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1.  Oral bacteria induce a differential activation of human immunodeficiency virus-1 promoter in T cells, macrophages and dendritic cells 
Oral microbiology and immunology  2009;24(5):401-407.
The human immunodeficiency virus (HIV) can integrate into T cells, macrophages and dendritic cells resulting in a latent infection. Reports have also demonstrated that various microbial and host cell factors can trigger HIV reactivation leading to HIV recrudescence, potentially undermining highly active antiretroviral therapies.
This study evaluated the capacity of oral bacteria associated with chronic periodontal infections to stimulate HIV promoter activation in various cell models of HIV latency.
T cells (1G5) challenged with oral bacteria demonstrated a dose–response of HIV promoter activation with a subset of the bacteria, as well as kinetics that were generally similar irrespective of the stimuli. Direct bacterial challenge of the T cells resulted in increased activation of approximately 1.5- to 7-fold over controls. Challenge of macrophages (BF24) indicated different kinetics for individual bacteria and resulted in consistent increases in promoter activation of five fold to six fold over basal levels for all bacteria except Streptococcus mutans. Dendritic cells showed increases in HIV reactivation of 7- to 34-fold specific for individual species of bacteria.
These results suggested that oral bacteria have the capability to reactivate HIV from latently infected cells, showing a relationship of mature dendritic cells > immature dendritic cells > macrophages ≥ T cells. Expression of various pattern recognition receptors on these various cell types may provide insight into the primary receptors/signaling pathways used for reactivation by the bacteria.
PMCID: PMC4131714  PMID: 19702954
dendritic cells; human immunodeficiency virus latency; human immunodeficiency virus type 1 promoter; macrophages; oral bacteria; T cells
2.  Evaluating two methods for fingerprinting genomes of Actinobacillus actinomycetemcomitans 
Oral microbiology and immunology  1993;8(6):337-343.
The arbitrary primer polymerase chain reaction (AP-PCR) and Southern blot restriction fragment length polymorphism (RFLP) were used to genotype the periodontal pathogen A. actinomycetemcomitans. Total genomic DNA from 73 strains was extracted by conventional methods. Three random-sequence 10-base oligonucleotide primers were chosen for AP-PCR. The amplified DNA products were separated electrophoretically in a 1% agarose gel containing ethidium bromide and the banding patterns were compared among different strains. For RFLP analysis, DNA was digested with EcoRI, separated on a 0.8% agarose gel and transferred to a nylon membrane. The membrane was probed with a previously characterized 5.2 kilobases (kb) DNA fragment cloned from A. actinomycetemcomitans strain Y4. The probe was labeled with digoxigenin, and hybridized fragments were detected with anti-digoxigenin antibody. AP-PCR produced 4–10 DNA bands in the 0.5–5 kb regions and distinguished 9, 13 or 17 genotypes, depending on the specific primer used. Southern blot RFLP analysis revealed 12 hybridization patterns consisting of 1 or 2 DNA fragments (2–23 kb). The addition of the Southern blot analysis to the AP-PCR analysis gave rise to a total of 30 DNA profiles among the 73 A. actinomycetemcomitans study strains. The results indicate that both AP-PCR and Southern blot analysis are useful in clonal analysis of A. actinomycetemcomitans.
PMCID: PMC3534794  PMID: 7908736
Actinobacillus actinomycetemcomitans; DNA fingerprinting; polymerase chain reaction; restriction fragment length polymorphism; DNA probe; Southern blot
3.  Insertional inactivation of binding determinants of Streptococcus crista CC5A using Tn916 
Oral microbiology and immunology  1995;10(4):220-226.
Intermicrobial binding plays an important role in the ecology of the oral cavity because it represents one mechanism by which specific bacteria colonize dental plaque. The formation of “corncobs”, a morphologically distinct microbial unit composed of Streptococcus crista and Fusobacterium nucleatum, is a highly specific binding interaction that depends on the presence of polar tufts of fimbriae on the streptococci. We have used a genetic approach to examine the role of streptococcal cell surface components involved in the binding of S. crista to F. nucleatum. Such binding may be an important component of corncob formation. A method for the genetic transformation of S. crista was used to transfer the broad host range transposon, Tn916, into the bacteria. Cells were grown to early log phase in brain heart infusion broth containing 10% fetal calf serum. The competent cells were mixed with purified DNA from pDL916, a plasmid construct consisting of Tn916 and the streptococcal/Escherichia coli shuttle vector pDL278. Over 300 transformants were screened for a reduction in binding to F. nucleatum. Five of the transformants showed a change in binding ranging from 59% to 29% of the positive control values. Southern blots revealed that the binding-deficient transformants contained the Tn916 element integrated into one of 4 different sites in the chromosome. The transposon, integrated into 4 different sites, appeared to be stable in the absence of selective pressure. Based on these findings, it appears that some strains of S. crista are naturally competent and that insertional inactivation methods can be used to facilitate the study of binding receptors in this group of oral streptococci.
