Temperature-Induced Unfolding Pathway of Staphylococcal Enterotoxin B:Insights from Circular Dichroism and Molecular Dynamics Simulation

In this study,circular dichroism(CD)and molecular dynamics(MD)simulation were used to investigate the thermal unfolding pathway of staphylococcal enterotoxin B(SEB)at temperatures of 298–371 and 298–500 K,and the relationship between the experimental and simulation results were explored.Our computational findings on the secondary structure of SEB showed that at room temperature,the CD spectroscopic results were highly consistent with the MD results.Moreover,under heating conditions,the changing trends of helix,sheet and random coil obtained by CD spectral fitting were highly consistent with those obtained by MD.In order to gain a deeper understanding of the thermal stability mechanism of SEB,the MD trajectories were analyzed in terms of root mean square deviation(RMSD),secondary structure assignment(SSA),radius of gyration(R_(g)),free energy surfaces(FES),solvent-accessible surface area(SASA),hydrogen bonds and salt bridges.The results showed that at low heating temperature,domain Ⅰ without loops(omitting the mobile loop region)mainly relied on hydrophobic interaction to maintain its thermal stability,whereas the thermal stability of domain Ⅱ was mainly controlled by salt bridges and hydrogen bonds.Under high heating temperature conditions,the hydrophobic interactions in domain Ⅰ without loops were destroyed and the secondary structure was almost completely lost,while domain Ⅱ could still rely on salt bridges as molecular staples to barely maintain the stability of the secondary structure.These results help us to understand the thermodynamic and kinetic mechanisms that maintain the thermal stability of SEB at the molecular level,and provide a direction for establishing safer and more effective food sterilization processes.

Assessment of the inhibitory effects of sodium nitrite, nisin, potassium sorbate, and sodium lactate on Staphylococcus aureus growth and staphylococcal enterotoxin A production in cooked pork sausage using a predictive growth model

This study was conducted to analyze the effects of sodium nitrite,nisin,potassium sorbate,and sodium lactate against Staphylococcus aureus(S.aureus)growth and staphylococcal enterotoxins(SEs)production in cooked pork sausage by inoculating sausage samples containing preservative with an S.aureus strain producing staphylococcal enterotoxin A(SEA)and then storing them at 37℃ for 36 h.Samples were analyzed every 3 h to count the S.aureus colonies and to detect SEA.The modified Gompertz model was used to describe S.aureus growth in the samples under various conditions,and the preservatives with a significant antimicrobial effect were selected.In addition,the antimicrobial effects of the selected preservatives under various concentrations were tested.Results showed that sodium nitrite,nisin,and potassium sorbate had a weak effect against S.aureus growth and had no effect against SEA production,whereas sodium lactate could significantly inhibit S.aureus growth and SEA production.Moreover,the antimicrobial effect of sodium lactate was concentration-dependent,wherein sodium lactate concentration<12 g/kg showed no inhibitory effect,but when the concentration was increased to 24 g/kg,sodium lactate could effectively inhibit S.aureus growth and SEA production,and at 48 g/kg,sodium lactate had a significant inhibitory effect.

