Antigenic diversity of IgA receptors in Streptococcus pyogenes

Abstract In a previous study, group A and group B streptococcal IgA receptors were shown to differ serologically, in agreement with their known structural unrelatedness. The present study was undertaken to serologically compare the IgA binding epitopes of group A streptococcal strains representing various serotypes by the use of antisera to this species. It was found that blocking antibodies occurred in antisera to IgA binding but not to non-binding strains and that binding of IgA to a streptococcal strain was generally blocked by antiserum to the homologous type. However, cross-testing of a panel of 11 IgA binding strains, representing various M and T serotypes, with 10 different antisera to group A streptococci, demonstrated that IgA receptors were inhibited to a highly variable degree and that inhibition patterns were unique for each type. Comparing solubilized IgA receptors of various strains in immunoblot experiments, a variation in the molecular mass, between approximately 35 and 45 kDa, emerged. The IgA binding epitopes, analogous to protective sites of streptococcal M-protein, thus exhibited hypervariability which may suggest that IgA binding also plays a key role for evading host immune defence mechanisms.     

Electrotransformation of Streptococcus pyogenes with plasmid and linear DNA

Electrotransformation was used to introduce both plasmid and linear DNA into Streptococcus pyogenes. The method was optimized using strain NZ131, for which transformation frequencies up to 10(7) per micrograms of plasmid DNA were obtained. A linear fragment of DNA, containing the streptokinase gene (ska) in which an internal fragment had been replaced with an erythromycin resistance gene (erm), was transformed into strain NZ131 with a frequency of 10(3) per micrograms DNA. The introduction of linear DNA into S. pyogenes by electrotransformation should be useful for future genetic analyses as well as targeted gene replacement.     

Replication origin of Streptococcus pyogenes, organization and cloning in heterologous systems

The origin of DNA replication (oriC) of Streptococcus pyogenes, group A streptococci (GAS), has been cloned in Escherichia coli and reintroduced by transformation into other GAS strains. Transformation frequencies into GAS strains with oriC-carrying plasmids occurred with unusually high frequencies. However, the oriC-containing plasmids in the new recipients were found to be unstable and had a tendency to integrate into the chromosome, even when a recA GAS strain was used as a recipient. The GAS oriC was able to direct the replication of autonomous plasmids in group B streptococcal recipients. The chromosomal organization of the oriC region of GAS relative to other bacterial species appears to be similar to oriC of Bacillus subtilis and other Gram-positive microorganisms.     

化脓链球菌中野生型和突变体FtsB蛋白的铁色素结合特性

目的:进一步比较化脓链球菌中野生型和Y137A、W204A突变型FtsB蛋白的铁色素结合特性,确定铁色素结合位点。方法:制备野生型和Y137A、W204A突变型FtsB蛋白,采用ICP-MS和ITC比较其铁色素结合能力;Na2SO4还原实验比较铁色素的还原速率;CD热变性和盐酸胍化学变性实验比较其铁色素结合稳定性。结果:Y137A、W204A突变型FtsB蛋白的铁色素结合能力和结合稳定性均低于野生型蛋白,铁色素的还原速率均高于野生型蛋白,可见Tyr137和Trp204是FtsB蛋白重要的铁色素结合位点。结论:进一步确定了Tyr137和Trp204氨基酸残基在FtsB与铁色素结合中的重要作用,为深入研究细菌中的铁色素转运机理及开发疫苗候选物奠定了一定的理论基础。     

化脓性链球菌脂蛋白FtsB的基因克隆、表达、纯化与功能研究

体外克隆化脓性链球菌5005的tsb基因,表达并纯化FtsB蛋白,并对其铁色素结合特性进行初步研究.首先通过PCR方法从基因组中扩增ftsb基因,将其连接到pGEX4T-1载体上,转入大肠杆菌DH5a中大量扩增,将测序正确的重组载体转化到表达宿主大肠杆菌BL21进行表达,优化诱导表达条件.利用亲和层析方法纯化表达产物,并对FtsB的铁色素结合特性进行研究,同时通过DEPC封闭组氨酸实验对其结合位点进行初步研究.成功构建原核表达载体pGEX-ftsb,获得分子量约33 kD的野生型FtsB蛋白,产率为30 mg/L.紫外-可见光谱研究表明FtsB和铁色素结合后在450 nm处出现紫外吸收峰,DEPC封闭组氨酸实验证明FtsB中组氨酸不参与铁色素的结合.对FtsB蛋白进行铁色素结合特性和结合位点进行研究,为进一步研究细菌中的铁色素转运机理及开发疫苗候选物和药靶奠定一定的理论基础.     

