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R J Lamont

Publications and source records attributed to R J Lamont.

At least 19 recordsLinked to original sources
PubMed2001

Inhibition of epithelial cell apoptosis by Porphyromonas gingivalis.

Porphyromonas gingivalis is periodontal pathogen that is capable of invading gingival epithelial cells (GECs). Apoptotic responses of primary cultures of GECs to P. gingivalis were investigated with a DNA fragmentation ELISA assay. P. gingivalis induced a transient increase in GEC DNA fragmentation; however, after prolonged incubation GECs did not undergo apoptosis. Furthermore, P. gingivalis blocked apoptosis in GECs following stimulation with camptothecin. Immunoblotting of GECs with Bcl-2 or Bax antibodies showed that P. gingivalis up-regulated Bcl-2 levels in GECs, whereas Bax levels were transiently elevated and declined after 24 h stimulation. Streptococcus gordonii did not affect levels of either molecule. RT-PCR demonstrated that induction of Bcl-2 occurs at the transcriptional level. The results suggest that P. gingivalis can inhibit apoptosis in GECs by up-regulation of the anti-apoptotic molecule Bcl-2. The prevention of host cell apoptosis may represent a strategy for P. gingivalis survival within invaded GECs.

Apoptosis↗
PubMed2001

Searching the Porphyromonas gingivalis genome with peptide fragmentation mass spectra.

An approach is described for genomic database searching based on experimentally observed proteolytic fragments, e.g., isolated from 1D or 2D gels or analyzed directly, that can be applied to unfinished prokaryotic genomic data in the absence of annotations or previously assigned open reading frames (ORFs). This variation on the database search is in contrast to the more familiar use of peptide mass spectral fragmentation data to search fully annotated inferred protein databases, e.g., OWL or SWISS-PROT. We compared the SEQUEST search results from a six reading frame translation of the Porphyromonas gingivalis genome DNA sequence with those from computationally derived ORFs created using publicly available genomics software tools. The ORF approach eliminated many of the artifacts present in output from the six reading frame search. The method was applied to uninterpreted tandem mass spectrometric data derived from proteins secreted by the periodontal pathogen Porphyromonas gingivalis in response to the gingival epithelial cell environment, a model system for the study of host-pathogen interactions relevant to human periodontal disease.

Bacterial Proteins↗
PubMed2001

Regulation of Streptococcus gordonii sspB by the sspA gene product.

Streptococcus gordonii expresses two related adhesins, SspA and SspB, the genes for which are adjacent on the chromosome and are regulated independently. Although the adhesins are functionally similar, the sspA promoter is more active than that of sspB. In this study we show an additional role for SspA in the control of sspB activity. Gel shift and DNA footprinting assays demonstrate that the SspA protein binds to the sspB promoter and protects a region 233 to 264 bp upstream of the predicted -35 promoter element. The responsiveness of the sspB promoter to SspA was investigated with a promoter-cat reporter. Expression of the sspB promoter was reduced by over 60% in an SspA-deficient mutant of S. gordonii. These results indicate that expression of S. gordonii sspB is positively regulated by the sspA gene product.

Adhesins, Bacterial↗
PubMed2001

Association of mitogen-activated protein kinase pathways with gingival epithelial cell responses to Porphyromonas gingivalis infection.

Mitogen-activated protein (MAP) kinase pathways are key factors in host signaling events and can also play important roles in the internalization of pathogenic bacteria by host cells. Porphyromonas gingivalis, a periodontal pathogen, can efficiently invade human gingival epithelial cells (GECs). In this study, we examined the activation of MAP kinase pathways in GECs infected with P. gingivalis. c-Jun N-terminal kinase (JNK) was activated after 5 min of infection with P. gingivalis, whereas noninvasive Streptococcus gordonii did not have a significant effect on JNK activation. In contrast, extracellular signal-regulated kinase (ERK) 1/2 was downregulated in a dose-dependent manner by P. gingivalis, but not by S. gordonii, after a 15-min exposure. Nonmetabolically active P. gingivalis cells were unable to modulate MAP kinase activity. U0126, a specific inhibitor of MEK1/2 (ERK1/2 kinase), and toxin B, a specific inhibitor of Rho family GTPases, had no effect on P. gingivalis invasion. Genistein, a tyrosine protein kinase inhibitor, blocked uptake of P. gingivalis. The transcriptional regulator NF-kappaB was not activated by P. gingivalis. These results suggest that P. gingivalis can selectively target components of the MAP kinase pathways. ERK1/2, while not involved in P. gingivalis invasion of GECs, may be downregulated by internalized P. gingivalis. Activation of JNK is associated with the invasive process of P. gingivalis.

