Biomedical subjects

Howard K Kuramitsu

Publications and source records attributed to Howard K Kuramitsu.

At least 19 recordsLinked to original sources

PubMed2006

Role of bacteriocin immunity proteins in the antimicrobial sensitivity of Streptococcus mutans.

Bacteria utilize quorum-sensing systems to modulate environmental stress responses. The quorum-sensing system of Streptococcus mutans is mediated by the competence-stimulating peptide (CSP), whose precursor is encoded by the comC gene. A comC mutant of strain GS5 exhibited enhanced antimicrobial sensitivity to a wide variety of different agents. Since the addition of exogenous CSP did not complement this phenotype, it was determined that the increased tetracycline, penicillin, and triclosan sensitivities resulted from repression of the putative bacteriocin immunity protein gene, bip, which is located immediately upstream from comC. We further demonstrated that the inactivation of bip or smbG, another bacteriocin immunity protein gene present within the smb operon in S. mutans GS5, affected sensitivity to a variety of antimicrobial agents. Furthermore, both the bip and smbG genes were upregulated in the presence of low concentrations of antibiotics and were induced during biofilm formation relative to in planktonic cells. These results suggest, for the first time, that the antimicrobial sensitivity of a bacterium can be modulated by some of the putative bacteriocin immunity proteins expressed by the organism. The implications of these observations for the evolution of bacteriocin immunity protein genes as well as for potential new chemotherapeutic strategies are discussed.

Anti-Bacterial Agents↗

PubMed2006

Virulence properties of cariogenic bacteria.

The importance of Streptococcus mutans in the etiology of dental caries has been well documented. However, there is growing recognition that the cariogenic potential of dental plaque may be determined by the composite interactions of the total plaque bacteria rather than solely the virulence properties of a single organism. This study will examine how the interactions of S. mutans with other biofilm constituents may influence the cariogenicity of plaque samples. In order to begin to investigate the effects of nonmutans streptococci on the cariogenic potential of S. mutans, we have examined the effects of Streptococcus gordonii on the virulence properties of the former organisms. These studies have indicated that S.gordonii can attenuate several potential virulence properties of S. mutans including bacteriocin production, genetic transformation, and biofilm formation. Therefore, modulation of the interactions between plaque bacteria might be a novel approach for attenuating dental caries initiation.

Journal Article↗

PubMed2006

A universal stress protein of Porphyromonas gingivalis is involved in stress responses and biofilm formation.

Porphyromonas gingivalis is recognized as one of the major periodontal pathogens in subgingival plaque, which is implicated in the progression of chronic periodontal disease. We analyzed the role of upsA in P. gingivalis 381 and its uspA-deficient mutant CW301 under various stress conditions. In general, the uspA mutant was less tolerant to a variety of environmental stresses relative to the parental strain. In addition, gene expression of uspA is upregulated during biofilm formation. Biofilm formation of the uspA mutant was also less than that of strain 381. In conclusion, the uspA gene affecting the stress responses of P. gingivalis is required for optimal biofilm formation.

Anti-Bacterial Agents↗

PubMed2006

A pleiotropic regulator, Frp, affects exopolysaccharide synthesis, biofilm formation, and competence development in Streptococcus mutans.

Exopolysaccharide synthesis, biofilm formation, and competence are important physiologic functions and virulence factors for Streptococcus mutans. In this study, we report the role of Frp, a transcriptional regulator, on the regulation of these traits crucial to pathogenesis. An Frp-deficient mutant showed decreased transcription of several genes important in virulence, including those encoding fructosyltransferase (Ftf), glucosyltransferase B (GtfB), and GtfC, by reverse transcription and quantitative real-time PCR. Expression of Ftf was decreased in the frp mutant, as assessed by Western blotting as well as by the activity assays. Frp deficiency also inhibited the production of GtfB in the presence of glucose and sucrose as well as the production of GtfC in the presence of glucose. As a consequence of the effects on GtfB and -C, sucrose-induced biofilm formation was decreased in the frp mutant. The expression of competence mediated by the competence-signaling peptide (CSP) system, as assessed by comC gene transcription, was attenuated in the frp mutant. As a result, the transformation efficiency was decreased in the frp mutant but was partially restored by adding synthetic CSP. Transcription of the frp gene was significantly increased in the frp mutant under all conditions tested, indicating that frp transcription is autoregulated. Furthermore, complementation of the frp gene in the frp mutant restored transcription of the affected genes to levels similar to those in the wild-type strain. These results suggest that Frp is a novel pleiotropic effector of multiple cellular functions and is involved in the modulation of exopolysaccharide synthesis, sucrose-dependent biofilm formation, and competence development.

