Transcriptome and metabolome analyses of Streptococcus gordonii DL1 under acidic conditions.

OBJECTIVES: Streptococcus gordonii is associated with the formation of biofilms, especially those that comprise dental plaque. Notably, S. gordonii DL1 causes infective endocarditis (IE). Colonization of this bacterium requires a mechanism that can tolerate a drop in environmental pH by producing acid via its own sugar metabolism. The ability to survive acidic environmental conditions might allow the bacterium to establish vegetative colonization even in the endocardium due to inflammation-induced lowering of pH, increasing the risk of IE. At present, the mechanism by which S. gordonii DL1 survives under acidic conditions is not thoroughly elucidated. The present study was thus conducted to elucidate the mechanism(s) by which S. gordonii DL1 survives under acidic conditions. METHODS: We analyzed dynamic changes in gene transcription and intracellular metabolites in S. gordonii DL1 exposed to acidic conditions, using transcriptome and metabolome analyses. RESULTS: Transcriptome analysis revealed upregulation of genes involved in heat shock response and glycolysis, and down regulation of genes involved in phosphotransferase systems and biosynthesis of amino acids. The most upregulated genes were a beta-strand repeat protein of unknown function (SGO_RS06325), followed by copper-translocating P-type ATPase (SGO_RS09470) and malic enzyme (SGO_RS01850). The latter two of these contribute to cytoplasmic alkalinization. S. gordonii mutant strains lacking each of these genes showed significantly reduced survival under acidic conditions. Metabolome analysis revealed that cytoplasmic levels of several amino acids were reduced. CONCLUSIONS: S. gordonii survives the acidic conditions by recovering the acidic cytoplasm using the various activities, which are regulated at the transcriptional level.

The bacterial replication origin BUS promotes nucleobase capture.

Genome duplication is essential for the proliferation of cellular life and this process is generally initiated by dedicated replication proteins at chromosome origins. In bacteria, DNA replication is initiated by the ubiquitous DnaA protein, which assembles into an oligomeric complex at the chromosome origin (oriC) that engages both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) to promote DNA duplex opening. However, the mechanism of DnaA specifically opening a replication origin was unknown. Here we show that Bacillus subtilis DnaAATP assembles into a continuous oligomer at the site of DNA melting, extending from a dsDNA anchor to engage a single DNA strand. Within this complex, two nucleobases of each ssDNA binding motif (DnaA-trio) are captured within a dinucleotide binding pocket created by adjacent DnaA proteins. These results provide a molecular basis for DnaA specifically engaging the conserved sequence elements within the bacterial chromosome origin basal unwinding system (BUS).

Phylloquinone is preferable over menadione as a growth factor for Porphyromonas gingivalis.

OBJECTIVES: Porphyromonas gingivalis is the etiological agent of chronic periodontitis. Menadione (vitamin K3) and phylloquinone (vitamin K1) are well-known growth factors for P. gingivalis, while menadione is widely used in growth experiments. Here we attempted to determine the differences in phylloquinone and menadione in P. gingivalis growth experiments, which have not been well studied to date. METHODS: We investigated the effects of menadione and phylloquinone on the growth of two W83 strains and seven ATCC 33277 strains of P. gingivalis. RESULTS: The ATCC 33277 strains grew well with phylloquinone at 2.9 µM in a complex medium (nutrient medium) and at 29 µM in two minimal media. In contrast, the W83 strains grew well without menadione or phylloquinone in three different culture media. Menadione at 2.9 µM, the conventionally used concentration for culturing P. gingivalis, supported the growth of most ATCC 33277 strains but inhibited the growth of some W83 and ATCC 33277 strains. Furthermore, menadione at 14.5 µM frequently inhibited cell growth, while phylloquinone at 145 µM promoted cell growth. CONCLUSIONS: These results indicate that menadione and phylloquinone act as growth factors for ATCC 33277 but that menadione also can inhibit P. gingivalis growth. Thus, we propose that phylloquinone be used instead of menadione in P. gingivalis growth experiments requiring vitamin K.