In vitro compared activity of telithromycin and azithromycin against northwest Italian isolates of Streptococcus pyogenes and Streptococcus pneumoniae with different erythromycin susceptibility

Aims: This study compared the in vitro activity of telithromycin with that of azithromycin against 438 Streptococcus pyogenes and 198 Streptococcus pneumoniae, isolated over the period 2005–2007 from specimens of different human origin obtained in three Piemonte Region’s hospitals. Methods and Results: The determination of antimicrobial activity was evaluated by the microdilution broth method and the erythromycin‐resistant (Ery‐R) phenotypes by the triple‐disc test. Exactly 78·8% of S. pyogenes and 69·2% of S. pneumoniae were erythromycin‐susceptible (Ery‐S). Concerning S. pyogenes, telithromycin was active against M and inducible MLS_B, subtype‐C, phenotypes but not against constitutive MLS_B strains. Telithromycin acted well against all S. pneumoniae, irrespective of their mechanism of macrolide‐resistance. On the contrary, the Ery‐R isolates, both S. pyogenes and S. pneumoniae, were resistant to azithromycin. Conclusions: Our results indicate that macrolide resistance in streptococci still persist in northwest Italy (21·2% of S. pyogenes and 30·8% of S. pneumoniae) and that telithromycin is confirmed as being extremely active even against recent clinical Ery‐R streptococcal isolates. Significance and Impact of the Study: The present study emphasizes that an active surveillance of the phenotype distribution and antibacterial resistance in streptococci is essential in guiding the effective use of empirical treatment option for streptococcal infections, also at regional level.     

Isolation of a new superantigen with potent mitogenic activity to murine T cells from Streptococcus pyogenes

Abstract A mitogenic substance on murine lymphocytes was detected in the culture supernate of Streptococcus pyogenes type 12 strain. This substance had a molecular weight of 28 000 and p I 9.2, and was designated as S. pyogenes mitogen (SPM). The proliferative response of C3H/HeN spleen cells began at 1 ng ml⁻¹ and reached a maximal response at 100 ng ml⁻¹ of SPM for 4 days culture. Anti-Thy 1.2 mAb and complement-treated spleen cells abrogated the proliferative response to any dose of SPM. Although the anti-major histocompatibility complex class I mAbs had no blocking effect on proliferation by SPM, this proliferation was substantially inhibited by the addition of either anti-I-A or anti-I-E mAb, and complete inhibition was produced by the addition of both mAbs. Fixed antigen-presenting cells still induced T cell proliferation by SPM. A significant expansion of T cells bearing Vβ13 T-cell receptor was observed up to 73% among the Thy1.2⁺ cells in cultures stimulated with SPM, indicating expansion in a Vβ-specific manner. Immunoblotting of IEF-separated proteins showed that anti-streptococcal pyrogenic exotoxin (SPE) C reacted with a protein of p I 6.9 and anti-SPEB did not show any reactivity. SPEA was reported to expand Vβ8.1 and 8.2 bearing murine T cells, and SPM did not. SPM also exhibited potent mitogenic activity on human T cells and Vβ21⁺ T cells were selectively expanded. These results lead to the conclusion that SPM was neither SPEA, B nor C, but a new protein belonging to a group of streptococcal superantigens with activity on not only human but also murine lymphocytes.     

