Biomedical subjects

Richard J Lamont

Publications and source records attributed to Richard J Lamont.

At least 19 recordsLinked to original sources

PubMed2006

Porphyromonas gingivalis genes involved in community development with Streptococcus gordonii.

Porphyromonas gingivalis, one of the causative agents of adult periodontitis, develops biofilm microcolonies on substrata of Streptococcus gordonii but not on Streptococcus mutans. P. gingivalis genome microarrays were used to identify genes differentially regulated during accretion of P. gingivalis in heterotypic biofilms with S. gordonii. Thirty-three genes showed up- or downregulation by array analysis, and differential expression was confirmed by quantitative reverse transcription-PCR. The functions of the regulated genes were predominantly related to metabolism and energy production. In addition, many of the genes have no current known function. The roles of two upregulated genes, ftsH (PG0047) encoding an ATP-dependent zinc metallopeptidase and ptpA (PG1641) encoding a putative tyrosine phosphatase, were investigated further by mutational analysis. Strains with mutations in these genes developed more abundant biofilms with S. gordonii than the parental strain developed. ftsH and ptpA may thus participate in a regulatory network that constrains P. gingivalis accumulation in heterotypic biofilms. This study provided a global analysis of P. gingivalis transcriptional responses in an oral microbial community and also provided insight into the regulation of heterotypic biofilm development.

ATP-Dependent Proteases↗

PubMed2006

A Porphyromonas gingivalis haloacid dehalogenase family phosphatase interacts with human phosphoproteins and is important for invasion.

Haloacid dehalogenase (HAD) family phosphatases are widespread in prokaryotes and are generally involved in metabolic processes. Porphyromonas gingivalis, an invasive periodontal pathogen, secretes the HAD family phosphoserine phosphatase SerB653 when in contact with gingival epithelial cells. Here we characterize the structure and enzymatic activity of SerB653 and show that a SerB653 allelic replacement mutant of P. gingivalis is deficient in internalization and persistence in gingival epithelial cells. In contrast, mutation of a second HAD family serine phosphatase of P. gingivalis (SerB1170), or of a serine transporter, did not affect invasion. A pull-down assay identified GAPDH and heat-shock protein 90 as potential substrates for SerB653. Furthermore, exogenous phosphatase regulated microtubule dynamics in host cells. These data indicate that P. gingivalis has adapted a formerly metabolic enzyme to facilitate entry into host cells by modulating host cytoskeletal architecture. Our findings define a virulence-related role of a HAD family phosphatase and reveal an invasin of an important periodontal pathogen.

Alleles↗

PubMed2006

Streptococcus gordonii utilizes several distinct gene functions to recruit Porphyromonas gingivalis into a mixed community.

Dental plaque biofilm formation proceeds through a developmental pathway initiated by the attachment of pioneer organisms, such as Streptococcus gordonii, to tooth surfaces. Through a variety of synergistic interactions, pioneer organisms facilitate the colonization of later arrivals including Porphyromonas gingivalis, a potential periodontal pathogen. We have investigated genes of S. gordonii required to support a heterotypic biofilm community with P. gingivalis. By screening a plasmid integration library of S. gordonii, genes were identified that are crucial for the accumulation of planktonic P. gingivalis cells into a multispecies biofilm. These genes were further investigated by specific mutation and complementation analyses. The biofilm-associated genes can be grouped into broad categories based on putative function as follows: (i) intercellular or intracellular signalling (cbe and spxB), (ii) cell wall integrity and maintenance of adhesive proteins (murE, msrA and atf), (iii) extracellular capsule biosynthesis (pgsA and atf), and (iv) physiology (gdhA, ccmA and ntpB). In addition, a gene for a hypothetical protein was identified. Biofilm visualization and quantification by confocal microscopy confirmed the role of these genes in the maturation of the multispecies community, including biofilm architectural development. The results suggest that S. gordonii governs the development of heterotypic oral biofilms through multiple genetic pathways.

Bacterial Adhesion↗

PubMed2006

Expression of the short fimbriae of Porphyromonas gingivalis is regulated in oral bacterial consortia.

