of zero to three modified from the grading system of Pulsinelli and colleagues in which: 0 = normal, 1 = few neurons damaged, 2 = many neurons damaged, and 3 = all neurons damaged. During the onset of heat stroke, as compared to those of normothermia controls, the heat stroke rats displayed a higher value of colonic temperature or neuronal damage score, as well as a lower value of local cerebral blood flow or mean arterial blood pressure. In addition, compared to those of normothermic, control rats, the heat stroke rats had increased interleukin-1 and tumor necroting factor production in the diencephalon, brain stem and cortex. The heat stroke-induced neuronal damage and diminished local cerebral blood flow in different brain structures, as well as the systemic hypotension, were attenuated in animals pretreated with IL-1ra (200 micrograms/kg, iv) 30 min before the onset of heat stroke. The results indicate that IL-1ra attenuates the heat stroke-induced cerebral neuronal damage by reducing cerebral ischemia in rats.

Effects of Oligopeptide Permease in Group A Streptococcal Infection

ABSTRACT The oligopeptide permease (Opp) of group A streptococci (GAS) is a membrane-associated protein and belongs to the ATP-binding cassette transporter family. It is encoded by a polycistronic operon containing oppA, oppB, oppC, oppD, and oppF. The biological function of these genes in GAS is poorly understood. In order to understand more about the effects of Opp on GAS virulence factors, an oppA isogenic mutant was constructed by using an integrative plasmid to disrupt the opp operon and confirmed by Southern blot hybridization. No transcript was detected in the oppA isogenic mutant by Northern blot analysis and reverse transcriptase PCR. The growth curve for the oppA isogenic mutant was similar to that for wild-type strain A-20. The oppA isogenic mutant not only decreased the transcription of speB, speX, and rofA but also increased the transcription of speF, sagA (streptolysin S-associated gene A), slo (streptolysin O), pel (pleotrophic effect locus), and dppA (dipeptide permease). No effects on the transcription of emm, sda, speJ, speG, rgg, and csrR were found. The phenotypes of the oppA mutant were restored by the oppA revertant and by the complementation strain. The oppA mutant caused less mortality and tissue damage than the wild-type strain when inoculated into BALB/c mice via an air pouch. Based on these data, we suggest that the opp operon plays an important role in the pathogenesis of GAS infection.

Oxidative stress and metal ions regulate a ferritin-like gene, dpr, in Streptococcus pyogenes

Bacteria encounter oxidative stress by exposure to reactive oxygen species (ROS) present in the aerobic environment and during immune responses. In Streptococcus pyogenes, Dpr has been identified as a stress protein conferring resistance to hydrogen peroxide and multiple other stresses. The expression of Dpr is under perR (peroxide stress response regulator) control. However, the exact molecular mechanism of PerR regulation of Dpr is not clear. In this study, a perR deletion mutant was constructed by double cross-over mutagenesis. The profile of Dpr expression, performed by Western blot assay, revealed growth-phase dependency under normal culture conditions. Dpr expression decreased under iron-restricted conditions, whereas iron, zinc, nickel, and hydrogen peroxide induced its expression. The perR mutant does not induce Dpr as well when exposed to environmental signals. PerR binds the promoter region of dpr. Increased iron and hydrogen peroxide concentrations decreased PerR binding to the promoter region of dpr, suggesting that regulation of Dpr by environmental signals is mediated by PerR directly.

Molecular mimicry between streptococcal pyrogenic exotoxin B and endothelial cells

