Impact of monocytic cells on recovery of uncultivable bacteria from atherosclerotic lesions.

OBJECTIVE: Epidemiological evidence suggests that infections may contribute to atherogenesis. However, with the exception of Chlamydophila pneumoniae, cultivable bacteria have not been recovered from atherosclerotic lesions. Therefore, we aimed at developing an approach to recover uncultivable bacteria from atherectomy tissues. METHODS: We cultured homogenates from atherectomy specimens from seven nonseptic patients undergoing surgery for arterial obstruction either alone or together with THP-1 monocyte-like cells. We performed 16S rDNA analysis, biochemical tests, random amplification of polymorphic DNA PCR analysis, quantitative polymerase chain reaction (qPCR) and immunohistofluorescence to identify the cultivated bacteria. Wilcoxon signed-rank tests were used to determine whether THP-1 treatment yielded a higher number of isolates than did the untreated controls. RESULTS: We recovered more bacteria from cocultures of atherectomy specimens with THP-1 cells than atherectomy specimens cultured alone. On average, tissue homogenates incubated with THP-1 cells versus control yielded 124 vs. 22 colony-forming units, a median of 140 vs. 7, respectively (P = 0.02). We recovered 872 isolates of limited number of species, including Propionibacterium acnes, Staphylococcus epidermidis and Streptococcus infantis and the fastidious anaerobe Porphyromonas gingivalis, and confirmed its presence in tissue using double immunofluorescence imaging. qPCR demonstrated the presence of ≥3.5 × 10(3) P. gingivalis genomes per gram of atheromatous tissue. CONCLUSIONS: These results indicate that viable previously uncultivable bacterial species are present within atheromas. Our results suggest revisiting the hypothesis that infections may have a causative role in atherosclerotic inflammation and have implications for research regarding novel diagnostics and treatments for cardiovascular disease.

The effect of monoclonal antibody and route of immunization on the humoral immune response against Porphyromonas gingivalis.

Immunomodulation mediated by exogenous antibodies has been proposed as a vaccine strategy to improve immune protection against pathogenic microorganisms and suggested to contribute to protection following passive immunization. To test whether a monoclonal antibody directed against an adhesion epitope of the periodontal pathogen Porphyromonas gingivalis could influence the humoral immune response following mucosal immunization, BALB/c mice were immunized orally or intranasally with P. gingivalis alone or P. gingivalis coated with monoclonal antibody 61BG1.3. Differences in antigenic specificity of anti- P. gingivalis serum immunoglobulin G (IgG) were demonstrated between groups of mice that received monoclonal antibody-coated P. gingivalis versus those that received P. gingivalis alone by either route of immunization. Binding of monoclonal antibody 61BG1.3 to P. gingivalis prior to immunization did not influence the serum IgG subclass distribution. However, minor differences in subclass distribution were observed between the various routes of mucosal immunization. These results support the hypothesis that specific monoclonal antibody bound to a bacterial vaccine can alter the quality of the humoral immune response to that organism.

Expression and immunogenicity of hemagglutinin A from Porphyromonas gingivalis in an avirulent Salmonella enterica serovar typhimurium vaccine strain.

Porphyromonas gingivalis is a major etiologic agent of periodontitis, a chronic inflammatory disease that ultimately results in the loss of the supporting tissues of the teeth. Previous work has demonstrated the usefulness of avirulent Salmonella enterica serovar Typhimurium strains as antigen delivery systems for protective antigens of pathogens that colonize or cross mucosal surfaces. In this study, we constructed and characterized a recombinant S. enterica serovar Typhimurium avirulent vaccine strain which expresses hemagglutinin A and carries no antibiotic resistance markers. HagA, a major virulence-associated surface protein, is a potentially useful immunogen that contains an antigenic epitope which, in humans, elicits an immune response that is protective against subsequent colonization by P. gingivalis. The hagA gene, including its promoter, was cloned into a balanced-lethal Salmonella vector and transferred to the vaccine strain. Heterologous expression of HagA was demonstrated in both Escherichia coli JM109 and S. enterica serovar Typhimurium vaccine strain chi4072. The HagA epitope was present in its native configuration as determined by immunochemistry and immunoelectron microscopy. Purified recombinant HagA was recognized by sera from mice immunized with the S. enterica serovar Typhimurium vaccine strain. The HagA-specific antigen of the vaccine was also found to be recognized by serum from a periodontal patient. This vaccine strain, which expresses the functional hemagglutinin protein, induces a humoral immune response against HagA and may be useful for developing a protective vaccine against periodontal diseases associated with P. gingivalis.

