current trends in antimicrobial agent research: chemo- and ... - biofilm - food fraud... · ismail...
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Ismail Fliss and Riadh Hammami
Current trends in antimicrobial agent research:chemo- and bioinformatics approaches
Antimic 2017, Québec, 21-23 juin
A Multidisciplinary Team
Amina SghairiFrançois Bédard
Riadh Hammami Sylvie Rebuffat Éric Biron Muriel Subirade
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Chemical additives and salts: very controversial barriers
Prohibition of antibiotics as a growth promotor
Very few new antibioticsResistance phenomenon
MAPs
Sustainable alternative
Natural
Safe (resistance)
Why the urgent need for antimicrobial peptides (AMP)?
AMPs: Widely distributed compounds through Nature
o Key components of innate, non-adaptive immune defense systems of multicellular organisms.
o Set of cationic and amphiphilic peptides, 20 to 50 amino acids, broad spectrum of antibacterial and antifungal activity.
o Great structural diversityo Mechanism of action: permeabilization of the cell membrane by a
detergent-like effect or by pore formation.
Plant AMPs
Microbial AMPs
Milk AMPs
Fish AMPS
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AMPs: Ubiquitous active compounds
o Small cysteine-rich AMPs
o Bacteriocins (nisin)
o Latoferrin, lactoperoxidase
o Cecropin family
• Narrow spectrum
• MIC in nM
• Non-toxic
• Thermal stability
• Mode of action (multiple)
• Food grade (GRAS)
• Non-ribosomal synthesis
• Large spectrum
• MIC in μM
• Toxic
Past
Antibiotics Vs AMPs
Future
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Enzymeribsome
Antibiotics
A
C
D E
Bβ-lactams, vancomycin
Fluoroquinolones, rifamycins
Daptomycin, lipopetides
macrolides
tetracyclines
aminoglycosides
trimethoprim,
sulfonamides
nisin, nukacin ISK-1
pediocin, nisin A, dysgalacticin
colicin, microcin B17
microcin J25
Protein synthesis
Replication and
transcription
Cell wall
synthesis
Perturbation de
membrane
composition
Perturbation of
membrane efflux
pediocin PA-1
Septum formation
benzamides,
N-heterocycles,
phenols
garvicin A,
lactoccin 972
Mode of action
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PEPTIDES
ANTIMICROBIENS
Extraction/purificationChimio-and bioinformatics
Recombinant organisms
• Identification
• Characterization
• Extraction
• Purification
• Development of databases
• Sequence analysis
• Development of new analogues
• Modeling and molecular dynamics
• Molecular interactions (in silico)
• Synthesis
• Identification de genetic
• Cloning
• Expression
• Production (microcin J25)
1 2
3
Bacterials Plants
Animals
Strategies for the production of AMPs
• Random Methods• Time consuming and
expensive• Variables (tests used)• Low yields
• Rapid• Inexpensive• Good correlation with
biological activity• High yields
Bioinformatics analysis tools and methods
o Bioinformatics: Concept using mathematics, statistics, computer science, technology and molecular biology
o Bioinformatic methods: include data collection, information management, sequence analysis, molecular interactions and advanced systems simulations.
oDatabases
oGenomics tools
oBioinformatics tools
oPrograms
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Contain genomic, proteomic and functionalinformation
Classification of bioinformatics tools and methods
o Thousands of peptides have been identified
o Very few having known biological activities, structures or sequences
o Several information absent or dispersed in the scientific literature (not available to potential users).
o The majority of the sequences are listed in UniProtKB / Swiss-Prot: universal and non-specific database.
o Urgent need for:
o Collect, filter and analyze these information
o Store in more specialized resources to improve efficiency of use
Why databases for AMPs?
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Database contents and tools• Literature
•Microbiology
•Physicochemical
•Taxonomy
Query
•BLAST
• FASTA
• SSEARCHHomology Search
•ClustalW
•MUSCLE
•T-Coffee
Multiple Sequence Alignment
•Various Physicochemical parameters
•Wheel representation
•Hydropathy
Physicochemical profile
•2D: NPS@
•3D: Modeller
Structure Prediction
• Sequences
• Structures
•Database dumpData Recovery
A database is a set of structured largecollection of data organized especiallyfor rapid search and retrieval by acomputer.
