WO2013025099A1 - Substrats pour la détection de périopathogènes de la cavité buccale - Google Patents

Substrats pour la détection de périopathogènes de la cavité buccale Download PDF

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WO2013025099A1
WO2013025099A1 PCT/NL2012/050565 NL2012050565W WO2013025099A1 WO 2013025099 A1 WO2013025099 A1 WO 2013025099A1 NL 2012050565 W NL2012050565 W NL 2012050565W WO 2013025099 A1 WO2013025099 A1 WO 2013025099A1
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Prior art keywords
substrate
gingivalis
lysine
arginine
amino acid
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PCT/NL2012/050565
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Floris Jacob Bikker
Wendy Esmeralda Kaman-Van Zanten
John Philip HAYS
Engelmundus Cornelis Ignatius Veerman
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Vereniging Voor Christelijk Hoger Onderwijs, Wetenschappelijk Onderzoek En Patiëntenzorg
Stichting Vu-Vumc
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Publication of WO2013025099A1 publication Critical patent/WO2013025099A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06086—Dipeptides with the first amino acid being basic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06086—Dipeptides with the first amino acid being basic
    • C07K5/06095—Arg-amino acid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody

Definitions

  • the present invention relates to Porphyromonas gingivalis specific substrates useful in the detection of Porphyromonas gingivalis.
  • the substrates may be
  • periopathogens used as indicators for infection by periopathogens, in particular those infecting the oral cavity and are also useful as indicators of periodontitis and peri- implant disease.
  • Periodontitis is an inflammation of the periodontium, the tissues that
  • Severe periodontitis affects at least 10% of the general population (35). It is caused by micro-organisms that adhere to and
  • Periodontitis involves progressive loss of the alveolar bone around the teeth, and if left untreated, can lead to the
  • Periodontitis include Porphyromonas gingivalis, Aggregatobacter
  • Perio-pathogens notably P. gingivalis, secrete proteases acting as virulence
  • periodontal patients in cases of low prevalence of perio- pathogens, removal of microbial plaque and calculus and non-surgical cleaning below the gumline is often sufficient to restore periodontal health, especially if the periodontal pockets are shallower than 4-5 mm (51) If non-surgical therapy is unsuccessful in managing signs of disease activity, periodontal surgery, to definitively remove calculus and manage surgical management of bony irregularities which have resulted from the disease process to reduce pockets, may be necessary. Though, additional periodontal disease treatments, such as antibiotic treatment may be necessary for successful therapy.
  • antibiotic treatment may be crucial (13,40).
  • One object of the disclosure is to provide a method for detecting the presence of a periopathogen, preferably Porphyromonas gingivalis, and/or its virulence factors in a sample comprising incubating said sample with a substrate and detecting cleavage of said substrate, wherein said substrate comprises i) a dipeptide consisting of amino acids Xi and X2 and ii) a set of markers that distinguish whether said substrate is intact or cleaved; wherein, Xi is an arginine or lysine, X2 is a charged amino acid selected from arginine, aspartic acid, glutamic acid, histidine, and lysine, and Xi or X2 is a D-amino acid.
  • a periopathogen preferably Porphyromonas gingivalis, and/or its virulence factors in a sample
  • said substrate comprises i) a dipeptide consisting of amino acids Xi and X2
  • a further object of the disclosure is to provide a method for determining whether an individual is at risk of developing or having an oral disease, preferably peri-implant disease or periodontitis, comprising incubating a sample obtained from an individual at risk thereof with a substrate and detecting cleavage of said substrate, wherein said substrate comprises i) a dipeptide consisting of amino acids Xi and X2 and ii) a set of markers that distinguish whether said substrate is intact or cleaved; wherein, Xi is an arginine or lysine, X2 is a charged amino acid selected from arginine, aspartic acid, glutamic acid, histidine, and lysine, and Xi or X2 is a D-amino acid.
  • the sample is obtained from the oral cavity, preferably said sample is saliva or subgingival plaque.
  • a further object of the disclosure is to provide a substrate comprising i) a dipeptide consisting of amino acids Xi and X2 and ii) a set of markers that distinguish whether said substrate is intact or cleaved; wherein, Xi is an arginine or lysine, X2 is a charged amino acid selected from arginine, aspartic acid, glutamic acid, histidine, and lysine, Xi or X2 is a D-amino acid, and wherein said substrates is cleavable by a Porphyromonas gingivalis protease.
  • Xi is arginine.
  • X2 is the D-amino acid.
  • Xi and X2 are selected from Arg-DAsp, Arg-DGlu, Arg-DHis, Arg-DLys, or Arg- DArg.
  • the set of markers is a fluorescent label and a quencher for said fluorescent label.
  • the substrate comprises FITC linked to aminohexonic acid and DABCYL linked to lysine.
  • the substrate does not comprise a dipeptide of charged L-amino acids, wherein the N-terminal L-amino acid is an arginine, preferably an arginine or lysine.