Butadienes↗
PubMed2001

Intra- and interspecies regulation of gene expression by Actinobacillus actinomycetemcomitans LuxS.

The cell density-dependent control of gene expression is employed by many bacteria for regulating a variety of physiological functions, including the generation of bioluminescence, sporulation, formation of biofilms, and the expression of virulence factors. Although periodontal organisms do not appear to secrete acyl-homoserine lactone signals, several species, e.g., Porphyromonas gingivalis, Prevotella intermedia, and Fusobacterium nucleatum, have recently been shown to secrete a signal related to the autoinducer II (AI-2) of the signal system 2 pathway in Vibrio harveyi. Here, we report that the periodontal pathogen Actinobacillus actinomycetemcomitans expresses a homolog of V. harveyi luxS and secretes an AI-2-like signal. Cell-free conditioned medium from A. actinomycetemcomitans or from a recombinant Escherichia coli strain (E. coli AIS) expressing A. actinomycetemcomitans luxS induced luminescence in V. harveyi BB170 >200-fold over controls. AI-2 levels peaked in mid-exponential-phase cultures of A. actinomycetemcomitans and were significantly reduced in late-log- and stationary-phase cultures. Incubation of early-log-phase A. actinomycetemcomitans cells with conditioned medium from A. actinomycetemcomitans or from E. coli AIS resulted in a threefold induction of leukotoxic activity and a concomitant increase in leukotoxin polypeptide. In contrast, no increase in leukotoxin expression occurred when cells were exposed to sterile medium or to conditioned broth from E. coli AIS(-), a recombinant strain in which luxS was insertionally inactivated. A. actinomycetemcomitans AI-2 also induced expression of afuA, encoding a periplasmic iron transport protein, approximately eightfold, suggesting that LuxS-dependent signaling may play a role in the regulation of iron acquisition by A. actinomycetemcomitans. Finally, A. actinomycetemcomitans AI-2 added in trans complemented a luxS knockout mutation in P. gingivalis by modulating the expression of the luxS-regulated genes uvrB and hasF in this organism. Together, these results suggest that LuxS-dependent signaling may modulate aspects of virulence and the uptake of iron by A. actinomycetemcomitans and induce responses in other periodontal organisms in mixed-species oral biofilm.

Aggregatibacter actinomycetemcomitans↗
PubMed2001

Discrete protein determinant directs the species-specific adherence of Porphyromonas gingivalis to oral streptococci.

For pathogens to survive in the human oral cavity, they must identify a suitable niche in the complex multispecies biofilm that exists on oral tissues. The periodontal pathogen Porphyromonas gingivalis adheres to Streptococcus gordonii by interacting with a specific region of the streptococcal SspB polypeptide, designated BAR. However, it does not adhere to Streptococcus mutans, which expresses SpaP, a highly conserved homolog of SspB. Comparison of the predicted secondary structure of BAR with the corresponding region of SpaP suggested that the substitution of Asn for Gly1182 and Val for Pro1185 in SspB may confer a unique local structure that is not conserved in SpaP. A synthetic peptide of 26 amino acids that encompassed residues 1167 to 1193 of SspB promoted avid adherence of P. gingivalis, whereas a peptide derived from the region corresponding to BAR in SpaP was inactive. Substitution of Gly1182 and Pro1185 for Asn1182 and Val1185 in SspB by site-specific mutation generated proteins that were predicted to assume an SpaP-like secondary structure, and the purified proteins did not promote P. gingivalis adherence. Furthermore, Enterococcus faecalis strains expressing the site-specific mutants did not support adherence of P. gingivalis cells. In contrast, P. gingivalis adhered efficiently to E. faecalis strains expressing intact SspB or SspB-SpaP chimeric proteins containing BAR. These results suggest that a region of SspB consisting of 26 amino acids is sufficient to mediate the adherence of P. gingivalis to S. gordonii and that the species specificity of adherence arises from its interaction with a discrete structural determinant of SspB that is not conserved in SpaP.

