1. La Thérapie par les
Bactériophages
Approche par la Médecine Dentaire
BIF7000 – UQAM 2013
•Patricia Morales
•Natalia Samus
•Igor Lemos
•Deraldo Duran (Lisbon University)

2. BACKGROUND
Problématique
Medécine Dentaire

3. BACKGROUND
Problématique
Streptococcus mutans
• Most important bacteria
associated with tooth
decay,
• Naturally present in the
human oral microbiota

4. BACKGROUND
Problématique
Healthy teeth

5. BACKGROUND
Problématique
Dental caries
Periodontal diseases and dental caries are the
most prevalent chronic diseases, affecting
children, adolescents, adults and the elderly. In
fact, dental caries affects 60% to 90% of
schoolchildren and the vast majority of adults in
most industrialized countries.
http://www.phac-aspc.gc.ca/publicat/cdicmcbc/30-4/preface-eng.php#ref_05

6. BACKGROUND
Problématique
Dental caries
Endodontic therapy
• In certain cases,
endodontic therapy
may be necessary for
the restoration of a
tooth

7. BACKGROUND
Problématique
Dental caries
Endodontic therapy
Prosthetic teeth
Dental surgery

8. BACKGROUND
Problématique
Streptococcus mutans 80% - 90%

9. BACKGROUND
Problématique
Efficacy of dental floss in
addition to a toothbrush
??????????????
X
In fact, dental caries affects 60% to 90% of
schoolchildren and the vast majority of adults in
most industrialized countries.

10. BACKGROUND
Phage therapy
Synthetic
Biology
Bioinformatics
+
Genetic engineering
+
Mol Biology

11. Phage Therapy
Phage therapy is the therapeutic use of
bacteriophages to treat pathogenic bacterial
infections
1:n
Lysin
Streptococcus mutans

12. Phage Therapy
1 - Bacteriophage
X
1:n
n - Bacteries
Chain Reaction

13. Phage Therapy
Solution
1 - Bacteriophage
1:1
1 - Bacteria
No Chain Reaction

14. SOLUTION
Synthetic
Bacteriophage
Engineered dna
PROMOTER+LYSIN
1:1
Lysin
Streptococcus mutans

15. SOLUTION
Synthetic
Bacteriophage
Toothpaste

16. Lysin destroys a cell wall of grampositive bacteria (Streptococcus)
Lysin
Hydrolysis of (1->4)-beta-linkages
between N-acetylmuramic acid
and N-acetyl-D-glucosamine
residues in a peptidoglycan
Peptidoglycan

17. PHAST(PHAge Search Tool) - web server designed
to identify, annotate and graphically display prophage sequences
within bacterial genomes or plasmids
10947 Genomes Databases for download:
vIrus DB (57.902 Mb). Last update: Nov 1 2013
Prophage and virus DB (58.039 Mb). Last update: Nov 1 2013
Bacteria DB (2.626 Gb). Last update: Nov 1 2013
PHAST result DB (tar zip format) (104.167 Mb). Last update: Nov 1 2013

18. Pathologic Streptococci
(compete genom)
NC number
strain
PHAGE #
Str. mutans
NC_004350.1
UA159
1
Str. mutans
AP010655.1
NN2025
0
Str. mutans
CP003686.1
GS-5
0
Str. criceti
AEUV00000000
HS-6
-
Str. ratti
-
-
-
Str. sobrinus
-
-
-
Str. downei
AEKN00000000.1
F0415
1
Str. ferus
-
-
-
Str. macacae
AEUW00000000
NCTC 11558
-
Allan L. Delisle, Ming Guo, Natalia I. Chalmers, et al Appl Environ Microbiol. 2012 Apr;78(7):2264-71

19. Streptococcus mutans
UA159 complete genome
Genes
de phages

20. Streptococcus mutans UA159
genome, phage genes

21. Streptococcus mutans UA159,
genes of phages
#
CDS_POSITION
BLAST_HIT
E-VALUE
SEQUENCE
1
complement(1777567..17
78640)
PHAGE_Entero_phiV10: O-acetyltransferase; SMU_1884c;
phage(gi89152472)
4e-16
Click
2
complement(1781127..17
81393)
PHAGE_Human__2: EBNA-1; SMU_1889c; phage(gi139424506)
5e-06
Click
3
complement(1781999..17
82835)
PROPHAGE_Escher_MG1655: IS150 transposase B;
SMU_1893c; phage(gi16131429)
8e-74
Click
4
complement(1782829..17
83347)
PROPHAGE_Ralsto_GMI1000: ISRSO11-transposase ORFA
protein; SMU_1894c; phage(gi17546156)
2e-07
Click
5
complement(1783662..17
83964)
PHAGE_Staphy_PT1028: ORF011; SMU_1896c;
phage(gi66395175)
4e-07
Click
6
1784304..1784786
PHAGE_Bacill_SPBc2: ABC transporter; SMU_1897;
phage(gi9630145)
3e-07
Click
7
1784881..1786392
PHAGE_Bacill_SPBc2: ABC transporter; SMU_1898;
phage(gi9630145)
5e-62
Click
8
1786502..1787515
PHAGE_Megavi_lba: hypothetical protein; SMU_1900;
phage(gi448825457)
1e-12
Click
9
complement(1788212..17
89234)
PHAGE_Equid__9: envelope glycoprotein J; SMU_1904c;
phage(gi216905924)
5e-05
Click