PMCID: PMC3534806  PMID: 8602334
Streptococcus crista; Fusobacterium nucleatum; corncob; transposon; transformation; dental plaque
4.  Epithelial cell invasion by Actinobacillus actinomycetemcomitans strains from restriction fragment-length polymorphism groups associated with juvenile periodontitis or carrier status 
Oral microbiology and immunology  1998;13(6):341-347.
The epithelial cell invasiveness of Actinobacillus actinomycetemcomitans strains of different restriction fragment-length polymorphism (RFLP) groups associated with disease conversion and asymptomatic carrier status in localized juvenile periodontitis was examined. Twenty clinical isolates were studied for their ability to invade KB monolayers, using the quantitative gentamicin killing assay. Five isolates were found to be invasive; five were not invasive; and the other 10 did not invade better than an invasion negative control Haemophilus aphrophilus strain ATCC 19415. Using probe-specific DNA fingerprinting, 11 strains were assigned to RFLP group II (disease–associated); 4 to RFLP type XIII (carrier status-associated); and the others to groups III, IV, V and VII. Eight isolates, all RFLP group II, were leukotoxin producers as determined by PCR amplification of the lkt promoter region. No correlation was found between invasiveness and RFLP group. Leukotoxin production was more associated with noninvasive than invasive strains.
PMCID: PMC3528405  PMID: 9872109
Actinobacillus actinomycetemcomitans; invasion; KB epithelial cell; restriction fragment-length polymorphism; leukotoxin
5.  Association of a novel high molecular weight, serine-rich protein (SrpA) with fibril-mediated adhesion of the oral biofilm bacterium Streptococcus cristatus 
Oral microbiology and immunology  2005;20(3):131-140.
The surface of the oral plaque bacterium Streptococcus cristatus is decorated with a lateral tuft of fibrils. The fibrillar tuft functions in the adhesion of S. cristatus to heterologous bacterial species in the plaque biofilm. The tuft typically consists of a densely packed fringe of shorter fibrils 238 ± 19 nm long with longer, less abundant fibrils 403 ± 66 nm long projecting through the fringe of short fibrils. The two types of fibrils in the tufts of S. cristatus have been refractory to biochemical separation, complicating their characterization. A hexadecane partition assay was used to enrich for subpopulations of S. cristatus CR311 (type strain NCTC 12479) having distinct fibrillar morphotypes. Negative staining in the TEM revealed that cells of a hydrophobic subpopulation of S. cristatus (CR311var1) carried only the long fibrils (395 ± 32 nm). A hydrophilic subpopulation of S. cristatus (CR311var3) consisted of mixed morphotypes having no fibrils or remnant short fibrils (223 ± 49 nm). No long fibrils were observed on any cells in the CR311var3 subpopulation. The CR311var3 morphotype, unlike the wild-type strain and CR311var1, was not able to form corncobs with either Corynebacterium matruchotii or Fusobacterium nucleatum. Variant CR311var3 did not express the novel gene srpA, which encodes a high molecular weight (321,882 Da) serine-rich protein, SrpA. The SrpA protein contains two extensive repeat motifs of 17 and 71 amino acids and a gram-positive cell wall anchor consensus sequence (LPNTG). The unusual properties of SrpA most closely resemble those of Fap1, the fimbrial-associated adhesin protein of Streptococcus parasanguis. The association of long fibrils, high surface hydrophobicity, ability to form corncob formations, and expression of the srpA gene suggest that SrpA is a long fibril protein in S. cristatus.
PMCID: PMC3523328  PMID: 15836513
corncobs; fibrils; SrpA; Streptococcus cristatus; tufts
6.  Intra-oral colonization of macaque monkeys by Actinobacillus actinomycetemcomitans 
Actinobacillus actinomycetemcomitans was acquired by captive Macaca fascicularis 3 to 6 months after birth, and all monkeys aged over 6 months harbored detectable levels. This microorganism was most frequently isolated from the gingival plaque of the incisor (and other) teeth compared with other oral sites. Strains were leukotoxic by bioassay and Western blot analysis. Antibodies in macaque serum contained neutralized the leukotoxin of a human A. actinomycetemcomitans strain. High titres of maternal neutralizing anti-leukotoxin antibodies were detected in neonates; the titre then fell rapidly so that by 6 months the antibody titer was zero. Antileukotoxin antibody production was detected after 6 months of age, rapidly reaching a high level within 2 years after birth. The presence of leukotoxic strains of A. actinomycetemcomitans in the gingival region did not appear to be correlated with an increase in susceptibility to periodontal disease.
PMCID: PMC3516870  PMID: 2628866
non-human primates; Actinobacillus actinomycetemcomitans; leukotoxin; colonization; maternal antibody
7.  Scavenger receptor A is expressed by macrophages in response to Porphyromonas gingivalis, and participates in TNF-α expression 
Oral microbiology and immunology  2009;24(6):456-463.