Adhesins, Bacterial↗
PubMed2001

Signaling system in Porphyromonas gingivalis based on a LuxS protein.

The luxS gene of quorum-sensing Vibrio harveyi is required for type 2 autoinducer production. We identified a Porphyromonas gingivalis open reading frame encoding a predicted peptide of 161 aa that shares 29% identity with the amino acid sequence of the LuxS protein of V. harveyi. Conditioned medium from a late-log-phase P. gingivalis culture induced the luciferase operon of V. harveyi, but that from a luxS insertional mutant did not. In P. gingivalis, the expression of luxS mRNA was environmentally controlled and varied according to the cell density and the osmolarity of the culture medium. In addition, differential display PCR showed that the inactivation of P. gingivalis luxS resulted in up-regulation of a hemin acquisition protein and an arginine-specific protease and reduced expression of a hemin-regulated protein, a TonB homologue, and an excinuclease. The data suggest that the luxS gene in P. gingivalis may function to control the expression of genes involved in the acquisition of hemin.

Amino Acid Sequence↗
PubMed2000

Environmental conditions modulate the expression of the sspA and sspB genes in Streptococcus gordonii.

The SspA and SspB surface proteins of Streptococcus gordonii are multifunctional adhesins encoded by tandemly arranged genes. The transcriptional regulation of the sspA and sspB genes was investigated by generating chromosomal promoter- cat gene fusions and measuring CAT enzyme activity. The sspA promoter was found to be three-fold more active than the sspB promoter. In addition, sspA transcriptional activity increased throughout growth, whereas sspB activity decreased in stationary phase. Promoter activity of both sspA and sspB was regulated in response to temperature, pH and osmolarity; however the two promoters showed a different pattern of regulation. Changes in promoter activity were reflected in levels of surface protein and in adherence of S. gordonii to Porphyromonas gingivalis, a phenotypic property dependent on Ssp proteins. The results show that S. gordonii strain DL1 differentially regulates sspA and sspB transcription in response to oral environmental cues, suggesting that the SspA and SspB polypeptides may have distinct functional roles in cell adherence to oral substrates.

Adhesins, Bacterial↗
PubMed2000

Dental plaque formation.

Dental plaque is a complex biofilm that accumulates on the hard tissues (teeth) in the oral cavity. Although over 500 bacterial species comprise plaque, colonization follows a regimented pattern with adhesion of initial colonizers to the enamel salivary pellicle followed by secondary colonization through interbacterial adhesion. A variety of adhesins and molecular interactions underlie these adhesive interactions and contribute to plaque development and ultimately to diseases such as caries and periodontal disease.

Adhesins, Bacterial↗
PubMed2000

Subgingival colonization by Porphyromonas gingivalis.

Porphyromonas gingivalis, a gram-negative anaerobe, is a major causative agent in the initiation and progression of severe forms of periodontal disease. In order to cause periodontal disease, P. gingivalis must colonize the subgingival region, a process that involves several distinct steps and multiple gene products. The organism must first navigate within the oral fluids in order to reach the hard or soft tissues of the mouth. Retention and growth of bacteria on these surfaces is facilitated by a repertoire of adhesins including fimbriae, hemagglutinins and proteinases. Once established subgingivally, P. gingivalis cells participate in intercellular communication networks with other oral prokaryotic cells and with eukaryotic cells. The establishment of these multiple interactive interfaces can lead to biofilm formation, invasion of root dentin and internalization within gingival epithelial cells. The resulting bacterial and host cellular locations, products and fate contribute to the success of P. gingivalis in colonizing the periodontal region.

