Immunoglobulin G (IgG) class, but Not IgA or IgM, antibodies to peptides of the Porphyromonas gingivalis chaperone HtpG predict health in subjects with periodontitis by a fluorescence enzyme-linked immunosorbent assay.

Chaperones are molecules found in all cells and are critical in stabilization of synthesized proteins, in repair/removal of defective proteins, and as immunodominant antigens in innate and adaptive immunity. Subjects with gingivitis colonized by the oral pathogen Porphyromonas gingivalis previously demonstrated levels of anti-human chaperone Hsp90 that were highest in individuals with the best oral health. We hypothesized that similar antibodies to pathogen chaperones might be protective in periodontitis. This study examined the relationship between antibodies to P. gingivalis HtpG and clinical statuses of healthy and periodontitis-susceptible subjects. We measured the humoral responses (immunoglobulin G [IgG], IgA, and IgM) to peptides of a unique insert (P18) found in Bacteroidaceae HtpG by using a high-throughput, quantitative fluorescence enzyme-linked immunosorbent assay. Indeed, higher levels of IgG class anti-P. gingivalis HtpG P18 peptide (P < 0.05) and P18alpha, consisting of the N-terminal 16 amino acids of P18 (P < 0.05), were associated with better oral health; these results were opposite of those found with anti-P. gingivalis whole-cell antibodies and levels of the bacterium in the subgingival biofilm. When we examined the same sera for IgA and IgM class antibodies, we found no significant relationship to subject clinical status. The relationship between anti-P18 levels and clinical populations and individual subjects was found to be improved when we normalized the anti-P18alpha values to those for anti-P18gamma (the central 16 amino acids of P18). That same ratio correlated with the improvement in tissue attachment gain after treatment (P < 0.05). We suggest that anti-P. gingivalis HtpG P18alpha antibodies are protective in periodontal disease and may have prognostic value for guidance of individual patient treatment.

Serum antibodies to Porphyromonas gingivalis chaperone HtpG predict health in periodontitis susceptible patients.

BACKGROUND: Chaperones are ubiquitous conserved proteins critical in stabilization of new proteins, repair/removal of defective proteins and immunodominant antigens in innate and adaptive immunity. Periodontal disease is a chronic inflammatory infection associated with infection by Porphyromonas gingivalis that culminates in the destruction of the supporting structures of the teeth. We previously reported studies of serum antibodies reactive with the human chaperone Hsp90 in gingivitis, a reversible form of gingival disease confined to the oral soft tissues. In those studies, antibodies were at their highest levels in subjects with the best oral health. We hypothesized that antibodies to the HSP90 homologue of P. gingivalis (HtpG) might be associated with protection/resistance against destructive periodontitis. METHODOLOGY/PRINCIPAL FINDINGS: ELISA assays using cloned HtpG and peptide antigens confirmed gingivitis subjects colonized with P. gingivalis had higher serum levels of anti-HtpG and, concomitantly, lower levels of attachment loss. Additionally, serum antibody levels to P. gingivalis HtpG protein were higher in healthy subjects compared to patients with either chronic or aggressive periodontitis. We found a negative association between tooth attachment loss and anti-P. gingivalis HtpG (p = 0.043) but not anti-Fusobacterium nucleatum (an oral opportunistic commensal) HtpG levels. Furthermore, response to periodontal therapy was more successful in subjects having higher levels of anti-P. gingivalis HtpG before treatment (p = 0.018). There was no similar relationship to anti-F. nucleatum HtpG levels. Similar results were obtained when these experiments were repeated with a synthetic peptide of a region of P. gingivalis HtpG. CONCLUSIONS/SIGNIFICANCE: OUR RESULTS SUGGEST: 1) anti-P. gingivalis HtpG antibodies are protective and therefore predict health periodontitis-susceptable patients; 2) may augment the host defence to periodontitis and 3) a unique peptide of P. gingivalis HtpG offers significant potential as an effective diagnostic target and vaccine candidate. These results are compatible with a novel immune control mechanism unrelated to direct binding of bacteria.

HtpG, the Porphyromonas gingivalis HSP-90 homologue, induces the chemokine CXCL8 in human monocytic and microvascular vein endothelial cells.