Bacterial Proteins↗

PubMed2006

Porphyromonas gingivalis vesicles enhance attachment, and the leucine-rich repeat BspA protein is required for invasion of epithelial cells by "Tannerella forsythia".

The human oral cavity harbors more than 500 species of bacteria. Periodontitis, a bacterially induced inflammatory disease that leads to tooth loss, is believed to result from infection by a select group of gram-negative periodontopathogens that includes Porphyromonas gingivalis, Treponema denticola, and "Tannerella forsythia" (opinion on name change from Tannerella forsythensis pending; formerly Bacteroides forsythus). Epithelial cell invasion by periodontopathogens is considered to be an important virulence mechanism for evasion of the host defense responses. Further, the epithelial cells with invading bacteria also serve as reservoirs important in recurrent infections. The present study was therefore undertaken to address the epithelial cell adherence and invasion properties of T. forsythia and the role of the cell surface-associated protein BspA in these processes. Further, we were interested in determining if P. gingivalis, one of the pathogens frequently found associated in disease, or its outer membrane vesicles (OMVs) could modulate the epithelial cell adherence and invasion abilities of T. forsythia. Here we show that epithelial cell attachment and invasion by T. forsythia are dependent on the BspA protein. In addition, P. gingivalis or its OMVs enhance the attachment and invasion of T. forsythia to epithelial cells. Thus, interactions between these two bacteria may play important roles in virulence by promoting host cell attachment and invasion.

Antigens, Bacterial↗

PubMed2005

Synergistic biofilm formation by Treponema denticola and Porphyromonas gingivalis.

Biofilm formation is an important step in the etiology of periodontal diseases. In this study, in vitro biofilm formation by Treponema denticola and Porphyromonas gingivalis 381 displayed synergistic effects. Confocal microscopy demonstrated that P. gingivalis attaches to the substratum first as a primary colonizer followed by coaggregation with T. denticola to form a mixed biofilm. The T. denticola flagella mutant as well as the cytoplasmic filament mutant were shown to be essential for biofilm formation as well as coaggregation with P. gingivalis. The major fimbriae and Arg-gingipain B of P. gingivalis also play important roles in biofilm formation with T. denticola.

Adhesins, Bacterial↗

PubMed2005

Contact-dependent regulation of a Tannerella forsythia virulence factor, BspA, in biofilms.

Tannerella forsythia is one of the periodontal organisms implicated in the development of periodontal diseases. The surface associated and secreted protein, BspA (encoded by the bspA gene), of this bacterium is an important virulence factor. The present study was carried out to examine the regulation of the bspA gene during biofilm growth and contact stimuli encountered in interbacterial interactions. The expression levels of the bspA transcript were determined by real-time RT-PCR approach. The levels of bspA transcript were found to be significantly reduced as a result of contact stimulus and in biofilm cells relative to planktonic cells. The results of our study suggest that the likely downregulation of the BspA protein in biofilms and following contact may have implications in pathogenesis as a plausible mechanism of evasion of host immune responses.

Antigens, Bacterial↗

PubMed2005

Mechanisms of Porphyromonas gingivalis-induced monocyte chemoattractant protein-1 expression in endothelial cells.

Monocyte chemoattractant protein-1 (MCP-1) is expressed in vascular endothelial cells of inflamed gingival tissues and plays an important role in periodontal pathogenesis. Endothelial cells produce high levels of MCP-1 in response to Porphyromonas gingivalis, an important periodontal pathogen. The present study investigated the mechanisms involved in MCP-1 production by human umbilical vein endothelial cells (HUVEC) following infection with P. gingivalis. In contrast to P. gingivalis, Bacteroides forsythus only weakly stimulated MCP-1 production while Treponema denticola could not induce MCP-1 in HUVEC. The MCP-1 production was independent of endogenous interleukin (IL)-1alpha as IL-1 receptor antagonist treatment did not reduce MCP-1 production by P. gingivalis. Meanwhile, antioxidant treatment and inhibition of NAD(P)H oxidase significantly reduced MCP-1 production. Pharmacological inhibition of p38 mitogen-associated protein (MAP) kinase, c-Jun N-terminal kinase (JNK), nuclear factor-kappaB (NF-kappaB) or activator protein-1 (AP-1) also substantially attenuated P. gingivalis-induced MCP-1 expression by HUVEC. Indeed, activation of NF-kappaB and AP-1 was observed in P. gingivalis-infected HUVEC. These results suggest that MCP-1 expression is upregulated in P. gingivalis-infected endothelial cells via reactive oxygen species, p38 MAP kinase, JNK, NF-kappaB, and AP-1.