Calcium ions and vitamin B12 are growth factors for Porphyromonas gingivalis.

BACKGROUND: Porphyromonas gingivalis is a causative agent of chronic periodontitis. Standard strains of P. gingivalis, such as W83 and ATCC 33277, proliferate in minimal medium when protein is added as the energy source and hemin and menadione are added as growth factors. Nevertheless, minimal medium containing bovine serum albumin sometimes fails to support growth. HIGHLIGHTS: The proliferation of two W83 strains and seven ATCC 33277 strains in various minimal media was investigated. Previously, we determined that calcium chloride (CaCl2) was a growth factor for W83NM, a W83 strain. In this study, we found that vitamin B12 enhanced the proliferation of W83NM in a minimal medium with cultures from the fourth passage but not from the first to the third passage. Therefore, using fourth-passage cultures, we assessed the proliferation of two W83 and seven ATCC 33277 strains in minimal media and the effects of CaCl2 and vitamin B12. Surprisingly, the nine P. gingivalis strains all differed with respect to their proliferation in minimal media, and protein products used as energy sources showed product-to-product and lot-to-lot heterogeneity. Even though strains or protein products were different, we found CaCl2-dependent growth in nine strains and vitamin B12-dependent growth in seven strains. CONCLUSION: These results suggest that calcium ions and vitamin B12 are novel growth factors for P. gingivalis.

Hemagglutinating properties of a Streptococcus gordonii strain expressing sialic acid-binding adhesin homolog with low binding site similarity to that of strain DL1.

OBJECTIVES: The Hsa adhesin of Streptococcus gordonii strain DL1 was previously identified as a hemagglutinin that binds specifically to sialoglycoconjugates. We recently found that among oral streptococcal species, S. gordonii strains most frequently express Hsa homologs on the bacterial cell surface. However, the effect of amino acid sequence diversity of nonrepetitive region 2 (NR2), a putative binding site of Hsa, on antigenicity and hemagglutinating (HA) properties is unclear due to difficulties in DNA sequencing the NR2 coding region. The aim of this study was to elucidate the similarity of the low NR2 antigenicity Hsa homolog of strain NDU1118 to that of strain DL1 and the association of the homolog with HA properties of the strain. METHODS: The hsa homolog of NDU1118 was sequenced using a long-read next-generation sequencer, and the Hsa homolog was assessed by alignment analysis of the deduced amino acid sequences. The hsa mutant of NDU1118 was generated by insertion of the erythromycin resistance gene. The HA properties of the wild type and the hsa mutant were assessed with human erythrocytes. RESULTS: The NR2 amino acid sequence of the NDU1118 Hsa homolog was almost identical to that of the S. gordonii M99 Hsa homolog, also known as GspB, and less similar to that of DL1 Hsa. The hsa mutation of NDU1118 induced reduction of HA activity in untreated erythrocytes, but surprisingly increased lactose-inhibitable HA activity in neuraminidase-treated erythrocytes. CONCLUSIONS: The results suggest the existence of an adhesin other than the Hsa homolog on the cell surface of NDU1118.

Expression and diversity of the sialic acid-binding adhesin and its homologs associated with oral streptococcal infection.

