Myriam Bélanger

Expression of Multiple Outer Membrane Protein Sequence Variants from a Single Genomic Locus of Anaplasma phagocytophilum

ABSTRACT Anaplasma phagocytophilum is the causative agent of an emerging tick-borne zoonosis in the United States and Europe. The organism causes a febrile illness accompanied by other nonspecific symptoms and can be fatal, especially if treatment is delayed. Persistence of A. phagocytophilum within mammalian reservoir hosts is important for ensuring continued disease transmission. In the related organism Anaplasma marginale, persistence is associated with antigenic variation of the immunoprotective outer membrane protein MSP2. Extensive diversity of MSP2 is achieved by combinatorial gene conversion of a genomic expression site by truncated pseudogenes. The major outer membrane protein of A. phagocytophilum, MSP2(P44), is homologous to MSP2 of A. marginale, has a similar organization of conserved and variable regions, and is also encoded by a multigene family containing some truncated gene copies. This suggests that the two organisms could use similar mechanisms to generate diversity in outer membrane proteins from their small genomes. We define here a genomic expression site for MSP2(P44) in A. phagocytophilum. As in A. marginale, the msp2(p44) gene in this expression site is polymorphic in all populations of organisms we have examined, whether organisms are obtained from in vitro culture in human HL-60 cells, from culture in the tick cell line ISE6, or from infected human blood. Changes in culture conditions were found to favor the growth and predominance of certain msp2(p44) variants. Insertions, deletions, and substitutions in the region of the genomic expression site encoding the central hypervariable region matched sequence polymorphisms in msp2(p44) mRNA. These data suggest that, similarly to A. marginale, A. phagocytophilum uses combinatorial mechanisms to generate a large array of outer membrane protein variants. Such gene polymorphism has profound implications for the design of vaccines, diagnostic tests, and therapy.

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.

Autophagy: A Highway for Porphyromonas gingivalis in Endothelial Cells

P. gingivalis, an important periodontal pathogen associated with adult periodontitis and a likely contributing factor to atherosclerosis and cardiovascular disease, traffics in endothelial cells via the autophagic pathway. Initially, P. gingivalis rapidly adheres to the host cell surface followed by internalization via lipid rafts and incorporation of the bacterium into early phagosomes. P. gingivalis activates cellular autophagy to provide a replicative niche while suppressing apoptosis. The replicating vacuole contains host proteins delivered by autophagy that are used by this asaccharolytic pathogen to survive and replicate within the host cell. When autophagy is suppressed by 3-methyladenine or wortmannin, internalized P. gingivalis transits to the phagolysosome where it is destroyed and degraded. Therefore, the survival of P. gingivalis depends upon the activation of autophagy and survival of the endothelial host cell, but the mechanism by which P. gingivalis accomplishes this remains unclear.

Invasion of human coronary artery endothelial cells by Streptococcus mutans OMZ175

INTRODUCTION Dissemination of oral bacteria into the bloodstream has been associated with eating, oral hygiene, and dental procedures; including tooth extraction, endodontic treatment, and periodontal surgery. Recently, studies identified Streptococcus mutans, the primary etiological agent of dental caries, as the most prevalent bacterial species found in clinical samples from patients who underwent heart valve and atheromatous plaque surgery. METHODS By using antibiotic protection assays, we tested the capacity of 14 strains of S. mutans to invade primary human coronary artery endothelial cells (HCAEC). RESULTS Serotype e strain B14 and serotype f strain OMZ175 of S. mutans were able to efficiently invade HCAEC. Among the tested strains, serotype f S. mutans OMZ175 was the most invasive, whereas strains of serotype c S. mutans, the most prevalent serotype in dental plaque, were not invasive. Based on its high invasion rate, we further investigated the invasive properties of serotype f OMZ175. Using transmission electron microscopy and antibiotic protection assays we demonstrate that S. mutans OMZ175 is capable of attaching to the HCAEC surface, entering the cells and surviving in HCAEC for at least 29 h. DISCUSSION Our findings highlight a potential role for S. mutans in the pathogenesis of certain cardiovascular diseases.

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.

