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Adhesion-related interactions of Actinomyces and Streptococcus biofilm bacteria
Umeå University, Faculty of Medicine, Odontology, Cariology.
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Adhesion of bacteria is a key event in biofilm formation and is mediated by bacterial adhesins recognising host or bacterial partner receptors. In oral biofilm formation, primary Actinomyces and Streptococcus colonizers adhere to salivary pellicle proteins such as proline-rich proteins (PRPs) as well as to mucosal surfaces. Subsequently, Actinomyces and Streptococcus strains and other bacteria, such as Veillonella, Fusobacterium and Porphyromonas, adhere to each other. The nature of this community is highly important for the health or disease status, although specific pathogenic species may also have been implicated.

The aim of this thesis was to study key players in early oral colonisation, Actinomyces and Streptococcus species, and more specifically the nature of their adhesins and ligands. A further aim was to study the function of the salivary PRP proteins and an innate peptide derived thereof on bacterial adhesion, proliferation and regulation of pH, i.e. key factors in biofilm formation.

In paper I and II, adhesion, proliferation and pH affecting features of the RGRPQ (arginine-glycine-arginine-proline-glutamine) peptide, derived from PRP-1, were demonstrated. By use of an alanine-scan (I), motifs for adhesion inhibition and desorption of Actinomyces naeslundii, and proliferation stimulation, ammonia production and inhibition of sucrose induced pH drop by Streptococcus gordonii were indicated. The RGRPQ peptide also stimulated S. gordonii colonisation in vivo. In paper II, a more sophisticated quantitative structure-activity relationship (QSAR) study, using statistical molecular design (SMD) and multivariate modelling (partial least squares projections to latent structures, PLS), further narrowed down the RGRPQ peptide motifs. The R and Q amino acids were crucial for activity. For proliferation a hydrophobic and large size third position amino acid was crucial, while adhesion inhibition and desorption needed a small hydrophilic second position amino acid. All functions depended on a low polarity hydrophobic fourth position. Accordingly, activities could be optimized separately, with decreased function in the others.

In paper III and IV, focus was on the bacterial adhesins and their binding epitopes. The genes for FimA major subunit proteins of type-2 fimbriae were sequenced from A. naeslundii genospecies 1 and 2 and Actinomyces odontolyticus, each with unique carbohydrate binding specificities (III). Three major subtypes of FimA proteins were found that correlated with binding specificity, including a novel fimA gene in A. odontolyticus. All subtypes contained a pilin, LPXTG and E box motif. In paper IV, multiple PRP binding patterns for Actinomyces and Streptococcus strains were mapped using a hybrid peptide construct. The two most deviating binding groups deviated in type-1 fimbriae mediated binding to milk and saliva protein ligands.

In conclusion, differences in bacterial adhesins and their ability to utilise salivary proteins may render bacteria tropism for different niches. Peptides derived from protein receptors, such as RGRPQ, may be important modulators of biofilm formation, giving commensal bacteria a competitive edge in the bacterial community.

Place, publisher, year, edition, pages
Umeå: Odontologi , 2006. , 45 p.
Series
Umeå University odontological dissertations, ISSN 0345-7532 ; 92
Keyword [en]
biofilm, Actinomyces, Streptococcus, adhesion
National Category
Dentistry
Identifiers
URN: urn:nbn:se:umu:diva-860ISBN: 91-7264-111-8 (print)OAI: oai:DiVA.org:umu-860DiVA: diva2:144769
Public defence
2006-09-29, Sal B, Tandläkarhögskolan 9 tr, 90187 Umeå, Umeå, 13:00 (English)
Opponent
Supervisors
Available from: 2006-09-08 Created: 2006-09-08 Last updated: 2009-10-01Bibliographically approved
List of papers
1. A host-derived pentapeptide enhancing host-bacteria commensalisms and communication
Open this publication in new window or tab >>A host-derived pentapeptide enhancing host-bacteria commensalisms and communication
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2006 (English)In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 74, no 11, 6293-6299 p.Article in journal (Refereed) Published
Abstract [en]

