Structures of two cell wall-associated polysaccharides of a Streptococcus mitis biovar 1 strain. A unique teichoic acid-like polysaccharide and the group O antigen which is a C-polysaccharide in common with pneumococci.

The cell wall of Streptococcus mitis biovar 1 strain SK137 contains the C-polysaccharide known as the common antigen of a closely related species Streptococcus pneumoniae, and a teichoic acid-like polysaccharide with a unique structure. The two polysaccharides are different entities and could be partially separated by gel chromatography. The structures of the two polysaccharides were determined by chemical methods and by NMR spectroscopy. The teichoic acid-like polymer has a heptasaccharide phosphate repeating unit with the following structure: The structure neither contains ribitol nor glycerol phosphate as classical teichoic acids do, thus we have used the expression teichoic acid-like for this polysaccharide. The following structure of the C-polysaccharide repeating unit was established: where AAT is 2-acetamido-4-amino-2,4, 6-trideoxy-D-galactose. It has a carbohydrate backbone identical to that of one of the two structures of C-polysaccharide previously identified in S. pneumoniae. C-polysaccharide of S. mitis is characterized by the presence, in each repeating unit, of two residues of phosphocholine and both galactosamine residues in the N-acetylated form. Immunochemical analysis showed that C-polysaccharide constitutes the Lancefield group O antigen. Studies using mAbs directed against the backbone and against the phosphocholine moiety of the C-polysaccharide revealed several different patterns of these epitopes among 95 S. mitis and Streptococcus oralis strains tested and the exclusive presence of the group O antigen in the majority of S. mitis biovar 1 strains.     

Mutations in the tacF gene of clinical strains and laboratory transformants of Streptococcus pneumoniae: impact on choline auxotrophy and growth rate

The nutritional requirement that Streptococcus pneumoniae has for the aminoalcohol choline as a component of teichoic and lipoteichoic acids appears to be exclusive to this prokaryote. A mutation in the tacF gene, which putatively encodes an integral membrane protein (possibly, a teichoic acid repeat unit transporter), has been recently identified as responsible for generating a choline-independent phenotype of S. pneumoniae (M. Damjanovic, A. S. Kharat, A. Eberhardt, A. Tomasz, and W. Vollmer, J. Bacteriol. 189:7105-7111, 2007). We now report that Streptococcus mitis can grow in choline-free medium, as previously illustrated for Streptococcus oralis. While we confirmed the finding by Damjanovic et al. of the involvement of TacF in the choline dependence of the pneumococcus, the genetic transformation of S. pneumoniae R6 by using S. mitis SK598 DNA and several PCR-amplified tacF fragments suggested that a minimum of two mutations were required to confer improved fitness to choline-independent S. pneumoniae mutants. This conclusion is supported by sequencing results also reported here that indicate that a spontaneous mutant of S. pneumoniae (strain JY2190) able to proliferate in the absence of choline (or analogs) is also a double mutant for the tacF gene. Microscopic observations and competition experiments during the cocultivation of choline-independent strains confirmed that a minimum of two amino acid changes were required to confer improved fitness to choline-independent pneumococcal strains when growing in medium lacking any aminoalcohol. Our results suggest complex relationships among the different regions of the TacF teichoic acid repeat unit transporter.     

Allelic variation of polymorphic locus lytB, encoding a choline-binding protein, from streptococci of the mitis group

