Joseph Aduse-Opoku

Bio: Joseph Aduse-Opoku is an academic researcher from King's College London. The author has contributed to research in topics: Porphyromonas gingivalis & Protease. The author has an hindex of 28, co-authored 48 publications receiving 2562 citations. Previous affiliations of Joseph Aduse-Opoku include University of London & Queen Mary University of London.

Molecular genetics and nomenclature of proteases of Porphyromonas gingivalis

Abstract: The strategies used by bacterial pathogens to establish and maintain themselves in the host represent one of the fundamental aspects of microbial pathogenesis. Characterization of these strategies and the underlying molecular machinery offers new opportunities both to our understanding of how organisms cause disease in susceptible individuals and to the development of novel therapeutic measures designed to undermine or interfere with these determinants of successful survival. With respect to the microbial aetiology of the periodontal diseases, a growing body of evidence suggests that the proteolytic enzymes of Porphyromonas gingivalis represent key survival and, by extrapolation, virulence determinants of this periodontal bacterium. This in turn has led to international efforts to characterize these enzymes at the gene and protein level. Approximately 20 protease genes of P. gingivalis with different names and accession numbers have been deposited in the gene databases and a correspondingly heterogeneous nomenclature system is employed for the products of these genes in the literature. However, it is evident, through comparison of these gene sequences and through gene inactivation studies, that the genetic structure of the proteases of this organism, particularly those with specificity for arginyl and lysyl peptide bonds, is less complicated than originally thought. The major extracellular and surface associated arginine specific protease activity is encoded by 2 genes which we recommend be designated rgpA and rgpB (arg-gingipains A & B). Similarly we recommend that the gene encoding the major lysine specific protease activity is designated kgp (lys-gingipain). These three genes, which account for all the extracellular/surface arginine and lysine protease activity in P. gingivalis, belong to a family of sequence-related proteases and haemagglutinins.

Cysteine proteases of Porphyromonas gingivalis.

Abstract: The cysteine proteases of Porphyromonas gingivalis are extracellular products of an important etiological agent in periodontal diseases. Many of the in vitro actions of these enzymes are consistent with the observed deregulated inflammatory and immune features of the disease. They are significant targets of the immune responses of affected individuals and are viewed by some as potential molecular targets for therapeutic approaches to these diseases. Furthermore, they appear to represent a complex group of genes and protein products whose transcriptional and translational control and maturation pathways may have a broader relevance to virulence determinants of other persistent bacterial pathogens of human mucosal surfaces. As a result, the genetics, chemistry, and virulence-related properties of the cysteine proteases of P. gingivalis have been the focus of much research effort over the last ten years. In this review, we describe some of the progress in their molecular characterization and how their putative biological roles, in relation to the in vivo growth and survival strategies of P. gingivalis, may also contribute to the periodontal disease process.

LuxS-dependent quorum sensing in Porphyromonas gingivalis modulates protease and haemagglutinin activities but is not essential for virulence.

Abstract: Porphyromonas gingivalis is a Gram-negative black-pigmented obligate anaerobe implicated in the aetiology of human periodontal disease. The virulence of P. gingivalis is associated with the elaboration of the cysteine proteases Arg-gingipain (Rgp) and Lys-gingipain (Kgp), which are produced at high bacterial cell densities. To determine whether quorum sensing plays a role in the regulation of Rgp and Kgp, biosensors capable of detecting either N-acylhomoserine lactone (AHLs) or the luxS-dependent autoinducer (AI-2) quorum-sensing signalling molecules in spent culture supernatants were first employed. While no AHLs could be detected, the Vibrio harveyi BB170 biosensor was activated by spent P. gingivalis W50 culture supernatants. The P. gingivalis luxS gene was cloned and demonstrated to restore AI-2 production in the Escherichia coli luxS mutant DH5α. Mutation of luxS abolished AI-2 production in P. gingivalis. Western blotting using antibodies raised against the recombinant protein revealed that LuxS levels increased throughout growth even though AI-2 activity was only maximally detected at the mid-exponential phase of growth and disappeared by the onset of stationary phase. Similar results were obtained with E. coli DH5α transformed with luxS, suggesting that AI-2 production is not limited by a lack of LuxS protein. Analysis of Rgp and Kgp protease activities revealed that the P. gingivalis luxS mutant produced around 45% less Rgp and 30% less Kgp activity than the parent strain. In addition, the luxS mutant exhibited a fourfold reduction in haemagglutinin titre. However, these reductions in virulence determinant levels were insufficient to attenuate the luxS mutant in a murine lesion model of P. gingivalis infection.