  • the substrate has the formula:
  • Xi is an arginine or lysine
  • X2 is a charged amino acid selected from arginine, aspartic acid, glutamic acid, histidine, and lysine
  • Xi or X2 is a D-amino acid
  • Y is any amino acid and a and b are from 0-10
  • Li is an optional linker
  • L2 is an optional linker
  • Markerl and Marker2 are markers that distinguish whether said substrate is intact or cleaved.
  • Markerl is FITC
  • Li is aminohexonic acid
  • Marker2 is DABCYL
  • a and b are 0.
  • a further object of the disclosure is to provide a biosensor comprising a solid support and the substrate according to the invention.
  • the biosensor is in the form of a test strip.
  • Bacterial culture has long been considered the "gold standard” diagnostic method to detect and quantify the microflora colonizing the oral cavities and to create an antibiogram. Anaerobic culture is specific in its ability to distinguish species. However, it has limitations compared to the PCR: it is time
  • PCR rapid-time PCR
  • bacterial DNA needs to be extracted and isolated from the matrix, which can be also costly and laborious.
  • specificity of PCR can be problematic; the oral and subgingival microflora is extensive and diverse (Keijser et al., 2008), cross-reactivity with other (unknown) species is conceivable.
  • the PCR method does not provide evidence on pathogen viability and bacterial activity and may be less effective in samples in which bacterial DNA has been degraded, such as in saliva.
  • a further disadvantage of both PCR and bacterial culture is that a medical practitioner cannot obtain the results of the test during a single patient visit.
  • One objective of the present invention to overcome the above disadvantages.
  • the present invention is based on the identification of substrates that react specifically with P. gingivalis.
  • Bacterial enzymes such as proteases, have been used as biomarkers for quick and sensitive identification of microorganisms in clinical samples (Manafi et al., 1991). Many of these enzymes are released into the surrounding microenviroment and are accessible for detection based on sensitive fluorogenic and/or luminogenic substrates. The cleavage of these substrates indicates the presence of a protease.
  • P. gingivalis secretes a variety of proteases acting as virulence factors that allow these bacteria to invade the host tissues.
  • Human secretions or fluids such as human saliva or serum also contain proteases and can therefore interfere with diagnostic assays using cleavable substrates.
  • substrates which consist of D- amino acids are only cleaved by bacterial derived proteases (WO2010/059051).
  • diagnostic methods using the substrates provided herein are easy to perform and require a minimum of experimental steps due to the specific character of the substrates.
  • time- consuming enzyme pre-enrichment or purification steps are not required, thus offering the potential to development of a chair-side test.
  • substrates that are specifically cleaved by P. gingivalis proteases we have been able to detect with high specificity the presence of P.
  • the present diagnostic methods substantially reduce the false positive rate in comparison to the commercially available test strips currently used as a point-of-care tests for P. gingivalis, T. denticola, T. forsythensis (e.g., BANA test strips; U.S. Patent No. 5, 115, 735).
  • Periopathogens of the oral cavity are bacteria involved in the development of peri-implant diseases and periodontal diseases.
  • the procedure to replace lost natural teeth by implants offers an unwanted opportunity for bacterial colonization.
  • peri-implant epithelium may be colonized, and infected, with increased numbers and proportions of oral bacterial species, in analogy to the increase of microorganisms in deep periodontal pockets adjacent to natural teeth.
  • peri-implant diseases The inflammatory lesions that develop in the tissue around dental implants are collectively recognized as peri-implant diseases. Manifestation of peri- implant diseases represents a widespread problem.
  • One target bacterial species in peri-implant diseases is Porphyromonas gingivalis (Botero et al., 2005).
  • the presence or absence of P. gingivalis in peri-implant sites may be indicative of a pathogenic microflora, possibly one that is causing peri- implantitis, i.e. infection of tissue and bone around the dental implant. Therefore, the presence of P. gingivalis can be used as marker for a potentially pathogenic microflora.
  • one aspect of the disclosure provides a method for detecting the presence of a periopathogen and/or its virulence factors in a sample, comprising incubating said sample with one or more of the substrates disclosed herein and detecting cleavage of said substrate.
  • P. gingivalis specific proteases most likely Arg-gingipain and/or Lys-gingipain, are responsible for the substrate cleavage.
  • These cysteine peptidases are major virulence factors of P. gingivalis and are known to degrade host proteins like immunoglobulins G, transferrin, fibronectin and anti-microbial peptides present in saliva.
  • the presence of one of the proteases indicates the presence of P. gingivalis in the sample and in turn indicates the presence of or likelihood of colonization of a periopathogen.
  • a periopathogen refers to potentially pathogenic microflora of the oral cavity and includes, e.g., Porphyromonas gingivalis, Aggregatobacter actinomycetemcomitans, Prevotella intermedia, Bacterioides forsythius and Treponema denticola.
  • virulence factors in a sample, even if the sample itself does not comprise a periopathogen. This may occur, e.g., if a bacterial infection is concentrated within a dental implant.
