Agglutination of Streptococcus suis by sialic acid-binding lectins.

The 35 Streptococcus suis capsular-type reference strains as well as 45 field strains of type 2 were tested with sialic acid-binding lectins from Sambucus nigra (SNA I), Triticum vulgaris, Maackia amurensis, Homarus americanus, and Limax flavus. Only types 1, 1/2, 2, 14, 15, and 16 agglutinated with SNA I and/or the T. vulgaris lectin. All field strains agglutinated only with SNA I. Reaction with SNA I was probably due to the sialic acid moiety since it disappeared after sialidase treatment. These results confirm the presence of sialic acid in S. suis with the possible terminal sequence N-acetylneuraminic acid-alpha(2,6)GalNAc.     

Contribution of sialic acid-binding adhesin to pathogenesis of experimental endocarditis caused by Streptococcus gordonii DL1

An insertional mutation in hsa, the gene encoding the sialic acid-binding adhesin of Streptococcus gordonii DL1, resulted in a significant reduction of the infection rate of the organism and an inflammatory reaction in the rat aortic valve with experimental endocarditis, suggesting that the adhesin contributes to the infectivity of the organism for heart valves.     

Protoplast formation and localization of enzymes in Streptococcus mitis.

Cells of Streptococcus mitis ATCC 903 were converted to stable protoplasts by the cell wall-degrading M-1 enzyme of the mutanolysin complex isolated from Streptomyces globisporus. Over 90% of total glucokinase (EC 2.7.1.2), aminopeptidase (EC 3.4.11.1), and dextranglucosidase (EC 3.2.1.70) was recovered in the cytoplasmic fraction, whereas over 20% of total invertase (beta-fructofuranosidase: EC 3.2.1.26) was released during protoplast formation. ATPase (EC 3.6.1.3). chymotrypsin-like protease (EC 3.4.21.1), arginine aminopeptidase (EC 3.4.11.6), and lactate dehydrogenase (EC 1.1.1.27) were detected in Triton X-100 extracts of the cytoplasmic membrane fraction by crossed immunoelectrophoresis in combination with enzyme-staining procedures. By these methods, NADH dehydrogenase (EC 1.6.99.3), aminopeptidase, and lactate dehydrogenase were detected in the cytoplasmic fraction. Aminopeptidases in the cytoplasmic fraction differed from this activity in the membrane fractions in electrophoretic mobility and substrate specificity.     

Induction of capacitation in human spermatozoa in vitro by an endometrial sialic acid-binding protein

A Ca²⁺-dependent sialic acid-binding protein (SABP) of humanendometrium, which specifically bound to human sperm head plasmamembrane in vitro, was found to increase the percentage motilityand acrosome-reacted pattern of uncapacitated spermatozoa. Theprotein was synthesized in the endometrium and secreted intothe uterine fluid. This intra-uterine factor, which is apparentlyadvantageous in vitro in inducing human sperm capacitation,may play a significant role in promoting the postrelease maturationof ejaculated spermatozoa by enhancing ⁴⁵Ca uptake into spermatozoaby a pathway which is insensitive to calcium-channel blockers.However, the ⁴⁵Ca uptake could be enhanced on exposure to thedivalent cation ionophore A23187 and inhibited in the presenceof the calmodulin inhibitor trifluoperazine. The SABP also inducesan increase in intracellular Ca²⁺ in spermatozoa, as seen byFURA-2 AM studies. Furthermore, overlay studies show human SABPto be a Ca²⁺-binding protein. The data presented here suggestthat SABP induces invitro sperm capacitation and the subsequentacrosome reaction by increasing intracellular Ca²⁺ concentration.     