The Mfa1 protein of Porphyromonas gingivalis is the structural subunit of the short fimbriae and mediates coadhesion between P. gingivalis and Streptococcus gordonii. We utilized a promoter-lacZ reporter construct to examine the regulation of mfa1 expression in consortia with common oral plaque bacteria. Promoter activity of mfa1 was inhibited by S. gordonii, Streptococcus sanguinis and Streptococcus mitis. In contrast, Streptococcus mutans, Streptococcus cristatus, Actinomyces naeslundii, Actinobacillus actinomycetemcomitans and Fusobacterium nucleatum did not affect mfa1 expression. Expression of SspA/B, the streptococcal receptor for Mfa1, was not required for regulation of mfa1 promoter activity. Proteinaceous molecule(s) in oral streptococci may be responsible for regulation of Mfa1 expression. Porphyromonas gingivalis is capable of detecting heterologous organisms, and responds to selected organisms by specific gene regulation.

Actinomyces↗

PubMed2006

Structural characterization of peptide-mediated inhibition of Porphyromonas gingivalis biofilm formation.

Porphyromonas gingivalis is a periodontal pathogen whose primary niche is the anaerobic environment of subgingival dental plaque, but initial colonization of the oral cavity is likely to occur on supragingival surfaces that already support robust biofilm communities. Our studies have shown that P. gingivalis adheres to Streptococcus gordonii through interaction of the minor fimbrial antigen Mfa1 with a specific region of the streptococcal SspB polypeptide (residues 1167 to 1193) designated BAR. We show that a synthetic peptide comprising the BAR sequence potently inhibits P. gingivalis adherence to S. gordonii (50% inhibitory concentration = 1.3 microM) and prevents the development of P. gingivalis biofilms. However, a retroinverso peptide that possessed the same side chain topology as that of BAR was inactive, suggesting that interactions of Mfa1 with the peptide backbone of BAR are important for binding. A conformationally constrained analog of BAR inhibited P. gingivalis adherence and biofilm formation but at a lower specific activity than that of BAR. Therefore, to further define the structural features of the Mfa1-BAR interaction, we functionally screened combinatorial libraries of BAR in which active site residues (Asn1182, Thr1184, and Val1185) were replaced with each of the 19 common amino acids. Peptides containing positively charged amino acids at position 1182 or hydrophobic residues at position 1185 bound P. gingivalis more efficiently than did control peptides containing Asn and Val at these positions, suggesting that electrostatic and hydrophobic interactions may contribute to Mfa1-SspB binding. In contrast, replacement of Pro or Gly at these positions was detrimental to adherence, suggesting that perturbation of the BAR secondary structure influences activity. The net effect of substitutions for Thr1184 was less pronounced either positively or negatively than that at the other sites. These results define physicochemical characteristics of the interacting interface of Mfa1 and SspB and suggest that peptides or peptidomimetics with greater specific inhibitory activity than that of BAR can be developed. These compounds may represent potential therapeutics that target some of the first molecular interactions that allow P. gingivalis to colonize the oral cavity.

Adhesins, Bacterial↗

PubMed2006

The Actinobacillus actinomycetemcomitans ribose binding protein RbsB interacts with cognate and heterologous autoinducer 2 signals.