利用固定化金属亲和层析和质谱技术鉴定化脓性链球菌铜结合蛋白

化脓性链球菌是一种常见的高致病性革兰氏阳性菌,每年都在全球范围内引发大量的感染现象与病死案例。金属蛋白在微生物的代谢以及毒性构成中的作用已经逐渐得到重视。通过固定化金属离子亲合层析技术对化脓性链球菌中铜结合蛋白进行分离,配合质谱技术的使用,共鉴定出21个铜结合蛋白,这些蛋白分别在翻译、代谢、离子通道等各个生理进程中发挥相应作用。     

Molecular cloning, purification and immunological responses of recombinants GroEL and DnaK from Streptococcus pyogenes

To better understand the roles of heat shock proteins in streptococcal diseases, the groEL and dnaK genes from Streptococcus pyogenes were cloned and their products (GroEL and DnaK) and derivatives (F2GroEL, F3GroEL and C1DnaK) purified as His-tagged fusion proteins. Western blot analysis of the purified proteins with sera from individuals with streptococcal diseases demonstrated that 29 out of 36 sera tested were reactive with GroEL and eight recognized DnaK. Rabbit antiserum against myosin recognized both GroEL and DnaK. Antibodies raised against purified F2GroEL and DnaK reacted with myosin in the ELISA but not in a Western immunoblot. These data indicate that the S. pyogenes GroEL and DnaK may be important immunogens during streptococcal infections. Furthermore, we provide evidence of an immunogenic relatedness of the GroEL and DnaK proteins with myosin that could play a role in the pathogenesis of streptococcal non-suppurative sequelae.     

Limited repertoire of the C-terminal region of the M protein in Streptococcus pyogenes

We have amplified genomic sequences (emm) that may encode M protein from strains of Streptococcus pyogenes using the polymerase chain reaction (PCR). Genomic DNA from 22 isolates representing 14 M serotypes was selected for the study. Primers which corresponded to the observed N-terminal signal sequence and the variable C-terminal sequences of emm6, emm49 and ennX were used. PCR products using emm6 and emm49 oligonucleotides were classified into two mutually exclusive groups which correspond to the presence or absence of serum opacity factor. These findings support the concept of limited heterogeneity in the C-terminal sequences of the M protein.     

Purification and biochemical characterization of a basic superantigen (SPEX/SMEZ3) from Streptococcus pyogenes

A potent basic superantigen (designated streptococcal pyrogenic exotoxin X, SPEX/SMEZ3) was purified to homogeneity from culture supernatants of a Streptococcus pyogenes scarlatina strain of type 12 (genotype speA(-), speC(-)) and characterized. Sequence alignments revealed SPEX to be an allele of the streptococcal mitogens type Z (SMEZ). The N-terminal amino acid sequence of SPEX was found with LEVDNNSLLR to be identical to the recently described acidic superantigen SMEZ. Although SPEX/SMEZ genes were present in all of the streptococcal strains tested, a toxin production could only be detected in a small number of strains. The produced toxin concentration in the culture supernatants of positive strains differed between 0 and 20 ng ml(-1). The purified SPEX stimulated human T-lymphocytes with Vbeta8 specificity at extremely low concentrations (lower than 100 pg ml(-1)).     

利用IMAC与LC-MS/MS技术分离和鉴定化脓链球菌中的锌结合蛋白

化脓链球菌是一种革兰氏阳性致病菌,能引起一系列疾病。运用固定化金属离子亲和层析技术(IMAC)富集化脓链球菌中锌结合蛋白,进行溶液酶解,利用先进的LC-MS/MS技术对富集的锌结合蛋白进行质谱分析。鉴定到48个在化脓链球菌中与锌结合相关蛋白,这些蛋白主要涉及到蛋白翻译、糖代谢、金属转运、氧化等重要生理过程。这些结果对于深入研究化脓链球菌的致病机制,寻找新的抗菌药物作用靶点和疫苗候选物具有重要意义。     

Binding of complement subcomponent C1q to Streptococcus pyogenes: evidence for interactions with the M5 and FcRA76 proteins

