Association of two recombinant proteases from the Porphyromonas gingivalis strain and feline periodontal disease (2023)

Veterinary Microbiology

Volume 71, Issues 1-2,

January 2000

, s. 69-80

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Sera from 40 domestic cats with varying degrees of periodontal disease were used to detect two recombinant functional proteases from cat strain VPB 3457Porphyromonas gingivalisexpressed inMI, Escherichia coliA recombinant protease (VPB 2856) was constructed.polymerase chain reactionand 91% DNA identityprtC collagenaseHuman strain genesPorphyromonas gingivaliswhile another protease (VPB 2814) was isolated from the selected sizebiblioteka genomowaand has an amino-terminal sequence with no significant identity to the deposited sequence. 13 of the 40 cats had serumantibody responseUse West VPB 2856Western blotdetection. All 13 cats had an overall periodontal grade of 3 or higher and greater than 1.68×105 colony-forming unit Porphyromonas gingivalisin canines and premolarsmouthAn example website. Fourteen of the 40 cats showed a serum antibody response to VPB 2814. Thirteen of these 14 cats had an overall periodontal grade of 3 or higher. Regression analysis of periodontitis general grade to serum antibody response showed that VPB 2856 and VPB 2856 (R2=0,351;P<0,001) i VPB 2814 (R2=0,247;P<0.001). Regression analysis of all colony-forming units of feline strainsP. gingivitisVPB 2856 (R2=0,662;P<0,001) i VPB 2814 (R2=0,531;P<0.001). These data strongly demonstrate that feline recombinant proteasesP. gingivitisexpressed inMI, Escherichia coliClones VPB 2856 and VPB 2814 are associated with periodontal disease in cats.

to introduce

ThoughPorphyromonas gingivalis(PgH) is considered the main periodontal pathogen in the human oral cavity, and although research has focused on numerous proteases and their involvement in the development of destructive periodontal disease, the mechanisms of destruction of periodontal tissue components remain unclear. Recently, the cloning, sequencing and structural characterization of genes encoding some proteases, as well as more detailed analyzes of recombinant proteases, have begun to shed light on this important area of ​​research.

Collagen is a major component of gingival tissue in humans and animals, and the reduced density of gingival collagen fibers (Page and Schroeder, 1973) is an important feature of adult periodontal disease in humans. The ability of PgH to specifically degrade collagen may confer a selective advantage over other oral flora. The collagenolytic activity of PgH may be the result of a combination of nonspecific proteases and true collagenases (Mayrand and Grenier, 1984). Lawson and Meyer (1992) purified an active 94 kDa precursor protease from strain PgH 1101, which was cleaved into 75 kDa, 56 kDa and 19 kDa forms. Protease cleavage of human type IV basement membrane collagen and synthetic collagen peptide substrate for eukaryotic collagenase. Bedi and Williams (1994) purified a 55 kDa protease from the medium of strain PgH 381 and found that it hydrolyzed acid-soluble collagen types I, III, IV and V in the human placenta and acid-soluble collagens in the rat tail. . The purified protease also hydrolyses complement component C3, fibrinogen, fibronectin, alpha1- Alpha antitrypsin2- Macroglobulin, apotransferrin and human serum albumin.

Takahashi et al. (1991) were the first to report cloning and expressionprtCgen zPorphyromonas gingivalisATCC 33277ItsKato et al. (1992) determined the nucleotide sequenceprtCAnd he deduced that its amino acid sequence corresponded to a 37.8 kDa protein. ThoughprtCThe gene product has no structural similarity to eukaryotic collagenases, is capable of degrading soluble and recombinant fibrillar type I collagen, thermally denatured type I collagen and azocol, but not synthetic collagenase substrates, and does not contain the consensus sequence of the HELGH peptide found in these enzymes. These features may reflect the unique properties of this collagenase or may question its status as a true collagenase. However, the inability of collagen hydrolytic enzymes to degrade native fibrous collagen does not rule out its involvement in the pathogenesis of destructive periodontal disease. The degradation of type IV collagen in the basement membrane may be important for subepithelial invasion (Papapanou et al., 1994; Lamont et al., 1995), while enzymes that degrade other nonfibrillar collagens may reduce tissue integrity or may act in conjunction with true bacterial or host collagenases.

