Ann Progulske-Fox

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 , …

Characterization and Pathogenic Significance of Vibrio vulnificus Antigens Preferentially Expressed in Septicemic Patients

ABSTRACT Many important virulence genes of pathogenic bacteria are preferentially expressed in vivo. We used the recently developed in vivo-induced antigen technology (IVIAT) to identify Vibrio vulnificus genes induced in vivo. An expression library of V. vulnificus was screened by colony blot analysis by using pooled convalescent-phase serum that had been thoroughly adsorbed with in vitro-expressed V. vulnificus whole cells and lysates. Twelve clones were selected, and the sequences of the insert DNAs were analyzed. The DNA sequences showed homologies with genes encoding proteins of diverse functions: these functions included chemotaxis (a methyl-accepting chemotaxis protein), signaling (a GGDEF-containing protein and a putative serine/threonine kinase), biosynthesis and metabolism (PyrH, PurH, and IlvC), secretion (TatB and plasmid Achromobacter secretion [PAS] factor), transcriptional activation (IlvY and HlyU), and the activity of a putative lipoprotein (YaeC). In addition, one identified open reading frame encoded a hypothetical protein. Isogenic mutants of the 12 in vivo-expressed (ive) genes were constructed and tested for cytotoxicity. Cytotoxic activity of the mutant strains, as measured by lactate dehydrogenase release from HeLa cells, was nearly abolished in pyrH, purH, and hlyU mutants. The intraperitoneal 50% lethal dose in mice increased by ca. 10- to 50-fold in these three mutants. PyrH and PurH seem to be essential for in vivo growth. HlyU appears to be one of the master regulators of in vivo virulence expression. The successful identification of ive genes responsible for the in vivo bacterial virulence, as done in the present study, demonstrates the usefulness of IVIAT for the detection of new virulence genes.

Porphyromonas gingivalis Traffics to Autophagosomes in Human Coronary Artery Endothelial Cells

ABSTRACT Porphyromonas gingivalis is a periodontal pathogen that also localizes to atherosclerotic plaques. Our previous studies demonstrated that P. gingivalis is capable of invading endothelial cells and that intracellular bacteria are contained in vacuoles that resemble autophagosomes. In this study, we have examined the trafficking of P. gingivalis 381 to the autophagic pathway. P. gingivalis 381 internalized by human coronary artery endothelial (HCAE) cells is located within vacuoles morphologically identical to autophagosomes. The progression ofP. gingivalis 381 through intracellular vacuoles was analyzed by immunofluorescence microscopy. Vacuoles containingP. gingivalis colocalize with Rab5 and HsGsa7p early after internalization. At later times, P. gingivaliscolocalizes with BiP and then progresses to a vacuole that contains BiP and lysosomal glycoprotein 120. Late endosomal markers and the lysosomal cathepsin L do not colocalize with P. gingivalis 381. The intracellular survival of P. gingivalis 381 decreases over 8 h in HCAE cells pretreated with the autophagy inhibitors 3-methyladenine and wortmannin. In addition, the vacuole containing P. gingivalis 381 lacks BiP but contains cathepsin L in the presence of wortmannin. These results suggest that P. gingivalis 381 evades the endocytic pathway to lysosomes and instead traffics to the autophagosome.

Bacterial interactions with the autophagic pathway.

Bacteria have evolved a variety of mechanisms to invade eukaryotic cells and survive intracellularly. Once inside, bacterial pathogens often modulate their phagosome to establish an intracellular niche for survival and replication. A subset of intracellular pathogens, including Brucella abortus, Legionella pneumophila and Porphyromonas gingivalis, are diverted from the endosomal pathway to the auto‐phagic pathway. Once within the autophagosome, each in some way presumably modifies this compartment to establish an environment necessary for its survival. Transit into autophagosomes represents an avenue by which to escape host defences. In this review, we examine the biochemical and morphological evidence for the survival of some bacterial pathogens by replicating within an autophagosome‐like compartment.