The choline-binding protein LytB, an N-acetylglucosaminidase of Streptococcus pneumoniae, is the key enzyme for daughter cell separation and is believed to play a critical pathogenic role, facilitating bacterial spreading during infection. Because of these peculiarities LytB is a putative vaccine target. To determine the extent of LytB polymorphism, the lytB alleles from seven typical, clinical pneumococcal isolates of various serotypes and from 13 additional streptococci of the mitis group (12 atypical pneumococci and the Streptococcus mitis type strain) were sequenced. Sequence alignment showed that the main differences among alleles were differences in the number of repeats (range, 12 to 18) characteristic of choline-binding proteins. These differences were located in the region corresponding to repeats 11 to 17. Typical pneumococcal strains contained either 14, 16, or 18 repeats, whereas all of the atypical isolates except strains 1283 and 782 (which had 14 and 16 repeats, respectively) and the S. mitis type strain had only 12 repeats; atypical isolate 10546 turned out to be a DeltalytB mutant. We also found that there are two major types of alternating repeats in lytB, which encode 21 and 23 amino acids. Choline-binding proteins are linked to the choline-containing cell wall substrate through choline residues at the interface of two consecutive choline-binding repeats that create a choline-binding site. The observation that all strains contained an even number of repeats suggests that the duplication events that gave rise to the choline-binding repeats of LytB involved two repeats simultaneously, an observation that is in keeping with previous crystallographic data. Typical pneumococcal isolates usually grew as diplococci, indicating that an active LytB enzyme was present. In contrast, most atypical isolates formed long chains of cells that did not disperse after addition of purified LytB, suggesting that in these strains chains were produced through mechanisms unrelated to LytB.     

Versatility of choline metabolism and choline-binding proteins in Streptococcus pneumoniae and commensal streptococci

The pneumococcal choline-containing teichoic acids are targeted by choline-binding proteins (CBPs), major surface components implicated in the interaction with host cells and bacterial cell physiology. CBPs also occur in closely related commensal species, Streptococcus oralis and Streptococcus mitis , and many strains of these species contain choline in their cell wall. Physiologically relevant CBPs including cell wall lytic enzymes are highly conserved between Streptococcus pneumoniae and S. mitis . In contrast, the virulence-associated CBPs, CbpA, PspA and PcpA, are S. pneumoniae specific and are thus relevant for the characteristic properties of this species.     

Taxonomic study of "tufted mitior" strains of streptococci (Streptococcus sanguinis biotype 11); recognition of a new genospecies

The taxonomic position of tufted strains of streptococci, phenotypically resembling Streptococcus mitis and previously referred to as 'tufted mitior' was investigated. By 16S rRNA sequence analysis, it was clear that the "tufted mitior" strains belonged to the mitis group of species within the genus Streptococcus. It was confirmed that these strains were taxonomically independent at the species level, sharing less than 43%, DNA-DNA similarity with all established species of the mitis group. However biochemical test data obtained, using three commercial identification kits (Rapid ID32 Strep, STREPTOGRAM, and Biolog GP-plate) together with in-house biochemical tests employing 4-MUF-linked fluorogenic substrates did not reveal sufficient differential tests with which to identify the "tufted mitior" strains unequivocally. From these data, we conclude that these "tufted mitior" strains represent a new taxon within the mitis group of the genus Streptococcus, and propose that they should be considered as a genospecies until differential phenotypic characteristics are found for their identification.     

Trimethylsilyl-sugar profiles of Streptococcus milleri and Streptococcus mitis

Seventy strains of 'viridans-group' streptococci were analysed gas chromatographically after preparation of trimethylsilyl ethers of their cellular sugars. The resulting profiles were evaluated as a possible aid to taxonomy. Glycerol, glucose, galactose, N-acetyl-glucosamine and N-acetylmuramic acid were found in all strains, in varying amounts. Rhamnose was the major neutral sugar in most strains, other than representatives of Streptococcus mitis, which invariably had ribose and usually anhydroribitol but no rhamnose. One strain of Strep. mitis possessed arabitol. Some strains of Strep. mitis and 'Strep. milleri' were alone in containing N-acetylgalactosamine. A combination of N-acetylgalactosamine and rhamnose in the absence of ribose was diagnostic for strains of 'Strep. milleri'.     