Two cysteine ​​proteases, designated gingipain-R and gingipain-K (Potempa et al., 1995), are involved in many of the damaging and evasive properties of PgH. These properties include disruption of polymorphonuclear leukocyte function (Kadowaki et al., 1994), degradation of acid-soluble collagen (types I and IV) (Kadowaki et al., 1994; Tokuda et al., 1998), immunoglobulin degradation of proteins and complement factors C3 and C5 (Kadowaki et al., 1994; Jagels et al., 1996) and may be involved in blood coagulation (Shah et al., 1992). Confusion about the function and identity of gingival proteases arose before DNA sequencing due to the many forms of proteases with different molecular weights (Pike et al., 1994, Potempa et al., 1995). Additional PgH proteases have stimulated research, including those produced byHiOtogoto i Kuramitsu, 1993tprGenes (Bourgeau et al., 1992). Both have been isolated, sequenced and characterized, but their role as virulence factors remains uncertain. They do not appear to share significant homology to each other or to gingivalysin.

Little is known about the proteases of the three characteristic felids of the genusporphyromonasLove and Binas (1995) showed that members of the cat familyPorphyromonas gingivalis(PGF),Porphyromonas salivariusIPorphyrodontoidyVarious SDS-stabilized proteases have been produced. The limited characterization of these proteases suggests that some of them may be cysteine ​​proteases.

Three strategies to isolate, characterize and identify two of these proteases are described herein. First, a size-selected genomic library was constructed from PgF VPB 3457 (Love et al., 1987) and screened to identifyEscherichia coliClones containing homologous genomic sequencesprtCgen PgH ATCC 33277ItsSecond, the known DNA sequence of the collagenase gene,prtC, reported by Kato et al. (1992) for designing primers for the amplification of the homologous collagenase gene PgF VPB 3457. Third, immunological methods are used for identificationMI, Escherichia coliClones expressing potentially important recombinant PgF VPB 3457 proteins.

partial excerpt

Collagenase gene primer design

Primers intended for amplificationprtCThe use of the PgF VPB 3457 gene with GenBank accession number M60404, which encodes the so-called collagenase PgH ATCC 33277ItsSense primer 5′-CGAAGCTTGAGCTATTTAACGGTGAAT-3' uses bases 45 to 63 while the antisense primer 5'-CCChamber of CommerceGAGAGGTGATAATTCG-3' uses the sequence from bases 1119 to 1103. The sense primer hasEnjoyThe dIII site (underlined) has been added to its 5' end to allow insertion of the gene in frame with the α peptide of the LacZ gene

Characterization of expressed recombinant proteins

Single-stranded DNA sequencing along 356 bases of the 1076 bp insert showed that 91% of the DNAprtCGen z PgH ATCC 33277ItsAnd confirmed the identity of the cloned product.

VPB 2856 expresses a recombinant protein with a molecular weight of approximately 37 kDa (Figure 1) - similar to the deduced amino acid sequenceprtCProduct genu PgH ATCC 33277Its(Kato i in., 1992).

N-terminal sequence of the recombinant protein in VPB 2856

N-terminal sequencing of a recombinant protein (Institute of TechnologyPXLSYLT) confirms it is compatible withprtCi to

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In this study, two genes from PgF VPB 3457 were used to clone and express functional protease activity. The strong positive correlation between serum antibody responses to these recombinant proteases and the amount of OPG and isolated PgF further underscores the importance of proteases in the pathogenesis of periodontal disease. This study further demonstrates the pathogenic potential of PgF by meeting two criteria considered by Haffajee and Socransky

Thank you

This work was partly funded by the Australian Research Council. The authors would like to thank Lana Patoka and Frank Taeker for the preparation of all culture media and Denise Wigney for professional assistance throughout the study. Thank you to the staff at Inner West and Annandale Veterinary Hospital for contacting me about this matter and the facility.


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    What is the role of Porphyromonas gingivalis in periodontal disease? ›

    P. gingivalis and Streptococcus gordonii are able to interact to form communities and subsequent colonization of the dental plaque. P. gingivalis benefits from its interaction and coaggregation in the subgingival plaque for its destructive effects on periodontal tissues (Kuboniwa et al., 2017).

    How does Porphyromonas gingivalis cause periodontitis? ›

    P . gingivalis is a gram-negative oral anaerobe and considered as a main etiological factor in periodontal diseases by producing a number of virulence factors and extracellular proteases such as lipopolysaccharide, fimbria, gingipain etc., resulting in destruction of periodontal tissues (7–11).

    How do you get rid of P. gingivalis? ›

    Treatment procedures of P. gingivalis–mediated diseases such as periodontitis and peri-implantitis focus on the eradication of oral pathogens at the site of infection, usually by surface debridement procedures followed by adjunctive therapies, including the use of antiseptics or/and antibiotics [61–66].