Variable carbohydrate modifications to the catalytic chains of the RgpA and RgpB proteases of Porphyromonas gingivalis W50.

Abstract: The irreversible tissue destruction which is characteristic of the destructive periodontal diseases is considered to be a consequence of the reaction by a susceptible host to a complex and variable microbial challenge presented by the subgingival plaque. Porphyromonas gingivalis, an anaerobic, gram-negative bacterium, is frequently isolated from the subgingival plaque of periodontal patients and is thought to be an important etiological agent in these conditions (28, 46). P. gingivalis produces several extracellular proteolytic enzyme activities with different peptide bond specificities which have a number of in vitro properties consistent with a role in the periodontal disease process (11). These include deregulatory effects on plasma cascades (21, 35, 49) and the specific and innate host defenses (45, 51), activation of matrix metalloproteases (13), degradation of connective tissue components (22), and interference with host cell function (37). Many of these actions have been shown to be a function of the activity of P. gingivalis proteases with specificity for Arg-x peptide bonds, and therefore there is some justification for regarding these enzymes as important virulence determinants in the periodontal diseases. The extracellular Arg-x protease activity of P. gingivalis W50 is composed of three enzyme species (HRgpA, RgpA, and mt-RgpA), all derived from rgpA. These designations replace our earlier nomenclature of RI, RIA, and RIB, all derived from prpR1 (1, 10, 41). HRgpA is a heterodimer in which the catalytic α chain (Mr ∼ 54,000) is noncovalently associated with an adhesin chain (β), derived from the initial RgpA translation product, which is capable of mediating binding to the erythrocyte surface and host macromolecules. RgpA is the free monomeric catalytic chain, and membrane-type RgpA (mt-RgpA) is a highly posttranslationally modified form of this chain (Mr ∼ 70,000 to 80,000) which is exclusively associated with the membrane fraction (1, 10, 41). Two additional proteases with Arg-x specificity, RgpB and mt-RgpB (formerly RIIA and RIIB), were detected in the culture supernatant of an rgpA isogenic mutant of P. gingivalis W50 (42). These two forms, which closely resemble the monomeric proteases derived from rgpA, are produced from a second gene, rgpB (prR2), which lacks the coding region for the adhesin chain. RgpB and mt-RgpB may correspond to proteases which are normally cell associated in the wild-type strain W50. These enzymes have not been demonstrated in the culture supernatants of strain W50. Analysis of the structures and properties of the RgpA and RgpB proteases of P. gingivalis has provided some insights into the molecular survival strategies adopted by an organism whose sole ecological site in the oral cavity is the microbial biofilm in the hostile environment of an inflamed periodontal pocket. For example, these enzymes have been described as extremely efficient C3 and C5 convertases whose activity leads to the fluid-phase inactivation of these key components of the host’s defensive armory (51). Furthermore, while a primary function of the β component of the HRgpA heterodimer may be to target the action of this isoform (39), analysis of the antibody response to this protease in humans or experimental animal models indicates that the β component may also have a role in subversion of the very significant, specific immune response of the colonized host (10, 17, 23). Shielding the catalytically active component of the molecule with a highly immunogenic protein partner may effectively divert the antibody response from regions of the molecule directly involved in proteolysis. A similar strategy has been described for Trypanosoma cruzi, in which a long C-terminal extension to the catalytic domain of the cysteine protease, cruzipain, has been suggested to perform this role (31). In this work, we wished to extend our previous immunochemical investigations (8, 10) to examine the structure and immunogenicity of each of the RgpA and RgpB proteases by the development of monoclonal antibodies (MAbs) to the catalytic chain of each of these enzymes. To avoid complications arising from the immunogenicity of the β component of HRgpA, these experiments were performed with the single-chain RgpA isoform. The results of the study demonstrate the presence of immunogenic, covalently linked carbohydrate additions to the catalytic chain of some of the enzymes of this family of proteases. Glycosylation of these bacterial proteins may have an influence on the stability of not only the resulting glycoconjugate but also their immune recognition.