Streptococcus gordonii, one of the early colonizers of oral biofilms, is involved in the development of dental caries, periodontal disease, and infective endocarditis. The Hsa adhesin of S. gordonii DL1 has the ability to bind strongly to the terminal sialic acid groups of host glycoproteins via the binding region, nonrepetitive region 2 (NR2), which is important for the pathogenicity of S. gordonii DL1. Low similarity with the NR2 of Hsa homologs among other streptococcal species has been reported. However, the reports have been limited to certain strains. This study attempted to assess frequency of the expression on the bacterial cell surface and to analyze the diversity of Hsa homologs among different wild strains of oral streptococci. We isolated 186 wild-type strains of oral streptococci from healthy volunteers and analyzed their hemagglutinating (HA) activity on human erythrocytes and their Hsa homologs and NR2 homologous regions by dot immunoblotting using anti-Hsa and anti-NR2 antisera, respectively. We found 30 strains reacted with anti-NR2 antiserum (NR2 positive) and determined the sequence of the NR2 regions. Many strains with high HA activity were also NR2 positive, suggesting that the NR2 region may be associated with HA activity. Among the NR2-positive strains, four different amino acid sequence patterns were observed, demonstrating diversity in the NR2 region. Notably, S. gordonii strains frequently possessed Hsa homologs and NR2-like antigens compared with other streptococci. It is speculated that the possessing frequency of Hsa homologs and the amino acid sequence of NR2 region may vary among streptococcal species.

Streptococcus gordonii DL1 evades polymorphonuclear leukocyte-mediated killing via resistance to lysozyme.

Streptococcus gordonii is an etiological bacterial agent of infective endocarditis. Although the pathogenesis mechanisms are not well understood, the interaction between streptococci and phagocytes is considered important for the development of infective endocarditis. Previous studies show that some S. gordonii strains, including DL1, survive in polymorphonuclear leukocytes (PMNs), whereas other strains such as SK12 are sensitive to PMN-dependent killing. In this study, we assessed the differences between the sensitivity of S. gordonii DL1 and S. gordonii SK12 to PMN-dependent killing. S. gordonii DL1 showed a higher survival when treated with PMNs than SK12. Both S. gordonii DL1 and S. gordonii SK12 showed high resistance to low pH condition. Compared to S. gordonii SK12, S. gordonii DL1 was sensitive to hydrogen peroxide. However, the resistance of S. gordonii DL1 to the tested bactericidal agents, especially lysozyme, was higher than that of SK12. Furthermore, we performed a bactericidal assay by treating a mixture of S. gordonii DL1 and SK12 with PMNs. S. gordonii DL1 did not enhance the survival of S. gordonii SK12 exposed to PMNs. These results indicated that S. gordonii DL1 is resistant to bactericidal agents that degrade bacteria in phagolysosomes. In addition, there was no secretory factor involved in the resistance to bactericidal agents. The findings of this study may help develop treatments for infective endocarditis caused by S. gordonii.

Role of Streptococcus intermedius phosphoglucosamine mutase in bacterial growth, cell morphology, and resistance to polymorphonuclear leukocyte killing.

OBJECTIVES: Streptococcus intermedius is a member of the anginosus group of streptococci, an oral commensal bacterium found in infected root canals, and the causative agent of deep-seated abscesses. This organism has slow clearance when phagocytosed within neutrophils. Here, we investigated the role of its phosphoglucosamine mutase (GlmM), an enzyme associated with peptidoglycan synthesis, in bacterial growth, cell morphology, and resistance to polymorphonuclear leukocyte killing. METHODS: The glmM-deletion (ΔglmM) mutant and the plasmid-borne complementation (ΔglmM/glmM) strain of S. intermedius were generated. The wild type, the ΔglmM mutant, and the ΔglmM/glmM strain were phagocytosed with human polymorphonuclear leukocytes (PMNs), and bacterial viability in PMNs was determined by LIVE/DEAD staining. Additionally, bacterial growth and cell morphology were also compared. RESULTS: The survival rate of the ΔglmM mutant was significant lower than that of the wild type. Although the difference in the survival rate of the ΔglmM/glmM strain compared to that of the wild type or the ΔglmM mutant was not significant, the rate appeared to be restored to the middle level. Compared to the wild type and the ΔglmM/glmM strain, the ΔglmM mutant showed reduced growth potential, a significant increase in the number of bacterial chains, and heterogeneous bacteria. CONCLUSIONS: GlmM is one of the factors responsible for the stable resistance of S. intermedius to clearance by PMNs.

Reassessment of minimal media reveals differences in growth among Porphyromonas gingivalis standard strains.