Porphyromonas gingivalis is a periodontopathic bacterium closely associated with generalized aggressive periodontal disease. Pattern recognition receptors (PRRs) participate in host response to this organism. It is likely that PRRs not previously recognized as part of the host response to P. gingivalis also participate in host response to this organism.
Methods and Results
Employing qRT-PCR, we observed increased msr1 gene expression at 2, 6, and 24h of culture with P. gingivalis strain 381. Flow cytometry revealed increased surface expression of SR-A protein by the 24h time point. Macrophages cultured with an attachment impaired P. gingivalis fimA- mutant (DPG3) expressed intermediate levels of SR-A expression. Heat-killed P. gingivalis stimulated SR-A expression similar to live bacteria, and purified P. gingivalis capsular polysaccharide stimulated macrophage SR-A expression, indicating that live whole organisms are not necessary for SR-A protein expression in macrophage response. As SR-A is known to play a role in lipid uptake by macrophages, we tested the ability of low-density lipoprotein (LDL) to influence the SR-A response of macrophages to P. gingivalis, and observed no effect of LDL on P. gingivalis-elicited SR-A expression. Lastly, we observed that SR-A knockout (SR-A−/−) mouse macrophages produced significantly more tumor necrosis factor (TNF)-α than wild type mouse macrophages cultured with P. gingivalis.
These data identify that SR-A is expressed by macrophages in response to P. gingivalis, and support that this molecule plays a role in TNF-α production by macrophages to this organism.
PMCID: PMC2792726  PMID: 19832797
8.  Differential virulence and innate immune interactions of Type I and II fimbrial genotypes of Porphyromonas gingivalis 
Oral microbiology and immunology  2009;24(6):478-484.
The fimA-encoded fimbriae of the periodontal pathogen Porphyromonas gingivalis display genetic diversity. Type I fimbriated P. gingivalis (Pg-I) has been most widely studied at the molecular level, whereas Pg-II is the most frequent isolate from severe periodontitis.
To investigate virulence differences between Types I and II fimbriae, we examined strains 33277 (Pg-I) and OMZ314 (Pg-II), reciprocal swap mutants (i.e., expressing the heterologous fimbrial type), and their respective FimA-deficient derivatives. These organisms were tested in a mouse periodontitis model and in interactions with mouse macrophages, a cell type that plays important roles in chronic infections.
Strain 33277 induced significantly more periodontal bone loss than OMZ314, and substitution of Type II fimbriae with Type I in OMZ314 resulted in a more virulent strain than the parent organism. However, the presence of Type II fimbriae was associated with increased proinflammatory and invasive activities in macrophages.
The inverse relationship between proinflammatory potential and ability to cause experimental periodontitis may suggest that an aggressive phenotype could provoke a host response that would compromise the persistence of the pathogen.
PMCID: PMC2883777  PMID: 19832800
9.  Expression of receptor activator of nuclear factor-κB ligand by B cells in response to oral bacteria 
Oral microbiology and immunology  2009;24(3):190-196.
We investigated receptor activator of nuclear factor-κB ligand (RANKL) expression by B lymphocytes during early and late aspects of the immune response to Aggregatibacter actinomycetemcomitans, a gram-negative, anaerobic bacterium associated with aggressive periodontal disease.
Expression of messenger RNA transcripts (tumor necrosis factor-α, Toll-like receptors 4 and 9, interleukins 4 and 10, and RANKL) involved in early (1-day) and late (10-day) responses in cultured rat splenocytes was examined by reverse transcription–polymerase chain reaction (RT-PCR). The immune cell distribution (T, B, and natural killer cells and macrophages) in cultured rat splenocytes and RANKL expression in B cells were determined by flow cytometric analyses. B-cell capacity for induction of osteoclast differentiation was evaluated by coculture with RAW 264.7 cells followed by a tartrate-resistant acid phosphatase (TRAP) activity assay.
The expression levels of interleukins 4 and 10 in cultured cells were not changed in the presence of A. actinomycetemcomitans until cultured for 3 days, and peaked after 7 days. After culture for 10 days, the percentages of B and T cells, the overall RANKL messenger RNA transcripts, and the percentage of RANKL-expressing immunoglobulin G-positive cells were significantly increased in the presence of A. actinomycetemcomitans. These increases were considerably greater in cells isolated from A. actinomycetemcomitans-immunized animals than from non-immunized animals. RAW 264.7 cells demonstrated significantly increased TRAP activity when cocultured with B cells from A. actinomycetemcomitans-immunized animals. The addition of human osteoprotegerin-Fc to the culture significantly diminished such increases.
This study suggests that B-lymphocyte involvement in the immune response to A. actinomycetemcomitans through upregulation of RANKL expression potentially contribute to bone resorption in periodontal disease.
