Denis Mayrand

Role of Gingipains in Growth of Porphyromonas gingivalis in the Presence of Human Serum Albumin

ABSTRACT Porphyromonas gingivalis, a bacterium associated with active chronic periodontitis lesions, produces several proteolytic enzymes that are thought to be involved in host colonization, perturbation of the immune system, and tissue destruction. The aim of the present study was to investigate the contribution of Arg- and Lys-gingipains produced by P. gingivalis to its growth. Although all of the proteins studied were degraded by P. gingivalis, only human serum albumin and transferrin supported growth during serial transfers in a chemically defined medium (CDM). Growth studies with site-directed gingipain-deficient mutants ofP. gingivalis revealed that inactivation of both gingipains prevents growth, whereas inactivation of either Arg- or Lys-gingipain activity extended the doubling times to 33 or 13 h, respectively, compared to 9 h for the parent strain. Growth of the mutants and the parent strain was similar when the CDM was supplemented with a protein hydrolysate instead of human serum albumin. Incubation of resting P. gingivalis ATCC 33277 cells with fluorophore-labeled albumin indicated that the proteolytic fragments generated by the gingipains were internalized by the bacterial cells. Internalization of fluorophore-labeled albumin fragments was reduced or completely inhibited in the proteinase-deficient mutants. Interestingly, gingival crevicular fluid samples from diseased periodontal sites contained low-molecular-mass albumin fragments, whereas samples from healthy sites did not. The critical role of proteinases in the growth of P. gingivalis was further investigated using specific Arg- and Lys-gingipain inhibitors. Adding the inhibitors to CDM containing albumin revealed that leupeptin (Arg-gingipain A and B inhibitor) was more efficient at inhibiting growth than cathepsin B inhibitor II (Lys-gingipain inhibitor). Our study suggests that Arg-gingipains and, to a lesser extent, Lys-gingipain play an important role in the growth of P. gingivalis in a defined medium containing a human protein as the sole carbon and nitrogen source.

Iron-chelating activity of tetracyclines and its impact on the susceptibility of Actinobacillus actinomycetemcomitans to these antibiotics.

ABSTRACT Three tetracyclines (tetracycline, doxycycline, and minocycline) were found to possess iron-chelating activity in a colorimetric siderophore assay. Determination of MICs indicated that the activity of doxycycline against the periodontopathogen Actinobacillus actinomycetemcomitans was only slightly influenced by the presence of an excess of iron that likely saturates the antibiotic. On the other hand, the MICs of doxycycline and minocycline were significantly lower for A. actinomycetemcomitans cultivated under iron-poor conditions than under iron-rich conditions.

Effect of Inactivation of the Arg- and/or Lys-Gingipain Gene on Selected Virulence and Physiological Properties of Porphyromonas gingivalis

ABSTRACT Proteolytic enzymes produced by Porphyromonas gingivalis are thought to play critical roles in the pathogenesis of periodontitis. The aim of this study was to investigate the effect of gingipain cysteine proteinase gene inactivation on selected pathological and physiological functions of P. gingivalis. Our results showed that Arg- and Lys-gingipain activities are critical components for the efficient growth of P. gingivalis in human serum. However, when the serum was supplemented with peptides provided as pancreatic casein hydrolysate, the gingipains did not appear to be essential for growth. The effect of gingipain gene inactivation on the susceptibility of P. gingivalis to serum bactericidal activity was investigated using standardized human serum. The wild-type strain, P. gingivalis ATCC 33277, was largely unaffected by the bactericidal activity of human serum complement. On the other hand, mutants lacking Arg-gingipain A, Arg-gingipain B, or Lys-gingipain activity were susceptible to complement. Since gingipains are mostly located on the outer membrane of P. gingivalis, inactivation of the genes for these enzymes may modify cell surface properties. We showed that gingipain-deficient mutants differed in their capacities to assimilate radiolabeled amino acids, cause hemolysis, express adhesins, hemagglutinate, and form biofilms. Lastly, the gingipains, more specifically Arg-gingipains, were responsible for causing major cell damage to human gingival fibroblasts. In conclusion, our study indicated that, in addition to being critical in the pathogenic process, gingipains may play a variety of physiological roles in P. gingivalis, including controlling the expression and/or processing of virulence factors. Mutations in gingipain genes thus give rise to pleiotropic effects.

Oral Microbial Heat-shock Proteins and Their Potential Contributions to Infections

The oral cavity is a complex ecosystem in which several hundred microbial species normally cohabit harmoniously. However, under certain special conditions, the growth of some micro-organisms with a pathogenic potential is promoted, leading to infections such as dental caries, periodontal disease, and stomatitis. The physiology and pathogenic properties of micro-organisms are influenced by modifications in environmental conditions that lead to the synthesis of specific proteins known as the heat-shock proteins (HSPs). HSPs are families of highly conserved proteins whose main role is to allow micro-organisms to survive under stress conditions. HSPs act as molecular chaperones in the assembly and folding of proteins, and as proteases when damaged or toxic proteins have to be degraded. Several pathological functions have been associated with these proteins. Many HSPs of oral micro-organisms, particularly periodontopathogens, have been identified, and some of their properties-including location, cytotoxicity, and amino acid sequence homology with other HSPs-have been reported. Since these proteins are immunodominant antigens in many human pathogens, studies have recently focused on the potential contributions of HSPs to oral diseases. The cytotoxicity of some bacterial HSPs may contribute to tissue destruction, whereas the presence of common epitopes in host proteins and microbial HSPs may lead to autoimmune responses. Here, we review the current knowledge regarding HSPs produced by oral micro-organisms and discuss their possible contributions to the pathogenesis of oral infections.