Bacteroidaceae Infections↗

PubMed2005

Genetic analysis of a unique bacteriocin, Smb, produced by Streptococcus mutans GS5.

A dipeptide lantibiotic, named Smb, in Streptococcus mutans GS5 was characterized by molecular genetic approaches. The Smb biosynthesis gene locus is encoded by a 9.5-kb region of chromosomal DNA and consists of seven genes in the order smbM1, -T, -F, -M2, -G, -A, -B. This operon is not present in some other strains of S. mutans, including strain UA159. The genes encoding Smb were identified as smbA and smbB. Inactivation of smbM1, smbA, or smbB attenuated the inhibition of the growth of the indicator strain RP66, confirming an essential role for these genes in Smb expression. Mature Smb likely consists of the 30-amino-acid SmbA together with the 32-amino-acid SmbB. SmbA exhibited similarity with the mature lantibiotic lacticinA2 from Lactococcus lactis, while SmbB was similar to the mersacidin-like peptides from Bacillus halodurans and L. lactis. We also demonstrated that Smb expression is induced by the competence-stimulating peptide (CSP) and that a com box-like sequence is located in the smb promoter region. These results suggest that Smb belongs to the class I bacteriocin family, and its expression is dependent on CSP-induced quorum sensing.

Bacteriocins↗

PubMed2005

Interactions between oral bacteria: inhibition of Streptococcus mutans bacteriocin production by Streptococcus gordonii.

Streptococcus mutans has been recognized as an important etiological agent in human dental caries. Some strains of S. mutans also produce bacteriocins. In this study, we sought to demonstrate that bacteriocin production by S. mutans strains GS5 and BM71 was mediated by quorum sensing, which is dependent on a competence-stimulating peptide (CSP) signaling system encoded by the com genes. We also demonstrated that interactions with some other oral streptococci interfered with S. mutans bacteriocin production both in broth and in biofilms. The inhibition of S. mutans bacteriocin production by oral bacteria was stronger in biofilms than in broth. Using transposon Tn916 mutagenesis, we identified a gene (sgc; named for Streptococcus gordonii challisin) responsible for the inhibition of S. mutans bacteriocin production by S. gordonii Challis. Interruption of the sgc gene in S. gordonii Challis resulted in attenuated inhibition of S. mutans bacteriocin production. The supernatant fluids from the sgc mutant did not inactivate the exogenous S. mutans CSP as did those from the parent strain Challis. S. gordonii Challis did not inactivate bacteriocin produced by S. mutans GS5. Because S. mutans uses quorum sensing to regulate virulence, strategies designed to interfere with these signaling systems may have broad applicability for biological control of this caries-causing organism.

Antibiosis↗

PubMed2005

LuxS-based signaling affects Streptococcus mutans biofilm formation.

Streptococcus mutans is implicated as a major etiological agent in human dental caries, and one of the important virulence properties of this organism is its ability to form biofilms (dental plaque) on tooth surfaces. We examined the role of autoinducer-2 (AI-2) on S. mutans biofilm formation by constructing a GS-5 luxS-null mutant. Biofilm formation by the luxS mutant in 0.5% sucrose defined medium was found to be markedly attenuated compared to the wild type. Scanning electron microscopy also revealed that biofilms of the luxS mutant formed larger clumps in sucrose medium compared to the parental strain. Therefore, the expression of glucosyltransferase genes was examined and the gtfB and gtfC genes, but not the gtfD gene, in the luxS mutant were upregulated in the mid-log growth phase. Furthermore, we developed a novel two-compartment system to monitor AI-2 production by oral streptococci and periodontopathic bacteria. The biofilm defect of the luxS mutant was complemented by strains of S. gordonii, S. sobrinus, and S. anginosus; however, it was not complemented by S. oralis, S. salivarius, or S. sanguinis. Biofilm formation by the luxS mutant was also complemented by Porphyromonas gingivalis 381 and Actinobacillus actinomycetemcomitans Y4 but not by a P. gingivalis luxS mutant. These results suggest that the regulation of the glucosyltransferase genes required for sucrose-dependent biofilm formation is regulated by AI-2. Furthermore, these results provide further confirmation of previous proposals that quorum sensing via AI-2 may play a significant role in oral biofilm formation.