Molecular mimicry between group A streptococcus and host antigens has important roles in the development of post-streptococcal sequelae, including glomerulonephritis and rheumatic heart disease (RHD). The etiology of RHD involves host cross-reactivity with M proteins and carbohydrate antigens. In this study, we show that anti-streptococcal pyrogenic exotoxin B (SPE B) antibodies exhibited characteristics of autoantibodies, which cross-react with endothelial cells. Immunoglobulin G (IgG) deposition and complement activation were observed in the heart valve of SPE B-immunized mice. In addition, apoptosis in the heart valve was detected in SPE B-immunized mice. An anti-SPE B monoclonal antibody (mAb) 10G showed cross-reactivity with human microvascular endothelial (HMEC-1) cells and mouse valve endothelial cells. Passive immunization with mAb 10G also caused IgG deposition, complement activation, and apoptotic cell death in the mouse heart valve. We conducted peptide array and ELISA using synthetic peptides to identify the SPE B antigenic epitope recognized by mAb 10G. Results showed that the major epitope of mAb 10G is localized to amino-acid residues 296–310 of SPE B (P7–8). The cross-reactivity of mAb 10G with endothelial cells was inhibited using P7–8 peptides for competition. These results suggest that anti-SPE B antibodies cross-react with endothelial cells, and that a dominant epitope is located within the amino-acid residues 296–310 of SPE B. Moreover, we found that mAb 10G can also bind to N-acetyl-β-D-glucosamine (GlcNAc) conjugated with bovine serum albumin (BSA), but not to BSA or M1 protein. Competition assay showed that the binding activity of mAb 10G with GlcNAc-BSA and P7–8 of SPE B was inhibited by pretreatment with GlcNAc-BSA or P7–8 peptides. Therefore, our results suggest that conformational molecular mimicry may exist between SPE B and GlcNAc.

The severity of Streptococcus pyogenes infections in children is significantly associated with plasma levels of inflammatory cytokines

Cytokines are intimately involved with the innate and adaptive immune response to bacterial infections. This study was designed to determine the expression of inflammatory cytokines in children by the severity of Streptococcus pyogenes (group A Streptococcus [GAS]) infections. The study population consisted of 16 invasive, 20 noninvasive, and 24 pharyngeal colonization, and 21 healthy controls. All children underwent the laboratory tests and cytokine measurement. GAS isolates were analyzed for emm gene typing. Patients with invasive GAS diseases had significantly higher interferon (IFN)-gamma, interleukin (IL)-1beta, IL-6, IL-8, IL-10, and IL-18 than those with noninvasive diseases, colonization, and healthy controls. There was no difference in tumor necrosis factor (TNF)-alpha, IL-12, and IL-2 levels among the groups. Elevated white blood cell counts and levels of C-reactive protein and C3 were detected only in patients with invasive diseases. emm1 and emm12 predominated in invasive disease and colonization. Children with invasive GAS infections exhibited significant up-regulation of plasma levels of IFN-gamma, IL-1beta, IL-6, IL-8, IL-10, and IL-18, and suppression of TNF-alpha and IL-12 during the acute phase of their illness. An exuberant cytokine response was associated with the severity of illness.

Molecular Analysis of Group A Streptococcal Isolates Associated with Scarlet Fever in Southern Taiwan between 1993 and 2002

ABSTRACT Collected between 1993 and 2002 at a Taiwanese university hospital, 77 group A streptococcus isolates associated with scarlet fever were grouped by emm typing and pulsed-field gel electrophoresis. The predominance of an emm1 clone before 1996 and the presence of genetically diverse emm1 and emm4 strains thereafter were found.

Solution structure and backbone dynamics of streptopain: insight into diverse substrate specificity.

Streptococcal pyrogenic exotoxin B (SPE B) is a cysteine protease expressed by Streptococcus pyogenes. The D9N, G163S, G163S/A172S, and G239D mutant proteins were expressed to study the effect of the allelic variants on their protease activity. In contrast to other mutants, the G239D mutant was ∼12-fold less active. The Gly-239 residue is located within the C-terminal S230-G239 region, which cannot be observed in the x-ray structure. The three-dimensional structure and backbone dynamics of the 28-kDa mature SPE B (mSPE B) were determined. Unlike the x-ray structure of the 40-kDa zymogen SPE B (proSPE B), we observed the interactions between the C-terminal loop and the active site residues in mSPE B. The structural differences between mSPE B and proSPE B were the conformation of the C-terminal loop and the orientation of the catalytic His-195 residue, suggesting that activation and inactivation of SPE B is involved in the His-195 side-chain rotation. Dynamics analysis of mSPE B and the mSPE B/inhibitor complexes showed that the catalytic and C-terminal loops were the most flexible regions with low order parameter values of 0.5 to 0.8 and exhibited the motion on the ps/ns timescale. These findings suggest that the flexible C-terminal loop of SPE B may play an important role in controlling the substrate binding, resulting in its broad substrate specificity.

