Streptococcus pneumoniae heat shock protein 70 does not induce human antibody responses during infection

Mouse monoclonal antibodies (mAbs) were developed against Streptococcus pneumoniae in search for potential common pneumococcal proteins as vaccine antigens. mAb 230,B-9 (IgG1) reacted by immunoblotting with a 70-kDa protein which was isolated by immunoaffinity chromatography and subsequent preparative electrophoresis. N-terminal amino acid sequencing showed homology to that of heat shock protein 70 (hsp70). The hsp70 epitope reactive with mAb 230,B-9 was found in all the pneumococci examined as well as in other streptococci and enterococci. The epitope was not expressed in several other examined Gram-positive or -negative bacteria. Pneumococcal hsp70 has by other investigators been proposed to be a vaccine candidate. Binding experiments using flow cytometry showed that the epitope was not surface-exposed on live exponential phase grown S. pneumoniae. Human patient sera did not react with affinity-purified pneumococcal hsp70. Therefore the pneumococcal hsp70 does not seem to be of special interest in a vaccine formulation. The human sera contained antibodies to high molecular proteins co-purified with hsp70. Some of these proteins could be the pneumococcal surface protein A.     

Protective effect of beta-glucan against systemic Streptococcus pneumoniae infection in mice

The antimicrobial effect of soluble beta-1,3-D-glucan from Sclerotinia sclerotiorum (SSG) was examined in mice experimentally infected intraperitoneally (i.p.) with Streptococcus pneumoniae serotypes 4 and 6B. SSG was administered i.p. either 3 days before challenge or 3-48 h after challenge. The number of bacteria in blood samples and the mouse survival rates were recorded. Pre-challenge SSG administration protected dose-dependently against both S. pneumoniae type 4 and 6B infections. SSG injected 24 h post-challenge had a curative effect against type 6B but not type 4 pneumococcal infection. The data demonstrate that SSG administered systemically protects against pneumococcal infection in mice.     

Atypical behaviour and survival of Streptococcus pyogenes L forms during intraperitoneal infection in rats

Groups of rats were injected intraperitoneally with cell wall-deficient (L) forms of Streptococcus pyogenes, with their parental (S) forms, as well as with a combined inoculum of both forms (S+L). Peritoneal exudate samples were harvested on days 1, 3, 7, 15 and 30 after challenge and were investigated by microbiological, electron microscopic, cytometric and biochemical methods. Parental S forms were isolated from peritoneal exudate samples up to day 15 post infection, while L form cultures were isolated until the end of the examined interval. Electron microscopic examination revealed continuous adhesion of L forms on the macrophage surface as well as intracellular persistence inside them. It was demonstrated that the intraperitoneal inflammatory response to L form infection was higher than to the other infections and the monocyte-macrophage populations were predominant. The established atypical behaviour and long survival of S. pyogenes L forms in the rat's peritoneum could explain some of the mechanisms of the pathogens' persistence as well as the reasons for chronic streptococcal infections.     

The exopolysaccharides produced by Streptococcus thermophilus Rs and Sts have the same repeating unit but differ in viscosity of their milk cultures

The polysaccharides produced by Streptococcus thermophilus Rs and Sts in skimmed milk consist of -Gal and -Rha in a molar ratio of 5:2. Linkage analysis and 1D/2D NMR (¹H and ¹³C) studies revealed that both polysaccharides have the same branched heptasaccharide repeating unit:

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Remarkably, the two strains differ in their effects on the viscosity of stirred milk cultures. The milk culture of S. thermophilus Rs is non-ropy and affords 135 mg/L polysaccharide with an average molecular mass of 2.6×10³ kDa. In contrast, the milk culture of S. thermophilus Sts is ropy and produces 127 mg/L polysaccharide with an average molecular mass of 3.7×10³ kDa. Permeability measurements of non-stirred milk cultures of both strains suggest that both strains have a similar effect on the protein–polysaccharide network. Therefore, the only clear difference between both strains, which may cause the difference in ropiness of the milk cultures, is the difference in molecular mass of the polysaccharide.     

Characterization of HetN, a protein involved in heterocyst differentiation in the cyanobacterium Anabaena sp. strain PCC 7120

AtlA is a major cell-lytic enzyme called autolysin in Streptococcus mutans . In this study, we identified the atlg gene-encoding autolysin (Atlg), consisting of 863 residues from Streptococcus sobrinus 6715DP, and confirmed lytic activity of recombinant Atlg by zymography of S. sobrinus cells. An atlA -inactivated mutant was constructed in S. mutans Xc, and the atlg gene product was characterized by plasmid complementation. Microscopic analysis, saliva-induced aggregation assay and autolysis assay of static cultures in air revealed that the atlg gene product partially complemented the role of AtlA. Furthermore, the capability of biofilm formation of the atlA- deficient mutant cultivated in air was restored by plasmid comprising the atlg gene. These findings suggest that Atlg may be involved in cell separation and biofilm formation in S. sobrinus .     