  • a saliva sample obtained from the infected individual may therefore not contain bacteria, or at least a significant amount of bacteria, but may still contain one or more virulence factors.
  • Virulence factors are molecules which are expressed and secreted by bacteria and include, e.g., enzymes, such as proteases, and toxins. These factors generally assist bacteria in the colonization of a host or in invading the host's immune response.
  • the main virulence factors of P. gingivalis are thought to be its extracellular cysteine proteases, known collectively as the gingipains. Most common are RgpA and RgpB (the Arg- gingipains) and Kgp (the Lys-gingipain).
  • the Arg- gingipains cleave at the carboxyl side of Arg residues and the Lys- gingipains cleave at the carboxyl side of Lys residues.
  • the virulence factors detected by the methods of the invention are proteases.
  • a method for determining the risk of developing or having an oral disease in an individual comprising incubating a sample obtained from an individual at risk thereof with a substrate as disclosed herein and detecting cleavage of said substrate.
  • an oral disease refers to a bacterial infection of the oral cavity and disorders resulting from said infection.
  • the oral disease is periodontal disease ⁇ e.g., gingivitis, adult periodontitis, early-onset
  • periodontitis or peri-implant disease.
  • Cleavage of a substrate as provided herein indicates that the sample contains P. gingivalis or a virulence factor thereof, such as a protease.
  • cleavage of a substrate of the invention is a useful indicator of the oral health of an individual and allows a skilled person to determine the risk of an individual of developing or having an oral disease.
  • sample in the methods described herein includes biological samples, e.g., a plaque sample, gingival fluid, serum, or tissue biopsy.
  • the sample is obtained from the oral cavity.
  • the sample is saliva or subgingival plaque, more preferably saliva.
  • saliva refers to an oral fluid and can be unstimulated or stimulated. Unstimulated saliva means that the subject will expectorate in a collection vessel without stimulation of salivary flow, whereas the production of saliva may be stimulated by chewing on a piece of paraffin film.
  • the present invention also encompasses methods wherein said sample is incubated with more than one of the cleavable substrates.
  • said sample is incubated with at least 2, at least 3, or at least 4 of the cleavable substrates provided herein.
  • the sample may be incubated with the different substrates either simultaneously, separately, or subsequently. Preferably, they are incubated separately.
  • the incubation conditions required for carrying out the methods described herein can be readily determined by a skilled person and include standard factors such as the time of incubation and the temperature at which the incubation is carried out.
  • the incubation is performed at a temperature of between 30-40°C, more preferably at around 37°C.
  • substrates comprising a dipeptide having a lysine or arginine residue are efficiently cleaved by P. gingivalis culture supernatant. Furthermore, substrates containing a D-amino acid in the cleavable dipeptide have high specificity with a low incidence of false positives (0% - 4%). P. gingivalis reactive substrates composed of L-amino acids have a much higher rate of false positives, namely 16% - 59%. Accordingly, in a further aspect of the disclosure, cleavable substrates are provided.
  • substrates are useful for detecting the presence of a periopathogen and/or its virulence factors in a sample and for determining the risk in an individual of developing or having an oral disease, as disclosed herein.
  • the disclosure provides substrates comprising i) a dipeptide consisting of amino acids Xi and X2, preferably both amino acids are charged and ii) a set of markers that distinguish whether said substrate is intact or cleaved.
  • the dipeptide is linked directly or indirectly to the set of markers.
  • At least one of the amino acids of the dipeptide is an arginine or lysine residue.
  • the other amino acid may vary and is preferably a charged amino acid, i.e., selected from arginine, aspartic acid, glutamic acid, histidine, and lysine, as shown in Table 1.
  • Xi is arginine or lysine, more preferably Xi is arginine, and X2 is a charged amino acid.
  • Xi is arginine and X2 is a charged amino acid.
  • X2 is the D-amino acid.
  • the dipeptide is Arg-DAsp, Arg-DGlu, Arg-DHis, Arg-DLys, or Arg-DArg.
  • Xi is lysine and X2 is a charged amino acid.
  • X2 is the D-amino acid. More preferably, the dipeptide is Lys- DAsp, Lys -DGlu, Lys DHis, Lys-DLys, or Lys-DArg, even more preferably the dipeptide is Lys-DLys.
  • the dipeptide containing a D-amino acid is selected from Arg-Asp, Arg-Glu, Arg-His, Arg-Lys, Arg-Arg, Lys-Lys, and Phe-Arg; more preferably the dipeptide is selected from Arg-Asp, Arg-Glu, Arg-His, Arg- Lys, Arg-Arg, Lys-Lys, even more preferably the dipeptide is selected from Arg-His, Arg-Lys, and Arg-Arg.
  • the amino acid in position 2 of the dipeptide is the D-amino acid.
