Skip to main content
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems
  • Log in
  • My alerts
  • My Cart

Main menu

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • Archive
    • Minireviews
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About IAI
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems

User menu

  • Log in
  • My alerts
  • My Cart

Search

  • Advanced search
Infection and Immunity
publisher-logosite-logo

Advanced Search

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • Archive
    • Minireviews
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About IAI
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
Bacterial Infections

Histopathological Studies on Virulence of Dipeptidyl Aminopeptidase IV (DPPIV) of Porphyromonasgingivalis in a Mouse Abscess Model: Use of a DPPIV-Deficient Mutant

Hisao Yagishita, Yumi Kumagai, Kiyoshi Konishi, Yukihiro Takahashi, Takaaki Aoba, Masanosuke Yoshikawa
Hisao Yagishita
Department of Pathology and
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Yumi Kumagai
Department of Microbiology, Nippon Dental University, Fujimi 1-9-20, Chiyoda-ku, Tokyo 102-8159, Japan
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Kiyoshi Konishi
Department of Microbiology, Nippon Dental University, Fujimi 1-9-20, Chiyoda-ku, Tokyo 102-8159, Japan
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Yukihiro Takahashi
Department of Microbiology, Nippon Dental University, Fujimi 1-9-20, Chiyoda-ku, Tokyo 102-8159, Japan
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Takaaki Aoba
Department of Pathology and
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Masanosuke Yoshikawa
Department of Microbiology, Nippon Dental University, Fujimi 1-9-20, Chiyoda-ku, Tokyo 102-8159, Japan
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
DOI: 10.1128/IAI.69.11.7159-7161.2001
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

ABSTRACT

To elucidate the role of dipeptidyl aminopeptidase IV (DPPIV) in the virulence of Porphyromonasgingivalis, mice were infected with either a wild-type strain or a DPPIV-deficient mutant using an abscess model. Histopathological analysis of the resulting lesions indicated that DPPIV participates in virulence through the destruction of connective tissue and the less effective mobilization of inflammatory cells.

Porphyromonas gingivalis, a gram-negative anaerobe, is thought to be a major etiological agent associated with adult periodontitis (1, 4, 6, 8). Recently, much interest has been focused on a possible implication of oral bacteria in systemic diseases. P. gingivalis has been suggested to be involved in aspiration pneumonia in the elderly and also to be responsible for the development of other systemic diseases, such as endocarditis and pulmonary infections, as a putative blood-borne pathogen (1, 2, 6, 10). However, little is known about the mechanisms by whichP. gingivalis penetrates the systemic circulation to cause infections at distant sites. The mouse abscess model has been used to investigate the virulence of P. gingivalis (6). Several components, such as trypsin-like proteases, are considered to be potential virulence factors on the basis of results obtained with this model, involving infection by strains harboring either wild-type or mutant structural genes for the putative virulence factors (5, 6). However, there is still a paucity of information about specific roles or the relative importance of the potential virulence factors of the bacterium.

Dipeptidyl aminopeptidase IV (DPPIV; EC 3.4.14.5 ) is a serine protease that cleaves X-Pro or X-Ala dipeptide from the N-terminal ends of polypeptide chains. Eukaryotic DPPIV has been postulated to be involved in various biological processes, such as T-cell activation, interaction with collagen and/or fibronectin, and degradation of biologically active peptides, including chemokines and others (3). However, the physiological and pathological functions of bacterial DPPIV have not yet been clarified. The gene (dpp) coding for DPPIV has been cloned from P. gingivalis W83, adpp null mutant (4351) has been constructed, and it has been shown that P. gingivalis DPPIV plays a role as a virulence factor (9).

In the present study, to assess the role of DPPIV in the virulence ofP. gingivalis, histopathological differences in the lesions caused by W83 and 4351 in a mouse abscess model were investigated. Ten BALB/c mice (male, 11 weeks old; Tokyo Laboratory Animal Science Co. Ltd., Tokyo, Japan) each were challenged withP. gingivalis W83 and 4351 by dorsal subcutaneous injection as described previously (9). Protocols conformed to the guidelines for the care and use of laboratory animals at Nippon Dental University. W83 and 4351 were harvested immediately after reaching the stationary phase, as determined by measurement of the optical densities, because the optical densities of both W83 and 4351 are proportional to viable cell numbers by this phase, as previously shown (9). No significant differences were found between the two strains in growth rates at the logarithmic phase, viable counts after reaching the stationary phase, and viability during exposure to the atmosphere. The dose of bacteria used for injection was determined as reported previously (5, 6). Phosphate-buffered saline was injected as a negative control.