The influences of hinge length and composition on the susceptibility of human IgA to cleavage by diverse bacterial IgA1 proteases

The influences of IgA hinge length and composition on its susceptibility to cleavage by bacterial IgA1 proteases were examined using a panel of IgA hinge mutants. The IgA1 proteases of Streptococcus pneumoniae, Streptococcus sanguis strains SK4 and SK49, Neisseria meningitidis, Neisseria gonorrhoeae, and Haemophilus influenzae cleaved IgA2-IgA1 half hinge, an Ab featuring half of the IgA1 hinge incorporated into the equivalent site in IgA1 protease-resistant IgA2, whereas those of Streptococcus mitis, Streptococcus oralis, and S. sanguis strain SK1 did not. Hinge length reduction by removal of two of the four C-terminal proline residues rendered IgA2-IgA1 half hinge resistant to all streptococcal IgA1 metalloproteinases but it remained sensitive to cleavage by the serine-type IgA1 proteases of Neisseria and Haemophilus spp. The four C-terminal proline residues could be substituted by alanine residues or transferred to the N-terminal extremity of the hinge without affect on the susceptibility of the Ab to cleavage by serine-type IgA1 proteases. However, their removal rendered the Ab resistant to cleavage by all the IgA1 proteases. We conclude that the serine-type IgA1 proteases of Neisseria and Haemophilus require the Fab and Fc regions to be separated by at least ten (or in the case of N. gonorrhoeae type I protease, nine) amino acids between Val(222) and Cys(241) (IgA1 numbering) for efficient access and cleavage. By contrast, the streptococcal IgA1 metalloproteinases require 12 or more appropriate amino acids between the Fab and Fc to maintain a minimum critical distance between the scissile bond and the start of the Fc.     

Trimethylsilyl-sugar profiles of Streptococcus milleri and Streptococcus mitis

Seventy strains of 'viridans-group' streptococci were analysed gas chromatographically after preparation of trimethylsilyl ethers of their cellular sugars. The resulting profiles were evaluated as a possible aid to taxonomy. Glycerol, glucose, galactose, N-acetyl-glucosamine and N-acetylmuramic acid were found in all strains, in varying amounts. Rhamnose was the major neutral sugar in most strains, other than representatives of Streptococcus mitis , which invariably had ribose and usually anhydroribitol but no rhamnose. One strain of Strep, mitis possessed arabitol. Some strains of Strep. mitis and ' Strep. milleri ' were alone in containing N-acetylgalactosamine. A combination of N-acetylgalactosamine and rhamnose in the absence of ribose was diagnostic for strains of ' Strep. milleri '.     

Evolution of Streptococcus pneumoniae and its close commensal relatives

Streptococcus pneumoniae is a member of the Mitis group of streptococci which, according to 16S rRNA-sequence based phylogenetic reconstruction, includes 12 species. While other species of this group are considered prototypes of commensal bacteria, S. pneumoniae is among the most frequent microbial killers worldwide. Population genetic analysis of 118 strains, supported by demonstration of a distinct cell wall carbohydrate structure and competence pheromone sequence signature, shows that S. pneumoniae is one of several hundred evolutionary lineages forming a cluster separate from Streptococcus oralis and Streptococcus infantis. The remaining lineages of this distinct cluster are commensals previously collectively referred to as Streptococcus mitis and each represent separate species by traditional taxonomic standard. Virulence genes including the operon for capsule polysaccharide synthesis and genes encoding IgA1 protease, pneumolysin, and autolysin were randomly distributed among S. mitis lineages. Estimates of the evolutionary age of the lineages, the identical location of remnants of virulence genes in the genomes of commensal strains, the pattern of genome reductions, and the proportion of unique genes and their origin support the model that the entire cluster of S. pneumoniae, S. pseudopneumoniae, and S. mitis lineages evolved from pneumococcus-like bacteria presumably pathogenic to the common immediate ancestor of hominoids. During their adaptation to a commensal life style, most of the lineages gradually lost the majority of genes determining virulence and became genetically distinct due to sexual isolation in their respective hosts.     

The teichoic acid (C-polysaccharide) synthesized by Streptococcus pneumoniae serotype 5 has a specific structure

The teichoic acid synthesized by Streptococcus pneumoniae serotype 5, also known as pneumococcal common antigen (C-polysaccharide), was purified. On the basis of compositional analysis, HPAEC-PAD analysis, MALDI-TOF mass spectrometry and NMR spectroscopy, made on the native polysaccharide and on the dephosphorylated repeating unit, the following structure is proposed: [structure: see text]. This C-polysaccharide (C-PS), differs from those previously described by the replacement of Glc by Gal in its repeating unit structure.     