PMCID: PMC2692067  PMID: 19416447
Aggregatibacter actinomycetemcomitans; B lymphocytes; immune response; periodontal disease; receptor activator of nuclear factor-κB ligand
10.  Comparative whole-genome analysis of Streptococcus mutans isolates within and among individuals of different caries status 
Oral microbiology and immunology  2009;24(3):197-203.
Genotypic analyses of Streptococcus mutans using fingerprinting methods depend on a few genetic loci being different but do not reveal the underlying genome-wide differences between strains. We used comparative genomic hybridization (CGH) with 70-mer oligonucleotide microarrays containing open reading frames (ORFs) from S. mutans UA159 to examine the genetic diversity of 44 isolates from nine children selected from a local study population in Eastern Iowa. Unique strains (clones) within each child initially identified by AP-PCR were confirmed by CGH. There was a wide range of variation in the hybridization patterns of the 1948 ORFs among test isolates examined. Between 87 and 237 ORFs failed to give a positive signal among individual isolates. A total of 323 of the UA159 ORFs were absent from one or more of the test strains. These 323 variable genes seemed to be distributed across the entire UA 159 genome and across all the predicted functional categories. This set of very close geographically and temporally collected S. mutans isolates had a degree of gene content variations as high as a global set of strains examined in a previous publication (Waterhouse et al., 2007). Comparing the frequency of these variable genes, the majority of which have unknown function, among strains of different origins (i.e. different caries status) could help determine their relevance in S. mutans cariogenicity.
PMCID: PMC2744306  PMID: 19416448
Dental Caries; S. mutans; CGH (comparative genomic hybridization)
11.  Invasion of human coronary artery endothelial cells by Streptococcus mutans OMZ175 
Oral microbiology and immunology  2009;24(2):141-145.
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.
By using antibiotic protection assays, we tested the capacity of 14 strains of S. mutans to invade primary human coronary artery endothelial cells (HCAEC).
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.
Our findings highlight a potential role for S. mutans in the pathogenesis of certain cardiovascular diseases.
PMCID: PMC2853226  PMID: 19239641
invasion; serotype f; Strepococcus mutans
12.  Correlations of oral bacterial arginine and urea catabolism with caries experience 
Alkali generation by oral bacteria plays a key role in plaque pH homeostasis and may be a major impediment to the development of dental caries. To determine if the capacity of oral samples to produce ammonia from arginine or urea was related to caries experience, the arginine deiminase system (ADS) and urease activity in saliva and dental plaque samples were measured in 45 adult subjects.
The subjects were divided into three groups according to caries status; 13 caries-free (CF) individuals (decayed, missing, and filled teeth = 0); 21 caries-active (CA) individuals (decayed teeth ≥ 4); and 11 caries-experienced (CE) individuals (decayed teeth = 0; missing and filled teeth > 0). Real-time polymerase chain reaction was used to quantify the proportion of certain acid- or alkali-producing organisms in the samples.
The amount of ammonia generated from the test substrates by plaque samples was generally higher than that produced by salivary samples in all groups. Significantly higher levels of salivary ADS activity and plaque urease activity were observed in CF subjects compared to CA subjects (P = 0.0004 and P = 0.014, respectively). The proportions of Streptococcus mutans from saliva and dental plaque of CA subjects were significantly higher than those from the CF group (P = 0.0153 and P = 0.0009, respectively). In the CA group, there was an inverse relationship between urease activity and the levels of S. mutans (P < 0.0001).
This study supports the theory that increased caries risk is associated with reduced alkali-generating capacity of the bacteria colonizing the oral cavity; providing compelling evidence to further our understanding of oral alkali-generation in health and disease.
PMCID: PMC2742966  PMID: 19239634
alkali; arginine; caries; plaque; saliva; urea
13.  Effects of oral commensal and pathogenic bacteria on human dendritic cells 
Oral microbiology and immunology  2009;24(2):96-103.
The oral cavity harbors a diverse and complex microbial community. Bacteria accumulate on both the hard and soft oral tissues in sessile biofilms and engage the host in an intricate cellular dialog, which normally constrains the bacteria to a state of commensal harmony. Dendritic cells (DCs) are likely to balance tolerance and active immunity to commensal microorganisms as part of chronic inflammatory responses. While the role played by DCs in maintaining intestinal homeostasis has been investigated extensively, relatively little is known about DC responses to oral bacteria.
In this study, we pulsed human monocyte-derived immature DCs (iDCs) with cell wall extracts from pathogenic and commensal gram-positive or gram-negative oral bacteria.
Although all bacterial extracts tested induced iDCs to mature and produce cytokines/chemokines including interleukin-12p40, tumor necrosis factor-α, and monocyte chemoattractant protein-1 (MCP-1), the most important factor for programming DCs by oral bacteria was whether they were gram-positive or gram-negative, not whether they were commensal or pathogenic. In general, gram-negative oral bacteria, except for periodontopathic Porphyromonas gingivalis, stimulated DC maturation and cytokine production at lower concentrations than gram-positive oral bacteria. The threshold of bacteria needed to stimulate chemokine production was 100-fold to 1000-fold lower than that needed to induce cytokines. In addition, very low doses of oral commensal bacteria triggered monocytes to migrate toward DC-derived MCP-1.