Summary of databases dealing with AMPs
Bactibase: web-accessible database for bacteriocins
o Created in 2007o 123 bacteriocins produced by both Gram-
positive and Gram-negative bacteria.
o Information is very easy to extract and allows rapid prediction of relationships structure/function and target organisms
o The BACTIBASE database is freely available at http://bactibase.pfba-lab.org
o 47742 visitors since January 2016
Gram-positive bacteriocins : a new structure-based classification 10 20 30 40 50 60 70
Group Genus Bacteriocin .........|.........|.........|.........|.........|.........|.........|....
G 1
a
Ac Actargadine ---------------SSGWVCTLTIECGT-----V---------ICAC-------------------------- -1
Bs Mersacidin ---------CTFTLPGGGGVCTLTSEC-----------------IC---------------------------- -1
La Lacticin 3147A1 --CSTNTFSLSDYWGNNGAWCTLTHEC-M-----A---------WCK--------------------------- 0
b
Lac Plantaricin Wa ---KCKWWNISCDLGNNGHVCTLSHEC-Q-----V---------SCN--------------------------- +2
La Bacteriocin J46 -------------KGGSGVIHTISHEVIYNSWNFVF--------TCCS-------------------------- +2
La Lacticin 481 -------------KGGSGVIHTISHECNMNSWQFVF--------TCCS-------------------------- +2
Sta Nukacin ISK1 -------------KKKSGVIPTVSHDCHMNSFQFVF--------TCCS-------------------------- +4
Ko Variacin ---------------GSGVIPTISHECHMNSFQFVF--------TCCS-------------------------- +1
St Streptococcin AFF22 --------------GKNGVFKTISHECHLNTWAFLA--------TCCS-------------------------- +3
St Streptococcin AM49 --------------GKNGVFKTISHECHLNTWAFLA--------TCCS-------------------------- +3
Ru Ruminococcin A --------------G-NGVLKTISHECNMNTWQFLF--------TCC--------------------------- +1
St Mutacin H29B ------------NRWWQGVVPTVSYECRMNSWQHVF--------F----------------------------- +2
St Mutacin 2 ------------NRWWQGVVPTVSYECRMNSWQHVF--------TCC--------------------------- +2
G 2
Stm Ancovenin ---------------------CV-QSCS--FGPLTW--------SCD-GNTK---------------------- 0
Stm Duramycin C ---------------------CA-NSCS--YGPLTW--------SCD-GNTK---------------------- 0
Stm Cinnamycin ---------------------CR-QSCS--FGPFTF--------VCD-GNTK---------------------- 0
G 3
En Bacteriocin 31 ------------ATYYGNGLYCNKQKCWVDWNKASREIG----KIIVNG-NVQHGPWAP--R------------ +2
Leu Leucocin C -------------KNYGNGVHCTKKGCSVDWGYAWTNIANNSVMNGLTG---GNAGWHN--------------- +4
Li Listeriocin 743A -------------KSYGNGVQCNKKKCWVDWGSAISTIGNNSAANWATG---GAAGW----KS----------- +4
Lac Sakacin P -------------KYYGNGVHCGKHSCTVDWGTAIGNIGNNAAANWATG---GNAGW-N--K------------ +4
En Mundticin -------------KYYGNGVSCNKKGCSVDWGKAIGIIGNNSAANLATG---GAAGW-S--K------------ +4
En Mundticin KS -------------KYYGNGVSCNKKGCSVDWGKAIGIIGNNSAANLATG---GAAGW----KS----------- +4
Lac Prebacteriocin 423 -------------KYYGNGVTCGKHSCSVNWGQAFSCSV----SHLAN-F--GHG------KC----------- +6
Leu Leucocin A -------------KYYGNGVHCTKSGCSVNWGEAFSAGV----HRLANG---GNGFW----------------- +4