OBJECTIVES: Porphyromonas gingivalis is one of the etiologic agents of chronic periodontitis. Our previous study showed that the use of minimal media for P. gingivalis allowed to isolate novel inhibitors of P. gingivalis growth. However, growth of P. gingivalis in minimal media was not always reproducible. METHODS: To explain this phenomenon, we analyzed the growth of seven wild-type ATCC 33277 strains and two wild-type W83 strains in 10 minimal media and three complex media. RESULTS: All nine strains grew in LF (Lactalbumin-Ferric chloride), GC (bovine γ-immunoglobulin G-Calcium chloride), and newly developed mC (milk-Casein) minimal media. Therefore, LF, GC, and mC could be used as minimal media for P. gingivalis. In contrast, other six minimal media containing bovine serum albumin (BSA) supported the growth of several less strains; among these, two media also showed lack of reproducibility in growth among ATCC 33277 strains. On the other hand, four ATCC 33277 strains grew similarly in all 13 media, but two W83 and other three ATCC 33277 strains grew differently in at least one medium. CONCLUSIONS: These results suggest that the lack of reproducibility of P. gingivalis growth on minimal media is caused by the presence of BSA, and by differences among the standard strains of P. gingivalis.

A screening system using minimal media identifies a flavin-competing inhibitor of Porphyromonas gingivalis growth.

Chronic periodontitis is caused by dysbiosis of human oral commensals and especially by increase in Porphyromonas gingivalis. Inhibitors of P. gingivalis growth are expected to serve as effective drugs for the periodontal therapy. In the present study, we isolated new growth inhibitors of P. gingivalis using minimal media for P. gingivalis. The minimal media included the previously reported Globulin-Albumin (GA) and the newly developed Lactalbumin-Ferric chloride (LF) and Globulin-Calcium chloride (GC); all supported growth of the wild-type strain of P. gingivalis but did not support the growth of a mutant defective for a type IX secretion system. GC contains CaCl2, indicating that P. gingivalis requires a calcium ion for growth. Using LF and GA, we screened about 100 000 compounds and identified 73 that strongly inhibited the growth of P. gingivalis. More than half of these candidates would not have been obtained if these minimal media had not been used in our screen. One of our candidate inhibitors was diphenyleneiodonium chloride (DPIC), which showed strong bactericidal activity against P. gingivalis. Excess amounts of flavin adenine dinucleotide or flavin mononucleotide suppressed the inhibitory activity of DPIC, suggesting that DPIC would be a novel potent growth inhibitor.

Contribution of Streptococcus gordonii Hsa Adhesin to Biofilm Formation.

Adhesion of oral mitis group streptococci, such as Streptococcus gordonii, to acquired pellicle on the tooth surface is the first step in oral biofilm formation. S. gordonii strain DL1 possesses an Hsa adhesin, which recognizes the terminal sialic acid of host sialoglycoconjugates. The aim of the present study was to investigate the role of the Hsa adhesin in biofilm formation. The biofilm-forming ability of a S. gordonii hsa mutant on microtiter plates pre-coated with saliva, fetuin, or mucin was significantly lower than that of wild-type strain DL1. In contrast, no significant difference in biofilm-forming ability was observed in plates pre-coated with bovine serum albumin, which does not contain sialic acid. The biofilm-forming ability of strain DL1 in saliva-coated microtiter plates was also significantly reduced when the plate was pre-treated with neuraminidase. The sialic acid-dependent biofilm-forming ability of different wild-type S. gordonii strains varied. However, Southern and western blot analyses showed that all the tested wild-type strains possessed and expressed hsa homologs, respectively. These results indicate that the binding of Hsa adhesin to sialoglycoconjugates is associated with biofilm formation of S. gordonii DL1, and imply variation in the contribution of Hsa and its homologs to S. gordonii biofilm formation.

Correction: Two Arginine Residues of Streptococcus gordonii Sialic Acid-Binding Adhesin Hsa Are Essential for Interaction to Host Cell Receptors.