Oral commensal and pathogenic bacteria do not differ qualitatively in how they program DCs. DC-derived MCP-1 induced in response to oral commensal bacteria may play a role, at least in part, in the maintenance of oral tissue integrity by attracting monocytes.
PMCID: PMC2733846  PMID: 19239635
commensal bacteria; dendritic cells; MCP-1
14.  Multiple components contribute to ability of saliva to inhibit influenza viruses 
Saliva is a potentially important barrier against respiratory viral infection but its mechanism of action is not well studied.
We tested the antiviral activities of whole saliva, specific salivary gland secretions, and purified salivary proteins against strains of influenza A virus (IAV) in vitro.
Whole saliva or parotid or submandibular/sublingual secretions from healthy donors inhibited IAV based on hemagglutination inhibition and neutralization assays. This differs from human immunodeficiency virus (HIV), for which only submandibular/sublingual secretions are reported to be inhibitory. Among purified salivary proteins, MUC5B, scavenger receptor cysteine-rich glycoprotein 340 (salivary gp-340), histatins, and human neutrophil defensins (HNPs) inhibited IAV at the concentrations present in whole saliva. In contrast, some abundant salivary proteins (acidic proline-rich proteins and amylase) had no activity, nor did several other less abundant salivary proteins with known activity against HIV (e.g. thrombospondin or serum leukocyte protease inhibitor). Whole saliva and MUC5B did not inhibit neuraminidase activity of IAV and viral neutralizing and aggregating activity of MUC5B was potentiated by the neuraminidase inhibitor oseltamivir. Hence, MUC5B inhibits IAV by presenting a sialic acid ligand for the viral hemagglutinin. The mechanism of action of histatins requires further study.
These findings indicate that saliva represents an important initial barrier to IAV infection and underline the complexity of host defense activity of oral secretions. Of interest, antiviral activity of saliva against IAV and HIV differs in terms of specific glandular secretions and proteins that are inhibitory.
PMCID: PMC2848456  PMID: 19121065
histatins; innate immunity; MUC5B
15.  Differential utilization of nuclear factor-κB signaling pathways for gingival epithelial cell responses to oral commensal and pathogenic bacteria 
Oral microbiology and immunology  2008;23(2):119-126.
Human β-defensin-2 (hBD-2) is an antimicrobial peptide, induced by bacterial stimuli and inflammation, that plays a role in mucosal and skin innate immune defense. The nuclear factor-κB (NF-κB) transcription factor family is important in innate and adaptive immune responses to bacteria and proinflammatory cytokines. NF-κB operates via the traditional IKKβ signaling, as well as an alternative pathway utilizing IKKα signaling, which is important in keratinocyte differentiation. Our previous studies showed that pathogenic, but not commensal, bacteria used NF-κB signaling in hBD-2 induction. The objective of this study was to understand which arm of the NF-κB pathway is involved in gingival epithelial cell responses to pathogenic bacteria, including hBD-2 induction.
Cultured oral epithelial cells were transfected with synthetic small interfering RNAs (siRNAs) specific for various steps in each pathway, namely IKKβ, TRAF6 and MyD88 in the canonical, and IKKα and TRAF3 in the alternative pathway, and subsequently stimulated with various oral bacteria.
The hBD-2 induction level was reduced to 21–61% in cells in which the alternative NF-κB pathway was blocked and subsequently stimulated with pathogenic bacteria, while cells in which the canonical pathway was blocked showed reduction to 78–99%. Cells stimulated with commensals showed little change in hBD-2 induction level regardless of the siRNA used. Microarray analysis showed that oral epithelia differentially regulated numerous innate immune markers in response to pathogens and commensals.
Our data suggest a role for the IKKα/TRAF3 pathway in NF-κB activation by pathogenic bacteria, while commensal bacteria do not utilize either NF-κB pathway, for hBD-2 induction.
PMCID: PMC2826319  PMID: 18279179
commensals; gingival epithelium; innate immunity; nuclear factor-κB; pathogens
16.  Distribution, Regulation and Role of the Agmatine Deiminase System in Mutans Streptococci 
The agmatine deiminase system was identified in seven strains of mutans streptococci. Genes encoding the AgDS of Streptococcus rattus FA-1 were sequenced and found to share homology with the agu genes of Streptococcus mutans UA159. Agmatine inhibited bacterial growth, suggesting that the AgDS degrades a deleterious substance into useful compounds.
PMCID: PMC2742967  PMID: 19121075
acid tolerance; biofilm; caries
17.  Proteomic analysis of endodontic infections by liquid chromatography–tandem mass spectrometry 
Oral microbiology and immunology  2009;24(4):347-352.