CXCL8 (interlukin 8, IL-8) has a diverse spectrum of biological activities including T cell, neutrophil and basophil chemotactic properties. It is produced by a wide variety of cell types and plays a significant role in the initiation of the acute inflammatory response. During inflammation, CXCL8 attracts and activates leukocytes at the site of infection leading to leukocyte infiltration, which can lead to tissue damage. Porphyromonas gingivalis, an aetiological agent of periodontitis, induces production of CXCL8 from several types of cells via its LPS and outer membrane proteins. Bacterial chaperones elicit a strong pro-inflammatory response in cells of the innate immune system. In P. gingivalis the htpG gene codes for the homologue of human Hsp90, a chaperone that associates with transcription factors, hormone receptors and protein kinases, affecting signal transduction pathways. CXCL8 mRNA and CXCL8 protein production was induced in monocytic/human microvascular vein endothelial cells treated with P. gingivalis cells or rHtpG protein. Blocking of receptors CD91 and TLR4 reduced the production of CXCL8 by rHtpG either using receptor-specific antibody or by siRNA silencing. Pre-incubation of P. gingivalis rHtpG preparations with human anti-HtpG significantly inhibited CXCL8 production. A P. gingivalis HtpG disruption mutant also induced less CXCL8 mRNA and protein. These results suggest that P. gingivalis HtpG might be involved in CXCL8-mediated immunopathogenesis.

The spectrum of antimicrobial activity of the bacteriocin subtilosin A.

BACKGROUND: Bacterocins are antimicrobial peptides produced by bacteria with a relatively narrow range of activity against closely related organisms. Subtilosin A is a bacteriocin produced by Bacillus subtilis that has activity against Listeria monocytogenes, which might indicate antimicrobial activity unusual for bacteriocins. OBJECTIVES: To examine the antimicrobial activity and factors affecting the activity of subtilosin A against a range of potentially pathogenic bacteria. METHODS: The peptide was purified from cultures of B. subtilis and the MIC determined for 18 species of bacteria using a microdilution methodology. The extent of capsule formation was determined using microscopic examination of cells mounted in India ink. Protease mutants of a susceptible bacteria and mild heat shock were used to examine the effect of environmental stress on subtilosin A activity. RESULTS: Subtilosin A proved to have antimicrobial activity against a wide range of bacteria including Gram-positive and Gram-negative bacteria and both aerobes and anaerobes. The peptide was less effective against capsulated forms of two Gram-negative bacteria than the non-capsulated strains of either. Heat shock but not protease activity also altered the effectiveness of the bacteriocin. CONCLUSIONS: Subtilosin A has limited antimicrobial activity against a number of human pathogens which, combined with its relative ineffectiveness against some capsulated pathogens, may limit its usefulness as a human therapeutic.

A comparison of the gingival health of children with Down syndrome to healthy children residing in an institution.

The purpose of this study was to compare the onset and severity of gingivitis in children with Down syndrome, when compared to a healthy control group of children. The subjects included 41 children with Down syndrome ages two to 14 years (mean age: 7.6 years) and 112 age-matched healthy controls. We assessed the gingival health of all subjects using the gingival inflammation (M-PMA) index and periodontal probing depth (PD). Children were divided into three age categories: <5 years (AI), 5 to <10 years (AII), and 10 to <17 years (AIII). Supragingival plaque was measured using the Oral Hygiene Index (OHI) and the subjects were screened with the BANA test (Perioscan-Oral-B). Measurement of the M-PMA index in the healthy children showed an age-related increase (F = 10.369, p < 0.001), and the M-PMA index at the younger age group <5 year (AI) was significantly lower than that for the other two age groups All or AIII (p < 0.005, p < 0.001). In contrast, the M-PMA index values at AI and AIII in the subjects with Down syndrome were significantly higher than those for healthy children (p < 0.001, p < 0.001). Both groups had an age-related increase in PD (F = 3.388, p < 0.05 & F = 10.806, p < 0.001), and PD at AIII was significantly higher than that at AI in both groups (p < 0.01, p < 0.001). The children with Down syndrome showed an age-related increase in the BANA test score (F = 3.452, p < 0.05), and the BANA test score at AIII was significantly higher than that at AI (p < 0.02). The BANA test score in the healthy children was not age-related but was significantly higher than that in the children with Down syndrome (p < 0.02, p < 0.05).

Induction of {beta}-defensin resistance in the oral anaerobe Porphyromonas gingivalis.