  • the substrates are depicted by formula:
  • Xi and/or X2 is an arginine or lysine and Xi or X2 is a D-amino acid;
  • Y is independently any amino acid and a and b are from 0-10;
  • Li is an optional linker, preferably Ahx; Li is an optional linker, preferably lysine;
  • Marker 1 and Marker2 are markers that distinguish whether said substrate is intact or cleaved.
  • the dipeptide Xi and X2 may be flanked by additional amino acids, depicted above in the formula as (Y)a and (Y)b.
  • Y may be any amino acid and the identity of amino acids N-terminal to the dipeptide is independent from the identity of amino acids C-terminal to the dipeptide.
  • the substrate may comprise the sequence Gly-Arg-DAsp-Pro linked to a set of markers.
  • the number of flanking amino acids, i.e., a and b are from 0-10, preferably from 0-5, more preferably from 0-2, and even more preferably 0.
  • a and b may be 0, but the substrate may contain additional amino acids in the form of a linker, such as a lysine linker.
  • the substrates disclosed herein comprise less than 30 amino acids, less than 20 amino acids, preferably less than 10 amino acids, and more preferably less than 5 amino acids.
  • the dipeptide is flanked on its C-terminus with a lysine residue acting as a linker. It is apparent to the skilled person that the additional amino acid(s) should not introduce an additional P. gingivalis specific cleavage site that can be cleaved by non-bacterial proteases.
  • the addition of an L-arginine-L- arginine dipeptide to the D-amino acid containing dipeptides described above would result in a substrate cleavable by human proteases found in saliva (Table 2).
  • the substrate consists of a single P.
  • the substrate does not comprise a dipeptide wherein at least one of the amino acids is an arginine or lysine and both amino acids are charged L-amino acids.
  • the disclosure also encompasses substrates that comprise more that one dipeptide of Xi and X2 as described herein. The addition of additional cleavable dipeptides specific for P. gingivalis in a single substrate may increase the sensitivity of the substrate.
  • the substrates comprise at least two cleavable dipeptides.
  • Detection of substrate cleavage is based on the use of markers that can distinguish between an intact and cleaved substrate.
  • Suitable molecular markers are well-known to one of skill in the art and include, e.g., the color changes of gold colloids by cysteinyl derivatives (see, e.g., Guarise, C. et al.,
  • a peptide having acetylated cysteines on both the N- and C-terminus interferes with the aggregation of nanometer- sized gold colloids and introduces a shift in visible color.
  • Cleaved peptides have only one acetylated cysteine terminus and lack of ability to introduce a color change.
  • the set of markers is a set of acetylated cysteine residues.
  • substrates using these markers include those having the formula: Marker 1 - (Y)a - Xi - X2 - (Y)b - Marker2; wherein Marker 1 and Marker2 are an acetylated cysteine residue.
  • substrates are provided as follows:
  • More preferred substrates are as follows:
  • a preferred method for detecting cleavage of a substrate by an enzyme is to label the substrate with two different dyes, where one serves to quench the fluorescence of the other dye by fluorescence energy transfer (FRET) when the molecules, for example, dyes or colorimetric substances are in close proximity, and is measured by detecting changes in fluorescence.
  • FRET fluorescence energy transfer
  • Fluorescence resonance energy transfer is the process of a distance-dependent excited state interaction in which the emission of one fluorescent molecule is coupled to the excitation of another. If the two molecules are in close proximity, then excitation of the donor molecule is transferred to the acceptor molecule without the emission of a photon. If a substrate as described herein is intact, then energy transfer is efficient. Cleavage of a substrate results in the donor and acceptor molecule no longer being in close proximity and energy transfer is inefficient. This results in either a decrease in the fluorescence of the acceptor or an increase in fluorescence of the donor.
  • the set of markers comprises a fluorecent label, preferably FITC, and a quencher for said label, preferably DABCYL.
  • Suitable donor-acceptor pairs and their respective excitation and emission wavelengths are known in the art.
  • Abz (2-aminobenzoyl) is usually used as a donor with Dnp (2,4-dinitropheny) or EDDnp (N-(2,4- dinitropheny)ethylenediamine) as acceptors, i.e., quenchers.
  • N-Me-Abz N- methyl-anthraniloyl
  • 5-FAM 5-carboxyfluorescein, single isomer
  • FITC fluorescein-5-isothiocyanate, isomers
  • Mca (7-methoxycoumarin-4- yl)acetyl) are usually used as donors with Dnp as the quencher.
  • the set of molecular markers is a donor-acceptor pair as described above, for example: Markerl is Abz and Marker2 is Dnp or EDDnp;
  • Markerl is Dnp or EDDnp and Marker2 is Abz;
  • Markerl is N-Me-Abz, 5-FAM, FITC, or Mca and Marker2 is Mca;
  • Marker2 is N-Me-Abz, 5-FAM, FITC, or Mca and Markerl is Dnp;
  • Markerl is EDANS, FITC, or Lucifer yellow and Marker2 is DABCYL;
  • Marker2 is EDANS, FITC, or Lucifer yellow and Markerl is DABCYL.