Identification and characterization of hsa, the gene encoding the sialic acid-binding adhesin of Streptococcus gordonii DL1

Oral colonization by Streptococcus gordonii, an important cause of subacute bacterial endocarditis, involves bacterial recognition of sialic acid-containing host receptors. The sialic acid-binding activity of this microorganism was previously detected by bacterium-mediated hemagglutination and associated with a streptococcal surface component identified as the Hs antigen. The gene for this antigen (hsa) has now been cloned in Escherichia coli, and its expression has been detected by colony immunoblotting with anti-Hs serum. Mutants of S. gordonii containing hsa inactivated by the insertion of an erythromycin resistance gene or deletion from the chromosome were negative for Hs-immunoreactivity, bacterium-mediated hemagglutinating activity, and adhesion to alpha 2-3-linked sialoglycoconjugates. The deletion in the latter mutants was complemented by plasmid-borne hsa, resulting in Hs antigen production and the restoration of cell surface sialic acid-binding activity. The hsa gene encodes a 203-kDa protein with two serine-rich repetitive regions in its 2,178-amino-acid sequence. The first serine-rich region occurs within the amino-terminal region of the molecule, between different nonrepetitive sequences that may be associated with sialic acid binding. The second serine-rich region, which is much longer than the first, is highly repetitive, containing 113 dodecapeptide repeats with a consensus sequence of SASTSASVSASE. This long repetitive region is followed by a typical gram-positive cell wall anchoring region at the carboxyl-terminal end. Thus, the predicted properties of Hsa, which suggest an amino-terminal receptor-binding domain attached to the cell surface by a molecular stalk, are consistent with the identification of this protein as the sialic acid-binding adhesin of S. gordonii DL1.     

Characterization of the fibrinogen binding domain of bacteriophage lysin from Streptococcus mitis

The binding of bacteria to human platelets is a likely central mechanism in the pathogenesis of infective endocarditis. Platelet binding by Streptococcus mitis SF100 is mediated in part by a lysin encoded by the lysogenic bacteriophage SM1. In addition to its role in the phage life cycle, lysin mediates the binding of S. mitis to human platelets via its interaction with fibrinogen on the platelet surface. To better define the region of lysin mediating fibrinogen binding, we tested a series of purified lysin truncation variants for their abilities to bind this protein. These studies revealed that the fibrinogen binding domain of lysin is contained within the region spanned by amino acid residues 102 to 198 (lysin(102-198)). This region has no sequence homology to other known fibrinogen binding proteins. Lysin(102-198) bound fibrinogen comparably to full-length lysin and with the same selectivity for the fibrinogen Aα and Bβ chains. Lysin(102-198) also inhibited the binding in vitro of S. mitis to human fibrinogen and platelets. When assessed by platelet aggregometry, the disruption of the lysin gene in SF100 resulted in a significantly longer time to the onset of aggregation of human platelets than that of the parent strain. The preincubation of platelets with purified lysin(102-198) also delayed the onset of aggregation by SF100. These results indicate that the binding of lysin to fibrinogen is mediated by a specific domain of the phage protein and that this interaction is important for both platelet binding and aggregation by S. mitis.     

Immunopathological activities of extracellular products of Streptococcus mitis, particularly a superantigenic fraction.

Previously, we prepared extracellular products, fractions F-1 and F-2 of Streptococcus mitis 108, an isolate from the tooth surface of an infant, and showed that F-1 exhibited inflammatory cytokine-inducing activities. In the present study, we present evidence that fraction F-2 induced human T-cell proliferation in the presence of irradiated human peripheral blood mononuclear cells and selectively activated T cells bearing V beta 2 and V beta 5.1 in the T-cell receptor. F-1, on the other hand, stimulated human gingival fibroblasts to support the T-cell proliferation in the same way as human gamma interferon or Prevotella intermedia lipopolysaccharide (LPS). Fraction F-1 also primed gingival fibroblasts to support the production of interleukin-2 and gamma interferon by the T cells upon stimulation with F-2. Human gingival fibroblasts stimulated with fraction F-1, like those stimulated by P. intermedia LPS and human gamma interferon, exhibited human leukocyte antigen (HLA)-DR mRNA expression and cell surface HLA-DR molecules as detected by enzyme-linked immunosorbent assay. An anti-HLA-DR monoclonal antibody inhibited T-cell proliferation in response to F-2, probably through inactivating the accessory function of HLA-DR-bearing fibroblasts. T cells activated with F-2 in the presence of irradiated peripheral blood mononuclear cells exhibited definite cytotoxic effects against fibroblasts and squamous carcinoma cells originating from human oral tissues. These findings are strongly suggestive of an association of extracellular products of viridans streptococci with pathogenesis of oral mucosal diseases, particularly those disorders in gingiva which are accompanied by heavy infiltration of T cells.     