Induction of resistance of oral anaerobes to the effects of human beta-defensin 1 (hbetaD-1) to hbetaD-4 was investigated by pretreating cells with either sublethal levels of defensins or environmental factors, followed by a challenge with lethal levels of defensins. Cultures of Porphyromonas gingivalis were (i) pretreated with defensins at 1 ng/ml, (ii) heated to 42 degrees C (heat stress), (iii) exposed to normal atmosphere (oxidative stress), or (iv) exposed to 1 mM hydrogen peroxide (peroxide stress). Samples (10 microl) were distributed among the wells of sterile 384-well plates containing hbetaD-1 to -4 (100 microg/ml). Plates were incubated at 37 degrees C for 36 h in an anaerobe chamber. Growth inhibition was determined by a system that measures the total nucleic acid of a sample with a DNA binding dye. The MICs of the four defensins for P. gingivalis were 3 to 12 microg/ml. We found that sublethal levels of the defensins and heat and peroxide stress, but not oxidative stress, induced resistance to 100 microg of defensin per ml in P. gingivalis. Resistance induced by sublethal levels of hbetaD-2 lasted 90 min, and the resistance induced by each defensin was effective against the other three. Multiple strains exposed to hbetaD-2 all evidenced resistance induction. Defensin resistance is vital to the pathogenic potential of several human pathogens. This is the first report describing the induction of defensin resistance in the oral periodontal pathogen P. gingivalis. Such resistance may have an effect on the ability of oral pathogens to persist in the mouth and to withstand innate human immunity.

Localizing antibody-defined immunoreactivity in Porphyromonas gingivalis HtpG recognized by human serum utilizing selective protein expression.

Earlier studies suggested that specific immunoreactive domains of the prokaryotic homologue of Hsp90, HtpG, might contribute to the virulence of the periodontal pathogen, Porphyromonas gingivalis (Pg) [J. Periodontol. 70 (1999) 1185]. Since serum antibodies to this protein appeared to be associated with oral health, we developed a rapid epitope-mapping system that could be tailored to detect antibodies against specific immunoreactive regions of the Pg HtpG protein. This paper describes the use of Caulobacter crescentus (Cc) and the creation of a Cc RsaA fusion protein library that defined specific regions of the Pg HtpG protein. The fusion protein library was used to identify immunoreactive regions in the Pg HtpG dominant in patient and control sera. The development of methods to rapidly localize dominant immunoreactive regions in protein antigens may prove useful for the development of screening tests, vaccines and therapeutics in periodontal and other infectious diseases.

Construction and characterization of a Porphyromonas gingivalis htpG disruption mutant.

Our previous reports implicated the Hsp90 homologue (HtpG) of Porphyromonas gingivalis (Pg) in its virulence in periodontal disease. We investigated the role of the HtpG stress protein in the virulence of Pg. This report describes the (i) expression of a recombinant Pg HtpG (rHtpG), (ii) generation and characterization of a polyclonal rabbit anti-Pg rHtpG antiserum, and (iii) construction of a Pg htpG isogenic mutant and evaluation of the growth, adherence and invasion properties compared to the wild-type parental strain. The disruption of the htpG gene did not significantly affect growth, and had no effect on Pg adherence to and invasion of cultured human cells.

Expression of CXCR4 and CXCL12 (SDF-1) in human prostate cancers (PCa) in vivo.

Human prostate cancers (PCa) express great variability in their ability to metastasize to bone. The identification of molecules associated with aggressive phenotypes will help to define PCa subsets and will ultimately lead to better treatment strategies. The chemokine stromal-derived factor-1 (SDF-1 or CXCL12) and its receptor CXCR4 are now known to modulate the migration and survival of an increasing array of normal and malignant cell types including breast, pancreatic cancers, glioblastomas, and others. The present investigation extends our previous investigations by determining the expression of CXCR4 and CXCL12 in humans using high-density tissue microarrays constructed from clinical samples obtained from a cohort of over 600 patients. These data demonstrate that CXCR4 protein expression is significantly elevated in localized and metastastic cancers. At the RNA level, human PCa tumors also express CXCR4 and message, but overall, they were not significantly different suggesting post-transcriptional regulation of the receptor plays a major role in regulating protein expression. Similar observations were made for CXCL12 message, but in this case more CXCL12 message was expressed by metastastic lesions as compared to normal tissues. PCa cell lines also express CXCL12 mRNA, and regulate mRNA expression in response to CXCL12 and secrete biologically active protein. Furthermore, neutralizing antibody to CXCL12 decreased the proliferation of bone homing LNCaP C4-2B and PC3 metastastic tumor cells. These investigations provide important new information pertaining to the molecular basis of how tumors may 'home' to bone, and the mechanisms that may account for their growth in selected end organs.