Comparison of Serological Detection Methods for Diagnosis of Ehrlichia canis Infections in Dogs

ABSTRACT We determined the value of four serological assays for the diagnosis of canine monocytic ehrlichiosis by comparing them to the indirect fluorescent-antibody assay “gold standard.” The specificity of Dip-S-Ticks was significantly lower than that of all of the other tests evaluated. The sensitivity of Dip-S-Ticks was significantly higher than that of Snap3Dx or the Snap Canine Combo. The sensitivity of the rMAP2 enzyme-linked immunosorbent assay (ELISA) was significantly higher than that of the Snap Canine Combo. The accuracy levels of the rMAP2 ELISA, Snap3Dx, Dip-S-Ticks, and Snap Canine Combo were 97.0, 89.8, 85.1, and 82.9%, respectively.

Porphyromonas gingivalis htrA is involved in cellular invasion and in vivo survival

HtrA is a heat-stress protein that functions both as a chaperone and as a serine protease. HtrA has been shown in several organisms to be involved in responses to stressful environmental conditions and involvement of HtrA in virulence has been reported in pathogenic species. A Porphyromonas gingivalis htrA mutant demonstrated no significant difference to the W83 parent strain when subjected to high temperature and pH values from 3 to 11. However, the htrA mutant showed increased sensitivity to H(2)O(2). Cell invasion assays indicated that the total interaction (adherence) with KB cells, human coronary artery endothelial cells and gingival epithelial cells (GEC) was the same for both the wild-type and the htrA mutant. However, the htrA mutant showed increased invasion in KB cells and GEC. Microarray experiments indicated that a total of 253 genes were differentially regulated in the htrA mutant, including a group of stress-related genes, which might be responsible for the observed decreased resistance to H(2)O(2). In animal experiments, a competition assay showed that the htrA mutant did not survive as well as the wild-type. In another in vivo assay, fewer mice infected with the htrA mutant died than mice infected with W83, suggesting that the htrA gene is virulence-related. These data indicate that the htrA gene in P. gingivalis does not relate to stress conditions such as high temperature and pH, but rather to H(2)O(2) stress. The htrA gene also appears to be important for virulence and survival in in vivo animal models.

Genetic Manipulation of Porphyromonas gingivalis

Porphyromonas gingivalis, an oral anaerobic bacterium, is an important etiological agent of periodontal disease and may contribute to cardiovascular disease, preterm birth, and diabetes as well. Therefore, genetic studies are of crucial importance in investigating molecular mechanisms of P. gingivalis virulence. Although molecular genetic tools have been available for many bacterial species for some time, genetic manipulations of Porphyromonas species were not developed until more recently and remain limited. In this unit, current molecular genetic approaches for mutant construction in P. gingivalis using the suicide vector pPR‐UF1 and the transposon Tn4351 are described, as are protocols for performing electroporation and conjugation. Furthermore, a technique to restore the wild‐type phenotype of the mutant by complementation using vector pT‐COW is provided. Finally, a description of a noninvasive reporter system allowing the study of gene expression and regulation in P. gingivalis completes this unit.

Porphyromonas gingivalis Strain Specific Interactions with Human Coronary Artery Endothelial Cells: A Comparative Study

Both epidemiologic and experimental findings suggest that infection with Porphyromonas gingivalis exacerbates progression of atherosclerosis. As P. gingivalis exhibits significant strain variation, it is reasonable that different strains possess different capabilities and/or mechanisms by which they promote atherosclerosis. Using P. gingivalis strains that have been previously evaluated in the ApoE null atherosclerosis model, we assessed the ability of W83, A7436, 381, and 33277 to adhere, invade, and persist in human coronary artery endothelial (HCAE) cells. W83 and 381 displayed an equivalent ability to adhere to HCAE cells, which was significantly greater than both A7436 and 33277 (P<0.01). W83, 381, and 33277 were more invasive than A7436 (P<0.0001). However, only W83 and A7436 were able to remain viable up to 48 hours in HCAE cell cultures, whereas 381 was cleared by 48 hours and 33277 was cleared by 24 hours. These differences in persistence were in part due to strain specific differences in intracellular trafficking. Both W83 and 381 trafficked through the autophagic pathway, but not A7436 or 33277. Internalized 381 was the only strain that was dependent upon the autophagic pathway for its survival. Finally, we assessed the efficacy of these strains to activate HCAE cells as defined by production of IL-6, IL-8, IL-12p40, MCP-1, RANTES, TNF-α, and soluble adhesion molecules (sICAM-1, sVCAM-1, and sE-selectin). Only moderate inflammation was observed in cells infected with either W83 or A7436, whereas cells infected with 381 exhibited the most profound inflammation, followed by cells infected with 33277. These results demonstrate that virulence mechanisms among different P. gingivalis strains are varied and that pathogenic mechanisms identified for one strain are not necessarily applicable to other strains.