Bacterial Proteins↗

PubMed2005

Inducible antisense RNA expression in the characterization of gene functions in Streptococcus mutans.

In order to examine gene function in Streptococcus mutans, we have recently initiated an antisense RNA strategy. Toward this end, we have now constructed and evaluated three Escherichia coli-S. mutans shuttle expression vectors with the fruA and scrB promoters from S. mutans, as well as the tetR-controlled tetO promoter from Staphylococcus aureus. Among these, the tetO/tetR system proved to be the most tightly controlled promoter. By using this shuttle plasmid system, modulation of gene function by inducible antisense RNA expression was demonstrated for comC antisense fragments of different sizes as well as for distinct gtfB antisense fragments. It was demonstrated that the size, but not the relative position, of an antisense DNA fragment is important in mediating the antisense phenomenon. Furthermore, by constructing and screening random DNA libraries with the tet expression shuttle system, 78 growth-retarded transformants harboring antisense DNA fragments were also identified. Almost all of them corresponded to homologous essential genes in other bacteria. In addition, a novel essential gene, the coaE gene, encoding dephospho-coenzyme A kinase, which is involved in the final step of coenzyme A catabolism in S. mutans, was identified and characterized. These results suggest that the antisense RNA strategy can be useful for identifying novel essential genes in S. mutans bacteria as well as further characterizing the physiology (including potential virulence factors) of these organisms.

Gene Library↗

PubMed2005

Identification and characterization of a novel adhesin unique to oral fusobacteria.

Fusobacterium nucleatum is a gram-negative anaerobe that is prevalent in periodontal disease and infections of different parts of the body. The organism has remarkable adherence properties, binding to partners ranging from eukaryotic and prokaryotic cells to extracellular macromolecules. Understanding its adherence is important for understanding the pathogenesis of F. nucleatum. In this study, a novel adhesin, FadA (Fusobacterium adhesin A), was demonstrated to bind to the surface proteins of the oral mucosal KB cells. FadA is composed of 129 amino acid (aa) residues, including an 18-aa signal peptide, with calculated molecular masses of 13.6 kDa for the intact form and 12.6 kDa for the secreted form. It is highly conserved among F. nucleatum, Fusobacterium periodonticum, and Fusobacterium simiae, the three most closely related oral species, but is absent in the nonoral species, including Fusobacterium gonidiaformans, Fusobacterium mortiferum, Fusobacterium naviforme, Fusobacterium russii, and Fusobacterium ulcerans. In addition to FadA, F. nucleatum ATCC 25586 and ATCC 49256 also encode two paralogues, FN1529 and FNV2159, each sharing 31% identity with FadA. A double-crossover fadA deletion mutant, F. nucleatum 12230-US1, was constructed by utilizing a novel sonoporation procedure. The mutant had a slightly slower growth rate, yet its binding to KB and Chinese hamster ovarian cells was reduced by 70 to 80% compared to that of the wild type, indicating that FadA plays an important role in fusobacterial colonization in the host. Furthermore, due to its uniqueness to oral Fusobacterium species, fadA may be used as a marker to detect orally related fusobacteria. F. nucleatum isolated from other parts of the body may originate from the oral cavity.

Adhesins, Bacterial↗

PubMed2005

Biofilm formation by the periodontopathic bacteria Treponema denticola and Porphyromonas gingivalis.

BACKGROUND: Periodontitis develops as a result of the interaction of the host with subgingival plaque bacteria. Both Porphyromonas gingivalis and Treponema denticola are frequently associated together in these oral biofilms. METHODS: The molecular basis for in vitro biofilm formation was investigated for P. gingivalis 381, T. denticola 35405, and mixtures of the two organisms using microtiter plate assays. In addition, the biofilms were examined following confocal laser scanning microscopy. RESULTS: P. gingivalis 381, but not T. denticola strains, formed biofilms in vitro. This property was dependent, in part, on the strain 381 fimA, ppk, and usp genes. Microarray and Northern blot analyses suggested that the expression of the ppk gene was required for maximal expression of the uspA gene. P. gingivalis 381 formed synergistic biofilms when incubated with T. denticola strains. This process was dependent upon the strain 381 rgpB and fimA genes as well as the T. denticola flgE and cfpA genes. CONCLUSIONS: P. gingivalis 381 formed synergistic biofilms with T. denticola 35405. These results may be relevant to the previous observations that the two organisms are frequently observed together in subgingival plaque with the spirochetes localized to the exterior of the oral biofilms. It is suggested that other such synergistic effects may also occur between other plaque bacteria.