STAPHYLOCOCCAL SCALDED SKIN SYNDROME: RETROSPECTIVE ANALYSIS OF 82 CASES

Objective To explore distinctive clinical manifestations and appropriate treatment, and assess prognosis of staphylococcal scalded skin syndrome （SSSS）. Methods A retrospective analysis was conducted of the data of 82 cases of SSSS hospitalized at Xinhua Hospital during the period from May 1993 to September 2003. Results The disease in all the 82 patients occurred in their first decade （mean 2.5 years）. Possible predisposing factors were found in 48 （58. 5% ）. Fever was present in 78 （95. 1% ）. Radial spokes of crusting around mouth were present in 80 （ 97. 6% ）. Erythema began on the face, especially around the mouth and eye in 63 （ 76. 8% ）. The course was acute in all cases and the eruptions quickly spread to the whole body within one day to two days. Of the 82 cases of SSSS, 47 were complete form of SSSS, 27 were abortive form of SSSS, and 8 were between the two forms. Staphylococcus aureus with positive staphylocoagulase was isolated from the possible primary infection sites including pharynx, eyelid, conjunctiva, nose, ear, and skin in 18 of 31 patients. Microbiological cultures of bullae and little pustulae developed after the onset were negative in 16 cases. All the 82 patients completely recovered after receiving antibiotic therapy （ ceftriaxone, oxacillin） alone or in combination with human immunoglobulin （IVIG） therapy. Additional IVIG therapy was used in those patients who had systemic involvements such as pneumonia, fever higher than 38. 5℃ or leukocytosis. Conclusion SSSS is a spectrum disease. Besides abortive and complete forms, presenting between the two forms a new form might be appeared in 8 cases who developed both scarlatiniform rash and flaccid bullae. The abortive form and complete form are usually misdiagnosed clinically. Radial spokes of crusting around mouth seem to be characteristic manifestation of SSSS. All the patients in this study had favorable prognosis after receiving prompt diagnosis and appropriate treatment.

Emergence of staphylococcal cassette chromosome mec type Ⅰ with high-level mupirocin resistance among methicillin-resistant Staphylococcus aureus

Objective: To investigate the molecular epidemiology and antimicrobial resistance patterns of methicillin-resistant Staphylococcus aureus(MRSA) among healthcare workers and patients.Methods: MRSA isolates were recovered from nasal swabs collected at a tertiary care hospital of Nepal and confirmed on the basis of Gram staining, conventional biochemical tests, and PCR amplification of mec A gene. PCRs were also used for detection of the different resistance genes and staphylococcal cassette chromosome(SCC) mec types.Antibiotic susceptibility patterns of isolates were assessed by disc diffusion method and minimum inhibitory concentrations were determined by E-test.Results: A total of 29 MRSA were isolated from 536 nasal swabs(5.4%) of health care workers and patients at a tertiary care hospital in Nepal. All isolates were susceptible to amikacin, gentamicin, vancomycin(minimal inhibitory concentrations < 2 mg/m L), tigecycline, tetracycline, nitrofurantoin, rifampicin, quinupristin-dalfopristin, and linezolid. Among the 29 MRSA isolates, resistance to erythromycin(72%), ciprofloxacin(75%), co-trimoxazole(62%), clindamycin(10%), and chloramphenicol(10%) was found, and fifteen isolates(51%)exhibited high-level mupirocin resistance(minimal inhibitory concentrations > 1 024 mg/m L).Fourteen isolates were found harboring the mup A gene and one isolate was found carrying the novel mup B gene. High prevalence(68%) of SCCmec I type was found, followed by SCCmec V(13%) and SCCmec III(3%) among all the MRSA isolates.Conclusions: We found the emergence of SCCmec type I with high-level mupirocin resistance among MRSA in Nepal. Data also suggest that MRSA SCCmec type V strain has spread from the community to the hospital.

Ultrafast solvation dynamics at internal sites of staphylococcal nuclease investigated by site-directed mutagenesis

Internal solvation of protein was studied by site-directed mutagenesis, with which an intrinsically fluorescent probe,tryptophan, is inserted into the desired position inside a protein molecule for ultrafast spectroscopic study. Here we review this unique method for protein dynamics research. We first introduce the frontiers of protein solvation, site-directed mutagenesis, protein stability and characteristics, and the spectroscopic methods. Then we present time-resolved spectroscopic dynamics of solvation dynamics inside cavities of active sites. The studies are carried out on a globular protein, staphylococcal nuclease. The solvation at sites inside the protein molecule＇s cavities clearly reveals characteristics of the local environment. These solvation behaviors are directly correlated to enzyme activity.