  • FITC and DABCYL are used as the FRET pairs.
  • Substrate cleavage can be detected at 520nm by using an excitation wavelength of 490nm.
  • FITC is preferably used in conjuction with aminohexonic acid
  • a lysine residue is preferably used as a linker for DABCYL.
  • the substrate consists of a cleavable dipeptide as disclosed herein, a set of markers that distinguish whether said substrate is intact or cleaved, and, optionally, linkers to link the dipeptide to the markers.
  • the substrate is selected from BikKam9, BikKamlO, BikKamll, BikKaml2, and BikKaml3 (see Table 1).
  • Markerl is FITC
  • Li is aminohexonic acid (Ahx) linked to the dipeptide depicted in Table 1, which is linked via a Lysine residue (L2) to Marker2, which is DABCYL.
  • no extra amino acids are introduced thus a and b from the formula (Y)a and (Y)b are both zero.
  • a further aspect of the disclosure provides a biosensor comprising a solid support and a substrate as disclosed herein.
  • Support materials include, for example, organic or inorganic polymers, biopolymers, natural and synthetic polymers, polysaccharides, paper, nitrocellulose, cotton, polyester, rayon, nylon, polyethersulfone, and polyethylene.
  • the support is in the form of a test strip having a semi-rigid support and may comprise, e.g., poly(vinyl chloride), polypropylene, polyester, and polystyrene.
  • to comprise and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded.
  • verb "to consist” may be replaced by "to consist essentially of meaning that a compound or adjunct compound as defined herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention.
  • an element means one element or more than one element.
  • the word “approximately” or “about” when used in association with a numerical value preferably means that the value may be the given value of 10 more or less 1% of the value.
  • An "individual” refers to a mammal including primates and domesticated animals such as dogs, cats, sheep, cattle, goats, pigs, mice rats, and rabbits. Preferably the individual is a human.
  • Example 1 Design of a substrate library.
  • the library consisted of fluorogenic substrate peptides, each comprising two amino acids where; i) both amino acids were L-amino acids, ii) the C-terminally located amino acid was a D-amino acid, and iii) both amino acids were D-amino acids. All peptides were flanked with an Ahx-linked FITC probe at the N-terminus and a lysine coupled Dabcyl (KDbc) quencher at the C-terminus.
  • the library consisted of 119 peptides in total ( Figure 3).
  • culture supernatant of P. gingivalis was incubated with all the FRET-peptides present within the library. Of the 119 peptides, eight peptides were cleaved by the P. gingivalis culture supernatant.
  • FRET- library peptides cleaved by P. gingivalis culture supernatant.
  • FITC Fluorescein isothiocyanate
  • Dbc Dabcyl
  • Aminohexonic acid (Ahx) and lysine (Lys) were used as linkers.
  • BikKam9-13 correspond to "A-E", respectively, as used in Tables 6-7.
  • the peptides were incubated with culture supernatants from eight different P. gingivalis strains and twelve other oral pathogens (Table 1). All 8 substrates were cleaved by all P. gingivalis strains tested except for BikKam9, which was not cleaved by the culture supernatant of P. gingivalis K2. Further, cleavage of the substrates varied in efficiency; BikKam9-10 showed the lowest cleavage activity, whereas BikKaml4-16 showed the highest cleavage activity (Table 2). It was found that the majority of the substrates cleaved by P. gingivalis enzymes were not degraded by any of the other oral pathogens tested, the only exception being BikKaml5, which was cleaved by the culture supernatant of T. forsythensis (Table 2).
  • RF Relative Fluorescence per minute
  • Example 3 Mapping the proteolytic characteristics of the P.
  • gingivalis the enzymes responsible for the cleavage of the substrates could be the P. gingivalis specific Arg-gingipain and Lys-gingipain peptidases. These gingipains are members of the cysteine peptidase family. Therefore we verified in addition whether; i) the addition of L-cysteine and DTT could stimulate cleavage and ii) inhibitors such as NEM and leupeptin could inhibit proteolytic activity.
  • Example 4 Proteolytic activity of the P. gingivalis gingipain knockout strains
  • BikKaml5 was 10 7 CFU/ml ( Figure 2).
  • BikKaml4 and BikKaml5 showed the best susceptibility with a limit of detection of 10 6 CFU/ml ( Figure 2).
  • Example 6 Screening of patient derived paper points using the P. gingivalis substrates
  • the applicability of the novel protease assay was examined by screening 72 paper points obtained from individuals suffering from periodontitis and were characterized for the presence of perio-pathogens by routine culture as a gold standard. Twenty-three of the paper points were P. gingivalis culture positive, varying in P. gingivalis concentration from 10 8 - 10 3 CFU/ml. All 72 paper points were analyzed using all 8 substrates. P. gingivalis could be detected to a concentration of 10 6 - 10 7 CFU/ml using BikKam9 and BikKamlO and no false positive activity was observed (Table 4).