    What is the shape and arrangement of Porphyromonas gingivalis? ›

    Porphyromonas gingivalis is a Gram-negative, rod-shaped, obligate anaerobe that obtains its metabolic energy from protein breakdown products, heme, and vitamin K for its growth. It is a pathobiont of the oral cavity that is distributed among the human population worldwide.

    What are the proteases of P. gingivalis? ›

    gingivalis, they have been suggested to play multiple roles in the pathogenic process of periodontitis. Indeed, P. gingivalis proteases hydrolyze a variety of serum and tissue proteins thus contributing to neutralize the immune defense system and to cause tissue destruction.

    What are the virulence factors of Porphyromonas gingivalis? ›

    Virulence factors of P. gingivalis are fimbriae, hemolysin, hemagglutinins, capsule, outer membrane vesicles (OMVs), lipopolysaccharides (LPS), and gingipains (Table 1). Fimbriae are thin, filamentous structures by most strains of P. gingivalis.

    What are the 2 main ways bacteria cause periodontal disease? ›

    Causes. Bacteria in the mouth infect tissue surrounding the tooth, causing inflammation around the tooth leading to periodontal disease. When bacteria stay on the teeth long enough, they form a film called plaque, which eventually hardens to tartar, also called calculus.

    What can Porphyromonas gingivalis cause? ›

    Porphyromonas gingivalis is a Gram-negative oral anaerobe that is involved in the pathogenesis of periodontitis, an inflammatory disease that destroys the tissues supporting the tooth, eventually leading to tooth loss.

    What is the primary cause of gingivitis and periodontal disease? ›

    Overview. Periodontal (gum) disease is an infection of the tissues that hold your teeth in place. It's typically caused by poor brushing and flossing habits that allow plaque—a sticky film of bacteria—to build up on the teeth and harden.

    How can I get rid of gum disease permanently? ›

    You will need to use small brushes (interproximal brushes) to clean in between your teeth daily, along with adopting good tooth brushing habits. Unfortunately, once you have gum disease, it's impossible to clean all the areas that are infected and you will need to go to a professional such as a hygienist or dentist.

    How do you know if you have P. gingivalis? ›

    There is a reliable test that can be done in any dental office to test for PG. It's called the Oral DNA test. It tests for the 11 different bacteria that cause periodontal disease, including PG.

    How does P. gingivalis enter the brain? ›

    The keystone periodontal pathogen, P. gingivalis (P.g), can enter the bloodstream during episodes of transient bacteremia and gain access to the brain via multiple routes including a leaky blood-brain barrier.

    Where is P. gingivalis found in the body? ›

    The major habitat of P. gingivalis is the subgingival sulcus of the human oral cavity. It relies on the fermentation of amino acids for energy production, a property required for its survival in deep periodontal pocket, where sugar availability is low (Bostanci and Belibasakis, 2012).

    What enzymes are secreted by P. gingivalis? ›

    Arg-gingipain (Rgp) and lys-gingipain (Kgp) are endopeptidase enzymes secreted by P. gingivalis. These gingipains serve many functions for the organism, contributing to its survival and virulence.

    How is Porphyromonas gingivalis transmitted? ›

    gingivalis into the oral cavity is thought to occur by transmission from infected individuals (77). Saliva is considered an important vector for transmission; however, the spouses and children of individuals with P. gingivalis do not always harbor the same genotype (203, 269).

    What are the 3 main proteases? ›

    Proteases fall into four main mechanistic classes: serine, cysteine, aspartyl and metalloproteases.

    Is Porphyromonas gingivalis positive or negative? ›

    Porphyromonas gingivalis is a Gram-negative oral anaerobe that is involved in the pathogenesis of periodontitis and is a member of more than 500 bacterial species that live in the oral cavity.

    What are 2 examples of protease? ›

    Protease Enzyme Examples
    Protease Enzyme NameFunction
    PepsinPresent in stomach and converts proteins to smaller peptides – proteoses and peptones
    RenninSecreted by chief cells of the stomach and curdles milk protein
    ThrombinInvolved in blood coagulation
    PlasminInvolved in blood coagulation
    12 more rows

    What are the effects of Porphyromonas gingivalis on atherosclerosis related cells? ›

    It can induce the dysfunction of endothelium, promote the formation of foam cells as well as the proliferation and calcification of vascular smooth muscle cells, and lead to the imbalance of regulatory T cells (Tregs) and T helper (Th) cells, ultimately promoting the occurrence and development of atherosclerosis.

    What bacteria causes Porphyromonas gingivalis? ›

    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.