Detection of Specific Strains and Variants of Streptococcus cremoris in Mixed Cultures by Immunofluorescence

Antisera against four different strains of Streptococcus cremoris were raised by injecting rabbits with washed suspensions of whole cells. These antisera interacted specifically with the corresponding strain in a mixture of up to nine different S. cremoris strains. The antisera could be used for analyzing the composition of mixed cultures containing these strains by immunofluorescence. Competition experiments were performed in batch and continuous cultures under amino acid limitation. A bacteriophage-sensitive variant of S. cremoris SK11 (SK1128) could be distinguished from a bacteriophage-resistant variant (SK1143) by the same immunofluorescence technique. The competition between the two variants and the stability of both variants in pure cultures were followed with the specific antibodies. Antibodies against the purified proteolytic system of S. cremoris Wg2 were used to determine the presence of proteases by immunofluorescence in several S. cremoris strains under different culture conditions. The described immunofluorescence methods can be used to analyze complex mixed starter cultures common in the dairy industry as the strains and variants present in these mixtures can be recognized microscopically.     

Inhibition of adhesion of viridans streptococci to fibronectin-coated hydroxyapatite beads by lipoteichoic acid

Fibronectin-coated hydroxyapatite (FnHA) beads were used in a model adhesion assay to isolate the lipoteichoic acid (LTA) mediated adhesion of oral streptococci. Representative strains of the commonly isolated viridans streptococci were incubated with FnHA beads in the presence and absence of exogenous LTA. The LTA inhibited the adhesion of all strains to a greater or lesser extent, but only a very few strains were inhibited by more than 90%. Strains of Streptococcus sanguis Type II and Streptococcus mitis which synthesize an amphiphile other than LTA were also inhibited. The findings provided circumstantial evidence for the involvement of LTA in the adhesion of this group of oral bacteria.     

Inhibition of adhesion of viridans streptococci to fibronectin-coated hydroxyapatite beads by lipoteichoic acid

Fibronectin-coated hydroxyapatite (FnHA) beads were used in a model adhesion assay to isolate the lipoteichoic acid (LTA) mediated adhesion of oral streptococci. Representative strains of the commonly isolated viridans streptococci were incubated with FnHA beads in the presence and absence of exogenous LTA. The LTA inhibited the adhesion of all strains to a greater or lesser extent, but only a very few strains were inhibited by more than 90%. Strains of Streptococcus sanguis Type II and Streptococcus mitis which synthesize an amphiphile other than LTA were also inhibited. The findings provided circumstantial evidence for the involvement of LTA in the adhesion of this group of oral bacteria.     

Binding of laminin to oral and endocarditis strains of viridans streptococci.

Attachment of bacteria to the host tissue is regarded as a crucial step in the development of many types of infections. Recent studies by us and others have shown that matrix proteins which serve as adhesion proteins for eucaryotic cells may also be recognized by some bacteria. In the present communication, we report that several strains of viridans streptococci are able to bind to laminin. Most strains isolated from blood and heart valves of patients with endocarditis expressed laminin receptors, whereas only a few of the strains isolated from the oral cavity recognized this protein. This observation indicates that laminin binding might be an important factor in the pathogenesis of viridans endocarditis. Laminin binding to two strains (Streptococcus mitis UAB594 and UAB597) isolated from patients with endocarditis was characterized further. The bacterial cells expressed a limited number of laminin receptors (4 X 10(2) to 1 X 10(3) per cell) which bound the protein in a high-affinity interaction (Kd, 40 to 80 nM). This receptor of S. mitis UAB594 was heat labile and could be solubilized from bacteria by brief digestion with trypsin. Solubilized receptors which competed with cell-bound receptors for 125I-laminin could be adsorbed on laminin-Sepharose but not on Sepharose substituted with fibrinogen or fibronectin. Comparison of laminin receptors from S. mitis with those previously described for Streptococcus pyogenes suggest that different sites in the laminin molecule are recognized by the two bacteria and hence that the corresponding receptor molecules are not identical.     