Salivary proline-rich proteins (PRPs) attach commensal Actinomyces and Streptococcus species to teeth. Here, gel filtration, mass spectrometry and Edman degradation were applied to show the release of a pentapeptide, RGRPQ, from PRP-1 upon proteolysis by Streptococcus gordond. Moreover, synthetic RGRPQ and derivatives were used to investigate associated innate properties and responsible motifs. The RGRPQ peptide increased 2.5-fold the growth rate of S. gordonii via a Q-dependent sequence motif and selectively stimulated oral colonization of this organism in a rat model in vivo. In contrast, the growth of Streptococcus mutans, implicated in caries, was not affected. While the entire RGRPQ sequence was required to block sucrose-induced pH-decrease by S. gordonii and S. mutans, the N-terminal Arg residue mediated the pH increase (i.e., ammonia production) by S. gordonii alone (which exhibits Arg catabolism to ammonia). Strains of commensal viridans streptococci exhibited PR-P degradation and Arg catabolism, whereas cariogenic species did not. The RGRPQ peptide mediated via a differential Q-dependent sequence motif, adhesion inhibition, and desorption of PRP-1-binding strains of A. naeslundii genospecies 2 (5 of 10 strains) but not of S. gordonii (n = 5). The inhibitable A. naeslundii strains alone displayed the same binding profile as S. gordond to hybrid peptides terminating in RGRPQ or GQSPQ, derived from the middle or C-terminal segments of PRP-1. The present findings indicate the presence of a host-bacterium interaction in which a host peptide released by bacterial proteolysis affects key properties in biofilm formation.

Place, publisher, year, edition, pages
Washington: American society for microbiology, 2006
Keyword
proline-rich proteins, candida albicans, oral bacteria, salivary agglutinin, apatitic surfaces, innate immunity, Dental caries, Gordonii, commensal, statherin
National Category
Immunology in the medical area Infectious Medicine Dentistry
Identifiers
urn:nbn:se:umu:diva-5291 (URN)10.1128/IAI.00068-06 (DOI)000241600500031 ()
External cooperation:
Available from: 2006-09-08 Created: 2006-09-08 Last updated: 2016-08-26Bibliographically approved
2. Multivariate design and evaluation of a set of RGRPQ-derived innate immunity peptides
Open this publication in new window or tab >>Multivariate design and evaluation of a set of RGRPQ-derived innate immunity peptides
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2006 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, Vol. 281, no 22, 15164-15171 p.Article in journal (Refereed) Published
Abstract [en]

Oral commensal Streptococcus gordonii proteolytically cleave the salivary PRP-1 polypeptide into an RGRPQ innate peptide. The Arg and Gln termini are crucial for RGRPQ-mediated ammonia production and proliferation by S. gordonii SK12 and adhesion inhibition and desorption by Actinomyces naeslundii T14V, respectively. Here we have applied (i) a multivariate approach using RGRPQ-related peptides varied at amino acids 2, 3, and 4 simultaneously and (ii) size and N- and C-terminal modifications of RGRPQ to generate structure activity information. While the N-terminal arginine motif mediated ammonia production independent of peptide size, other responses required more or less full-length peptide motifs. The motifs for adhesion inhibition and desorption were the same. The adhesion and proliferation motifs required similarly a hydrophobic/low polarity amino acid 4 but differentially a hydrophilic or hydrophobic character of amino acids 2/3, respectively; polar peptides with small/hydrophilic and hydrophilic amino acids 2 and 3, respectively, had high adhesion inhibition/desorption activity, and lipophilic peptides with large/hydrophobic amino acids 2 and 3 had high proliferation activity. Accordingly, while RIWWQ had increased proliferation but abolished adhesion/desorption activity, peptides designed with hydrophilic amino acids 2 and 3 were predicted to behave in the opposite way. Moreover, a RGRPQ mimetic for all three responses should mimic small hydrophilic, large nitrogen-containing, and hydrophobic/low polarity amino acids 2, 3, and 4, respectively. Peptides fulfilling these criteria were 1-1.6-fold improved in all three responses. Thus, both mimetics and peptides with differential proliferation and adhesion activities may be generated for evaluation in biofilm models.

