Characterization of <Emphasis Type="Italic">Streptococcus mutans</Emphasis> diversity by determining restriction fragment-length polymorphisms of the <Emphasis Type="Italic">gtfB</Emphasis> gene of isolates from 5-year-old children and their mothers

The diversity between Streptococcus mutans clinical isolates from 5-year-old children and their mothers in two South African ethnic groups was investigated. The gtfB gene encoding for glucosyltransferase (EC 2.4.1.5), an enzyme responsible for the synthesis of extracellular polysaccharides was characterized by PCR-RFLP with HaeIII restriction enzyme digestion. Forty-seven children were examined for dental caries and 128 S. mutans clinical isolates cultured from samples of their saliva and plaque and from the saliva of their mothers. Thirty-three children had active caries (70%) and the remainder (n = 14) were caries-free. Caries prevalence was significantly different (p = 0.02) between black African and coloured children, but no differences were found between gtfB amplitypes by caries or ethnic grouping. Thirty-four (27%) of the S. mutans clinical isolates investigated did not ferment melibiose. Melibiose-negative phenotypes (n = 10) isolated from four families showed gtfB RFLP patterns identical to each other. Mothers and children harboured between one and three amplitypes. GtfB amplitypes were identical in 17 families (17/47), of which nine only were identical to S. mutans reference strains. The percentage match between S. mutans amplitype from mothers and their children was low (13%) in the caries-free group compared to children with caries (44%). RFLP analysis of the gtfB gene showed the diversity of S. mutans genotypes within two South African populations that were acquired from mothers and other sources.     

Inhibition of Family II Pyrophosphatases by Analogs of Pyrophosphate and Phosphate

Imidodiphosphate (the pyrophosphate analog containing a nitrogen atom in the bridge position instead of oxygen) is a potent inhibitor of family II pyrophosphatases from Streptococcus mutans and Streptococcus gordonii (inhibition constant Ki approximately 10 microM), which is slowly hydrolyzed by these enzymes with a catalytic constant of approximately 1 min(-1). Diphosphonates with different substituents at the bridge carbon atom are much less effective (Ki = 1-6 mM). The value of Ki for sulfate (a phosphate analog) is only 12 mM. The inhibitory effect of the pyrophosphate analogs exhibits only a weak dependence on the nature of the metal ion (Mn, Mg, or Co) bound in the active site.     

Complementary exploration of the action pattern of hyaluronate lyase from Streptococcus agalactiae using capillary electrophoresis, gel-permeation chromatography and viscosimetric measurements

Hyaluronic acid (HA) was treated with hyaluronate lyase (GBS HA lyase, E.C. 4.2.2.1, from Streptococcus agalactiae strain 4755), and the products have been analyzed by capillary electrophoresis (CE-UV and online CE-ESIMS), gel-permeation chromatography (GPC) and viscosimetric measurements. The resulting electropherograms showed that the enzyme produced a mixture of oligosaccharides with a 4,5-unsaturated uronic acid nonreducing terminus. More exhaustive degradation of HA led to increasing amounts of di-, tetra-, hexa-, octa- and decasaccharides. Using CE, linear relationships were found between peak area of the observed oligosaccharides and reaction time. Determination of viscosity at different stages of reaction yielded an initial rapid decrease following Michaelis-Menten theory. A reaction time-dependent change in the elution position of the HA peak due to partial digestion of HA with GBS hyaluronate lyase has been observed by GPC. These results indicated that the HA lyase under investigation is an eliminase that acts in a nonprocessive endolytic manner, as at all stages of digestion a mixture of oligosaccharides of different size were found. For GBS HA lyase from Streptococcus agalactiae strain 3502, previously published findings reported an action pattern that involves an initial random endolytic cleavage followed by rapid exolytic and processive release of unsaturated disaccharides. Our results suggest that differences between the two enzymes from distinct S. agalactiae strains (GBS strains 4755 and 3502) have to be considered.     

Identification and characterization of bacterial-binding property in the type III repeat domain of fibronectin

To characterize fibronectin binding with Granulicatella adiacens, a causative agent of infective endocarditis, monoclonal antibodies were generated against human fibronectin and selected for their capacity to inhibit the fibronectin binding of the organism. Thermolysin and lysyl-endopeptidase digests of fibronectin were characterized by Western blot. The epitope of inhibitory monoclonal antibody was found in the central portion of fibronectin known as the cell-binding domain, and not in the N-terminal portion known to be the binding region of most microbial species, e.g., Staphylococcus aureus and Streptococcus pyogenes. While these two species could bind to both the N-terminal and central portion, Escherichia coli and G. adiacens bind only to the latter. Excess amounts of free fibronectin in the solution inhibited the bacterial adherence to the N-terminal fibronectin fragment, but not to the central region, thereby suggesting the central region plays a significant role for in vivo bacterial colonization in the presence of high concentrations of soluble fibronectin.     