The influence of biosurfactants released by S. mitis BMS on the adhesion of pioneer strains and cariogenic bacteria

The influence of Streptococcus mitis BMS biosurfactants on the adhesion of eight pioneer and four cariogenic oral bacterial strains was, for a first screening, examined in a microtiter plate assay. The adhesion to pellicle-coated wells of three cariogenic strains was inhibited >70% by the biosurfactants, while only one pioneer strain showed >70% reduction. The reduction for the other strains did not exceed 50%. Subsequently, adhesion of Streptococcus mutans ATCC 25175 and Streptococcus sobrinus HG 1025, both cariogenic strains, and Actinomyces naeslundii T14V-J1 and Streptococcus oralis J22, two pioneer strains, to biosurfactants-coated enamel with and without a salivary pellicle was studied in a parallel plate flow chamber. A biosurfactants coating to enamel with or without a pellicle caused a reduction in the number of adhering cariogenic organisms, although no such reduction was observed for the pioneer strains. Consequently, it is concluded that S. mitis BMS biosurfactants may play a protective role against adhesion of cariogenic bacteria.     

Chemical composition of Streptococcus mutans cell walls and their susceptibility to Flavobacterium L-11 enzyme

The susceptibility to a cell wall lytic L-11 enzyme from Flavobacterium sp. and the quantitative and/or qualitative composition of the cell walls of some strains of cariogenic Streptococcus mutans and a non-cariogenic strain of Streptococcus mitis were determined. The purified cell walls of S. mutans strains HS-1 (serotype a), BHT (b), NCTC10449 (c), C67-1 (c), C67-25 (c), OMZ 176 (d), MT703 (e), MT557 (f), OMZ65 (g), and AHT (g), and S. mitis CHT contained glutamic acid, alanine, and lysine as well as muramic acid and glucosamine as a peptidoglycan component. Besides these amino acids, significant amounts of threonine were detected in strains HS-1, OMZ65, and AHT cell walls, and considerable amounts of aspartic acid and/or threonine as well as several other amino acids in OMZ176, OMZ65, and CHT cell walls. Rhamnose was a common special component of the cell walls of S. mutans strains BHT, NCTC10449, MT703, B2 (e), MT557, and AHT, and S. mitis CHT. An additional sugar component, glucose, was detected in the cell walls of all of these strains except BHT, and galactose was found in BHT, AHT, and CHT cell walls. Galactosamine was present in S. mitis CHT cell walls. Varying amounts of phosphorus were detected in the cell walls of all the strains examined. The cell walls of all these streptococcal strains except MT703, 6715, and AHT were susceptible to the lytic action of the L-11 enzyme to various extents. No consistent relationship was observed between the amino acid and sugar composition of these cell walls and their susceptibility to the L-11 enzyme. The chemical composition of these cell walls is discussed in terms of the serological classification of S. mutans.     

Autolysis in strains of viridans streptococci.

Seven strains of viridans streptococci of the species Streptococcus sanguis, S. mutans and S. mitis were investigated for autolysis. The effect of pH, salt concentration and temperature on the autolytic process was studied in Na2HPO4/NaH2PO4 buffer. Whole cells and walls of all strains autolysed most rapidly at pH values above 7. Autolysis of whole cells of S. sanguis and one strain of S. mitis (ATCC15909) was maximal in 0-05 TO 0-2 M buffer, while the two S. mutans strains and S. mitis ATCC15912 showed maximal autolysis in 0-5 and 1-0 M buffers. Cultures harvested in the stationary phase of growth possessed only slightly decreased autolytic activity compared with those from the exponential phase. Whole cells autolysed more rapidly at 37 degrees C Than at 45 degrees C and 10 degrees C. Autolysis of isolated walls of three strains of S. mitis (ATCC903, ATCC15909 and ATCC15912) was maximal at pH 7-0 AND 7-5 and in 1-0 M buffers. Streptococcus mitis ATCC15909 also showed maximal lysis in 0-01 M and 0-5 M buffers. An endopeptidase action of the autolytic system of S. mitis ATCC15912 was indicated by the progressive release of soluble amino groups during autolysis of the walls. No release of reducing groups was observed. Several free amino acids were released during autolysis of these walls, alanine, lysine and glutamic acid being in greatest quanitity.     