[This corrects the article DOI: 10.1371/journal.pone.0154098.].

Two Arginine Residues of Streptococcus gordonii Sialic Acid-Binding Adhesin Hsa Are Essential for Interaction to Host Cell Receptors.

Hsa is a large, serine-rich protein of Streptococcus gordonii DL1 that mediates binding to α2-3-linked sialic acid termini of glycoproteins, including platelet glycoprotein Ibα, and erythrocyte membrane protein glycophorin A, and band 3. The binding of Hsa to platelet glycoprotein Ibα contributes to the pathogenesis of infective endocarditis. This interaction appears to be mediated by a second non-repetitive region (NR2) of Hsa. However, the molecular details of the interaction between the Hsa NR2 region and these glycoproteins are not well understood. In the present study, we identified the amino acid residues of the Hsa NR2 region that are involved in sialic acid recognition. To identify the sialic acid-binding site of Hsa NR2 region, we prepared various mutants of Hsa NR2 fused with glutathione transferase. Fusion proteins harboring Arg340 to Asn (R340N) or Arg365 to Asn (R365N) substitutions in the NR2 domain exhibited significantly reduced binding to human erythrocytes and platelets. A sugar-binding assay showed that these mutant proteins abolished binding to α2-3-linked sialic acid. Furthermore, we established S. gordonii DL1 derivatives that encoded the corresponding Hsa mutant protein. In whole-cell assays, these mutant strains showed significant reductions in hemagglutination, in platelet aggregation, and in adhesion to human leukocytes. These results indicate that the Arg340 and Arg365 residues of Hsa play an important role in the binding of Hsa to α2-3-linked sialic acid-containing glycoproteins.

[Adhesins of oral streptococci].

Oral streptococci comprise a numerically prominent group of oral bacteria that occur primarily on the human tooth surface as members of the biofilm community, commonly referred to as dental plaque. These streptococci are not only causative of dental caries and are primers for colonization of periodontopathic bacteria, but also well known for their ability to colonize damaged heart valves, identified most frequently as primary etiological agents of infective endocarditis. A number of streptococcal cell surface components are known to contribute to colonization of the tooth surface including putative adhesins recognizing host sialic acid (sialic acid-binding adhesins). Interactions mediated by these adhesins include the attachment of these bacteria to saliva-coated hydroxyapatite and their adhesion to erythrocytes, both of which are abolished or reduced by sialidase pretreatment of the corresponding host sialoglycoconjugate receptors. The sialic acid-binding adhesin on Streptococcus gordonii, an early colonizer on the tooth surface, has been molecularly analyzed. The adhesin, Hsa (203-kDa protein), consists of an N-terminal non repetitive region (NR1) including a signal sequence, a relatively short serine-rich region (SR1), a second non repetitive region (NR2), a long serine-rich region (SR2) containing 113 dodecapeptide repeats accounting for 75% of the whole protein, and a C-terminal cell wall anchoring domain. Therefore, it has been suggested that NR2, the putative sialic acid-binding domain of Hsa, is presented on the bacterial surface at the end of a long molecular stalk formed by SR2. The present review deals with the function and pathogenicity of oral streptococcal adhesins.

The U-box-type ubiquitin ligase PRP19ß regulates astrocyte differentiation via ubiquitination of PTP1B.