    What is the contribution of Porphyromonas gingivalis lipopolysaccharide to periodontitis? ›

    Porphyromonas gingivalis is a key bacterium in chronic periodontitis, which is associated with several chronic inflammatory diseases. Lipopolysaccharides from P. gingivalis (Pg LPS) can activate multiple cell types via the production of pro-inflammatory cytokines.

    What is 3 infection caused by bacteria getting under the gum tissue and destroying the gums and bone? ›

    Periodontal disease occurs when the toxins found in plaque begin to irritate or inflame the gingiva (gum tissue). The resulting bacterial infection often known as gingivitis, can eventually lead to the destruction of the gum tissue and underlying bone.

    Can you reverse periodontal disease? ›

    Periodontitis can't be reversed, only slowed down, while gingivitis can be reversed. This is why it's important to catch it in its early stages and prevent it from moving on to periodontitis.

    What are the two strains of bacteria found to assist with periodontal health? ›

    Some oral strains of lactobacilli and streptococci [36] and bifidobacteria [37] have been reported to have in vitro inhibitory activity against periodontal pathogens, while others are more active against mutans streptococci.

    What chemical released by P. gingivalis has been implicated in Alzheimer's disease? ›

    gingivalis increased the production of amyloid beta, a component of the amyloid plaques whose accumulation contributes to Alzheimer's.

    What is the relationship between Porphyromonas gingivalis and systemic disease? ›

    P. gingivalis induce a chronic inflammation, this status could lead to infection, that is correlated with systemic diseases as diabetes, preterm birth, stroke, and CVD. P. gingivalis stimulate chronic inflammation and plaque accumulation and has a role on Toll-like receptors signaling.

    What is the mode of transmission of gingivalis? ›

    gingivalis has been identified, and oral-oral contact is believed to be its mode of transmission.

    What are 3 causes of periodontal disease? ›

    Risk factors

    Poor oral health care habits. Smoking or chewing tobacco. Hormonal changes, such as those related to pregnancy or menopause.

    What is the white stringy stuff in my mouth after I brush my teeth? ›

    This sticky, disgusting layer of film is called oral thrush, and it's normal to want to rid your mouth of the foul substance as quickly as possible! Read on to learn more from your dentist about what causes oral thrush, along with some measures you can take to address it and maintain good oral health.

    How can I rebuild my teeth and gums naturally? ›

    Here are five natural remedies for receding gums that you can try from the comfort of your home.
    1. Use a Salt Water Rinse. Saltwater is a great, natural tool to use as an oral rinse. ...
    2. Drink Green Tea. ...
    3. Try Practicing Oil Pulling. ...
    4. Rinse with a Hydrogen Peroxide Solution. ...
    5. Maintain Thorough Oral Hygiene.
    Sep 15, 2021

    What toothpaste helps with periodontal disease? ›

    Use Corsodyl Complete Protection Toothpaste, which physically removes the build of plaque bacteria along the gum line, helping to keep the seal between your gums and teeth tight. When used to brush twice daily it is 4x more effective* than a regular toothpaste at removing the main cause of bleeding gums.

    What toothpaste works best for gingivitis? ›

    Look for a toothpaste that carries the American Dental Association (ADA) Seal of Acceptance for gingivitis and plaque control.
    • Colgate Total. ...
    • Oral-B Gum Protection. ...
    • Crest Gum Detoxify and Pro-Health Advanced. ...
    • Meridol. ...
    • Paradontax. ...
    • Lacalut Aktiv. ...
    • Zymbion Q10.
    May 23, 2022

    When is it too late to reverse gum disease? ›

    It's never too late to seek treatment for gum disease, and the degree of treatment you require will depend on how advanced it is.

    What is the function of the Porphyromonas? ›

    Porphyromonas gingivalis (P. gingivalis) is the most important pathogenic bacteria for periodontal disease. It can produce outer membrane vesicles (OMVs) and release them into the environment, playing an important role in its pathogenesis.

    What is the mechanism of action of Porphyromonas gingivalis? ›

    P. gingivalis LPS causes a highly innate immune response through host receptors, which is toll-like receptor-2 (TLR-2) and TLR-4 on the host cell surface, leading to secrete interleukin-1 (IL-1), IL-6, IL-8, and TNF-α in host cells [12], [13], [14], [15].

    What is P. gingivalis and its role in systemic diseases? ›

    P. gingivalis induce a chronic inflammation, this status could lead to infection, that is correlated with systemic diseases as diabetes, preterm birth, stroke, and CVD. P. gingivalis stimulate chronic inflammation and plaque accumulation and has a role on Toll-like receptors signaling.


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