Bacterial Proteins↗

PubMed2004

A 43-kDa protein of Treponema denticola is essential for dentilisin activity.

A protease of Treponema denticola, dentilisin, is thought to be part of a complex with 43- and 38-kDa proteins. A sequence encoding a 43-kDa protein was located in the 3' region of the prcA gene upstream of the dentilisin gene (prtP). The 43-kDa protein was apparently generated from digestion of PrcA. To clarify the function of the protein, we constructed a mutant of the 43-kDa protein following homologous recombination. The mutant lacked detectable dentilisin activity. Immunoblot analysis demonstrated that the dentilisin protein was degraded in the mutant. The results of real-time polymerase chain reaction suggested that prtP mRNA expression in the mutant was somewhat decreased compared with the wild-type strain. These data suggest that the 43-kDa protein is involved in the stabilization of the dentilisin protein.

Bacterial Proteins↗

PubMed2004

Sequence analysis of plasmid pTS1 isolated from oral spirochetes.

The naturally occurring plasmid pTS1, identified previously in several species of oral spirochetes, has now been completely sequenced. Analysis of the four open reading frames identified on the plasmid suggests the presence of genes involved in replication and mobilization.

Amino Acid Sequence↗

PubMed2004

Genetic analysis of Treponema denticola ATCC 35405 biofilm formation.

Treponema denticola is a major aetiological organism implicated in periodontal disease. The interaction of T. denticola with other oral bacteria, in particular Porphyromonas gingivalis, in biofilm formation is thought to be an important step in the onset of periodontal disease. The interaction between T. denticola and P. gingivalis has been examined using a panel of T. denticola mutants and their effects on mixed biofilm formation tested in a static biofilm model. T. denticola ATCC 35405 did not form detectable biofilms on various inert surfaces. However, the spirochaete was demonstrated to form a biofilm with preattached P. gingivalis 381. T. denticola cfpA, which lacks the cytoplasmic filament, was unable to produce a mixed biofilm with P. gingivalis. A T. denticola flgE mutant which lacks the flagella hook protein and is therefore non-motile displayed a reduced, but readily detectable, ability to form a mixed biofilm as did the T. denticola mutant which does not possess the major outer sheath protein (Msp). The T. denticola lrrA mutant was only moderately defective in forming mixed biofilms with P. gingivalis. However, the T. denticola methyl-accepting chemotaxis protein (DmcA) did not appear to play a major role in mixed biofilm formation. In contrast, T. denticola lacking the PrtP protein for prolyl-phenylalanine-specific protease, showed an increased ability to form mixed biofilms and a prolonged viability in the biofilm.

Bacterial Proteins↗

PubMed2004

Interaction of Porphyromonas gingivalis with low-density lipoproteins: implications for a role for periodontitis in atherosclerosis.

OBJECTIVE: The association of periodontitis with atherosclerosis has been suggested from epidemiological studies. Recently, we have reported that macrophages stimulated by Porphyromonas gingivalis formed foam cells in the presence of low-density lipoproteins (LDL). In this study, we examined the direct interactions between LDL and P. gingivalis. METHODS: We investigated the aggregation of LDL with P. gingivalis and its outer membrane vesicles (OMVs), degradation of the apo B-100 protein of LDL by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analyses, as well as the effects of protease inhibitors or activators on the mobility of LDL by agarose gel shift assays. The binding of P. gingivalis or its OMVs with LDL was demonstrated by western blot analysis. We also examined whether or not the aggregated LDL induced foam cell formation from murine macrophages. RESULTS: LDL was aggregated in a dose-dependent manner with P. gingivalis and its OMVs. Moreover, degradation of the apo B-100 protein of LDL was directly demonstrated in the presence of P. gingivalis or its OMVs. Furthermore, the gel shift assays indicated that the mobility of LDL was increased in the presence of P. gingivalis. This alteration was attenuated in the presence of the protease inhibitors TLCK and leupeptin and increased in the presence of reducing agents. Moreover, LDL was bound to specific proteins of P. gingivalis suggesting that these proteins may also play a role in aggregation. Finally, the aggregated LDL induced murine macrophages to form foam cells. CONCLUSIONS: These results suggest that P. gingivalis may stimulate foam cell formation, in part, by aggregating LDL by proteolysis of apo B-100.

Adhesins, Bacterial↗