Endodontic infections are very prevalent and have a polymicrobial etiology characterized by complex interrelationships between endodontic microorganisms and the host defenses. Proteomic analysis of endodontic infections can provide global insights into the invasion, pathogenicity mechanisms, and multifactorial interactions existing between root canal bacteria and the host in the initiation and progression of apical periodontitis. The purpose of this study was to apply proteomic techniques such as liquid chromatography–tandem mass spectrometry (LC–MS/MS) for the identification of proteins of bacterial origin present in endodontic infections.
Endodontic specimens were aseptically obtained from seven patients with root canal infections. Protein mixtures were subjected to tryptic in-solution digestion and analysed by reverse-phase nano-LC–MS/MS followed by a database search.
Proteins, mainly of cell wall or membrane origin, from endodontic bacteria especially Enterococcus faecalis, Enterococcus faecium, Porphyromonas gingivalis, Fusobacterium nucleatum, and Treponema denticola were identified from all the samples tested. Identified proteins included adhesins, autolysins, proteases, virulence factors, and antibiotic-resistance proteins.
LC–MS/MS offers a sensitive analytical platform to study the disease processes in the root canal environment. The array of proteins expressed in endodontic infections reflects the complex microbial presence and highlights the bacterial species involved in the inflammatory process.
PMCID: PMC2744886  PMID: 19572900
bacteria; endodontics; liquid chromatography/mass spectroscopy; proteomics; virulence
18.  Evaluation of the humoral immune response to the cytolethal distending toxin of Aggregatibacter actinomycetemcomitans Y4 in subjects with localized aggressive periodontitis 
Oral microbiology and immunology  2009;24(2):116-123.
Cytolethal distending toxin (Cdt) is potentially one of several virulence factors of Aggregatibacter actinomycetemcomitans, the prime etiological agent of localized aggressive periodontitis (LAP). Little is known regarding the Cdt-specific antibody response in humans. The current study is a quantitative and qualitative evaluation of the toxin-specific antibody response in a cohort of LAP patients and age-, race- and sex-matched controls.
Ninety-five subjects provided a total of 692 serum samples. Sera were analysed by enzyme-linked immunosorbent assays to determine the titers of antibody against the intact Cdt holotoxin as well as the individual subunit proteins (CdtA, CdtB, and CdtC). Neutralization of growth inhibition mediated by Cdt was evaluated in a modified colony-forming assay using Chinese hamster ovary cells.
Fourteen of the 95 subjects exhibited significant serum Cdt-binding activity. There were no differences in the percentages of seropositive individuals or in the mean antibody titers between the control and LAP groups. Binding activity was detected against each of the three Cdt subunit proteins in all of the positive samples. Neutralization of Cdt-mediated growth inhibition was detected in samples from all of the seropositive subjects (range 20–75%).
Cdt, a recently identified A. actinomycetemcomitans virulence factor, is capable of inducing a neutralizing antibody response indicating that the toxin is produced during natural infection of humans. The failure of a vast majority (20 of 23) of the LAP subjects to mount a significant anti-Cdt response may in part explain their relative susceptibility to the disease.
PMCID: PMC2732719  PMID: 19239638
Aggregatibacter (Actinobacillus) actinomycetemcomitans; antibody; cytolethal distending toxin; localized aggressive periodontitis
19.  Analysis of neutrophil-derived antimicrobial peptides in gingival crevicular fluid suggests importance of cathelicidin LL-37 in the innate immune response against periodontogenic bacteria 
Oral microbiology and immunology  2008;23(4):328-335.
During periodontitis, an innate immune response to bacterial challenge is primarily mediated by neutrophils. We compared neutrophilic content and the level of neutrophil-derived antimicrobial peptides in gingival crevicular fluid (GCF) in two clinical forms of severe periodontitis.
GCF was collected from 14 patients with aggressive periodontitis, 17 patients with chronic periodontitis, and nine healthy subjects. Samples were analyzed for periodontopathogen load using real-time polymerase chain reactions. The amounts of myeloperoxidase and α-defensins (HNP1–3) were determined by enzyme-linked immunosorbent assay, and the level of cathelicidin (hCAP18/LL-37) was assayed by Western blot.
Myeloperoxidase concentration was not correlated with levels of LL-37 and HNP1–3 in samples from patients, compared to controls. The amount of HNP1–3 was twofold and fourfold higher in patients with aggressive and chronic periodontitis, respectively. Those with chronic disease had significantly elevated amounts of mature LL-37. The increased concentration of both peptides in chronic periodontitis correlated with the load of Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola.
The lack of a correlation between LL-37, HNP1–3, and myeloperoxidase content suggests that neutrophils are not the sole source of these bactericidal peptides in the GCF of patients with periodontitis; and that other cells contribute to their local production. The bacterial proteases of P. gingivalis, T. forsythia, and T. denticola might degrade hCAP18/LL-37, because the 11-kDa cathelicidin-derived fragment was present in GCF collected from pockets infected with these bacteria. Collectively, it appears that a local deficiency in LL-37 can be considered as a supporting factor in the pathogenesis of severe cases of periodontitis.