  • BikKamll-13 showed a somewhat higher sensitivity (70 - 74%), however these substrates were also cleaved by a few of the P. gingivalis culture negative samples (Table 4).
  • microbiological culture diagnosis Some of the paper points analyzed contained, besides P. gingivalis, other oral pathogens such as P. micros, P. intermedia or F. nucleatum nucleatum. No association was observed between the false positives and the presence of other oral pathogens (data not shown).
  • Table 4 Screening of patient derived paper points using P. gingivalis substrates a .
  • the demographic and clinical characteristics of the subjects included in the study are presented in Table 5.
  • the Mucositis, Implantitis and Cs groups had higher values in age when compared with the Cni and Cr groups.
  • Forty-nine females and 48 males participated in the current study. The great majority of subjects (85%) were Caucasian. Of the 97 participants, 18% were current smokers; Cni group showed the highest rate of smokers. Further, almost 30% of this study population had a previous history of periodontal disease.
  • All the study subjects had an average of at least 15 teeth present, with the averages ranging from a minimum of 15.5 for the Implantitis group and a maximum of 24.9 for the Cni group.
  • the average number of implants was 2.7.
  • the Cni group had no dental implants by definition and served as a reference group having an edentulous area, potentially suitable for implant therapy.
  • Values represent numbers (%) of subjects or means ⁇ SD.
  • Mucositis peri-implant mucositis group; Implantitis, peri-implantitis group; Cni, periodontal healthy with no implant group; Cr, periodontal healthy with recent implant placement; Cs, steadily healthy implant for at least 6 months; BOP, bleeding on probing; PPD, probing pocket depth; 9 > female; male.
  • Table 6 depicts the prevalence of P. gingivalis by anaerobic culture, real-time PCR and FRET technique for the 97 subgingival plaque samples.
  • P. gingivalis --positive samples were detected more often in peri-implant diseased patients than in the healthy control groups.
  • the subgingival plaque samples from 6 of 20 Implantitis patients were cultured positive for the presence of P. gingivalis.
  • Peri-implant mucositis group also contributed with 1 P. gingivalis--positive sample. Only 4 (7%) out of 57 samples revealed the presence of P. gingivalis when Cni, Cr, and Cs where grouped together.
  • PCR had a higher rate of P. gingivalis prevalence for the Cni group than for the other groups. We speculated that this occurred because of the high number of patients with a previous history of periodontal disease in this group. This finding supported the notion that the residual pockets acted as reservoirs for colonization of the implant surfaces.
  • Table 7 shows the prevalence of P. gingivalis by anaerobic culture, real-time PCR and FRET techniques for the salivary samples.
  • Culture technique gave the lowest prevalence for the P. gingivalis- iected samples among all different groups. For instance, P. gingivalis was not found by bacterial culture in any of the 19 Cs group subjects. Differently, PCR and FRET technique seemed more capable to detect P. gingivalis in saliva. In fact, 6 and 4 P. gingivalis-positive samples were detected by PCR in the Mucositis and Implantitis groups, respectively, while FRET detected larger numbers of P. girigivalis -positive samples, respectively 8 (40%) and 6 (30%) positive samples in the two peri- implant diseased patients' groups.
  • Table 6 Results from P. gin,givalis--positive subgingival plaque samples of the selected sites in the various study groups: prevalence in values represents numbers (%) of subjects or mean total CFU/ml xlO 6 ⁇ SD in case of culture or counts/ml xlO 6 ⁇ SD in case of PCR or relative fluorescence/min ⁇ SD in case of FRET.
  • Table 7 Results from P. gingivalis-positive salivary samples in the various study groups: prevalence in values represents numbers (%) of subjects or mean total CFU/ml xl06 ⁇ SD in case of culture or counts/ml xl06 ⁇ SD in case of PCR or relative fluorescence/min ⁇ SD in case of FRET.
  • Porphyromonas gingivalis The mean total counts, mean counts of P.g. species and mean of enzyme activity for each enzyme were calculated only including P.g. positive individuals. Samples with enzyme activity (RF/min) value > 5 were considered positive to the detection of P. gingivalis. Table 6
  • BikKam 12 (RF/min) 8.24 59.70 ⁇ 74.34 10.82 0 0
  • BikKam 10 (RF/min) 0.54 ⁇ 1.46 -0.44 ⁇ 1.43 4.08 ⁇ 5.51 -4.75 ⁇ 10.32 1.34 ⁇ 3.10 BikKam 11 (RF/min) 5.01 ⁇ 3.87 4.80 ⁇ 5.90 8.65 ⁇ 8.52 -0.81 ⁇ 10.70 4.03 ⁇ 3.07 BikKam 12 (RF/min) 3.30 ⁇ 1.41 3.31 ⁇ 3.30 9.19 ⁇ 8.83 15.80 ⁇ 27.00 2.55 ⁇ 1.54 BikKam 13 (RF/min) 5.27 ⁇ 2.73 8.55 ⁇ 5.44 1.03 ⁇ 8.80 10.20 ⁇ 11.50 6.34 ⁇ 4.20 P.g. positive for the three techniques: 0 1 1 0 0 P.g. positive for culture and PCR: 0 1 1 2 0 P.g. positive for culture and FRET: 0 1 1 0 0 P.g. positive for PCR and FRET: 2 3 6 2 2
  • Example 9 Comparison of anaerobic culture, real-time PCR and FRET assays.