Clindamycin is neuroprotective in experimental Streptococcus pneumoniae meningitis compared with ceftriaxone

In animal models of Streptococcus pneumoniae meningitis, rifampin is neuroprotective in comparison to ceftriaxone. So far it is not clear whether this can be generalized for other protein synthesis-inhibiting antimicrobial agents. We examined the effects of the bactericidal protein synthesis-inhibiting clindamycin (n = 12) on the release of proinflammatory bacterial components, the formation of neurotoxic compounds and neuronal injury compared with the standard therapy with ceftriaxone (n = 12) in a rabbit model of pneumococcal meningitis. Analysis of the CSF and histological evaluation were combined with microdialysis from the hippocampal formation and the neocortex. Compared with ceftriaxone, clindamycin reduced the release of lipoteichoic acids from the bacteria (p = 0.004) into the CSF and the CSF leucocyte count (p = 0.011). This led to lower extracellular concentrations of hydroxyl radicals (p = 0.034) and glutamate (p = 0.016) in the hippocampal formation and a subsequent reduction of extracellular glycerol levels (p = 0.018) and neuronal apoptosis in the dentate gyrus (p = 0.008). The present data document beneficial effects of clindamycin compared with ceftriaxone on various parameters linked with the pathophysiology of pneumococcal meningitis and development of neuronal injury. This study suggests neuroprotection to be a group effect of bactericidal protein synthesis-inhibiting antimicrobial agents compared with the standard therapy with beta-lactam antibiotics in meningitis.     

一株口腔链球菌新种—寡发酵链球菌产过氧化氢特性的研究

从健康人口腔中分离的寡发酵链球菌(Streptococcus oligofermentans)能够产生大量的过氧化氢,可能具有抑制致病菌的潜力。为了研究该细菌产过氧化氢的特性,检测了其在不同生长时期和从不同底物产过氧化氢的能力。结果表明,寡发酵链球菌从对数生长早期就开始产过氧化氢,在对数生长后期及稳定期过氧化氢产量达到最高,随后下降。在PYG培养基中,寡发酵链球菌所产的过氧化氢主要来源于大豆蛋白胨和酵母提取物;而代谢终产物乳酸也可作为过氧化氢产生的底物。对3种可能与过氧化氢生成有关的氧化酶的酶活测定表明,寡发酵链球菌具有乳酸氧化酶(LOX)及NADH氧化酶(NOX)的活性,说明其过氧化氢的产生主要依赖于这两种酶的活力。     

变形链球菌UA159及其基因缺陷菌生长特性变化的初步研究

目的探讨变形链球菌密度信号系统相关基因缺陷后其生长特性的变化情况。方法分别配制BHI液体培养基,含2%葡萄糖的BHI液体培养基,含2%蔗糖的BHI液体培养基,然后将细菌接种于上述3个营养环境中生长,采用722S型可见光分光光度计,进行细菌生长曲线的测定。结果在3种不同的营养环境中,变形链球菌UA159△ComD基因缺陷菌株的生长速度最快,变形链球菌UA159菌株变形链球菌UA159△LuxS基因变异株次之,变形链球菌UA159野生菌株最慢。结论在本实验中,无论那种密度感应信号系统被阻断后,均会影响变形链球菌的生长。     

Crystal Structure of the Minor Pilin FctB Reveals Determinants of Group A Streptococcal Pilus Anchoring

Cell surface pili are polymeric protein assemblies that enable bacteria to adhere to surfaces and to specific host tissues. The pili expressed by Gram-positive bacteria constitute a unique paradigm in which sortase-mediated covalent linkages join successive pilin subunits like beads on a string. These pili are formed from two or three distinct types of pilin subunit, typically encoded in small gene clusters, often with their cognate sortases. In Group A streptococci (GAS), a major pilin forms the polymeric backbone, whereas two minor pilins are located at the tip and the base. Here, we report the 1.9-Å resolution crystal structure of the GAS basal pilin FctB, revealing an immunoglobulin (Ig)-like N-terminal domain with an extended proline-rich tail. Unexpected structural homology between the FctB Ig-like domain and the N-terminal domain of the GAS shaft pilin helps explain the use of the same sortase for polymerization of the shaft and its attachment to FctB. It also enabled the identification, from mass spectral data, of the lysine residue involved in the covalent linkage of FctB to the shaft. The proline-rich tail forms a polyproline-II helix that appears to be a common feature of the basal (cell wall-anchoring) pilins. Together, our results indicate distinct structural elements in the pilin proteins that play a role in selecting for the appropriate sortases and thereby help orchestrate the ordered assembly of the pilus.     

Synthesis of a core disaccharide from the Streptococcuspneumoniae type 23F capsular polysaccharide antigen

The synthesis of methyl α-l-rhamnopyranosyl-(1→2)-β-d-galactopyranoside and methyl α-l-rhamnopyranosyl-(1→2)-3-(glycer-2-yl-phosphate)-β-d-galactopyranoside disaccharides from the Streptococcuspneumoniae type 23F capsular polysaccharide is reported. A simple protecting group strategy was followed using commercially available monosaccharides and phosphorylating reagents. H-Phosphonate and phosphoramidite coupling chemistries were explored for introducing the phosphodiester. Hydrazine hydrate was found to be a mild and efficient deacetylating agent, which was required to avoid phosphate migration during the deprotection of the phosphodiester functionalized disaccharide.     

Co-operative inhibition by fluoride and zinc of glucosyl transferase production and polysaccharide synthesis by mutans streptococci in suspension cultures and biofilms

Fluoride and zinc, alone or in combination at concentrations of 0.2 mM, inhibited production-secretion of glucosyltranferases by Streptococcus mutans UA159 growing in suspension cultures. Inhibition did not involve growth inhibition or starvation. Fluoride and zinc also inhibited glucan production, especially insoluble glucan, in fed-batch biofilms. Inhibition of biofilms appeared to be associated with starvation as indicated by markedly decreased ATP pools and iodophilic polysaccharide levels in biofilm cells. As insoluble glucans are important for virulence of mutans streptococci, the inhibitory actions of fluoride and zinc could significantly affect cariogenicity.