Adhesins, Bacterial↗
PubMed2000

Regulation of the Porphyromonas gingivalis fimA (Fimbrillin) gene.

In common with many bacterial virulence genes, the fimbrillin (fimA) gene of Porphyromonas gingivalis is modulated in response to environmental fluctuation. The trans-acting components that comprise the regulatory system for transcriptional activity of the fimA gene in P. gingivalis were investigated. Three major proteins were found to bind to the upstream region of the fimA promoter. One of these proteins was fimbrillin itself, and the other two were a major arginine protease (Rgp) and lysine protease (Kgp). Production of these proteins was necessary for maximal fimA transcription. An exogenous fimA promoter-lacZ reporter was inactive when introduced into a strain of P. gingivalis carrying a mutation in the indigenous fimA gene. Furthermore, fimA mRNA levels were significantly decreased in rgp and kgp mutant strains. These data indicate that P. gingivalis has evolved multiple levels of control of fimbrial gene expression to enhance its survival in hostile environments.

Amino Acid Sequence↗
PubMed2000

Identification of a Porphyromonas gingivalis receptor for the Streptococcus gordonii SspB protein.

Colonization of the plaque biofilm by the oral pathogen Porphyromonas gingivalis is favored by the presence of antecedent organisms such as Streptococcus gordonii. Coadhesion between P. gingivalis and S. gordonii can be mediated by the SspB protein of S. gordonii; however, the P. gingivalis cognate receptor for this protein has not been identified. In this study, we identified a surface protein of P. gingivalis that interacts with the SspB protein. Coprecipitation between P. gingivalis outer membrane proteins and purified SspB protein demonstrated that a 100-kDa P. gingivalis protein bound to SspB. The 100-kDa protein also bound to an engineered strain of Enterococcus faecalis that expresses the SspB protein on the cell surface. Monospecific polyclonal antibodies to the 100-kDa protein inhibited the binding between P. gingivalis and S. gordonii in a dose-dependent manner up to 86%. Amino acid sequencing of the 100-kDa protein showed homology to a protein previously identified as the P. gingivalis minor fimbria. The minor fimbrial protein may exist as a complex with a hemagglutinin-like protein since the genes encoding these proteins are adjacent on the chromosome and are cotranscribed. Thus, the P. gingivalis receptor for S. gordonii SspB is a 100-kDa protein that structurally may be a minor fimbria-protein complex and functionally effectuates coadhesion.

Adhesins, Bacterial↗
PubMed2000

Intergeneric communication in dental plaque biofilms.

Dental plaque is a complex biofilm that accretes in a series of discrete steps proceeding from a gram-positive streptococcus-rich biofilm to a structure rich in gram-negative anaerobes. This study investigated information flow between two unrelated plaque bacteria, Streptococcus cristatus and Porphyromonas gingivalis. A surface protein of S. cristatus caused repression of the P. gingivalis fimbrial gene (fimA), as determined by a chromosomal fimA promoter-lacZ reporter construct and by reverse transcription-PCR. Signaling activity was associated with a 59-kDa surface protein of S. cristatus and showed specificity for the fimA gene. Furthermore, P. gingivalis was unable to form biofilm microcolonies with S. cristatus. Thus, S. cristatus is capable of modulating virulence gene expression in P. gingivalis, consequently influencing the development of pathogenic plaque.

Antibiosis↗
PubMed1999

Fluorescence image analysis of the association between Porphyromonas gingivalis and gingival epithelial cells.

We have developed a fluorescence imaging technique using a DNA-binding dye to visualize, over time, the physical interactions between Porphyromonas gingivalis and human gingival epithelial cells in vitro. The results extend previous observations of P. gingivalis invasion of gingival epithelial cells based on indirect measurements. An intracellular location for P. gingivalis was established by optical sectioning of images in the z-plane. Kinetic analysis showed that P. gingivalis invasion of epithelial cells is a rapid and efficient process, reaching completion after 12 min. Imaging of infected monolayers revealed that over 90% of a population of gingival epithelial cells contained bacteria. Furthermore, only vital bacteria were capable of invasion, and intracellular bacteria congregated in the perinuclear region of the epithelial cells. P. gingivalis remained inside the epithelial cells over a 24 h period and induced rearrangement of the actin cytoskeleton along with alteration of the size and shape of the epithelial cells. These findings provide direct evidence that entry rates of P. gingivalis into gingival epithelial cells are high and rapid, and that internalized bacteria initially localize in a specific region of the epithelial cells.