Half of the animals injected with either W83 or 4351 were euthanatized 3 days after injection. Four of the remaining five mice inoculated with W83 and two of the five inoculated with 4351 were surviving at 3 days after inoculation but died by 6 days. Surviving animals (one mouse injected with W83 and three mice injected with 4351) were euthanatized 14 days after inoculation. Autopsy of the animals was conducted to examine whether infectious lesions had spread to adjacent internal organs or to distant places. Blood specimens were obtained from their tails 2 days after injection. The numbers of viable bacteria were counted by spreading blood specimens on brain heart infusion (Difco, Detroit, Mich.) agar plates supplemented with 5 μg of hemin (Sigma-Aldrich, St. Louis, Mo.)/ml, 0.5 μg of menadione (Sigma-Aldrich)/ml, and 5% defibrinated horse blood (Nippon Bio-supp. Center, Tokyo, Japan), followed by anaerobic incubation of the plates.

Skin specimens, including those from dorsal, lateral, and ventral sites, as well as internal organs (i.e., lungs, spleen, liver, kidneys, heart, and intestines) were dissected for pathological analysis. All tissue specimens were fixed in 4% paraformaldehyde in 0.1 M sodium cacodylate buffer (pH 7.3) and then embedded in paraffin. Serially cut sections (4 μm thick) were stained with hematoxylin-eosin for routine diagnosis and Elastica van Gieson (EVG) and Azan stains for detection of collagen and fibrous structures. Inflammatory cells were identified by their morphology. For skin specimens, two sections from peripheral areas and two sections from the central, most-severe areas of lesions from every animal were examined. Two fields were randomly selected from within the most-severe areas of lesions from each of four animals inoculated with W83 and 4351 after euthanasia at 3 days. The numbers of inflammatory cells and bacteria in the fields were counted at ×400 and ×1,000 magnifications, respectively.

Macroscopic observations revealed no local abscesses or no signs of illness after injection of phosphate-buffered saline. At autopsy of seriously ill animals challenged with W83 or 4351 and euthanatized at day 3, hemorrhage of intra-abdominal cavities and/or mesentery was detected. Lesions caused by W83 or 4351 on the dorsal surface and abdominal area were similar to those described previously (9). In short, all mice exhibited no severe skin lesions, except for redness, at the site of injection on the dorsal surface. Animals challenged with W83 exhibited a large abscess which covered the abdominal area and which progressed to ulceration with associated gangrenous skin necrosis and subcutaneous inflammation. In contrast, mice inoculated with 4351 developed merely a small focus of abscess on the abdomen which was resolved or substantially healed by the end of the observation period, with an increase in body weight beginning at 6 days after injection.

Upon histological examination, congestion of lungs, spleen, liver, and kidneys was found. Hepatic cells were hyperplastic, with intraparenchymal focal accumulation of mononuclear cells but without gross necrotic patches or large areas of inflammation. Neither ulceration nor massive inflammatory reactions were found at the injection site. During the development of abscess, the inflammatory infiltrate, comprising mainly inflammatory cells, spread between the cutaneous trunk muscle and the external oblique muscle toward the abdominal region. Bacteria were detected in the spreading infiltrate.

The pathological features of the abdominal regions were markedly different depending on the strain injected. Inoculation of W83 evoked an accumulation of (1.0 ± 0.5) × 103inflammatory cells (n = 8)/mm2together with (2.1 ± 0.6) × 105bacteria (n = 8)/mm2; the fasciae appeared to be partially collapsed, giving rise to diffuse penetration of inflammatory cells into the underlying musculature (Fig.1). In sharp contrast, challenge with 4351 induced an accumulation of (5.0 ± 1.0) × 103 inflammatory cells (n = 8)/mm2 above the fasciae together with (5.4 ± 3.6) × 104 bacteria (n = 8)/mm2; almost no inflammatory cells penetrated into the deep muscular layer, and there was no apparent destruction of the fasciae (Fig. 1). The differences in the numbers of both inflammatory cells and bacteria evoked by W83 and 4351 were statistically significant, with a P value of <0.001 for inflammatory cells and a P value of <0.02 for bacteria, as determined by unpaired nonparametric analysis (Mann-Whitney test).

Fig. 1.
  • Open in new tab
  • Download powerpoint
Fig. 1.

Micrographs of lesions dissected 3 days after injection from the abdomens of mice challenged with W83 and 4351 and stained with hematoxylin-eosin and EVG stains. The sections presented here are derived from the most-severe areas of lesions taken from one each of five animals inoculated with W83 and 4351. Scale bars, 50 μm.