PMCID: PMC2565510  PMID: 18582333
defensins; bactericidal peptide; cathelicidin LL-37; crevicular fluid; periodontitis; periodontopathogenic bacteria
20.  The host cytokine response to Porphyromonas gingivalis is modified by gingipains 
Clinical studies indicate that primary proinflammatory cytokines, such as interleukin-1β (IL-1β) are elevated in the gingival crevice around teeth with periodontitis but the secondary cytokines and chemokines, IL-6 and IL-8, are not. The human gingival epithelial cells (HGECs) lining the gingival sulcus respond to perturbation by microbes of dental plaque by releasing a wide range of cytokines. Porphyromonas gingivalis, a putative periodontal pathogen, possesses numerous virulence factors some of which directly impact on the host response. In the present study, we sought to determine how P. gingivalis influences the inflammatory cytokine responses.
HGECs were challenged with P. gingivalis and other putative periodontal pathogens, and the resultant production of IL-1β, IL-6, and IL-8 was assayed by enzyme-linked immunosorbent assay (ELISA). Culture supernatants and recombinant human cytokines were challenged with live P. gingivalis wild-type and gingipain-deficient strains and the resultant cytokine profile was assessed by ELISA and Western blot.
We show here that primary HGECs challenged with live P. gingivalis result in high levels of IL-1β but not the related secondary cytokines IL-6 and IL-8. We further demonstrate that cytokine response differences are the result of the action of P. gingivalis proteases, with lysine gingipain being the most effective.
We conclude that P. gingivalis, through lysine gingipain, can subvert the protective host proinflammatory response by direct cytokine degradation. Changes in the crevicular cytokine profile have consequences in periodontal disease pathogenesis that should be considered in the development of diagnostic and therapeutic modalities.
PMCID: PMC2717190  PMID: 19121064
cytokine degradation; epithelial cells; gingipains; Porphyromonas gingivalis
21.  Oral Candida infection and colonization in solid organ transplant recipients 
Oral microbiology and immunology  2009;24(3):249-254.
Oral Candida carriage and infection have been reported to be associated with a greater risk for systemic infection in transplant recipients; however, a systematic analysis of the oral Candida titers and species has not been previously conducted. The objectives of this study were to determine the prevalence of oropharyngeal candidiasis, the oral carrier status, Candida titers and species in this population.
Ninety kidney and heart transplant subjects and 72 age-matched healthy controls were included. Swabs from the oral mucosa and a standardized amount of unstimulated saliva were plated on Chromagar™ Candida, and colony-forming units per millilitre were calculated. Initial speciation was based on colony color and was confirmed by standard germ tube, biotyping, or polymerase chain reaction assays.
Infection with C. albicans was detected in seven transplant subjects and none of the controls. The transplant group had significantly higher oral Candida titers than the control group. There were no statistically significant relationships between the dose or type of immunosuppressants and oral Candida titers or infection. A significantly higher percentage of transplant subjects were colonized by more than one species, compared with control subjects. The most frequent species combination in transplant subjects was C. albicans and C. glabrata. C. glabrata was isolated from 13.5% of transplant carriers and none of the controls.
Increased oral Candida infection and carriage titers were found in the transplant population. Although the majority of transplant patients were colonized by C. albicans, C. glabrata appears to emerge as the second most prevalent species.
PMCID: PMC2699610  PMID: 19416456
Candida albicans; Candida glabrata; transplant; oral candidiasis
22.  Detection of cytolethal distending toxin activity and cdt genes in Actinobacillus actinomycetemcomitans isolates from geographically diverse populations 
Oral microbiology and immunology  2002;17(4):231-238.
A cytolethal distending toxin (CDT) found in Actinobacillus actinomycetemcomitans inhibits the eukaryotic cell cycle, which may contribute to the pathogenic potential of the bacterium. The presence of the cdtABC genes and CDT activity were examined in 40 clinical isolates of A. actinomycetemcomitans from Brazil, Kenya, Japan and Sweden. Thirty-nine of 40 cell lysates caused distension of Chinese hamster ovary cells. At least one of the cdt genes was detected in all strains examined. The three cdt genes were detected, by PCR, in 34 DNA samples. DNA from one strain from Kenya did not yield amplicons of the cdtA and cdtB genes and did not express toxic activity. Restriction analysis was performed on every amplicon obtained. PCR-RFLP patterns revealed that the three cdt genes were conserved. These data provided evidence that the cdt genes are found and expressed in the majority of the A. actinomycetemcomitans isolates. Although a quantitative difference in cytotoxicity was observed, indicating variation in expression of CDT among strains, no clear relationship between CDT activity and periodontal status was found.