Leu Leucocin BTa11a -------------KYYGNGVHCTKSGCSVNWGEAFSAGV----HRLANG---GNGFW----------------- +4
Leu Mesentericin Y105 -------------KYYGNGVHCTKSGCSVNWGEAASAGI----HRLANG---GNGFW----------------- +4
Lac Plantaricin C19 -------------KYYGNGLSCSKKGCTVNWGQAFSCGV----NRVATA---GHGK------------------ +6
Lac Bavaricin MN ------------TKYYGNGVYCNSKKCWVDWGQAAGGIG----QTVVXGW--LGGAIPG--K------------ +3
Car Divercin V41 ------------TKYYGNGVYCNSKKCWVDWGQASGCIG----QTVVGGW--LGGAIPG--KC----------- +3
Bs Coagulin A -------------KYYGNGVTCGKHSCSVDWGKATTCIINNGAMAWATG---GHQGTH---KC----------- +6
Car Divergicin M35 ------------TKYYGNGVYCNSKKCWVDWGTAQGCI------DVVIG--QLGGGIPGKGKC----------- +3
En Enterocin A --------TTHSGKYYGNGVYCTKNKCTVDWAKATTCIA----GMSIGGF--LGGAIPG--KC----------- +3
En Enterocin CRL35 -------------KYYGNGVSCNKKGCSVDWGKAIGIIGNNSAANLATG---GAAGW----KS----------- +4
Pe Pediocin PA1 -------------KYYGNGVTCGKHSCSVDWGKATTCIINNGAMAWATG---GHQGNH---KC----------- +6
Car Pisciocin V1a -------------KYYGNGVSCNKNGCTVDWSKAIGIIGNNAAANLTTG---GAAGWN---KG----------- +3
Car Piscicolin 126 -------------KYYGNGVSCNKNGCTVDWSKAIGIIGNNAAANLTTG---GAAGWN---KG----------- +3
Lac Bavaricin A -------------KYYGNGVHXGKHSXTVDWGTAIGNIGNNAAANXATG---XNAGG----------------- +3
En Enterocin SEK4 ------------ATYYGNGVYCNKQKCWVDWSRARSEIIDRGVKAYVNGFTKVLGGIGG--R------------ +5
La Lactococcin MMFII -------------TSYGNGVHCNKSKCWIDVSELETYKA----GTVSN--PK--DILW--------------- +1
En Enterocin P -----------ATRSYGNGVYCNNSKCWVNWGEAKENIA----GIVISGW---ASGLAGMGH------------ +2
Car Carnobacteriocin BM1 ------------AISYGNGVYCNKEKCWVNKAENKQAIT----GIVIGGW---ASSLAGMGH------------ +3
Car Pisciocin V1b ------------AISYGNGVYCNKEKCWVNKAENKQAIT----GIVIGGW---ASSLAGMGH------------ +3
Lac Curvacin A ------------ARSYGNGVYCNNKKCWVNRGEATQSII----GGMISGW---ASGLAGM-------------- +3
Lac Sakacin A ------------ARSYGNGVYCNNKKCWVNRGEATQSII----GGMISGW---ASGLAGM-------------- +3
Car Carnobacteriocin B2 -------------VNYGNGVSCSKTKCSVNWGQAFQERYTAGINSFVSG---VASGAGSIGRRP---------- +4
G 4
a Sta Epidermin ---------------IASKFICTP-GCAK-TGSFN---------SYCC-------------------------- +2
Sta Gallidermin ---------------IASKFLCTP-GCAK-TGSFN---------SYCC-------------------------- +2
St Mutacin 1140 ---------------FKSWSLCTP-GCAR-TGSFN---------SYCC-------------------------- +2
St Mutacin BNy266 ---------------FKSWSFCTP-GCAK-TGSFN---------SYCC-------------------------- +2
St Streptin ----------------GSRYLCTPGSCWK-LVCFTTT--------VK--------------------------- +3
b La Nisin A ---------------ITSISLCTP-GCK--TGALMGCNM----KTATCHCSIHVSK------------------ +2
La Nisin Z ---------------ITSISLCTP-GCK--TGALMGCNM----KTATCNCSIHVSK------------------ +4
Bs Subtilin ---------------WKSESLCTP-GCV--TGALQTCFL----QTLTCNC--KISK------------------ +2
G5 Sta Epicidin 280 --------------SLGPAIKATRQVCPKATRFVTV--SCK-KSDCQ--------------------------- +5
Sta Pep 5 --------------------KT-----LKATRLFTV--SCKGKNGCK--------------------------- +8