Porphyromonas gingivalis virulence factors and invasion of cells of the cardiovascular system.

Our laboratory is interested in the genes and gene products involved in the interactions between Porphyromonas gingivalis (Pg) and the host. These interactions may occur in either the periodontal tissues or other non-oral host tissues such as those of the cardiovascular system. We have previously reported the cloning of several genes encoding hemagglutinins, surface proteins that interact with the host tissues, and are investigating their roles in the disease process. Primary among these is HagA, a very large protein with multiple functional groups that have significant sequence homology to protease genes of this species. Preliminary evidence indicates that an avirulent Salmonella typhimurium strain containing hagA is virulent in mice. These data indicate that HagA may be a key virulence factor of Pg. Additionally, we are investigating the invasion of primary human coronary artery endothelial cells (HCAEC) by Pg because of the recent epidemiological studies indicating a correlation between periodontal disease (PD) and coronary heart disease (CHD). We found that some, but not all, strains of Pg are able to invade these cells. Scanning electron microsopy of the infected HCAEC demonstrated that the invading organisms initially attached to the host cell surface as aggregates and by a "pedestal"-like structure. By transmission electronmicroscopy it could be seen that internalized bacteria were present within multimembranous compartments localized with rough endoplasmic reticulum. In addition, invasion of the HCAEC by Pg resulted in an increase in the degradation of long-lived cellular proteins. These data indicate that Pg are present within autophagosomes and may use components of the autophagic pathway as a means to survive intracellularly. However, Pg presence within autophagosomes in KB cells could not be observed or detected. It is therefore likely that Pg uses different invasive mechanisms for different host cells. This and the role of HagA in invasion is currently being investigated further.

The number of direct repeats in hagA is variable among Porphyromonas gingivalis strains.

The coding sequence for the surface protein hemagglutinin A (HagA) of Porphyromonas gingivalis 381 has previously been shown to contain four direct 1.35-kb repeats, designated repHA. This study was performed to determine if the number of repHA units in hagA is consistently 4 or if allelic polymorphism exists among strains and/or upon multiple passage of P. gingivalis. To this end, primers which were homologous to the regions directly 5' and 3' of the repeat domain in hagA were synthesized. PCR conditions which allowed amplification of the 8.4-kb repeat region between the primers in P. gingivalis 381 were established. Genomic DNA templates from 13 other P. gingivalis strains and 9 fresh clinical isolates from patients were analyzed under the same conditions as used above. Analysis of these PCR products demonstrated that the strains tested had different numbers (two to four) of repHA units in the respective hagA genes. The PCR products of 8.4, 7.0, and 5.7 kb represent four, three, and two repeats, respectively. One strain from each group (381, four repeats; W83, three repeats; and AJW4, two repeats) was also tested to determine if the number of repeats remained invariant upon passaging onto solid medium. No variability in the number of repeats in hagA within a strain was detected after 18 passages. P. gingivalis 381 was chosen for further testing in a mouse abscess model to determine if conditions of in vivo growth would select for deletions or duplications of the repeated sequences. Five days after infection, no change in the number of repeats was detected in cells recovered from either nonimmunized or preimmunized mice. This data indicates an interstrain variability of the number of repeat units and hence a size variability of the HagA protein of P. gingivalis, but unlike some surface antigens of other pathogenic species, the number of repeats remains relatively stable given the conditions of growth tested here.

The cytosolic glycoprotein FP21 of Dictyostelium discoideum is encoded by two genes resulting in a polymorphism at a single amino acid position.

FP21 is a glycoprotein within the cytosolic compartment of Dictyostelium which carries an unusual carbohydrate modification(s) including the sugars fucose, galactose and N-acetylglucosamine. The soluble pool of FP21 from crude extracts resolves chromatographically into two fractions that differ in their glycosylation. Previous gene-mapping studies indicating the existence of two loci suggested that the FP21 fractions might be encoded by different genes. To address this issue, the two genes were cloned and sequenced, leading to the prediction that the protein products would differ by only a single amino acid, Ser or Ala, at codon 39. Protein sequence data on CNBr fragments of purified FP21 showed that both gene products are found in both fractions of the soluble pool. After further purification, the two fractions were no longer chromatographically resolvable, and there was no evidence for charge heterogeneity as determined by 2-D gel electrophoresis of whole cells. Thus, the initial separation of the different soluble subpopulations of this protein appears to be due to distinct molecular complexes, possibly related to differential glycosylation, and is not the result of the genetically-encoded amino acid polymorphism.