  • Table 8 Comparison of the prevalence of P. gingivalis by (A) anaerobic culture and real-time PCR, (B)by anaerobic culture and FRET assays, and (C) by realtime PCR and FRET assays for subgingival plaque samples. Table 8A
  • Sensitivity 67°/ 6; Specificity, 100% (anaerobic culture as gold
  • Sensitivity 58%; Specificity, 99% (real-time PCR as gold standard)
  • Table 9 Comparison of the prevalence of P. gingivalis by (A) anaerobic culture and real-time PCR, (B) anaerobic culture and FRET assays, and (C) by realtime PCR and FRET assays for salivary samples. Table 9A
  • Sensitivity 83°/ ⁇ ; Specificity, 72% (anaerobic culture as gold standard)
  • Sensitivity 63/ ⁇ ; Specificity, 75% (anaerobic culture as gold
  • Example 10 Comparison of detection of P. gingivalis in saliva and subgingival plaque samples.
  • the bacterial isolates used in this study are Actinomyces odontolyticus, Agrobacter actinomycetemcomitans (NTCT9710), Porphyromonas gingivalis W50, P. gingivalis K- (381), P. gingivalis Kl (W83), P. gingivalis K2 (X-2), P. gingivalis K3 (A7A1-28), P. gingivalis K4 (ATCC 49417), P. gingivalis K5 (HG1690), P. gingivalis K6 (HG1691), P.
  • gingivalis K7 (34-4), Prevotella intermedia (ATCC 25611), Prevotella nigrescens (NCTC 9336), Tannerella forsythensis (ATCC 43037), Peptostreptococcus micros (ATCC 33270),
  • Fusobacterium nucleatum periodontium ATCC 33693
  • Fusobacterium nucleatum nucleatum ATCC 25586
  • Fusobacterium nucleatum polymorphum ATCC 10953
  • Streptococcus mitis I SK 95
  • Streptococcus oralis SK 14757
  • Actinomyces naeslundii ATCC 12104.
  • the mutant P. gingivalis strains used were ARgpA (W501), ARgpB (D7) and AKgp (K1A) and were kindly provided by Dr. J. Potempa (Jagiellonian University, Poland). The generation of these protease-deficient strains has been described previously (1).
  • Bacteria were grown in 15 mL Brain Heart Infusion (BHI) medium (BioTrading, Mijdrecht, The Netherlands) under anaerobic conditions with 5% CO2 at 37°C. All P. gingivalis cultures were supplemented with hemin-menadione (Sigma, Zwijndrecht, The Netherlands) After 72 hr of culturing, the bacteria were pelleted by centrifugation for 10 min at 10,000 rpm. Supernatant, containing secreted enzymes, was sterilized by filtration through a 0.22 ⁇ filter
  • the 119 novel fluorogenic substrates were purchased at PepScan Presto B.V. (Lelystad, The Netherlands) with a purity > 90% (30,46). The identity of the substrates was confirmed by mass spectrometry.
  • the P. gingivalis positive substrates were denoted as 'BikKams' (Table 1). Assays were performed in Blackwell, clear bottom 96-well plates (Corning, Lowell, USA). Proteolytic activity was determined by incubating 16 ⁇ substrate with 50 ⁇ filtered culture supernatant or 50 microliter paper point vial buffer at 37 °C. Filtered BHI medium was used as a negative control. Plates were read for 60 minutes with 2 min intervals on a fluorescence microplate reader (Fluorstar Galaxy,
  • RF Relative Fluorescence
  • P. gingivalis strain W50 culture supernatant was prepared as described above. Culture supernatant was incubated with 16 ⁇ of each substrate in de presence of variable concentrations of five different chemicals which are known to influence the cleavage of gingipains (12; 17). Fluorescence was measured as described previously. Compounds used were leupeptin, dithiothreitol (DTT), N- ethylmaleimide (NEM), L-cysteine and glycyl-glycine. All chemicals were obtained from Sigma (Zwijndrecht, The Netherlands).
  • P. gingivalis W50 was cultured in BHI medium supplemented with hemin- menadione for 72 hr under anaerobic conditions with 5% CO2 at 37 °C.
  • the number of bacteria was determined by plating 10-fold serial dilutions on Trypticase Soy Agar (TSA) plates (BioTrading, Mijdrecht, The Netherlands). Plates were incubated at 37 °C under anaerobic conditions with 5% CO2 and bacteria were enumerated after 3 days incubation.