Binding of C1q, the first component of the complement system, to some human pathogens has been earlier reported. In the present study, direct binding of C1q to group A streptococci (GAS) of various serotypes as well as some other Gram-positive and Gram-negative species was demonstrated. The interaction between C1q and GAS was investigated more in detail. In hot neutral extracts of a number of GAS strains two components of 64 and 52 kDa, respectively, bound C1q; alkaline and SDS extracts yielded the 52 kDa component as the main C1q-binding substance. Trypsin treatment of the SDS extracts of two GAS strains suggested the C1q-binding component(s) to be of protein nature. C1q-binding material purified from the SDS extract of an avirulent strain, type T27, was separated in 12% SDS-PAGE and probed in Western blot with human C1q and fibrinogen, conjugated to horse radish peroxidase (HRP) as well as rabbit IgG antibodies complexed to HRP (PAP system). The 52 kDa component was non-reactive with fibrinogen or rabbit IgG. However, C1q-binding components purified from the alkaline extracts of two M-positive strains revealed strong binding of either fibrinogen (type M5) or both fibrinogen and rabbit IgG (type M76); the molecular mass of these components, 55 kDa and 43–40 kDa, respectively, was in agreement with the reported molecular mass of the M5 and FcRA76 proteins. Our findings suggest that C1q may interact with GAS through certain M-family proteins as well as by a so far unidentified surface factor of protein nature occurring in most GAS strains. The involvement of M-family proteins, regarded as virulence factors of these organisms, may suggest the interaction of GAS with C1q as biologically important.     

Insertional inactivation of virR in Streptococcus pyogenes M49 demonstrates that VirR functions as a positive regulator of ScpA, FcRA, OF, and M protein

Abstract Several reports have shown that Streptococcus pyogenes strains which produce opacity factor (OF+) have diverged significantly from OF⁻ serotypes. This study questions whether several surface proteins of an OF+ culture are regulated by the positive regulatory protein VirR, in a manner similar to OF~ strains. Interruption of the virR region of an OF⁺ S. pyogenes (strain CS101, M type 49) was performed using a temperature-sensitive plasmid containing a fragment of virR . Interruption of the virR region produced cultures with (indétectable amounts of M49 and ScpA proteins, and reduced the yield of FcRA protein. In addition, mutants had a significant reduction in detectable opacity factor. These results suggest that virR functions as a positive regulator of a variety of surface proteins in OF⁺ strains.     

Effects of penicillin and erythromycin on adherence of invasive and noninvasive isolates of Streptococcus pyogenes to laminin

This study investigated the possible relationship between the invasiveness of group AStreptococcus (GAS) strains and their abilities to adhere tolaminin and assessed the effects of subinhibitory concentrations of penicillin anderythromycin on the ability of GAS to adhere to laminin. The adherence of noninvasiveand highly invasive isolates of GAS to laminin was significantly higher than theadherence displayed by isolates of low invasiveness. Antibiotic treatment causedsignificant reductions in adherence to laminin in all three groups of strains.Penicillin was more successful in reducing the adherence abilities of the tested GASstrains than erythromycin.     

酿脓链球菌Cas9核酸酶的重组表达、分离纯化及酶学特性

【背景】Cas9核酸酶是一种RNA引导的核酸内切酶,可与单链向导RNA (single-guide RNA,sgRNA)形成稳定的核糖核蛋白复合物,识别和切割特定的核苷酸片段。由于其具备高灵活性和高效率的特点,目前已经成为基础科学研究领域和临床治疗方法中使用最广泛的基因编辑工具。【目的】为Cas9核酸酶的合理开发和利用提供理论依据。【方法】利用大肠杆菌表达系统表达野生型酿脓链球菌(Streptococcus pyogenes) Cas9核酸酶,经硫酸铵沉淀和镍柱亲和层析两步纯化获得较高纯度表达产物,并对其热稳定性、pH稳定性、金属离子的影响等酶学特性进行研究。【结果】经高密度发酵后,大肠杆菌湿菌重达191.0 g/L。纯化后酿脓链球菌Cas9核酸酶的比酶活达641.29 U/mg,纯化倍数为16.02,收率为46.40%。Cas9核酸酶在25-42°C保温2 h后剩余酶活保持在65%以上,而在45°C保温15 min后全部失活;其在pH 6.0-10.0范围内稳定性较高,剩余酶活大于68%,在pH9.0时稳定性最高;0.5-20.0mmol/L浓度范围内的Mg²⁺...