Autoinducer 2 (AI-2) produced by the oral pathogen Actinobacillus actinomycetemcomitans influences growth of the organism under iron limitation and regulates the expression of iron uptake genes. However, the cellular components that mediate the response of A. actinomycetemcomitans to AI-2 have not been fully characterized. Analysis of the complete genome sequence of A. actinomycetemcomitans (www.oralgen.lanl.gov) indicated that the RbsB protein was related to LuxP, the AI-2 receptor of Vibrio harveyi. To determine if RbsB interacts with AI-2, the bioluminescence of the reporter strain V. harveyi BB170 (sensor 1-, sensor 2+) was determined after stimulation with partially purified AI-2 from A. actinomycetemcomitans or conditioned medium from V. harveyi cultures in the presence and absence of purified six-His-tagged RbsB. RbsB efficiently inhibited V. harveyi bioluminescence induced by both A. actinomycetemcomitans AI-2 and V. harveyi AI-2 in a dose-dependent manner, suggesting that RbsB competes with LuxP for AI-2. Fifty percent inhibition occurred with approximately 0.3 nM RbsB for A. actinomycetemcomitans AI-2 and 15 nM RbsB for V. harveyi AI-2. RbsB-mediated inhibition of V. harveyi bioluminescence was reversed by the addition of 50 mM ribose, suggesting that A. actinomycetemcomitans AI-2 and ribose bind at the same site of RbsB. The RbsB/AI-2 complex was thermostable since A. actinomycetemcomitans AI-2 could not be recovered by heating. This was not due to heat inactivation of A. actinomycetemcomitans AI-2 since signal activity was unaffected by heating in the absence of RbsB. Furthermore, an isogenic A. actinomycetemcomitans mutant that was unable to express rbsB was deficient in depleting A. actinomycetemcomitans AI-2 from solution relative to the wild-type organism. Inactivation of rbsB also influenced the ability of the organism to grow under iron-limiting conditions. The mutant strain attained a cell density of approximately 30% that of the wild-type organism under iron limitation. In addition, real-time PCR showed that the expression of afuABC, encoding a major ferric ion transporter, was reduced by approximately eightfold in the rbsB mutant. This phenotype was similar to that of a LuxS-deficient mutant of A. actinomycetemcomitans that is unable to produce AI-2. Together, our results suggest that RbsB may play a role in the response of A. actinomycetemcomitans to AI-2.

Aggregatibacter actinomycetemcomitans↗

PubMed2006

LuxS involvement in the regulation of genes coding for hemin and iron acquisition systems in Porphyromonas gingivalis.

The periodontal pathogen Porphyromonas gingivalis employs a variety of mechanisms for the uptake of hemin and inorganic iron. Previous work demonstrated that hemin uptake in P. gingivalis may be controlled by LuxS-mediated signaling. In the present study, the expression of genes involved in hemin and iron uptake was determined in parent and luxS mutant strains by quantitative real-time reverse transcription-PCR. Compared to the parental strain, the luxS mutant showed reduced levels of transcription of genes coding for the TonB-linked hemin binding protein Tlr and the lysine-specific protease Kgp, which can degrade host heme-containing proteins. In contrast, there was up-regulation of the genes for another TonB-linked hemin binding protein, HmuR; a hemin binding lipoprotein, FetB; a Fe(2+) ion transport protein, FeoB1; and the iron storage protein ferritin. Differential expression of these genes in the luxS mutant was maximal in early-exponential phase, which corresponded with peak expression of luxS and AI-2 signal activity. Complementation of the luxS mutation with wild-type luxS in trans rescued expression of hmuR. Mutation of the GppX two-component signal transduction pathway caused an increase in expression of luxS along with tlr and lower levels of message for hmuR. Moreover, expression of hmuR was repressed, and expression of tlr stimulated, when the luxS mutant was incubated with AI-2 partially purified from the culture supernatant of wild-type cells. A phenotypic outcome of the altered expression of genes involved in hemin uptake was impairment of growth of the luxS mutant in hemin-depleted medium. The results demonstrate a role of LuxS/AI-2 in the regulation of hemin and iron acquisition pathways in P. gingivalis and reveal a novel control pathway for luxS expression.

Bacterial Proteins↗

PubMed2006

Intercellular spreading of Porphyromonas gingivalis infection in primary gingival epithelial cells.

Porphyromonas gingivalis, an important periodontal pathogen, is an effective colonizer of oral tissues. The organism successfully invades, multiplies in, and survives for extended periods in primary gingival epithelial cells (GECs). It is unknown whether P. gingivalis resides in the cytoplasm of infected cells throughout the infection or can spread to adjacent cells over time. We developed a technique based on flow cytofluorometry and fluorescence microscopy to study propagation of the organism at different stages of infection of GECs. Results showed that P. gingivalis spreads cell to cell and that the amount of spreading increases gradually over time. There was a very low level of propagation of bacteria to uninfected cells early in the infection (3 h postinfection), but there were 20-fold and 45-fold increases in the propagation rate after 24 h and 48 h, respectively, of infection. Immunofluorescence microscopy of infected cells suggested that intercellular translocation of P. gingivalis may be mediated through actin-based membrane protrusions, bypassing the need for release of bacteria into extracellular medium. Consistent with these observations, cytochalasin D treatment of infected cells resulted in significant inhibition of bacterial spreading. This study shows for the first time that P. gingivalis disseminates from cell to cell without passing through the extracellular space. This mechanism of spreading may allow P. gingivalis to colonize oral tissues without exposure to the humoral immune response.