THE EFFECT OF SUPERANTIGEN STAPHYLOCOCCALENTEROTOXIN B AND D-GALACTOSAMINE ON BALB/CMOUSE HEPATOCYTES

Objective To observe the role of superantigen staphylococcal enterotoxin B(SEB) andD - galactosamine (D - GalN) on Balb/c mouse hepatocytes and its mechanism. Methods After Balb/c mice wereinjected intraperitoneally with SEB, D- GalN or both, blood samples were collected and livers were removed at 2,6, 12, 24h. Patterns of hepatocellular death were studied morphologically and biochemically, circulating cytokines(TNF, IFN-γ) were determined, and mice mortality within 24h was assessed. Results SEB could induce thetypical apoptotic changes of hepatocytes morphologically and biochemically. The mechanism is probably associatedwith the production and release of Cytokines (such as TNF, IFN- γ, etc).D - GalN could induce hepatocytesapoptosis and degeneration at the same time. Besides this, we confirmed hepatocytes of the mice which wereadministered SEB and D - GalN developing apoptosis at 2, 6h, but after 12h hepatocytes were characterized bysevere injury, the mice mortality within 24h is 50%. Conclusion SEB or D - GalN alone could induce the typicalapoptotic changes of hepatocytes. SEB+D-GalN developed hepatocytes apoptosis in the early stage and necrosisin the later. It suggests that there is some relationship between hepatic cell apoptosis and necrosis, and massivehepatocyte apoptosis is the probably initiating step of acute hepatic necrosis in mice.

Spectroscopic Characterization of Staphylococcal Nuclease Mutants with Tryptophan at Internal Sites

Tryptophan （Trp） is an intrinsic fluorescent probe for detecting the site-specified dynamics inside/outside protein. It is found that the Trp can easily be inserted in desired sites of protein, which affects the integrity of the overall structure. To evaluate this effect, we design thirteen double point mutants of staphylococcal nuclease, each of which has a single Trp residue planted at an internal site. The studies on Trp fluorescence, ANS-binding fluorescence, far- and near-UV CD spectra, and enzymatic activity are carried out. It is found that the mutation at the hydrophobic core of protein generates molten globular state conformation, which is a loose structure compared to their original compactness in wild type （WT）. Its enzyme activity and surface hydrophobicity are also affected. The studies show that by proper site designing and external binding, Trp mutagenesis is a suitable method for carrying out the study on site specified dynamics of proteins.

Effect and Mechanism of Superantigen Staphylococcal Enterotoxin Therapy for Mouse Gastric Tumor

The anti-tumor effect and mechanism of the staphylococcal enterotoxin A (SEA) were studied. The mouse gastric tumor model was produced by subcutaneously inoculating gastric tumor ceils (MGC80-3). The experimental group was treated with SEA, and the control group was treated with normal saline. The percentage of tumor generation and tumor mass was measured. The results showed that the percentage of the tumor generation in the SEA-treated mice was lower than in the control group, but there was no significant difference (P>0. 05). However, the tumor mass in the experimental group was significantly lighter than in the control group, with the difference being very significant (P<0. 001). There were more CD4+ T cells and CD8+ T cells in the tumor of the mice treated with SEA than those of the control group. SEA has an obvious anti-tumor effect on mice gastric tumor. The mechanism might be that SEA induces the effect of superantigen-dependent cell mediated cytotoxicity to the tumor cells.

The Akt Pathway Inhibitor Degeulin Prevents Staphylococcal Enterotoxin B Induced Splenocyte Proliferation and Inflammation

Staphylococcal Enterotoxin B (SEB) is considered a potential biological weapon. It is toxic by both inhalation and ingestion. Effects of ingestion include fever, vomiting and diarrhoea, while inhalation may additionally result in chest pain, dyspnoea, pulmonary oedema and respiratory failure. Severe exposure may be fatal and treatment relies on symptomatic support. At a cellular level, SEB up-regulates T-cell proliferation leading to a pathological inflammatory response. Deguelin, a rotenoid isolated from the African plant Mundulea sericea (Leguminosae), has been shown to reduce cellular proliferation by inhibiting the phosphoinositide 3-kinase/Akt (PI3K/Akt) signalling pathway. Using isolated murine splenocytes, we have demonstrated that treatment with deguelin reduces SEB inducing T cell proliferation by 60%. Deguelin treatment also decreased IL-2 and CCL2 secretion by splenocytes exposed to SEB. We demonstrate that targeting cellular proliferation can significantly reduce inflammation after SEB exposure and suggest that anti-proliferatives may have a role as potential generic medical counter measures if superantigens are used as biological weapons.