Quantitative reverse transcription polymerase chain reaction analysis of Porphyromonas gingivalis gene expression in vivo.

An etiological relationship between periodontitis, a significant oral health problem, and the anaerobe Porphyromonas gingivalis may be related to the expression of a variety of putative virulence factors. The objective of the experiments described here was to develop a quantitative reverse transcription polymerase chain reaction (QRT-PCR) method to examine P. gingivalis gene expression in human dental plaque from periodontitis subjects. PCR primers and probes for six target genes representing putative virulence factors were chosen and evaluated in vitro for specificity. A potential cross-reactivity level of only 10 copies/10(7) whole genomic equivalents was occasionally observed with non-P. gingivalis microbes. P. gingivalis cells stressed in vitro by a 5 degrees C temperature increase showed a rapid rise in the mRNA associated with the molecular chaperons (htpG, dnaK, groEL), SOD (sodA) and gingipain (rgp-1) genes. We examined the stability of bacterial RNA in plaque specimens and found no significant difference in the amount of RNA obtained before or after storage 3 months in a stabilizing buffer (p=0.786, t-test). Sixty-five percent of plaque samples obtained from two clinical locations contained P. gingivalis; there was a mean level of gene expression (fold increase) for all samples tested for groEL, dnaK, htpG, sodA, PG1431 and rgp-1 of 0.84+/-2.03 to 7.85+/-10.0. ANOVA showed that the levels of stress gene transcription for dnaK and htpG were significantly elevated (p<0.05) at diseased sites; groEL gene transcription approached statistically significant elevation (p=0.059). We found correlations between probing depth and increased transcription of groEL, htpG and rgp-1 and between attachment loss and htpG. When sorted by disease status, we detected correlations between disease status and elevated expression of dnaK and htpG.

Pesquisa de bactérias bucais em amostras de placa subgengival de indivíduos com o periodonto normal e de portadores de periodontite através da técnica do "slot immunoblot" / Assessment of oral bacteria in subgingival dental plaque from healthy subjects and periodontitis patients utilizing the slot immunoblot technique

Dentre alguns dos microrganismos considerados significantemente associados com a periodontite, estäo os bacilos Gram negativos, como a P. gingivalis, P. intermedia, A. actinomycetemcomitans (A.a.) e B. forsythus. Utilizando a técnica do "slot immunoblot" (SIB), procurou-se avaliar a presença e o grau de infecçäo desses microrganismos em amostras de placa subgengival de 4 sítios de 10 indivíduos com periodonto normal e de 27 portadores de periodontite do adulto. No grupo normal, a prevalência de indivíduos com pelo menos um sítio positivo a uma das bactérias variou de 90 por cento para o B. forsythus, 70 por cento para a P. gingivalis, 60 por cento para a P. intermedia e 50 por cento para o A. a., enquanto 100 por cento dos pacientes exibiram pelo menos 1 sítio positivo para todos os microrganismos. As porcentagens de sítios positivos no grupo de pacientes e no grupo sadio foram, respectivamente, 93,5 por cento x 40 por cento para a P. gingivalis, 91,6 por cento x 45 por cento para a P. intermedia, 98,0 por cento x 47,5 por cento para o B. forsythus e 81,4 por cento x 27,5 por cento para o A. a. Foi observada diferença estatisticamente significante entre os dois grupos para os escores médios de detecçäo do A. a. (p<0,001) e das demais bactérias (p<0,0001). O teste de Spearman revelou correlaçäo significante entre a profundidade de sondagem média dos 4 sítios amostrados (ó3 mm para o grupo normal e ò4 mm para o grupo da periodontite) e escores médios para os mesmos sítios de todas as bactérias (p<0,001). O trabalho demonstra que a intensidade de colonizaçäo dos microorganismos nos pacientes, significantemente diferente daquela dos normais, é compatível com um estado de doença