W83 caused a spreading type of infection, often accompanied by massive lysis of epithelial and subcutaneous tissues. These lesions generally degraded into gangrenous necrosis of the skin. Staining with EVG or Azan stain revealed a considerable loss of collagen fibers and associated supportive fibrous structures in the dermis and fasciae. In contrast, lesions caused by 4351 appeared far less destructive in connective tissues than those caused by W83 (Fig. 1). A gradual shift from the acute inflammatory phase to the reparative phase was observed in lesions caused by 4351 within 14 days after injection; this shift featured the formation of a subcutaneous granulomatous focus accompanied by blood capillary formation and a number of inflammatory cells and macrophages, leading to the lesions being resolved.

The histopathological differences in lesions caused by W83 and 4351 are likely to be consistent with the findings obtained by in vitro assays using purified DPPIV. DPPIV was found to participate in the degradation of type I collagen together with tissue- or inflammatory cell-derived matrix metalloproteinases 1 and 8 (unpublished data). Furthermore, it has been demonstrated that eukaryotic DPPIV cleaves C-C and C-X-C chemokines at the C terminus of the proline residue on the penultimate position of the N terminus and that truncated chemokines have activity as chemotactic inhibitors (3). Previous studies proved that recombinant DPPIV of P. gingivaliswas also capable of cleaving two synthetic substrates possessing the same N-terminal sequences to human RANTES and human MCP1 (9). On the basis of these data, P. gingivalis DPPIV is likely to cleave chemokines similarly, leading to the disturbance in the mobilization of inflammatory cells and thus in the host defense mechanism. It is suggested that in lesions caused by W83, a less effective accumulation of inflammatory cells provokes an increase in the level of bacteria, most likely resulting in more-severe tissue destruction by proteases produced by P. gingivalis, i.e., trypsin-like proteases and DPPIV.

Between 103 and 106bacteria/ml of blood were detected in animals injected with W83 but not 4351 when the plates were incubated anaerobically. All colonies on plates were black pigmented, specifically, P. gingivalis. DPPIV is not essential for bacterial growth in vitro, since the rates of growth of W83 and 4351 are similar (9). In contrast, DPPIV may be required for P. gingivalis to survive in vivo. This property of DPPIV meets one of the modern criteria for virulence factors (7), and the difference in the virulence of W83 and 4351 is likely attributable to the lower viability of 4351 in vivo, as implied by the difficulty in isolating the mutant from blood. P. gingivalisDPPIV also may be involved in dissemination of the bacteria through the destruction of connective tissue, so that bacteria can enter the bloodstream. This process may confer upon the bacterium a mechanism to provoke systemic diseases as a blood-borne pathogen.

We are currently attemting to develop an inoculation model using the oral cavity of mice to investigate activities of DPPIV in the pathogenesis of periodontitis and other systemic diseases.

ACKNOWLEDGMENTS

We thank Ayako Yajima for skillful assistance in animal experiments.

This work was supported by grants-in-aid for scientific research 07457071 and 08307004 from the Ministry of Education, Science, Culture and Sports, Tokyo, Japan.

Notes

Editor: V. J. DiRita

FOOTNOTES

    • Received 31 January 2001.
    • Returned for modification 24 April 2001.
    • Accepted 23 July 2001.
  • Copyright © 2001 American Society for Microbiology