Actins↗

PubMed2006

Role of the Porphyromonas gingivalis InlJ protein in homotypic and heterotypic biofilm development.

The oral pathogen Porphyromonas gingivalis expresses a homolog of the internalin family protein InlJ. Inactivation of inlJ reduced monospecies biofilm formation by P. gingivalis. In contrast, heterotypic P. gingivalis-Streptococcus gordonii biofilm formation was enhanced in the InlJ-deficient mutant. The results indicate a nuanced role for InlJ in regulating biofilm accumulations of P. gingivalis.

Bacterial Proteins↗

PubMed2006

Microarray analysis of human epithelial cell responses to bacterial interaction.

Host-pathogen interactions are inherently complex and dynamic. The recent use of human microarrays has been invaluable to monitor the effects of various bacterial and viral pathogens upon host cell gene expression programs. This methodology has allowed the host response transcriptome of several cell lines to be studied on a global scale. To this point, the great majority of reports have focused on the response of immune cells, including macrophages and dendritic cells. These studies revealed that the immune response to microbial pathogens is tailored to different microbial challenges. Conversely, the paradigm for epithelial cells has--until recently--held that the epithelium mostly served as a relatively passive physical barrier to infection. It is now generally accepted that the epithelial barrier contributes more actively to signaling events in the immune response. In light of this shift, this review will compare transcriptional profiling data from studies that involved host-pathogen interactions occurring with epithelial cells. Experiments that defined both a common core response, as well as pathogen-specific host responses will be discussed. This review will also summarize the contributions that transcriptional profiling analysis has made to our understanding of bacterial physio-pathogensis of infection. This will include a discussion of how host transcriptional responses can be used to infer the function of virulence determinants from bacterial pathogens interacting with epithelial mucosa. In particular, we will expand upon the lessons that have been learned from gastro-intestinal and oral pathogens, as well as from members of the commensal flora.

Animals↗

PubMed2006

Mass spectrometry-based proteomics and its application to studies of Porphyromonas gingivalis invasion and pathogenicity.

Porphyromonas gingivalis is a Gram-negative anaerobe that populates the subgingival crevice of the mouth. It is known to undergo a transition from its commensal status in healthy individuals to a highly invasive intracellular pathogen in human patients suffering from periodontal disease, where it is often the dominant species of pathogenic bacteria. The application of mass spectrometry-based proteomics to the study of P. gingivalis interactions with model host cell systems, invasion and pathogenicity is reviewed. These studies have evolved from qualitative identifications of small numbers of secreted proteins, using traditional gel-based methods, to quantitative whole cell proteomic studies using multiple dimension capillary HPLC coupled with linear ion trap mass spectrometry. It has become possible to generate a differential readout of protein expression change over the entire P. gingivalis proteome, in a manner analogous to whole genome mRNA arrays. Different strategies have been employed for generating protein level expression ratios from mass spectrometry data, including stable isotope metabolic labeling and most recently, spectral counting methods. A global view of changes in protein modification status remains elusive due to the limitations of existing computational tools for database searching and data mining. Such a view would be desirable for purposes of making global assessments of changes in gene regulation in response to host interactions during the course of adhesion, invasion and internalization. With a complete data matrix consisting of changes in transcription, protein abundance and protein modification during the course of invasion, the search for new protein drug targets would benefit from a more comprehensive understanding of these processes than what could be achieved prior to the advent of systems biology.

Bacterial Proteins↗

PubMed2005

Oral microbial communities in sickness and in health.

The relationship between humans and their oral microflora begins shortly after birth and lasts a lifetime. Up until fairly recently, the associations between the host and oral bacteria were considered in terms of a multiplicity of single species interactions. However, it is becoming more apparent that the oral microbes comprise a complex community, and that oral health or disease depends on the interface between the host and the microbial community as a whole. Although it is important to continue studies of the pathogenic properties of specific microbes, these are relevant only in the context of the properties of the community within which they reside. Understanding the microbial communities that drive sickness or health is a key to combating human oral diseases.