Bovicin HC5 inhibits wasteful amino acid degradation by mixed ruminal bacteria in vitro

Streptococcus bovis HC5 produces a broad spectrum lantibiotic (bovicin HC5) that inhibits pure cultures of hyper ammonia-producing bacteria (HAB). Experiments were preformed to see if: (1) S. bovis HC5 cells could inhibit the deamination of amino acids by mixed ruminal bacteria taken directly from a cow, (2) semi-purified bovicin was as effective as S. bovis HC5 cells, and 3) semi-purified and the feed additive monensin were affecting the same types of ammonia-producing ruminal bacteria. Because purified and semi-purified bovicin HC5 was as effective as S. bovis HC5 cells, it appeared that bovicin HC5 was penetrating the cell membranes of HAB before it could be degraded by peptidases and proteinases. Mixed ruminal bacteria that were successively transferred and enriched nine times with trypticase did not become significantly more resistant to either bovicin HC5 (50 AU mL⁻¹) or monensin (5 μM), and amplified rDNA restriction analysis indicated that bovicin HC5 and monensin appeared to be selecting against the same types of bacteria.     

肺炎链球菌SP0306蛋白的表达纯化及结晶研究

SP0306蛋白是肺炎链球菌TIGR4菌株中的一种假想的转录因子,但其蛋白三维结构及生物学功能尚未明了,生物信息学分析提示其可能调控碳水化合物代谢相关基因的表达。成功构建了SP0306蛋白的全长表达载体PET28a-sp0306,利用大肠杆菌BL21(DE3)菌株进行原核表达,获得了以可溶形式表达的目的蛋白。经Ni-NTA柱亲和层析及DEAE阴性离子交换层析纯化后,获得了高纯度的目的蛋白。采用悬滴气相扩散法获得了质量较好的SP0306蛋白晶体,并初步进行了晶体X射线衍射,为其最终的三维结构解析及生物学功能研究奠定了基础。     

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。     

2型猪链球菌MocR家族转录调控因子SSU0562基因敲除突变体的构建及毒力分析

目的:构建2型猪链球菌强毒株05ZYH33中MocR家族转录调控因子SSU0562基因敲除的突变株,探索SSU0562基因缺失对细菌基本生物学特性和毒力的影响。方法:构建左右两侧为SSU0562基因上下游的同源序列,中间部分为壮观霉素抗性基因(Spcr)的基因敲除质粒,通过同源重组的方法筛选SSU0562基因敲除突变株Δ0562。对突变株与野生株的基本生物学特性进行系统的比较分析,并且将小鼠作为动物感染的模型来研究突变株的毒力。结果:组合PCR的分析及基因测序结果均表明Spcr完全取代了S.suis2中SSU0562基因位点,表明基因敲除突变体Δ0562构建成功,反转录PCR(RT-PCR)证实了突变株Δ0562中SSU0562基因在转录水平的缺失;在溶血活性、生长速率及对小鼠的致病力方面,突变株Δ0562与野生株05ZYH33相比均无显著差别,然而革兰染色实验显示突变株Δ0562的成链能力明显减弱。结论:猪链球菌强毒株05ZYH33的毒力并未因SSU0562基因的缺失而发生显著性改变,表明SSU0562基因并非猪链球菌的毒力决定因子,但很有可能参与猪链球菌成链能力的调控。     

多重聚合酶链反应与荚膜肿胀试验对肺炎链球菌血清分型的比较研究

为评估多重聚合酶链反应（PCR ）对肺炎链球菌血清分型的可行性,分别采用多重PCR和荚膜肿胀试验对568株肺炎链球菌进行血清分型,并对分型结果进行比较分析。结果显示,568株肺炎链球菌中,213株通过荚膜肿胀试验分出16个血清群,主要有血清群19（23．1％,131／568）、6（5．3％,30／568）、23（1．6％,9／568）、14（1．4％,8／568）、9（1．1％,6／568）、15（1．1％,6／568）等,分型率为37．5％（213／568）;356株通过多重PCR分出21个血清群,主要有血清群19（27．8％,158／568）、23（8．5％,48／568）、6（7．4％,42／568）、14（4．4％,25／568）、3（4．2％,24／568）、15（3．5％,20／568）等,分型率为62．7％（356／568）。荚膜肿胀试验鉴定出血清群4和18,但多重PCR未能鉴定;多重PCR鉴定出血清群5、12、35、16、17和22,但荚膜肿胀试验未能鉴定。多重PCR与荚膜肿胀试验对19F、19A血清型的鉴定无显著差异。结果提示,这2种方法对肺炎链球菌血清分型结果有差别,多重PCR的分型率高于荚膜肿胀试验。对来源复杂的标本进行肺炎链球菌血清分型,2种方法可相互补充,以提高分型率。