Are dental diseases examples of ecological catastrophes

Abstract: Dental diseases are among the most prevalent and costly diseases affecting industrialized societies, and yet are highly preventable. The microflora of dental plaque biofilms from diseased sites is distinct from that found in health, although the putative pathogens can often be detected in low numbers at normal sites. In dental caries, there is a shift towards community dominance by acidogenic and acid-tolerant Gram-positive bacteria (e.g. mutans streptococci and lactobacilli) at the expense of the acid-sensitive species associated with sound enamel. In contrast, the numbers and proportions of obligately anaerobic bacteria, including Gram-negative proteolytic species, increase in periodontal diseases. Modelling studies using defined consortia of oral bacteria grown in planktonic and biofilm systems have been undertaken to identify environmental factors responsible for driving these deleterious shifts in the plaque microflora. Repeated conditions of low pH (rather than sugar availability per se) selected for mutans streptococci and lactobacilli, while the introduction of novel host proteins and glycoproteins (as occurs during the inflammatory response to plaque), and the concomitant rise in local pH, enriched for Gram-negative anaerobic and asaccharolytic species. These studies emphasized (a) significant properties of dental plaque as both a biofilm and a microbial community, and (b) the dynamic relationship existing between the environment and the composition of the oral microflora. This research resulted in a novel hypothesis (the ‘ecological plaque hypothesis’) to better describe the relationship between plaque bacteria and the host in health and disease. Implicit in this hypothesis is the concept that disease can be prevented not only by directly inhibiting the putative pathogens, but also by interfering with the environmental factors driving the selection and enrichment of these bacteria. Thus, a more holistic approach can be taken in disease control and management strategies.

Outer-membrane vesicles from Gram-negative bacteria: biogenesis and functions

Abstract: In this Review, Schwechheimer and Kuehn describe recent developments in elucidating the mechanisms of biogenesis and cargo selection of the outer-membrane vesicles (OMVs) produced by Gram-negative bacteria. They also discuss the functions of OMVs in bacterial physiology and during pathogenesis. Outer-membrane vesicles (OMVs) are spherical buds of the outer membrane filled with periplasmic content and are commonly produced by Gram-negative bacteria. The production of OMVs allows bacteria to interact with their environment, and OMVs have been found to mediate diverse functions, including promoting pathogenesis, enabling bacterial survival during stress conditions and regulating microbial interactions within bacterial communities. Additionally, because of this functional versatility, researchers have begun to explore OMVs as a platform for bioengineering applications. In this Review, we discuss recent advances in the study of OMVs, focusing on new insights into the mechanisms of biogenesis and the functions of these vesicles.

Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors

Abstract: Porphyromonas gingivalis, the keystone pathogen in chronic periodontitis, was identified in the brain of Alzheimer's disease patients. Toxic proteases from the bacterium called gingipains were also identified in the brain of Alzheimer's patients, and levels correlated with tau and ubiquitin pathology. Oral P. gingivalis infection in mice resulted in brain colonization and increased production of Aβ1-42, a component of amyloid plaques. Further, gingipains were neurotoxic in vivo and in vitro, exerting detrimental effects on tau, a protein needed for normal neuronal function. To block this neurotoxicity, we designed and synthesized small-molecule inhibitors targeting gingipains. Gingipain inhibition reduced the bacterial load of an established P. gingivalis brain infection, blocked Aβ1-42 production, reduced neuroinflammation, and rescued neurons in the hippocampus. These data suggest that gingipain inhibitors could be valuable for treating P. gingivalis brain colonization and neurodegeneration in Alzheimer's disease.

Life Below the Gum Line: Pathogenic Mechanisms of Porphyromonas gingivalis

Abstract: Porphyromonas gingivalis, a gram-negative anaerobe, is a major etiological agent in the initiation and progression of severe forms of periodontal disease. An opportunistic pathogen, P. gingivalis can also exist in commensal harmony with the host, with disease episodes ensuing from a shift in the ecological balance within the complex periodontal microenvironment. Colonization of the subgingival region is facilitated by the ability to adhere to available substrates such as adsorbed salivary molecules, matrix proteins, epithelial cells, and bacteria that are already established as a biofilm on tooth and epithelial surfaces. Binding to all of these substrates may be mediated by various regions of P. gingivalis fimbrillin, the structural subunit of the major fimbriae. P. gingivalis is an asaccharolytic organism, with a requirement for hemin (as a source of iron) and peptides for growth. At least three hemagglutinins and five proteinases are produced to satisfy these requirements. The hemagglutinin and proteinase genes contain extensive regions of highly conserved sequences, with posttranslational processing of proteinase gene products contributing to the formation of multimeric surface protein-adhesin complexes. Many of the virulence properties of P. gingivalis appear to be consequent to its adaptations to obtain hemin and peptides. Thus, hemagglutinins participate in adherence interactions with host cells, while proteinases contribute to inactivation of the effector molecules of the immune response and to tissue destruction. In addition to direct assault on the periodontal tissues, P. gingivalis can modulate eucaryotic cell signal transduction pathways, directing its uptake by gingival epithelial cells. Within this privileged site, P. gingivalis can replicate and impinge upon components of the innate host defense. Although a variety of surface molecules stimulate production of cytokines and other participants in the immune response, P. gingivalis may also undertake a stealth role whereby pivotal immune mediators are selectively inactivated. In keeping with its strict metabolic requirements, regulation of gene expression in P. gingivalis can be controlled at the transcriptional level. Finally, although periodontal disease is localized to the tissues surrounding the tooth, evidence is accumulating that infection with P. gingivalis may predispose to more serious systemic conditions such as cardiovascular disease and to delivery of preterm infants.

Communication among Oral Bacteria

Abstract: Human oral bacteria interact with their environment by attaching to surfaces and establishing mixed-species communities. As each bacterial cell attaches, it forms a new surface to which other cells can adhere. Adherence and community development are spatiotemporal; such order requires communication. The discovery of soluble signals, such as autoinducer-2, that may be exchanged within multispecies communities to convey information between organisms has emerged as a new research direction. Direct-contact signals, such as adhesins and receptors, that elicit changes in gene expression after cell-cell contact and biofilm growth are also an active research area. Considering that the majority of oral bacteria are organized in dense three-dimensional biofilms on teeth, confocal microscopy and fluorescently labeled probes provide valuable approaches for investigating the architecture of these organized communities in situ. Oral biofilms are readily accessible to microbiologists and are excellent model systems for studies of microbial communication. One attractive model system is a saliva-coated flowcell with oral bacterial biofilms growing on saliva as the sole nutrient source; an intergeneric mutualism is discussed. Several oral bacterial species are amenable to genetic manipulation for molecular characterization of communication both among bacteria and between bacteria and the host. A successful search for genes critical for mixed-species community organization will be accomplished only when it is conducted with mixed-species communities.