Keyword
Actinomyces/immunology/pathogenicity, Amino Acid Sequence, Ammonia/metabolism, Bacterial Adhesion/drug effects, Drug Design, Humans, Immunity; Natural, Oligopeptides/chemistry/*immunology/pharmacology, Peptide Library, Peptides/chemistry/immunology, Quantitative Structure-Activity Relationship, Saliva/immunology, Salivary Proteins/chemistry/immunology, Streptococcus/immunology/pathogenicity
Identifiers
urn:nbn:se:umu:diva-10654 (URN)doi:10.1074/jbc.M511727200 (DOI)16595685 (PubMedID)
Note
Kemi, kemometri, odontologi, kariologiAvailable from: 2007-04-19 Created: 2007-04-19 Last updated: 2009-10-01Bibliographically approved
3. Sequence analyses of fimbriae subunit FimA proteins on Actinomyces naeslundii genospecies 1 and 2 and Actinomyces odontolyticus with variant carbohydrate binding specificities.
Open this publication in new window or tab >>Sequence analyses of fimbriae subunit FimA proteins on Actinomyces naeslundii genospecies 1 and 2 and Actinomyces odontolyticus with variant carbohydrate binding specificities.
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2006 (English)In: BMC Microbiology, ISSN 1471-2180, Vol. 43, no 6Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: Actinomyces naeslundii genospecies 1 and 2 express type-2 fimbriae (FimA subunit polymers) with variant Galbeta binding specificities and Actinomyces odontolyticus a sialic acid specificity to colonize different oral surfaces. However, the fimbrial nature of the sialic acid binding property and sequence information about FimA proteins from multiple strains are lacking. RESULTS: Here we have sequenced fimA genes from strains of A.naeslundii genospecies 1 (n = 4) and genospecies 2 (n = 4), both of which harboured variant Galbeta-dependent hemagglutination (HA) types, and from A.odontolyticus PK984 with a sialic acid-dependent HA pattern. Three unique subtypes of FimA proteins with 63.8-66.4% sequence identity were present in strains of A. naeslundii genospecies 1 and 2 and A. odontolyticus. The generally high FimA sequence identity (> 97.2%) within a genospecies revealed species specific sequences or segments that coincided with binding specificity. All three FimA protein variants contained a signal peptide, pilin motif, E box, proline-rich segment and an LPXTG sorting motif among other conserved segments for secretion, assembly and sorting of fimbrial proteins. The highly conserved pilin, E box and LPXTG motifs are present in fimbriae proteins from other Gram-positive bacteria. Moreover, only strains of genospecies 1 were agglutinated with type-2 fimbriae antisera derived from A. naeslundii genospecies 1 strain 12104, emphasizing that the overall folding of FimA may generate different functionalities. Western blot analyses with FimA antisera revealed monomers and oligomers of FimA in whole cell protein extracts and a purified recombinant FimA preparation, indicating a sortase-independent oligomerization of FimA. CONCLUSION: The genus Actinomyces involves a diversity of unique FimA proteins with conserved pilin, E box and LPXTG motifs, depending on subspecies and associated binding specificity. In addition, a sortase independent oligomerization of FimA subunit proteins in solution was indicated.

Keyword
biofilm, adhesion, fimbriae
National Category
Dentistry Dentistry
Identifiers
urn:nbn:se:umu:diva-12937 (URN)10.1186/1471-2180-6-43 (DOI)16686953 (PubMedID)
Available from: 2007-11-01 Created: 2007-11-01 Last updated: 2009-10-01Bibliographically approved
4. Actinomyces and Streptococcus species display differential binding epitopes on acidic proline-rich protein PRP-1
Open this publication in new window or tab >>Actinomyces and Streptococcus species display differential binding epitopes on acidic proline-rich protein PRP-1
Manuscript (Other academic)
Identifiers
urn:nbn:se:umu:diva-5294 (URN)
Available from: 2006-09-08 Created: 2006-09-08 Last updated: 2010-01-13Bibliographically approved

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