Cell Nucleus↗
PubMed1999

Promoter architecture of the Porphyromonas gingivalis fimbrillin gene.

Porphyromonas gingivalis fimbriae can mediate adherence to many of the available substrates in the oral cavity. Expression of P. gingivalis fimbriae is regulated at the transcriptional level by environmental signals, such as temperature and hemin concentration. The arrangement of the upstream promoter and regulatory sequences required for transcription and control of the fimbrial structural gene (fimA) was investigated. Primer extension analysis demonstrated that the transcriptional start site of the fimA gene is located 41 bp upstream from the translational start codon. A region (upf) spanning 648 bp upstream of the start codon to 44 bp downstream of the translational start site was cloned upstream of a promoterless lacZ reporter gene. A series of deletion and base substitution mutations were then generated in the upf region. The constructs were introduced into the chromosome of P. gingivalis, and promoter activity measured by assaying levels of beta-galactosidase. The results showed that fimA contains sequences resembling sigma70 promoter consensus sequences, consisting of a -10 region (TATGAC) located at -18 to -23 and a -35 region (TTGTTG) located at -41 to -46 from the transcriptional start point. The AT-rich upstream sequences spanning bases -48 to -85 and bases -90 to -240 were required for full expression of the fimA gene, indicating the existence of positive regulation regions. Moreover, the -48 to -64 region may constitute an UP element, contributing to promoter activity in P. gingivalis. Thus, our data suggest that the P. gingivalis fimA gene has a transcription complex consisting of -10 and -35 sequences, an UP element, and additional AT-rich upstream regulatory sequences.

Bacterial Proteins↗
PubMed1998

Biofilm formation by Porphyromonas gingivalis and Streptococcus gordonii.

Confocal scanning laser microscopy (CSLM) was used to visualize and quantify biofilm formation by the oral bacteria Streptococcus gordonii and Porphyromonas gingivalis. A saliva-coated glass coverslip under continuous bacterial challenge and conditions of low shear force was used to investigate attachment to the salivary pellicle and also the effect of cell-cell interactions on the extent of colonization and biofilm development. S. gordonii bound to the salivary pellicle and outcompeted P. gingivalis for attachment sites. Both P. gingivalis and S. gordonii failed to establish substantial biofilm formation independently. However, biofilm formation did occur subsequent to initial adherence of P. gingivalis to S. gordonii cells deposited on the salivary pellicle. The commensal species S. gordonii may, therefore, provide an attachment substrate for colonization and biofilm accretion by the potential pathogen, P. gingivalis.

Bacterial Adhesion↗
PubMed1998

Contact-dependent protein secretion in Porphyromonas gingivalis.

Porphyromonas gingivalis can induce its uptake by host epithelial cells; however, the nature and role of the P. gingivalis molecules involved in this invasion process have yet to be determined. In this study, modulation of secreted P. gingivalis proteins following association with gingival epithelial cells was investigated. Western immunoblot analysis showed that contact with epithelial cells or epithelial cell growth media induces P. gingivalis 33277 to secrete several proteins with molecular masses between 35 and 95 kDa. Secretion of the Arg-gingipain and Lys-gingipain proteases was repressed under these conditions. The contact-induced secreted protein profile was altered in Arg-gingipain-deficient and Lys-gingipain-deficient mutants, indicating a possible role for these proteases in the secretion pathway. The P. gingivalis contact-dependent protein secretion pathway differs to some extent from type III protein secretion pathways in enteric pathogens, as a gene homologous to the invA family genes was not detected in P. gingivalis. The secreted proteins of P. gingivalis may play a role in the interactions of the organism with host cells.

Adhesins, Bacterial↗