U-box protein PRP19ß, a splicing variant of PRP19α, suppresses neuronal differentiation and conversely promotes astrocyte differentiation as a neuron/glia switch molecule. However, the mechanistic basis of PRP19ß in astrocyte differentiation is not well understood. Here, we demonstrated that PRP19ß regulates the stability of protein tyrosine phosphatase 1B (PTP1B) via ubiquitination during N(6),2'-O-dibutyryl cyclic AMP (cAMP)-induced astrocyte differentiation of C6 cells. Only overexpression of PRP19ß conferred astrocyte properties at a certain level, and induced more astrocyte markers, glial fibrillary acidic protein (GFAP) and S100ß, in the presence of cAMP, whereas its down-regulation by antisense RNA showed a suppressive effect. In addition, ectopic expression of PRP19ß led to robust phosphorylation of signal transducer and activator of transcription 3 (STAT3) accompanying the reduction in PTP1B stability during astrocyte differentiation. Immunological analysis revealed that PRP19ß interacted with PTP1B and ubiquitinated PTP1B via its U-box region. Forced expression of the U-box deletion mutant of PRP19ß resulted in inhibition of astrocyte differentiation. Moreover, down-regulation of PTP1B by short hairpin (sh)RNA enhanced astrocyte differentiation, while forced expression of PTP1B showed an inhibitory effect. Thus, these results indicate that PRP19ß activates the gp130/Janus kinase (JAK)/STAT signaling pathway during astrocyte differentiation of C6 cells via PTP1B ubiquitination.

Involvement of crosstalk between Oct4 and Meis1a in neural cell fate decision.

Oct4 plays a critical role both in maintaining pluripotency and the cell fate decision of embryonic stem (ES) cells. Nonetheless, in the determination of the neuroectoderm (NE) from ES cells, the detailed regulation mechanism of the Oct4 gene expression is poorly understood. Here, we report that crosstalk between Oct4 and Meis1a, a Pbx-related homeobox protein, is required for neural differentiation of mouse P19 embryonic carcinoma (EC) cells induced by retinoic acid (RA). During neural differentiation, Oct4 expression was transiently enhanced during 6-12 h of RA addition and subsequently disappeared within 48 h. Coinciding with up-regulation of Oct4 expression, the induction of Meis1a expression was initiated and reached a plateau at 48 h, suggesting that transiently induced Oct4 activates Meis1a expression and the up-regulated Meis1a then suppresses Oct4 expression. Chromatin immunoprecipitation (ChIP) and luciferase reporter analysis showed that Oct4 enhanced Meis1a expression via direct binding to the Meis1 promoter accompanying histone H3 acetylation and appearance of 5-hydoxymethylcytosine (5hmC), while Meis1a suppressed Oct4 expression via direct association with the Oct4 promoter together with histone deacetylase 1 (HDAC1). Furthermore, ectopic Meis1a expression promoted neural differentiation via formation of large neurospheres that expressed Nestin, GLAST, BLBP and Sox1 as neural stem cell (NSC)/neural progenitor markers, whereas its down-regulation generated small neurospheres and repressed neural differentiation. Thus, these results imply that crosstalk between Oct4 and Meis1a on mutual gene expressions is essential for the determination of NE from EC cells.

Streptococcus gordonii promotes rapid differentiation of monocytes into dendritic cells through interaction with the sialic acid-binding adhesin.

Infective endocarditis is frequently attributed to oral streptococci. Although the pathogenetic mechanisms are not well understood, interaction between streptococci and phagocytes is thought to be important for infective endocarditis. In this study, HL-60 cell-derived monocytes were characterized following interaction with Streptococcus gordonii DL1. Exposure of monocytes to S. gordonii DL1 induced up-regulation of the dendritic cell (DC) markers CD83, CD1a, CD86, and interleukin-12, while monocyte markers PU.1 and MafB, which are typically present at low levels in mature DCs, were down-regulated. Interaction of HL-60-derived monocytes with S. gordonii DL1 was instructive for DC differentiation in the absence of released cytokines. Furthermore, neither the filtered culture medium of S. gordonii nor the hsa mutant, deficient in sialic acid-binding activity, was able to induce the differentiation of HL-60 cells. Taken together, these data suggest that monocytes stimulated with S. gordonii DL1 rapidly undergo monocyte-to-DC differentiation through interaction with the bacterial surface receptor Hsa and that this response may be the initial step in infective endocarditis by oral streptococci.

Implication of Akt-dependent Prp19 alpha/14-3-3beta/Cdc5L complex formation in neuronal differentiation.