  • the culture was serial diluted in BHI (10 9 , 10 8 , 10 7 and 10 6 CFU/ mL) and of each dilution 50 ⁇ , was used to test the sensitivity of the substrates. Cleavage of the substrates was catalyzed by the addition of 5 mM L-cysteine to each enzyme reaction.
  • peri-implant diseases The definition and diagnosis of peri-implant diseases was based on the clinical and radiographic criteria described previously (Zitzmann & Berglundh, 2008) i) Peri-implant mucositis: presence of inflammation in the mucosa at an implant with no signs of loss of supporting bone; ii) Peri-implantitis: in addition to inflammation in the mucosa, peri-implantitis is characterized by loss of supporting bone as evidenced on radiographs.
  • Controls included subjects who showed no radiographic signs of bone loss and/or displayed fewer than five pockets >5 mm with clinical attachment level (CAL) >2 mm at the same time (Armitage 1999), following periodontal measurements assessed within 3 months before sample collections.
  • the parameters assessed included number of teeth and implants present, pocket probing depth (PPD), gingival recession, and bleeding on probing (BOP) at six different sites (mesio-buccal, mid-buccal, disto-buccal, mesio-lingual, mid- lingual, disto-lingual) of each implant/tooth present, excluding third molars. Bleeding of the peri-implant mucosa was scored dichotomously as presence or absence upon completion of probing (Ainamo & Bay, 1975). Presence of gingivitis was not an exclusion criteria for a control.
  • Exclusion criteria for peri-implant diseased patients and healthy controls were: (I) recent history or presence of any acute infection; (II) tooth extraction and trauma less than 2 weeks preceding the sample; (III) systemic antibiotic treatment during the preceding 3 months, (IV) pregnancy and (V) systemic diseases that could possibly influence the condition of the periodontal tissues and subgingival microflora.
  • Smoking habits were defined as follows: current smokers were participants who smoked at least one cigarette per day, while non-smokers were defined as those who had never smoked in their life or as those who had stopped smoking. A previous history of periodontal disease did not represent an impediment to enter this study, but it was recorded.
  • the peri-implant site with the deepest inflamed pocket was selected for bacterial sampling.
  • disto-lingual or disto-palatal peri-implant site was selected in the Cr and Cs groups, while in the Cni group, a caries-free gingival sulcus adjacent to the edentulous candidate implant area was selected.
  • two sterile paper points PP; Absorbent Points # 504; Henry Schein U.K. Holdings Ltd., Southall,
  • gingivalis was identified on the basis of Gram's stain, anaerobic growth, the inability to ferment glucose, the production of indole, and a positive
  • CFU total colony-forming units
  • the DNA was extracted from 100 ⁇ of subgingival plaque sample or from 100 ⁇ of saliva samples using a commercial kit (MagNA Pure LC DNA isoalation kit III, Roche Diagnostics, Indianapolis, IN, USA) in MagNA Pure LC (Roche Diagnostics) according to the instructions provided by the manufacturer.
  • MagNA Pure LC DNA isoalation kit III Roche Diagnostics, Indianapolis, IN, USA
  • MagNA Pure LC Roche Diagnostics
  • PCR analysis with primers and probe specific for P. gingivalis (Boutaga et al., 2003), modified for LightCycler® 480 (Roche Diagnostics), were used in the current study.
  • PCR amplification reactions were carried out in a reaction mixture of 20 ⁇ consisting of 4 ⁇ sample lysate and 16 ⁇ of reaction mixture containing Lightcycler® PCR mix, PCR water and primers (5'- GCGCTCAACGTTCAGCC-3' forward and 5'-CACGAATTCCGCCTGC-3' reverse) and probe (LC610-CACTGAACTCAAGCCCGGCAGTTTCAA-BBQ) for P. gingivalis.
  • PCR was carried out in a Lightcycler® 480 thermal cycler.
  • the conditions for PCR amplification were as follows: initial denaturation at 95°C for 5 minutes, 45 amplification cycles; denaturation at 95°C for 10 seconds, annealing and extension at 60°C for 20 seconds, followed by one cycle of cooling at 40°C for 15 seconds.
  • FRET fluorescence resonance energy transfer
  • P. gingivalis K 1 W83
  • P. gingivalis K2 X-2
  • P. gingivalis K3 A7A1-28
  • P. gingivalis K4 ATCC 49417
  • P. gingivalis K5 HG1690
  • P. gingivalis K6 HG1691
  • P. gingivalis K7 34-4)

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Abstract

La présente invention concerne des substrats spécifiques de Porphyromonas gingivalis utiles dans la détection de Porphyromonas gingivalis. Les substrats peuvent être utilisés comme indicateurs pour l'infection par des périopathogènes, en particulier ceux infectant la cavité buccale et sont également utiles comme indicateurs d'une parodontite ou d'une maladie péri-implant.
PCT/NL2012/050565 2011-08-17 2012-08-14 Substrats pour la détection de périopathogènes de la cavité buccale WO2013025099A1 (fr)

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