A general procedure for the isolation of heat-shock proteins from periodontopathogenic bacteria

Abstract The isolation of heat-shock proteins (HSP) from whole cells of Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans and Bacteroides forsythus , which are recognized as periodontopathogens, was performed by affinity chromatography on adenosine 5′-triphosphate-agarose followed by preparative polyacrylamide gel electrophoresis under denaturing conditions. Analysis of the final material by silver nitrate staining of SDS-PAGE gels and Western immunoblotting using a commercial polyclonal antibody against HSP60 from Synechococcus sp. revealed homogenous protein preparations corresponding to GroEL-like proteins with apparent molecular mass of 68 kDa ( P. gingivalis ), 64 kDa ( A. actinomycetemcomitans ) and 67 kDa ( B. forsythus ). A second HSP was also isolated from P. gingivalis and B. forsythus , and recognized as a DnaK-like protein by immunoblotting with polyclonal antibody against DnaK (HSP70) from Escherichia coli . Our HSP isolation procedure is rapid and simple, and the purified proteins obtained may be used to determine their homology with other HSP previously characterized and to raise antibodies.

Monitoring the uptake of protein-derived peptides by Porphyromonas gingivalis with fluorophore-labeled substrates.

The aim of this study was to develop a simple method to quantify peptide uptake by the periodontopathogenic bacterium Porphyromonas gingivalis. After incubation of bacterial cells with self-quenched fluorescent bovine serum albumin (DQ™ Green BSA), the fluorescence measured in the supernatant of the assay mixture indicated the degree of protein degradation, whereas the fluorescence associated with the lysate of washed cells indicated the amount of BSA-derived fragments incorporated by the bacteria. The optimal conditions for uptake of fluorophore-labeled albumin fragments were found to be mid-log grown cells, 150 mM NaCl in phosphate buffer, pH 7, 37°C, and anaerobiosis. Among the protease inhibitors tested, 4-(2-aminoethyl)-benzene sulfonyl-fluoride hydrochloride (AEBSF) and cathepsin B inhibitor II caused a significant inhibition of the uptake of BSA-derived peptides. This assay was applicable for other commercially available fluorescent substrates. This simple method may be useful to investigate protein processing in proteolytic bacteria and for studying the effects of environmental parameters or cell treatments on the peptide uptake.

Stress response in Actinobacillus actinomycetemcomitans: induction of general and specific stress proteins.

Stress proteins are highly conserved proteins that are essential for cell survival. In this study, the induction of general and specific stress proteins in Actinobacillus actinomycetemcomitans cells subjected to different stress conditions was evaluated by two-dimensional SDS-PAGE analysis. Twenty-eight (up- or down)regulated proteins, including DnaK and GroEL proteins, were identified as general stress proteins. In addition, eighteen regulated proteins were classified as pH stress-specific proteins, ten as acid stress-specific proteins, five as alkaline stress-specific proteins, three as heat stress-specific proteins, and ten as acid/heat stress-specific proteins. Further proteomic studies are required to determine the exact nature of the proteins regulated during the stress response of A. actinomycetemcomitans.

Aminopeptidase activities in Peptostreptococcus spp. are statistically correlated to gelatin hydrolysis.

One hundred Peptostreptococcus isolates from five species were assessed for their ability to hydrolyze gelatin. Most Peptostreptococcus magnus (95.8%) and Peptostreptococcus micros isolates (79.0%) hydrolyzed gelatin in contrast to Peptostreptococcus asaccharolyticus (8.0%), Peptostreptococcus anaerobius (10.0%), and Peptostreptococcus prevotii isolates (16.7%). Gelatin hydrolysis in Peptostreptococcus magnus and Peptostreptococcus micros isolates correlated (r = 0.80; P = 0.0019) with more aminopeptidases produced than Peptostreptococcus asaccharolyticus, Peptostreptococcus anaerobius, or Peptostreptococcus prevotii. The five species were further classified into three groups using the extended Tukey test (P < 0.0001) based on the mean percentage of aminopeptidases produced by each species with Peptostreptococcus magnus and Peptostreptococcus micros belonging to group I, Peptostreptococcus asaccharolyticus and Peptostreptococcus prevotii belonging to group II, and Peptostreptococcus anaerobius forming group III. An analysis of possible proteolytic activity of four selected Peptostreptococcus magnus isolates indicated that only 5 of 11 substrates were hydrolyzed as compared to a control isolate of Porphyromonas gingivalis W83, which had a strong proteolytic profile. Therefore, gelatin hydrolysis by Peptostreptococcus spp., in particular Peptostreptococcus magnus and Peptostreptococcus micros, is probably due to a variety of aminopeptidases rather than proteinases.