Streptococcus tigurinus, a Novel Member of the Streptococcus mitis Group, Causes Invasive Infections

We recently described the novel species Streptococcus tigurinus sp. nov. belonging to the Streptococcus mitis group. The type strain AZ_3a(T) of S. tigurinus was originally isolated from a patient with infective endocarditis. According to its phenotypic and molecular characteristics, S. tigurinus is most closely related to Streptococcus mitis, Streptococcus pneumoniae, Streptococcus pseudopneumoniae, Streptococcus oralis, and Streptococcus infantis. Accurate identification of S. tigurinus is facilitated by 16S rRNA gene analysis. We retrospectively analyzed our 16S rRNA gene molecular database, which contains sequences of all clinical samples obtained in our institute since 2003. We detected 17 16S rRNA gene sequences which were assigned to S. tigurinus, including sequences from the 3 S. tigurinus strains described previously. S. tigurinus originated from normally sterile body sites, such as blood, cerebrospinal fluid, or heart valves, of 14 patients and was initially detected by culture or broad-range 16S rRNA gene PCR, followed by sequencing. The 14 patients had serious invasive infections, i.e., infective endocarditis (n = 6), spondylodiscitis (n = 3), bacteremia (n = 2), meningitis (n = 1), prosthetic joint infection (n = 1), and thoracic empyema (n = 1). To evaluate the presence of Streptococcus tigurinus in the endogenous oral microbial flora, we screened saliva specimens of 31 volunteers. After selective growth, alpha-hemolytic growing colonies were analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and subsequent molecular methods. S. tigurinus was not identified among 608 strains analyzed. These data indicate that S. tigurinus is not widely distributed in the oral cavity. In conclusion, S. tigurinus is a novel agent of invasive infections, particularly infective endocarditis.     

Clonal diversity of Streptococcus mitis biovar 1 isolates from the oral cavity of human neonates.

The clonal diversity of 101 isolates of the pioneer bacterium Streptococcus mitis biovar 1 obtained from the oral cavities of 40 human neonates 1 to 3 days, 2 weeks, and 1 month postpartum was examined by using rRNA gene restriction patterns. There was a high degree of genetic diversity, with the 101 isolates comprising 93 unique PvuII ribotypes. There were eight identical pairs of ribotype patterns, and seven of the eight pairs were obtained from individual neonates. Only one identical pair comprised isolates obtained from different neonates. In all but two cases, isolates with matching ribotypes were obtained at one visit. Two pairs of isolates with matching ribotype patterns were obtained from neonates on successive visits. The ribotype patterns of the isolates were examined by cluster analysis. The isolates forming each cluster were very similar, yet each cluster was well separated from its neighbors. When several isolates were obtained from individual neonates at a particular visit, in some instances they were contained in a single cluster, whereas in other cases each isolate was contained in a separate cluster. Isolates obtained from individual neonates on successive visits tended to be contained in different clusters. This high degree of diversity, which has been observed in other mucosal commensal bacteria, may serve as a mechanism for avoiding immune elimination of these bacteria.     

Functional analysis of the Streptococcus gordonii DL1 sialic acid-binding adhesin and its essential role in bacterial binding to platelets