Changing Epidemiology of Streptococcus pyogenes emm Types and Associated Invasive and Noninvasive Infections in Southern Taiwan

ABSTRACT A total of 242 isolates were recovered from 76 patients with invasive diseases, 89 with scarlet fever, and 77 with pharyngitis. The most frequent emm types were types 12 (43.4%), 4 (18.2%), and 1 (16.9%). emm12 reemerged in 2005 and peaked in 2007. emm11 was recovered only from patients with invasive disease.

Procaspase 8 and bax are up-regulated by distinct pathways in streptococcal pyrogenic exotoxin B-induced apoptosis

We have previously identified integrin αvβ3 and Fas as receptors for the streptococcal pyrogenic exotoxin B (SPE B), and G308S, a mutant of SPE B that binds to Fas only. In the current study we found that after binding to αvβ3, SPE B stimulated the tyrosine phosphorylation of JAK2 and STAT1. STAT1 tyrosine phosphorylation was inhibited by a JAK2 inhibitor, AG490, short interfering RNA (siRNA) silencing of JAK2, and anti-αVβ3 antibody. AG490 also decreased the binding of tyrosine-phosphorylated STAT1 to the procaspase 8 promoter, decreasing procaspase 8 expression, suggesting that SPE B up-regulates procaspase 8 expression via the JAK2/STAT1 pathway. Alternatively, both SPE B and G308S increased STAT1 phosphorylation at serine 727, which was inhibited by anti-Fas antibody, a p38 inhibitor, SB203580, and siRNA silencing of p38. In addition, SPE B and G308S increased binding of serine-phosphorylated STAT1 to the Bax promoter and Bax expression, which was decreased by SB203580. SPE B and G308S-stimulated Bax expression was also inhibited by anti-Fas antibody. These findings suggest that Fas mediate SPE B-induced Bax expression through p38. Silencing of JAK2 or p38 by siRNA blocked procaspase 8 expression, whereas only p38 siRNA decreased Bax expression. Furthermore, JAK2 inhibition and p38 inhibition reduced SPE B-induced apoptosis, but only p38 inhibition blocked G308S-induced apoptosis.

Streptococcal Pyrogenic Exotoxin B-Induced Apoptosis in A549 Cells Is Mediated through αvβ3 Integrin and Fas

ABSTRACT Our previous work suggested that streptococcal pyrogenic exotoxin (SPE) B-induced apoptosis is mediated through a receptor-like mechanism. In this study, we have identified αvβ3 and Fas as the SPE B receptors for this function. The SPE B fragment without the RGD motif and G308S, a SPE B mutant with the RSD motif, induced less apoptosis than did native SPE B, suggesting that the RGD motif is critical for SPE B-induced apoptosis. Fluorescein isothiocyanate-SPE B binding assays and immunoprecipitation analysis showed that SPE B specifically interacted with αvβ3. Anti-αvβ3 antibody partially inhibited SPE B-induced apoptosis but had no effect on G308S-induced apoptosis. In addition, Fas binding to SPE B was verified in an affinity column and an immunoprecipitation analysis. Anti-Fas antibody inhibited SPE B- and G308S-induced apoptosis in a dose-dependent manner, suggesting that Fas-mediated SPE B-induced apoptosis also occurs RGD independently. Both anti-αvβ3 and anti-Fas antibodies synergistically inhibited SPE B-induced apoptosis. The apoptotic cascades were activated by SPE B and G308S, with a little delay by the latter. After SPE B binding, the cell surface level of αvβ3, but not of Fas, was decreased. The decreased αvβ3 level was restored by treatment with the proteasome inhibitor MG132, suggesting a SPE B-mediated endocytosis of integrin αvβ3 via the ubiquitin-proteasome system. Taken together, our results demonstrate that SPE B-induced apoptosis is mediated through αvβ3 integrin and Fas in a synergistic manner.