Relatedness between Streptococcus pneumoniae and viridans streptococci: transfer of penicillin resistance determinants and immunological similarities of penicillin-binding proteins

The occurrence of highly variable penicillin-binding proteins (PBPs) in penicillin-resistant Streptococcus pneumoniae suggested that transfer of homologous genes from related species may be involved in resistance development. Antiserum and monoclonal antibodies raised against PBPs 1a and 2b from the susceptible S. pneumoniae R6 strain were used to identify related PBPs in 41 S. mitis, S. sanguis I and S. sanguis II strains mostly isolated in South Africa with MIC values ranging from less than 0.15 to 16 mg/ml. Furthermore, the possibility of genetic exchange was examined with 30 penicillin-resistant strains of this collection (MIC greater than 0.06 mg/ml) as donors using S. pneumoniae R6 as recipient in transformation experiments. The majority of S. mitis and S. sanguis II strains but none of the S. sanguis I strains could transform penicillin resistance genes into S. pneumoniae R6. All positive donor strains and all susceptible isolates of S. mitis and S. sanguis II strains contained PBPs which cross-reacted with the anti-PBP 1a and/or anti-PBP 2b antibodies. On the other hand, only five of the 14 S. sanguis I strains contained a PBP that reacted with one of the antibodies. This strongly suggested the presence of genes homologous to the pneumococcal PBP 1a and 2b genes in viridans streptococci, and documents that penicillin resistance determinants can be transformed from viridans streptococci into the pneumococcus.     

α-溶血性链球菌在健康人群口咽部分布及 生物拮抗作用

目的通过采集健康人群口咽部分泌物,分析上呼吸道中α-溶血性链球菌的分布状况,并对革兰阳性化脓性球菌进行生物拮抗试验,为进一步研究上呼吸道益生菌提供理论基础。方法随机自愿原则,用无菌咽拭子采集沈阳市年龄在3～75岁的300名健康人群咽后壁分泌物,对α-溶血性链球菌进行鉴定和定量分析。对致病菌的生物拮抗试验采用小缸杯法。结果定量分析显示不同年龄人群咽后壁的α-溶血性链球菌检出率均较高。在咽后壁菌群中α-溶血性链球菌构成比最多的是幼儿组,达到60.3%。其中唾液链球菌群在幼儿组所占比重最大;老年组人群格氏链球菌占比较大;儿童、青年、成人以缓症链球菌和口腔链球菌为主。对革兰阳性化脓性球菌的生物拮抗试验显示,1株婴儿链球菌婴儿亚种能够拮抗8株致病菌;4株分离菌只能拮抗1株病原菌,提示不同的菌株拮抗病原菌的能力差异较大。结论α-溶血性链球菌在人群中分布广,数量多,不同年龄人群的菌群构成存在差异。并且某些菌株显示出对致病菌较强的生物拮抗作用,推测这些菌株在呼吸道黏膜保护中起到重要作用,可作为上呼吸道益生菌的备选菌株。     

Physico-chemical and structural properties of the surfaces of Peptostreptococcus micros and Streptococcus mitis as compared to those of mutans streptococci, Streptococcus sanguis and Streptococcus salivarius.

The surface properties of nine Streptococcus mitis and four Peptostreptococcus micros strains from the oral cavity were examined and compared with a large group of oral streptococci. Zeta potential and contact angle measurements were employed to determine physico-chemical cell surface properties. In addition, elemental surface concentration ratios were obtained via X-ray photoelectron spectroscopy, and surface structures were examined with transmission electron microscopy. The S. mitis and P. micros strains were found to have higher isoelectric points, higher hydrophobicities and higher N/C surface concentration ratios than some other oral streptococci. The combined data suggest that both species possess large amounts of surface protein. All the S. mitis strains displayed abundant surface fibrils in negative staining, but the P. micros strains were devoid of surface appendages indicating that surface protein is present in different forms in the two species. The surfaces of S. mitis and P. micros type strains differed significantly from the other strains examined.