REFERENCES

  1. 1.↵
    1. Allaker R. P.,
    2. Hardie J. M.
    Oral infections Topley & Wilson's principles of bacteriology, virology and immunity 9th ed. Hausler W. J. Jr., Sassman M. 3 1998 373 390 Arnold London, Great Britain
    OpenUrl
  2. 2.↵
    1. Allen S. D.,
    2. Duerden B. I.
    Infections due to non-sporing anaerobic bacilli and cocci Topley & Wilson's principles of bacteriology, virology and immunity 9th ed. Hausler W. J. Jr., Sassman M. 3 1998 743 776 Arnold London, Great Britain
    OpenUrl
  3. 3.↵
    1. Augustyns K.,
    2. Bal G.,
    3. Thonus G.,
    4. Belyaev A.,
    5. Zhang X. M.,
    6. Bollaert W.,
    7. Lambeir A. M.,
    8. Durinx C.,
    9. Goosens F.,
    10. Haemers A.
    The unique properties of dipeptidyl-peptidase IV (DPPIV/CD26) and the therapeutic potential of DPPIV inhibitors.Curr. Med. Chem.61999311327
    OpenUrlPubMedWeb of Science
  4. 4.↵
    1. Cutler C. W.,
    2. Kalmer J. R.,
    3. Genco C. A.
    Pathogenic strategies of the oral anaerobe, Porphyromonas gingivalis.Trends Microbiol.319954551
    OpenUrlCrossRefPubMed
  5. 5.↵
    1. Fletcher H. M.,
    2. Schenkein H. A.,
    3. Morgan R. M.,
    4. Bailey K. A.,
    5. Berry C. R.,
    6. Macrina F. L.
    Virulence of a Porphyromonas gingivalis W83 mutant defective in the prtH gene.Infect. Immun.63199515211528
    OpenUrlAbstract/FREE Full Text
  6. 6.↵
    1. Genco C. A.,
    2. Van Dyke T.,
    3. Amar S.
    Animal models for Porphyromonas gingivalis-mediated periodontal disease.Trends Microbiol.61998444449
    OpenUrlCrossRefPubMedWeb of Science
  7. 7.↵
    1. Hensel M.,
    2. Shea J. E.,
    3. Gleeson C.,
    4. Jones M. D.,
    5. Dalton E.,
    6. Holden D. W.
    Simultaneous identification of bacterial virulence genes by negative selection.Science2691995400403
    OpenUrlAbstract/FREE Full Text
  8. 8.↵
    1. Holt S. C.,
    2. Ebersole J.,
    3. Felton J.,
    4. Brunsvold M.,
    5. Kornman K. S.
    Implantation of Bacteroides gingivalis in nonhuman primates initiates progression of periodontitis.Science23919885557
    OpenUrlAbstract/FREE Full Text
  9. 9.↵
    1. Kumagai Y.,
    2. Konishi K.,
    3. Gomi T.,
    4. Yagishita H.,
    5. Yajima A.,
    6. Yoshikawa M.
    Enzymatic properties of dipeptidyl aminopeptidase IV produced by the periodontal pathogen Porphyromonas gingivalis and its participation in virulence.Infect. Immun.682000716724
    OpenUrlAbstract/FREE Full Text
  10. 10.↵
    1. Read R. C.,
    2. Finch R. G.
    Bacterial infections of the respiratory tract Topley & Wilson's principles of bacteriology, virology and immunity 9th ed. Hausler W. J. Jr., Sassman M. 3 1998 319 345 Arnold London, Great Britain
    OpenUrl
View Abstract
PreviousNext
Back to top
Download PDF
Citation Tools
Histopathological Studies on Virulence of Dipeptidyl Aminopeptidase IV (DPPIV) of Porphyromonasgingivalis in a Mouse Abscess Model: Use of a DPPIV-Deficient Mutant
Hisao Yagishita, Yumi Kumagai, Kiyoshi Konishi, Yukihiro Takahashi, Takaaki Aoba, Masanosuke Yoshikawa
Infection and Immunity Nov 2001, 69 (11) 7159-7161; DOI: 10.1128/IAI.69.11.7159-7161.2001

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Print

Alerts
Sign In to Email Alerts with your Email Address
Email

Thank you for sharing this Infection and Immunity article.

NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

Enter multiple addresses on separate lines or separate them with commas.
Histopathological Studies on Virulence of Dipeptidyl Aminopeptidase IV (DPPIV) of Porphyromonasgingivalis in a Mouse Abscess Model: Use of a DPPIV-Deficient Mutant
(Your Name) has forwarded a page to you from Infection and Immunity
(Your Name) thought you would be interested in this article in Infection and Immunity.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
Histopathological Studies on Virulence of Dipeptidyl Aminopeptidase IV (DPPIV) of Porphyromonasgingivalis in a Mouse Abscess Model: Use of a DPPIV-Deficient Mutant
Hisao Yagishita, Yumi Kumagai, Kiyoshi Konishi, Yukihiro Takahashi, Takaaki Aoba, Masanosuke Yoshikawa
Infection and Immunity Nov 2001, 69 (11) 7159-7161; DOI: 10.1128/IAI.69.11.7159-7161.2001
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Top
  • Article
    • ABSTRACT
    • ACKNOWLEDGMENTS
    • Notes
    • FOOTNOTES
    • REFERENCES
  • Figures & Data
  • Info & Metrics
  • PDF

KEYWORDS

abscess
Bacteroidaceae Infections
Dipeptidyl Peptidase 4
Porphyromonas gingivalis

Related Articles

Cited By...

About

  • About IAI
  • Editor in Chief
  • Editorial Board
  • Policies
  • For Reviewers
  • For the Media
  • For Librarians
  • For Advertisers
  • Alerts
  • RSS
  • FAQ
  • Permissions
  • Journal Announcements

Authors

  • ASM Author Center
  • Submit a Manuscript
  • Article Types
  • Ethics
  • Contact Us

Follow #IAIjournal

@ASMicrobiology

       

ASM Journals

ASM journals are the most prominent publications in the field, delivering up-to-date and authoritative coverage of both basic and clinical microbiology.

About ASM | Contact Us | Press Room

 

ASM is a member of

Scientific Society Publisher Alliance

 

American Society for Microbiology
1752 N St. NW
Washington, DC 20036
Phone: (202) 737-3600

Copyright © 2021 American Society for Microbiology | Privacy Policy | Website feedback

Print ISSN: 0019-9567; Online ISSN: 1098-5522