22. Streptococcus downei F0415
genome, phage genes

23. Endolysin
70%

24. Putative endolysin,
phage M-102
Definition:
Streptococcus phage M102
Accession:
NC_012884
Length:
31147 bp
LOCUS_TAG
M102_gp20
ORF START
20879
ORF STOP
21394
STRAND
Forward
REGION
1
REGION intact or
incomplete
39.25
PROTEIN SEQUENCE
MLKKTLAILGLSASLLFVSAHANAHTSRLTLDQTNELYTRLAAEGRGVDT
DQVYGMQCVD
IDTDLTNNYVGIPISGNAIDLLDSARRAGYEIVPANKPPRAGDLFVMDTN
ALYGHPFGHT
GYVYKVNPDGSFETVEQNVGDDSNFYTGTVAKFMHRTRDYMLGYIRLA
YRK
HOMOLOG/ORTHOLOG
SPECIES
PHAGE_Strept_M102
HOMOLOG/ORTHOLOG
PROTEIN
putative endolysin; phage(gi242345591)
HOMOLOG/ORTHOLOG
EVALUE
1e-96

25. Result BLAST
endolysin phage M-102
•lysin [Streptococcus mutans]
•NCBI Reference Sequence: WP_002287207.1
•LOCUS WP_002287207 171 aa linear BCT 12-MAY-2013
•DEFINITION lysin [Streptococcus mutans].
•ACCESSION WP_002287207 VERSION WP_002287207.1 GI:488215999
•SOURCE Streptococcus mutans
•ORGANISM Streptococcus mutans Bacteria; Firmicutes; Bacilli; Lactobacillales;
Streptococcaceae; Streptococcus.
•COMMENT REFSEQ: This record represents a single, non-redundant, protein sequence which
may be annotated on many different RefSeq genomes from the same, or different, species.
•FEATURES Location/Qualifiers source 1..171 /organism="Streptococcus mutans"
/db_xref="taxon:1309" Protein 1..171 /product="lysin" /calculated_mol_wt=18727 Region
54..165 /region_name="CHAP" /note="CHAP domain; pfam05257" /db_xref="CDD:218523"
•ORIGIN 1
•mlkktltflg lsvgllivsp hanahtsrlt ldqtnelytr laaegrgvdt dqqygmqcvd 61 idtdltnnyv gvpisgnaid
lldsaraagy eivpaskppr agdlfvmdts tvyghpfght 121 gyiyrvnpdg sfetveqnvg ddsnlytgtv
akfmhrtrdy mlgyirlayr k

26. Phage PH-10
complete genome

27. Lysin A,
phage PH-10
Definition:
Streptococcus phage
PH10
Accession:
NC_012756
Length:
31276 bp
LOCUS_TAG
PH10_gp51
ORF START
29525
ORF STOP
30529
STRAND
Forward
REGION
1
REGION intact or
incomplete
39.53
PROTEIN SEQUENCE
MKKNDLFIDVSSHNGYDITGILEQMGTTNTIIKISESTSYMNPCLSAQVEQSNPIGFYHFAWFG
GDIEEAEREARYFLDNVPQKVKYLCLDYEDHASGDKQANTDACIRFMEILKENGYEPIYYSYKP
FTLNNIYYEQILEKFPNSLWIAGYGLNDGTADFEYFPSMDGIRWWQYSSNPYDKNIVLLDDEE
AKPKWKRNDTGWWYEYPDGSYPKEEWEKIDGTWYYFNERGYSIASRWLKDDGKWYYLKE
NGAMAVGWVLVNGKWYYLDASGAMVTGWVQYKDKLYHFKEENGEMSSKEL
VKVEGGWYYVNEDGSRSDKPALDVLPDGLIVTTK
HOMOLOG/ORTHOLOG
SPECIES
PHAGE_Strept_PH10
HOMOLOG/ORTHOLOG
PROTEIN
lysA; phage (gi238821368)

28. Str.
downei
Str.
mutants
Phages
M-102, PH-10
Phage
M-102
Protein LYSIN

29. Résultats
•En utilisant la base de données de PHAST nous avons trouvé
rapidement deux Streptococcus pathogènes (Str. mutants et
Str. downei) les quels contiennent gènes de phages dans ses
génomes.
•On a trouvé parmi ces deux bacteriophages specifiques de
streptococcus mutants (M-102 and PH-10), le gène qui
contiennent la protéine lysin, la plus active des protéines
antimicrobiennes.
•Ainsi les protéines de ces phages pourraient être concernés
comme des agents potentiels contre les streptocoques
pathogènes pour d'autres études.