Diversity and Distribution of Staphylococcal Chromosomal Cassettes Mec (SCCmec) Types I, II and III in Coagulase-Negative Staphylococcal Strains Isolated from Surfaces and Medico-Technical Materials of the University Hospital of Abomey-Calavi/Sô-Ava

The coagulase-negative staphylococci (CoNS) have long been considered to be low pathogenicity. The possibility of a horizontal transfer of resistance and virulence genes from S. aureus to CoNS could increase the pathogenicity of these bacteria. The objective of this work is to contribute to a better knowledge of the pathogenicity of (CoNS) strains isolated from surfaces and medico-technical materials of the University Hospital of Abomey-Calavi/Sô-Ava. Seventy strains of CoNS isolated from surfaces and medico-technical materials of the University Hospital of Abomey-Calavi were tested for methicillin resistance. The resistance to methicillin was evaluated phenotypically by the resistance of the strains to cefoxitin and then confirmed by the search for the mecA gene using PCR. The genes encoding staphylococcal chromosomal cassette (SCCmec) types I, II and III originally found in S. aureus were tested in CoNS by multiplex PCR using specific primers. All the strains studied showed resistance to methicillin. However, only 28.5% (20/70) carried the mecA gene. SCCmec was identified in only 17.14% (12/70) of these strains. Four strains carried mecA gene as well as one of the three types of SCCmec searched. SCCmec types I, II and III were identified in CoNS strains studied. SCCmec type I was the most frequent chromosomal cassette in mecA+ strains, only or in association with another SCCmec. The study also revealed methicillin-resistant strains carrying SCCmec lacking the mecA gene. Finally, 60% (12/20) of the strains were found to be non-typeable. Our results show that CoNS strains present a high resistance to methicillin and the source of this resistance in the CoNS of our study is not only the mecA gene. There is also a high diversity of SCCmec, justified by a large number of non-typeable CoNS strains. The mecA− SCCmec+ methicillin-resistant strains deserve to be sequenced for further studies.

Neonatal Staphylococcal Scalded Skin Syndrome:A Case Report

Objective:To summarize the clinical features,diagnosis,and treatment of neonatal staphylococcal scalded skin syndrome(SSSS).Methods:The clinical data with SSSS was analyzed,and the related literature was reviewed.Results:The acute onset of the disease was characterized by generalized erythema,epidermis exfoliation,skin non-touch,radial chapping around the mouth,and positive Nissl sign.The culture of binocular secretions and neck exudates showed Staphylococcus aureus and was diagnosed as SSSS.According to the results of drug sensitivity of secretions and exudates,vancomycin was selected for anti-infective treatment,and skincare and symptomatic support were given simultaneously.The child was cured and discharged after ten days of treatment.Conclusion:SSSS belongs to neonatal acute and critical illness.Improving etiological examination and timely targeted anti-infective treatment is the key to correct diagnosis and recovery.