Bacterial recognition of host sialic acid-containing receptors plays an important role in microbial colonization of the human oral cavity. The sialic acid-binding adhesin of Streptococcus gordonii DL1 was previously associated with the hsa gene encoding a 203-kDa protein. The predicted protein sequence consists of an N-terminal nonrepetitive region (NR1), including a signal sequence, a relatively short serine-rich region (SR1), a second nonrepetitive region (NR2), a long serine-rich region (SR2) containing 113 dodecapeptide repeats, and a C-terminal cell wall anchoring domain. In the present study, the contributions of SR1, NR2, and SR2 to Hsa-mediated adhesion were assessed by genetic complementation. Adhesion of an hsa chromosomal deletion mutant to sialic acid-containing receptors was restored by plasmids containing hsa constructs encoding Hsa that lacked either the N- or C-terminal portion of SR2. In contrast, hsa constructs that lacked the coding sequences for SR1, NR2, or the entire SR2 region failed to restore adhesion. Surface expression of recombinant Hsa was not affected by removal of SR1, NR2, or a portion of SR2 but was greatly reduced by complete removal of SR2. Wheat germ agglutinin, a probe for Hsa-specific glycosylation, reacted with recombinant Hsa lacking SR1, NR2, or SR2 but not with recombinant Hsa lacking both SR1 and SR2. Significantly, the aggregation of human platelets by S. gordonii DL1, an interaction implicated in the pathogenesis of infective endocarditis, required the expression of hsa. Moreover, neuraminidase treatment of the platelets eliminated this interaction, further supporting the hypothesis that Hsa plays an essential role in the bacterium-platelet interaction.     

抗醛糖还原酶单克隆抗体的制备与特性鉴定

目的:制备抗醛糖还原酶(AR)的单克隆抗体(mAb),并与本室制备的抗醛糖还原酶相似蛋白(ARL-1)mAb进行比较。方法:经RT-PCR获得AR基因,将基因插入pGEX-4T-1(His)6C中,构建重组质粒pGEX-4T-1(His)6C-AR,以重组质粒转化E.coliRosetta诱导表达GST-AR蛋白。以纯化的GST-AR蛋白免疫BALB/c小鼠,采用杂交瘤技术制备mAb。应用间接ELISA和Western blot方法对mAb进行筛选和鉴定。使用Clustalx和Antheprot软件,比较AR与ARL-1的同源性,表达GST-dAR[80~142氨基酸(aa)],与ARL-1差异较大;并分析AR的抗原性,表达GST-dA1(1~79aa)、GST-dA2(80~99aa)、GST-dA3(111~142aa)、GST-dA4(143~316aa)。利用AR全长及截短蛋白,采用Western blot分析制备的抗AR mAb识别AR抗原的部位。结果:获得3株稳定分泌抗AR mAb的杂交瘤细胞系ARB3、AR7B3G4和ARF10。3株抗GST-AR的mAb均为IgG1(κ型),腹水mAb效价为1∶4×105,细胞培养上清mAb效价为1∶1×104,3株mAb均可与胎盘组织中的AR蛋白起反应,而与GST-ARL-1和GST蛋白无交叉反应。它们分别为抗GST-dA1、GST-dA3和GST-dA4蛋白的mAb。结论:成功地制备了3株特异性抗AR mAb,可分别识别AR的1~79、111~142、143~316位氨基酸。将它们与抗ARL-1mAb联合应用,将有助于进一步研究AR与ARL-1蛋白的功能,并为深入探讨AR、ARL-1与相关疾病的关系及进行大规模的流行病学调查提供了有力的工具。     

Monoclonal antibody recognizing a species-specific protein from Streptococcus pneumoniae.

Monoclonal antibodies (MAbs) against a nonencapsulated strain (R36A) of Streptococcus pneumoniae were produced to aid in a search for antigens common to this species. By Western immunoblot analysis, a species-specific 37-kilodalton (kDa) protein was found in lysates of 24 different encapsulated strains of S. pneumoniae. Monoclonal antibodies against the 37-kDa antigen did not react with 55 heterologous strains representing 19 genera and 36 species of bacteria that can also cause acute lower respiratory tract disease. Immunogold staining suggests that the antigen is synthesized inside the pneumococcal cell. However, MAbs to the 37-kDa antigen bound whole cells in the enzyme-linked immunosorbent assay and the indirect immunofluorescence assay. Antibody-binding epitopes of the antigen are probably exposed on the outer surface of the pneumococcus cell wall. The effectiveness of the 37-kDa antigen as a useful diagnostic marker is under study.     