PMCID: PMC2548306  PMID: 12121473
Actinobacillus actinomycetemcomitans; cdt genes; cytolethal distending toxin; periodontitis
23.  Kinetics of KB and HEp-2 cell responses to an invasive, cytolethal distending toxin-producing strain of Actinobacillus actinomycetemcomitans 
Oral microbiology and immunology  2002;17(4):245-251.
The periodontal pathogen Actinobacillus actinomycetemcomitans produces cytolethal distending toxin (CDT), a complex multicomponent toxin that arrests the growth of many types of eukaryotic cell. The kinetics of the effects of CDT-containing extracts, from an invasive strain of this bacterium, were examined on epithelial-like cells routinely used in invasion studies. Both KB and HEp-2 cells were exquisitely sensitive to the effects of the CDT with TD50 of 30 and 300 pg of total bacterial protein, respectively. Initial cell morphology changes were relatively rapid, occurring within the first 13 h of exposure. CDT-treated KB cells increased in size to 4–5 times the size of untreated controls. Cytotoxicity was irreversible when attached cells were incubated, for a minimum of 120 min, with nanogram quantities of CDT-containing extract. As cultures aged, the cells became more resistant to the effects of the CDT-containing extracts. These findings have important implications for understanding the ability of A. actinomycetemcomitans to invade and multiply in epithelial cells.
PMCID: PMC1434795  PMID: 12121475
Actinobacillus actinomycetemcomitans; cytolethal distending toxin; invasion; KB cells; Hep-2 cells
24.  Oral and vaginal epithelial cell anti-Candida activity is acid labile and does not require live epithelial cells 
Oral microbiology and immunology  2005;20(4):199-205.
Background: Candida albicans is the causative agent of oral and vaginal candidiasis. Innate host defenses against C. albicans are important against each infection. Among these are oral and vaginal epithelial cells that have anti-Candida activity. The mechanism of action includes a requirement for cell contact with no role for soluble factors, and a putative role for carbohydrates based on the sensitivity of the activity to periodic acid.
Methods: Periodic acid treatment of epithelial cells as well as the property of partial resistance of antifungal activity to fixation was used to further dissect the mechanism of action.
Results: The results herein effectively now challenge a role for carbohydrates alone. Firstly, the putative carbohydrate(s) released into supernatants of periodic acid-treated epithelial cells could not compete with fresh epithelial cells for activity, and equivalent abrogation of activity was observed by periodic acid-treated cells irrespective of the amount of carbohydrate released. Instead, the similar abrogation of activity following treatment with other acids or when cocultured under acidic conditions suggests that the activity is acid-labile. Finally, while activity requires intact epithelial cells, it does not require live cells; activity was minimally affected by fixing epithelial cells prior to coculture where the majority of cells remained impermeable to Trypan blue but were defined as non–viable by positive nuclear staining with propidium iodide.
Conclusion: These results suggest that antifungal activity is dependent on contact by intact, but not necessarily live, epithelial cells through an acid-labile mechanism.
PMCID: PMC1361270  PMID: 15943762
Candida albicans; epithelial cells; innate immunity; oral mucosa; vaginal mucosa
25.  Antibiotic resistance in an in vitro subgingival biofilm model 
Oral microbiology and immunology  2007;22(5):333-339.
The purpose of this study was to utilize an in vitro biofilm model of subgingival plaque to investigate resistances in subgingival biofilm communities to antibiotics commonly used as adjuncts to periodontal therapy.
Biofilms were grown on saliva-coated hydroxyapatite supports in trypticasesoy broth for 4 h–10 days and then exposed for 48 h to either increasing twofold concentrations of tetracycline, amoxicillin, clindamycin, and erythromycin or therapeutically achievable concentrations of tetracycline, doxycycline, minocycline, amoxicillin, metronidazole, amoxicillin/clavulanate, and amoxicillin/metronidazole.
Concentrations necessary to inhibit bacterial strains in steady-state biofilms were up to 250 times greater than the concentrations needed to inhibit the same strains grown planktonically. In the presence of therapeutically available antibiotic concentrations, significantly higher proportions of the biofilms remained viable as the biofilms reached steady-state growth. The combinations of amoxicillin/clavulanate and amoxicillin/metronidazole were the most effective in suppressing growth. These combinations were particularly effective against biofilms up to and including 7 days of age and inhibited 90% or more of the bacteria present relative to untreated controls. As the biofilms approached steady state, these combinations were less effective with 50−60% of the bacteria retaining viability.
Most, but not all, species of subgingival bacteria are considerably more resistant in biofilms than in planktonic cultures. Resistance appeared to be age-related because biofilms demonstrated progressive antibiotic resistance as they matured with maximum resistance coinciding with the steady-state phase of biofilm growth.
PMCID: PMC2040071  PMID: 17803631
antibiotics; biofilm; planktonic; resistances; subgingival

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