PRP19alpha and CDC5L are major components of the active spliceosome. However, their association process is still unknown. Here, we demonstrated that PRP19 alpha/14-3-3beta/CDC5L complex formation is regulated by Akt during nerve growth factor (NGF)-induced neuronal differentiation of PC12 cells. Analysis of PRP19 alpha mutants revealed that the phosphorylation of PRP19 alpha at Thr 193 by Akt was critical for its binding with 14-3-3beta to translocate into the nuclei and for PRP19 alpha/14-3-3beta/CDC5L complex formation in neuronal differentiation. Forced expression of either sense PRP19 alpha or sense 14-3-3beta RNAs promoted NGF-induced neuronal differentiation, whereas down-regulation of these mRNAs showed a suppressive effect. The nonphosphorylation mutant PRP19 alpha T193A lost its binding ability with 14-3-3beta and acted as a dominant-negative mutant in neuronal differentiation. These results imply that Akt-dependent phosphorylation of PRP19 alpha at Thr193 triggers PRP19 alpha/14-3-3beta/CDC5L complex formation in the nuclei, likely to assemble the active spliceosome against neurogenic pre-mRNAs.

Contribution of phosphoglucosamine mutase to the resistance of Streptococcus gordonii DL1 to polymorphonuclear leukocyte killing.

Phosphoglucosamine mutase (GlmM; EC 5.4.2.10) catalyzes the interconversion of glucosamine-6-phosphate to glucosamine-1-phosphate, an essential step in the biosynthetic pathway leading to the formation of the peptidoglycan precursor uridine 5'-diphospho-N-acetylglucosamine. We have recently identified the gene (glmM) encoding the enzyme of Streptococcus gordonii, an early colonizer on the human tooth and an important cause of infective endocarditis, and indicated that the glmM mutation in S. gordonii appears to influence bacterial cell growth, morphology, and sensitivity to penicillins. In the present study, we assessed whether the glmM mutation also affects escape from polymorphonuclear leukocyte (PMN)-dependent killing. Although no differences in attachment to human PMNs were observed between the glmM mutant and the wild-type S. gordonii, the glmM mutation resulted in increased sensitivity to PMN-dependent killing. Compared with the wild type, the glmM mutant induced increased superoxide anion production and lysozyme release by PMNs. Moreover, the glmM mutant is more sensitive to lysozyme, indicating that the GlmM may be required for synthesis of firm peptidoglycans for resistance to bacterial cell lysis. These findings suggest that the GlmM contributes to the resistance of S. gordonii to PMN-dependent killing. Enzymes such as GlmM could be novel drug targets for this organism.

Binding of the Streptococcus gordonii DL1 surface protein Hsa to the host cell membrane glycoproteins CD11b, CD43, and CD50.

Infective endocarditis is frequently attributed to oral streptococci. The mechanisms of pathogenesis, however, are not well understood, although interaction between streptococci and phagocytes are thought to be very important. A highly expressed surface component of Streptococcus gordonii, Hsa, which has sialic acid-binding activity, contributes to infective endocarditis in vivo. In the present study, we found that S. gordonii DL1 binds to HL-60 cells differentiated into monocytes, granulocytes, and macrophages. Using a glutathione S-transferase (GST) fusion to the NR2 domain, which is the sialic acid-binding region of Hsa, we confirmed that the Hsa NR2 domain also binds to differentiated HL-60 cells. To identify which sialoglycoproteins on the surface of differentiated HL-60 cells are receptors for Hsa, intrinsic membrane proteins were assessed by bacterial overlay and far-Western blotting. S. gordonii DL1 adhered to 100- to 150-kDa proteins, a reaction that was abolished by neuraminidase treatment. These sialoglycoproteins were identified as CD11b, CD43, and CD50 by GST pull-down assay and immunoprecipitation with each specific monoclonal antibody. These data suggest that S. gordonii DL1 Hsa specifically binds to three glycoproteins as receptors and that this interaction may be the initial bacterial binding step in infective endocarditis by oral streptococci.