Purification and partial characterization of a 32-kilodalton sialic acid-specific lectin from Aspergillus fumigatus

Adherence of the opportunistic fungus Aspergillus fumigatus to the extracellular matrix components is considered a crucial step in the establishment of the infection. Given the high carbohydrate content of these glycoproteins and the role of carbohydrate-protein interactions in numerous adherence processes, the presence of a lectin in A. fumigatus was investigated. Different fungal extracts obtained by sonication or grinding in liquid nitrogen from resting or swollen conidia, as well as from germ tubes and mycelium, were tested by hemagglutination assays using rabbit erythrocytes. A lectin activity was recovered in all the extracts tested. However, sonication of resting conidia resulted in the highest specific activity. Purification of the lectin was achieved by gel filtration followed by ion-exchange and hydrophobic-interaction chromatographies. Analysis of the purified lectin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed an apparent molecular mass of 32 kDa, which is similar to that of the alkaline protease already identified from different strains of A. fumigatus. However, as evidenced by the use of an alkaline protease-deficient mutant, the two activities were supported by distinct proteins. In addition, hemagglutination inhibition experiments using different saccharides and glycoproteins demonstrated the specificity of the lectin for sialic acid residues. Together these results suggest that this lectin may contribute to the attachment of conidia to the extracellular matrix components through the recognition of the numerous terminal sialic acid residues of their carbohydrate chains.     

Population Dynamics of Streptococcus mitis in Its Natural Habitat

The purpose of this study was to examine the genetic structure of the typical commensal Streptococcus mitis biovar 1 in its natural habitat in the human oral cavity and pharynx and to investigate the role that selected microbial properties and host, spatial, and temporal factors play in determining the structure of the bacterial population. Consecutive samples were collected from buccal and pharyngeal mucosal surfaces of two infants, their four parents, and two elderly individuals over a period of approximately 1 year. A total of 751 isolates identified as S. mitis biovar 1 were typed by restriction endonuclease analysis (REA) and representative clones were typed by multilocus enzyme electrophoresis (MLEE). The genetic diversity of the S. mitis biovar 1 isolates collected from single infant hosts over a period of 9 to 10 months was found to be between 0.69 and 0.76, which is considerably higher than that previously observed for intestinal populations of Escherichia coli. The study provides evidence of the existence of both transient and persistent clones in adult individuals. In the two infants, however, none of 42 demonstrated clones were detected on more than a single occasion. Statistical calculations showed that the ability to persist was not distributed at random in the S. mitis biovar 1 population. However, neither immunoglobulin A1 protease activity nor the ability to bind alpha-amylase from saliva was a preferential characteristic of persistent genotypes. In contrast to current concepts of climax ecosystems, the species niche in the habitat appears to be maintained predominantly by a succession of clones rather than by stable strains. Several lines of evidence suggest that the major origin of "new" clones is the many other habitats in the respiratory tract that are occupied by this species.     

Role of sialic acid in saliva-induced aggregation of Streptococcus sanguis.

The ability of saliva to induce aggregation of Streptococcus sanguis was destroyed by treating the saliva with protease or neuraminidase. Loss of aggregating activity could be correlated with the appearance of free sialic acid. Clarified saliva contains an endogenous neuraminidase that modifies aggregating activity. Aggregation was inhibited by mixed ganglioside preparations but less effectively by acid-hydrolyzed gangliosides. The aggregating activity of S. sanguis was not related to the rhamnose or phosphorous content of the cell wall or to antigen a, b, c, d, or e.     