Purification and characterization of an alpha1,2,-L-fucosyltransferase, which modifies the cytosolic protein FP21,from the cytosol of Dictyostelium.

A novel fucosyltransferase (cFTase) activity has been enriched over 10(6)-fold from the cytosolic compartment of Dictyostelium based on transfer of [3H]fucose from GDP-[3H]fucose to Galbeta1,3 GlcNAc beta-paranitrophenyl (paranitrophenyl-lacto-N-bioside or pNP-LNB). The activity behaved as a single component during purification over DEAE-, phenyl-, Reactive Blue-4-, GDP-adipate-, GDP-hexanolamine-, and Superdex gel filtration resins. The purified activity possessed an apparent Mr of 95 X 10(3), was Mg2+-dependent with a neutral pH optimum, and exhibited a Km for GDP-fucose of 0.34 microM, a Km for pNP-LNB of 0.6 mM, and a Vmax for pN-P-LNB of 620 nmol/min/mg protein. SDS-polyacrylamide gel electrophoresis analysis of the Superdex elution profile identified a polypeptide with an apparent Mr of 85 X 10(3), which coeluted with the cFTase activity and could be specifically photolabeled with the donor substrate inhibitor GDP-hexanolaminyl-azido-125I-salicylate. Based on substrate analogue studies, exoglycosidase digestions, and co-chromatography with fucosylated standards, the product of the reaction with pNP-LNB was Fucalpha1, 2Galbeta1,3GIcNAcbeta-pNP. The cFTase preferred substrates with a Galbeta1,3linkage, and thus its acceptor substrate specificity resembles the human Secretor-type alpha1,2- FTase. Afucosyl isoforms of the FP21 glycoprotein, GP21-I and GP21-II, were purified from the cytosol of a Dictyostelium mutant and found to be substrates for the cFTase, which exhibited an apparent K(m) of 0.21 microM and an apparent V(max) of 460 nmol/min/mg protein toward GP21-II. The highly purified cFTase was inhibited by the reaction products Fucalpha1,2Galbeta1,3GlcNAcbeta-pNP and FP21-II. FP21-I and recombinant FP21 were not inhibitory, suggesting that acceptor substrate specificity is based primarily on carbohydrate recognition. A cytosolic location for this step of FP21 glycosylation is implied by the isolation of the cFTase from the cytosolic fraction, its high affinity for its substrates, and its failure to be detected in crude membrane preparations.

Characterization of FP21, a cytosolic glycoprotein from Dictyostelium.

FP21 is a glycoprotein which, when tracked by radioactivity in its fucosyl moiety, was previously detected in the cytosol of Dictyostelium cells after cell fractionation. This compartmentalization is confirmed by SDS-polyacrylamide gel electrophoresis/Western blotting of cell fractions using three different antibodies. Although a substantial fraction of FP21 is also detected in the particulate fraction using these new antibodies, particulate FP21 is released by disrupting protein-protein interactions, but not membrane disruption. Since purified FP21 is susceptible to aggregation, and purified nuclei do not contain FP21, particulate FP21 is also part of the cytosol. Additional compositional and structural information provides strong evidence that FP21 does not at any time traverse the rough endoplasmic reticulum. First, cDNAs spanning the entire coding region of the FP21 gene predict no hydrophobic motifs expected to promote membrane insertion, but do predict an NH2-terminal coiled coil domain which could explain aggregation. Second, monosaccharide composition analysis of the predominant glycoform of FP21 yields 2 mol of galactose, 1 mol of xylose, and 1 mol of fucose/mol of polypeptide; FP21 from a fucosylation-defective mutant contains 1 additional mol of xylose in place of fucose. Thus the N-glycosylation sequon present in FP21 is not utilized by oligosaccharyl transferase, which resides in the rough endoplasmic reticulum. These findings indicate that nascent FP21 remains in the cytosol after synthesis and is therefore glycosylated by unusual cytosolic xylosyl-, galactosyl-, and fucosyltransferases.