Bacteria↗

PubMed2005

Differential protein expression by Porphyromonas gingivalis in response to secreted epithelial cell components.

The human oral pathogen Porphyromonas gingivalis colonizes the gingival crevice and invades gingival epithelial cells. Multidimensional capillary high-performance liquid chromatography coupled with tandem mass spectrometry and two-dimensional gel electrophoresis were used to analyze the proteome of P. gingivalis as it adapts to a set of experimental conditions designed to reflect important features of an epithelial cell environment. 1014 proteins (46% of the total theoretical proteome) were identified in four independent analyses; 479 of these proteins showed evidence of differential expression after exposure of P. gingivalis to either conditioned epithelial cell growth medium or control conditions: i.e., they were only detected under one set of conditions. Moreover, 276 genes annotated as hypothetical were found to encode expressed proteins. Among the proteins up-regulated in the presence of epithelial cell components were a homolog of the internalin proteins of Listeria monocytogenes and subunits of the ATP-dependent Clp protease complex. Insertional inactivation of clpP, encoding the Clp proteolytic subunit, resulted in approximately a 50% reduction in invasion of P. gingivalis. These results suggest that adaptation to an epithelial cell environment induces a major shift in the expressed proteome of the organism. Furthermore, ClpP, that is up-regulated in this environment, is required for optimal invasive activity of P. gingivalis.

Bacterial Adhesion↗

PubMed2005

Distinct transcriptional profiles characterize oral epithelium-microbiota interactions.

Transcriptional profiling, bioinformatics, statistical and ontology tools were used to uncover and dissect genes and pathways of human gingival epithelial cells that are modulated upon interaction with the periodontal pathogens Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis. Consistent with their biological and clinical differences, the common core transcriptional response of epithelial cells to both organisms was very limited, and organism-specific responses predominated. A large number of differentially regulated genes linked to the P53 apoptotic network were found with both organisms, which was consistent with the pro-apoptotic phenotype observed with A. actinomycetemcomitans and anti-apoptotic phenotype of P. gingivalis. Furthermore, with A. actinomycetemcomitans, the induction of apoptosis did not appear to be Fas- or TNF(alpha)-mediated. Linkage of specific bacterial components to host pathways and networks provided additional insight into the pathogenic process. Comparison of the transcriptional responses of epithelial cells challenged with parental P. gingivalis or with a mutant of P. gingivalis deficient in production of major fimbriae, which are required for optimal invasion, showed major expression differences that reverberated throughout the host cell transcriptome. In contrast, gene ORF859 in A. actinomycetemcomitans, which may play a role in intracellular homeostasis, had a more subtle effect on the transcriptome. These studies help unravel the complex and dynamic interactions between host epithelial cells and endogenous bacteria that can cause opportunistic infections.

Aggregatibacter actinomycetemcomitans↗

PubMed2005

Short fimbriae of Porphyromonas gingivalis and their role in coadhesion with Streptococcus gordonii.

Porphyromonas gingivalis, one of the causative agents of adult periodontitis, attaches and forms biofilms on substrata of Streptococcus gordonii. Coadhesion and biofilm development between these organisms requires the interaction of the short fimbriae of P. gingivalis with the SspB streptococcal surface polypeptide. In this study we investigated the structure and binding activities of the short fimbriae of P. gingivalis. Electron microscopy showed that isolated short fimbriae have an average length of 103 nm and exhibit a helical structure with a pitch of ca. 27 nm. Mfa1, the major protein subunit of the short fimbriae, bound to SspB protein, and this reaction was inhibited by purified recombinant Mfa1 and monospecifc anti-Mfa1 serum in a dose-dependent manner. Complementation of a polar Mfa1 mutant with the mfa1 gene restored the coadhesion phenotype of P. gingivalis. Hence, the Mfa1 structural fimbrial subunit does not require accessory proteins for binding to SspB. Furthermore, the interaction of Mfa1 with SspB is necessary for optimal coadhesion between P. gingivalis and S. gordonii.