Genetic relationships between clinical isolates of Streptococcus pneumoniae, Streptococcus oralis, and Streptococcus mitis: characterization of "Atypical" pneumococci and organisms allied to S. mitis harboring S. pneumoniae virulence factor-encoding genes

The oral streptococcal group (mitis phylogenetic group) currently consists of nine recognized species, although the group has been traditionally difficult to classify, with frequent changes in nomenclature over the years. The pneumococcus (Streptococcus pneumoniae), an important human pathogen, is traditionally distinguished from the most closely related oral streptococcal species Streptococcus mitis and Streptococcus oralis on the basis of three differentiating characteristics: optochin susceptibility, bile solubility, and agglutination with antipneumococcal polysaccharide capsule antibodies. However, there are many reports in the literature of pneumococci lacking one or more of these defining characteristics. Sometimes called "atypical" pneumococci, these isolates can be the source of considerable confusion in the clinical laboratory. Little is known to date about the genetic relationships of such organisms with classical S. pneumoniae isolates. Here we describe these relationships based on sequence analysis of housekeeping genes in comparison with previously characterized isolates of S. pneumoniae, S. mitis, and S. oralis. While most pneumococci were found to represent a closely related group these studies identified a subgroup of atypical pneumococcal isolates (bile insoluble and/or "acapsular") distinct from, though most closely related to, the "typical" pneumococcal isolates. However, a large proportion of isolates, found to be atypical on the basis of capsule reaction alone, did group with typical pneumococci, suggesting that they have either lost capsule production or represent as-yet-unrecognized capsular types. In contrast to typical S. pneumoniae, isolates phenotypically identified as S. mitis and S. oralis, which included isolates previously characterized in taxonomic studies, were genetically diverse. While most of the S. oralis isolates did fall into a well-separated group, S. mitis isolates did not cluster into a well-separated group. During the course of these studies we also identified a number of potentially important pathogenic isolates, which were frequently associated with respiratory disease, that phenotypically and genetically are most closely related to S. mitis but which harbor genes encoding the virulence determinants pneumolysin and autolysin classically associated with S. pneumoniae.     

Substances that interfere with action of viridin B, a Streptococcus mitis bacteriocin

Several body fluids and various substances were examined for their effects on the action of viridin B, a Streptococcus mitis bacteriocin. Heme has been shown previously to interfere with viridin B action. In the present study, peroxidase and catalase, but not superoxide dismutase, interfered with the bacteriocin. Sulfhydryl-containing compounds had no effect. Trypsin, protease, and amylase exhibited interfering capacities. Of various body fluids tested, serum, saliva, and leukocyte lysate interfered with viridin B. The activity in leukocyte lysate was probably due to myeloperoxidase. Salivary activity was likely due to both peroxidase and amylase. Interfering activity in serum was neither complement dependent nor antibody mediated and was probably due to a combination of enzymatic activities. Temporal studies with these agents suggested more than one mechanism of interference with viridin B.     

N-acetylneuraminic acid transport by Streptococcus oralis strain AR3.

Streptococcus oralis has emerged as one of the most important organisms of the viridans streptococcus group in terms of infections and is recognised as an agent of infective endocarditis and, in immunocompromised patients, septicaemia. The mechanisms by which this organism proliferates in vivo are unknown. However, host-derived sialic acids -- including N-acetylneuraminic acid (NeuNAc) which is present in serum and cell-associated glycoproteins -- are a potential source of fermentable carbohydrate for bacterial proliferation, especially for sialidase-producing bacteria, including S. oralis. To further elucidate the role of NeuNAc in supporting growth, this study determined the ability of S. oralis strain AR3 (isolated from a patient with infective endocarditis) to transport NeuNAc and characterised the transport system. The transport of [14C]-labelled NeuNAc into S. oralis was monitored and this transport system was induced by growth of the bacteria in the presence of the N-acetylated sugars NeuNAc, N-acetylglucosamine and N-acetylmannosamine. The transport system followed typical Michaelis-Menten kinetics, with a Km of 21.0 microM and a Vmax of 2.65 nmoles of NeuNAc transported/min/mg of dry cell mass. NeuNAc transport was inhibited by the presence of exogenous N-glycolylneuraminic acid, a related sialic acid. Chlorhexidine, NaF and 2,4-dinitrophenol were potent inhibitors of the transport system, suggesting that the uptake of NeuNAc occurs via a proton motive force-dependent permease system. This is the first report of the mechanism by which NeuNAc transport occurs in pathogenic streptococci. This transport process may have relevance to the acquisition of a source of fermentable carbohydrate and thus bacterial proliferation in vivo.