A non-viral gene therapy for melanoma by staphylococcal enterotoxin A

Staphylococcal enterotoxin A(SEA)derived from Staphylococcus aureus,as a superantigen,shows potential for cancer immunotherapy,but systemic immunotoxicity restricts its clinical application.Targeted delivery of SEA to tumor site provides a promising option for reducing the systemic toxicity.Here,we constructed an iRGD peptide(H-[Cys-Arg-Gly-Asp-Lys-Gly-Pro-Asp-Cys]-NH_(2))modified nanoparticle(iDPP)to deliver plasmids encoding SEA for melanoma treatment.The iDPP/SEA nanocomplexes efficiently mediated SEA expression in B16-F10 cells in vivo and in vitro and induced the activation of lymphocytes and maturation of murine bone marrow-derived dendritic cells(BMDCs)in vitro.In the subcutaneous B16-F10 melanoma model,the iDPP/SEA nanocomplexes could effectively enhance immune response and T lymphocytes infiltration in tumor site after intravenous administration,thereby considerably decreased melanoma growth.Meanwhile,no obvious adverse effect was observed after intravenous administration of the iDPP/SEA nanocomplexes in vivo.Our findings demonstrated that gene therapy of SEA is a potential candidate for melanoma treatment.

Inhibition of squamous cancer growth in a mouse model by Staphylococcal enterotoxin B-triggered Th9 cell expansion

Currently, therapy for squamous cancer （SqC） is unsatisfactory. Staphylococcal enterotoxin B （SEB） has strong immune regulatory activity. This study tests the hypothesis that SEB enforces the effect of immunotherapy on SqC growth in a mouse model. C3H/HeN mice and the SqC cell line squamous cell carcinoma VII were used to create an SqC mouse model. Immune cell assessment was performed by flow cytometry. Real-time RT-PCR and western blotting were used to evaluate target molecule expression. An apoptosis assay was used to assess the suppressive effect of T helper-9 （Th9） cells on the SqC cells. The results showed that immunotherapy consisting of SEB plus SqC antigen significantly inhibited SqC growth in the mice. The frequency of Th9 cells was markedly increased in the SqC tissue and mouse spleens after treatment. SEB markedly increased the levels of signal transducer and activator of transcription 5 phosphorylation and the expression of histone deacetylase-1 （HDAC1） and PU.1 （the transcription factor of the interleukin 9 （IL-9） gene） in CD4^＋ T cells. Exposure to SqC-specific Th9 cells markedly induced SqC cell apoptosis both in vitro and in vivo. In conclusion, the administration of SEB induces Th9 cells in SqC-bearing mice, and theseTh9 cells inhibit SqC growth.

The staphylococcal nuclease prevents biofilm formation in Staphylococcus aureus and other biofilm-forming bacteria

The staphylococcal nuclease, encoded by the nucl gene, is an important virulence factor of Staphylococcus aureus. However, the physiological role of the nuclease has not been fully characterized. The current study observed that biofilm development could be prevented in staphylococcal nuclease-producing strains of S. aureus; however, when the nucl gene was knocked out, the ability to form a biofilm significantly increased. Scanning electron and confocal scanning laser microscopy were used to evaluate the role of the nucl gene in biofilm formation. Moreover, the nucl gene product, staphylococcal nuclease, and re- combinant NUC1 protein were found to have a visible effect on other biofilm-forming bacteria, such as Pseudomonas aeru- ginosa, Actinobacillus pleuropneurnoniae, and Haernophilus parasuis. The current study showed a direct relationship between staphylococcal nuclease production and the prevention of biofilm development. The findings from this study underscore the important role of staphylococcal nuclease activity to prevent biofilm formation in S. aureus. They also provided evidence for the biological role of staphylococcal nucleases in other organisms.

Crystallization and preliminary X-ray analysis of SNR 141--An N-terminal fragment of staphylococcal nuclease R

STAPHYLOCOCCAL nuclease(SNase A,EC 3.1.4.7),which hydrolyzes the phosphodiesterbond of DNA and RNA,and releases 3’-phosphate mononucleotides and dinucleotides,con-sists of 149 amino acid residues(MW=16 807)without sulfhydryl and disulfide groups.SNase A was originally derived from Staphylococcus aureus.Later the gene of the enzyme wascloned and inserted into several expression systems.Its crystal structure was detected

NaCl-induced conformational transition of the acid-unfolded state of staphylococcal nuclease

