Emil Kozarov

Human Atherosclerotic Plaque Contains Viable Invasive Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis

To the Editor: Because epidemiological evidence supports an association between cardiovascular and periodontal disease, we assessed whether periodontal pathogens were present in atherosclerotic lesions. To detect invasive bacteria, the natural tropism of the bacteria toward human tissues was exploited. Further, bacterial presence was demonstrated using quantitative polymerase chain reaction (Q-PCR). This confirms the presence of periodontal pathogens in atherosclerotic lesions, whereby the bacteria could contribute to the vascular pathology either directly through their cytotoxicity or indirectly by inducing or exacerbating inflammation. Cardiovascular disease (CVD) is the leading cause of death in the in the United States.1 According to the American Heart Association’s statistics from 2003, there were no previous symptoms in 50% of men and 63% of women who died suddenly from CHD. In a 10-year follow-up study, ≈25% of coronary deaths in males and 15% in females occurred in persons in the lowest two quintiles of the multivariate Framingham Heart Study risk scores.2 This and other data have led to an emerging paradigm shift from coronary heart disease having a purely hereditary/nutritional causation to possibly having an infectious component.3 Many epidemiological studies strongly suggest that periodontitis may be a risk factor for coronary heart disease (CHD).4 Serologically, edentulousness and serum IgG-antibodies to Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis in 1163 men were recently shown to be associated with CHD.5 In a larger prospective study of 6950 subjects, the same authors provide serological evidence that an infection caused by major periodontal pathogens is associated with future stroke.6 Previous studies have identified 16S rRNA of oral microbial pathogens, including P gingivalis and A actinomycetemcomitans , …

Hemagglutinin B is involved in the adherence of Porphyromonas gingivalis to human coronary artery endothelial cells.

ABSTRACT Porphyromonas gingivalis is a periodontopathogen that may play a role in cardiovascular diseases. Hemagglutinins may function as adhesins and are required for virulence of several bacterial pathogens. The aim of this study was to determine the role of hemagglutinin B (HagB) in adherence of P. gingivalis to human coronary artery endothelial (HCAE) cells. P. gingivalis strain 381, a P. gingivalis 381 HagB mutant, Escherichia coli JM109 expressing HagB (E. coli-HagB), and E. coli JM109 containing pUC9 (E. coli-pUC9) were tested for their ability to attach to HCAE cells. Inhibition assays were performed to determine the ability of purified recombinant HagB (rHagB) as well as antibodies to HagB, including the polyclonal antibody (PAb) A7985 and the monoclonal antibody (MAb) HL1858, to inhibit the attachment of P. gingivalis to HCAE cells. As expected, when the attachment of P. gingivalis and the HagB mutant were compared, no statistical significance was observed between the two groups (P = 0.331), likely due to the expression of the hagB homolog hagC. However, E. coli-HagB adhered significantly better to HCAE cells than did E. coli-pUC9, the control strain. In a competition assay, the presence of purified rHagB decreased bacterial adhesion of P. gingivalis or E. coli-HagB to HCAE cells. The presence of PAb A7985 or MAb HL1858 also significantly decreased attachment of P. gingivalis and E. coli-HagB to host cells. These results indicate that HagB is involved in the adherence of P. gingivalis to human primary endothelial cells.

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

Abstract.  Rafferty B, Jönsson D, Kalachikov S, Demmer RT, Nowygrod R, Elkind MSV, Bush Jr H, Kozarov E. (Columbia University Medical Center, New York, NY; and Weill Cornell Medical College, New York, NY; USA) Impact of monocytic cells on recovery of uncultivable bacteria from atherosclerotic lesions. J Intern Med 2011; 270: 273–280.

Purification and Characterization of an 1,2-L-Fucosyltransferase, Which Modifies the Cytosolic Protein FP21, from the Cytosol of Dictyostelium

A novel fucosyltransferase (cFTase) activity has been enriched over 106-fold from the cytosolic compartment of Dictyostelium based on transfer of [3H]fucose from GDP-[3H]fucose to Galβ1,3GlcNAcβ-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 M of 95 × 103, was Mg-dependent with a neutral pH optimum, and exhibited a K for GDP-fucose of 0.34 μM, a K for pNP-LNB of 0.6 mM, and a V for pNP-LNB of 620 nmol/min/mg protein. SDS-polyacrylamide gel electrophoresis analysis of the Superdex elution profile identified a polypeptide with an apparent M of 85 × 103, which coeluted with the cFTase activity and could be specifically photolabeled with the donor substrate inhibitor GDP-hexanolaminyl-azido-I-salicylate. Based on substate analogue studies, exoglycosidase digestions, and co-chromatography with fucosylated standards, the product of the reaction with pNP-LNB was Fucα1, 2Galβ1,3GlcNAcβ-pNP. The cFTase preferred substrates with a Galβ1,3 linkage, and thus its acceptor substrate specificity resembles the human Secretor-type α1,2-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 of 0.21 μM and an apparent V of 460 nmol/min/mg protein toward GP21-II. The highly purified cFTase was inhibited by the reaction products Fucα1,2Galβ1,3GlcNAcβ-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.

Expression and Immunogenicity of Hemagglutinin A from Porphyromonas gingivalis in an Avirulent Salmonella enterica Serovar Typhimurium Vaccine Strain

ABSTRACT 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 recombinantS. 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. ThehagA 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. entericaserovar Typhimurium vaccine strain χ4072. 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.

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.

View of statins as antimicrobials in cardiovascular risk modification

Atherosclerosis is a complex arterial pathological development underlying heart attack and stroke and a leading cause of death in developed and now also in developing countries. The primary processes that lead to the inflammatory lipid-laden proliferative lesion, obstructing the blood flow, and referred to as atherosclerotic plaque are dyslipidaemia and inflammation. Here, we will review one of the most efficient classes of drugs indicated for management of cardiovascular disease (CVD), statins. We will assess their pleiotropic effects that emerged from CVD applications, focusing this review specifically on plausible antimicrobial activity. Only recently gaining strength, the recognition of possible antibacterial activity may extend the statin applicability for vascular as well as to other critical inflammatory conditions.