Use of in vivo-induced antigen technology (IVIAT) to identify genes uniquely expressed during human infection with Vibrio cholerae

In vivo-induced antigen technology is a method to identify proteins expressed by pathogenic bacteria during human infection. Sera from 10 patients convalescing from cholera infection in Bangladesh were pooled, adsorbed against in vitro-grown El Tor Vibrio cholerae O1, and used to probe a genomic expression library in Escherichia coli constructed from El Tor V. cholerae O1 strain N16961. We identified 38 positive clones in the screen, encoding pili (PilA and TcpA), cell membrane proteins (PilQ, MshO, MshP, and CapK), methyl-accepting chemotaxis proteins, chemotaxis and motility proteins (CheA and CheR), a quorum-sensing protein (LuxP), and four hypothetical proteins. Analysis of immune responses to purified PilA and TcpA in individual patients demonstrated that the majority seroconverted to these proteins, confirming results with pooled sera. These results suggest that PilA and its outer membrane secretin, PilQ, are expressed during human infection and may be involved in colonization of the gastrointestinal tract. These results also demonstrate substantial immune responses to TcpA in patients infected with El Tor V. cholerae O1. In vivo-induced antigen technology provides a simple method for identifying microbial proteins expressed during human infection, but not during in vitro growth.

Sequence analysis of the cloned streptococcal cell surface antigen I/II

The gene spa P (formerly designated as spa P1) encoding the M r 185,000 surface antigen (I/II) of Streptococcus mutons, serotype c (NG5), has been sequenced. The gene (4683 bp) encodes a protein of 1561 amino acid residues including putative signal peptide (residues 1–38) and transmembrane (residues 1537–1556) sequences. The N‐terminal region (60–550) has alanine‐rich repeats and is predicted to be α‐helical. However, the C‐ternunal region (800–1540) is proline‐rich and favours an extended structure. Except for a short central variable region the sequences appear to be highly conserved for S. mutans serotype c. N‐Tenninal sequencing of separated antigen I and antigen II polypeptides suggests that the former represents the N‐terminal and the latter the C‐terminal portions of the intact antigen.

IVIAT: a novel method to identify microbial genes expressed specifically during human infections

In vivo induced antigen technology (IVIAT) is a novel technology that can quickly and easily identify in vivo induced genes in human infections, without the use of animal models. This technology is expected to facilitate the discovery of new targets for vaccines, antimicrobials and diagnostic strategies in a wide range of microbial pathogens.

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 TNFα‐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.

Human β-defensin 3 binds to hemagglutinin B (rHagB), a non-fimbrial adhesin from Porphyromonas gingivalis, and attenuates a pro-inflammatory cytokine response

Regulatory mechanisms in mucosal secretions and tissues recognize antigens and attenuate pro‐inflammatory cytokine responses. Here, we asked whether human β‐defensin 3 (HBD3) serves as an upstream suppressor of cytokine signaling that binds and attenuates pro‐inflammatory cytokine responses to recombinant hemagglutinin B (rHagB), a non‐fimbrial adhesin from Porphyromonas gingivalis strain 381. We found that HBD3 binds to immobilized rHagB and produces a significantly higher resonance unit signal in surface plasmon resonance spectroscopic analysis, than HBD2 and HBD1 that are used as control defensins. Furthermore, we found that HBD3 significantly attenuates (P<0.05) the interleukin (IL)‐6, IL‐10, granulocyte macrophage colony stimulating factor (GM‐CSF) and tumor‐necrosis factor‐α (TNF‐α) responses induced by rHagB in human myeloid dendritic cell culture supernatants and the extracellular signal‐regulated kinases (ERK 1/2) response in human myeloid dendritic cell lysates. Thus, HBD3 binds rHagB and this interaction may be an important initial step to attenuate a pro‐inflammatory cytokine response and an ERK 1/2 response.

Dentinal Tubule Disinfection Using Three Calcium Hydroxide Formulations

The objective of this study was to determine the antibacterial efficacy of three calcium hydroxide (CH) formulations using an in vitro model of Enterococcus faecalis dentinal tubule infection. CH mixed with water (CH), CH mixed with iodine-potassium iodide (CH+IKI), and CH mixed with iodoform and silicone oil (Metapex) were tested. Human cylindrical dentin specimens infected with E. faecalis were filled with disinfectants and incubated for 1 week. Dentin powder samples collected with ISO 018 burs showed a statistically significant reduction in E. faecalis for all three experimental groups in comparison with untreated control specimens. Statistically significant differences were also found between the three experimental groups. Metapex was the most effective dentinal tubule disinfectant, followed by CH+IKI and then CH. Similar results were observed at greater dentin tubule depths (ISO 021 burs) with the exception that intracanal treatment with CH resulted in significantly higher numbers of E. faecalis in comparison with untreated control specimens.