The acid-unfolded state(U_A)of staphylococcal nuclease(SNase)can fold into a state(Astate)in acidic solution of high NaCl concentration,that is,U_A→A.The transition curve of U_A→A inducedby the increasing of NaCl concentration was superimposed with that induced by the increasing of HClconcentration,indicating that the U_A → A transition is induced by the chloride anion being inclined to bind onsome parts of SNase molecules in A state.Circular dichroism(CD)spectra showed that A state hassubstantial secondary structure.Size exclusion chromatography measurement indicated that A state is compact involume.The A state can bind with 1-anilino-naphthalene-8-sulfonate(ANS),a fluorescent probe forhydrophobicity.These results indicated that A state of SNaseR is a molten globule-like state.Besides,ourresults showed that A state can be unfolded by guanidine hydrochloride(Gu · HCl),implying that thehydrophobic interaction between side chains in A state is responsible to its stabilization.

Complete genome sequence of methicillin-sensitive Staphylococcus aureus containing a heterogeneic staphylococcal cassette chromosome element

Staphylococcus aureus is a common human bacterium that sometimes becomes pathogenic,causing serious infections.A key feature of S.aureus is its ability to acquire resistance to antibiotics.The presence of the staphylococcal cassette chromosome(SCC) element in serotypes of S.aureus has been confirmed using multiplex PCR assays.The SCC element is the only vector known to carry the mecA gene,which encodes methicillin resistance in S.aureus infections.Here,we report the genome sequence of a novel methicillin-sensitive S.aureus(MSSA) strain:SCC-like MSSA463.This strain was originally erroneously serotyped as methicillin-resistant S.aureus in a clinical laboratory using multiplex PCR methods.We sequenced the genome of SCC-like MSSA463 using pyrosequencing techniques and compared it with known genome sequences of other S.aureus isolates.An open reading frame(CZ049;AB037671) was identified downstream of attL and attR inverted repeat sequences.Our results suggest that a lateral gene transfer occurred between S.aureus and other organisms,partially changing S.aureus infectivity.We propose that attL and attR inverted repeats in S.aureus serve as frequent insertion sites for exogenous genes.

Structural and biochemical characterization of the staphylococcal phage endolysin LysGH15

With the support by the National Natural Science Foundation of China,Prof.Han Wenyu’s laboratory at the College of Veterinary Medicine,Jilin University,reported the structural and biochemical characterization of staphylococcal phage endolysin LysGH15,which was published in Plos Pathogens(2014,10(5):e1004109).LysGH15demonstrates a wide lytic spectrum and strong lytic activity against methicillin-resistant

新生儿金黄色葡萄球菌型烫伤样皮肤综合征1例

新生儿金黄色葡萄球菌型烫伤样皮肤综合征（staphylococcal scalded skin syndrom,SSSS）极为少见,现将我科最近收治的1例报告如下。 患婴男,2天,孕32周顺产。出生时发现周身皮肤潮红,继之脐部、颜面部皮肤受摩擦处出现剥脱并蔓延至全身,伴纳差、易惊,于1991年8月19日入院。患儿与其母无用药史。羊水污染++、母会阴侧切处有脓性分泌物。家族中无同类病史。体检:T37.2℃,咽充血,全身皮肤呈烫伤样外观。糜烂处表皮松驰如卷烟纸样外翻卷起,并有大片痂皮脱落,剥脱面呈鲜红色,有少量渗液,皮肤Nikolsky征阳性。血白细胞 4.1×10~9/L,中性粒细胞0.38,淋巴细胞0.62。皮肤渗液培养为金黄色葡萄球菌,诊断为SSSS。立即给予新青霉素Ⅱ0.5g/d,氨苄青霉素0.5g/d静滴,新霉素乳剂外涂,并全身暴露放入消毒保暖箱中。

新生儿剥脱性皮炎(附6例临床分析)

新生儿剥脱性皮炎又名金葡菌烫伤样皮肤综合征(staphylococcal scaldedskin syndrom ssss)、葡萄球菌性中毒性表吱坏死松解症或 Ritter 氏病,此病国内报道较少;我院新生儿病房自1975年10月至1991年3月共收住6例,观报告如下.