Adhesins, Bacterial↗

PubMed2004

Calcium receptor message, expression and function decrease in differentiating keratinocytes.

Calcium-sensing receptor (CaSR) expression and function were studied in proliferating and differentiating cultured human gingival keratinocytes (HGKs). CaSR mRNA and protein were present in proliferating HGKs cultured in 0.03 mM [Ca(2+)] and decreased in cells induced to differentiate by culturing in 1.2 mM [Ca(2+)] for 2 days. CaSR protein was also detected in gingival tissue. Exposure to 10 mM extracellular [Ca(2+)] activated two sequential whole-cell currents. The first was a small, transient calcium release activated calcium current I(CRAC)-like current with an inwardly rectifying I-V curve. The second current was larger with a linear I-V curve. Both currents were significantly decreased in differentiating cells. Neither neomycin nor gadolinium induced changes in whole cell currents nor in intracellular [Ca(2+)], but neomycin inhibited the late large current. Extracellular Ca(2+) increased intracellular [Ca(2+)] of proliferating HGKs in a dose-dependent fashion. Comparison of the time-courses of the whole-cell currents and the intracellular [Ca(2+)] responses indicated both induced currents supported a Ca(2+) influx. Extracellular [Mg(2+)] changes did not affect intracellular [Ca(2+)]. La(3+) and 2-APB inhibited the whole cell current and intracellular [Ca(2+)] changes. The results indicate that the CaSR signaling response likely plays a major role in initiating Ca(2+) induced differentiation responses in HGKs.

Blotting, Western↗

PubMed2004

Calcium oscillations in gingival epithelial cells infected with Porphyromonas gingivalis.

The periodontal pathogen Porphyromonas gingivalis modulates epithelial cell signal transduction pathways including Ca2+ signaling, and internalizes within the host cell cytoplasm. Since nuclear and cytoplasmic [Ca2+] increases can induce different host cell responses, P. gingivalis-related [Ca2+] changes in these compartments were measured by digital fluorescent imaging microscopy. Non-deconvolved and deconvolved fura-2 images showed that P. gingivalis exposure caused human gingival epithelial cells cultured in physiologic [Ca2+] levels to undergo sustained oscillations of [Ca2+] in nuclear and cytoplasmic spaces. However, P. gingivalis invasion was not tightly correlated with intracellular [Ca2+] oscillations, since invasion could significantly precede, or even occur in the absence of, oscillations. [Ca2+] oscillations required a Ca2+ influx, which was completely inhibited by La3+ or 2-APB (2-aminoethoxydiphenyl borate), indicating Ca2+ entry was via a Ca(2+)-permeable channel. Ca2+ entry was likely not via a store-operated channel, since Ca2+ release from intracellular stores was not observed during cellular uptake of P. gingivalis. Hence, uptake of P. gingivalis in gingival epithelial cells induces oscillations in nuclear and cytoplasmic spaces by activating a Ca2+ influx through Ca2+ channels.

Biological Transport↗

PubMed2004

Porphyromonas gingivalis genes involved in fimA regulation.

Porphyromonas gingivalis is an important component of the complex plaque biofilm that is a direct precursor of periodontal disease. The major fimbriae are required for attachment to oral surfaces and are an important virulence factor. Fimbrillin (FimA) expression in P. gingivalis is inhibited by surface molecule of Streptococcus cristatus, an early colonizer of dental plaque. In this study, differential display PCR was used to identify P. gingivalis genes that are regulated in response to S. cristatus. Of several differentially expressed genes, pg2131 and pg2167 were upregulated by S. cristatus signaling molecules. A null mutant of pg2167 did not transcriptionally regulate fimA following exposure to S. cristatus. In fact, fimA transcription was enhanced in the pg2167 mutant, suggesting that pg2167 may act to repress fimA expression. In contrast, a mutation in pg2131 did not affect transcription of fimA in the presence of S. cristatus. However, production of fimbrillin was significantly diminished in the pg2131 mutant, implicating involvement in posttranscriptional regulation in fimbriation. These data suggest that P. gingivalis fimbriation is controlled by more than one regulation mechanism, involving both transcriptional and posttranscriptional processes.

Bacterial Proteins↗