G 6
Bs Cytolysin ----------------GDVHAQTTWPCATVGVSVAL---CP-TTKCTSQC------------------------ +1
La Lacticin 3147A2 --------------TTPATPAISILSAYISTNTCPT-------TKCTRAC------------------------ +2
Lac Plantaricin Wbeta ----------------SGIPCTIG---AAVAASIAV---CP-TTKCSKRCGKRKK------------------- +7
G7
Lac Acidocin J1132 beta --------------GNPKVAHCASQI----GRST------A-WGAVSGA------------------------- +3
Lac Plantaricin 1.25 beta ---KKKKKKVACTWGNAATAAASGAVXGILGGPTGALAGAI-WGVSQCASNNLHGMH----------------- +8
G8
Car Carnobacteriocin A ---DQMSDGVNYGKGSSLSKGGAKCGLGIVGGLATIPSGPLGWLAGAAGVINSCMK------------------ +2
En Enterocin B ENDHRMPNELN--RPNNLSKGGAKCGAAIAGGLFGIPKGPLAWAAGLANVYSKCN------------------- +4
G 9
Lac Gassericin T --------RNNWAANIGGVGGATVAGWALGNAVCGPACGFVGAHYVPIAWAGVTAATGGFGKIRK--------- +5
Lac Lacticin FlafA --------RNNWQTNVGGAVGSAMIGATVGGTICGPACAVAGAHYLPILWTAVTAATGGFGKIRK--------- +5
Lac Plantaricin S alfa --------RNKLAYNMGHYAG--------KATIFG-----------LAAWALLA-------------------- +4
G 1
0 Lac Acidocin LF221B ---NKWGNAVIGAATGATRGVSWCRGFG-PWGMTACALG----GAAIGGYLGYKSN------------------ +6
Lac Lacticin FlafX ---NRWGDTVLSAASGAGTGIKACKSFG-PWGMAICGVG----GAAIGGYFGYTHN------------------ +3
Lac LactobinA ---NRWTNAYSAALGCAVPGVKYGKKLGGVWGAVIGGVG----GAAVCGLAGYVRKG----------------- +6
G 1
1 Lac Lactocin 705 -------------GMSGYIQGIPDFLKGYLHGISAAN-------KHKKGRL----------------------- +6
Car Divergicin 750 -------------KGILGKLGVVQAGVDFVSGVWAG---------IKQSAKDHPNA------------------ +3
G 1 2 Bs Sublancin 168 --------------GLGKAQCAALWLQCASGGTIGCGGG----AVACQNYRQFCR------------------- +3
Lac PlnJ ---------------GAWKNFWSSLRKGFYDGEAGR--------AIRR-------------------------- +4
The classification of bacteriocins from G(+) is complicated by their heterogeneity and increasing numberClassification schemes have had to continuously evolve
MilkAmp: dairy derived AMPshttp://milkampdb.org/home.php
o Created in 2012o 371 entries: 9 hydrolysates,
299 AMPs, 23 peptides predicted as antimicrobial and 40 non-active peptides.
o Freely available at http://milkampdb.org/,
o Should be useful to develop biologically active peptides for both pharmaceutical and food sectors
PhytAmp: Plant derived AMPshttp://phytamp.hammamilab.org/main.php
o 24 janv. 2012271 plant AMPs
o Expected to grow quickly with the rapid development of genomic and proteomic
o https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2686510/
Genomics based bioinformatics tools
Computational tools dedicated to: .o Development of whole-genome sequencing and expression
profiling technologies;
o Identify homologs of known genes (comparing new sequences with sequences of biochemically characterized active peptides);
o Identify genes that encode AMPs
o Large target-screening approaches and provide new insight into antimicrobial mechanisms of action (MOAs)
Structure-based drug design methods: QSAR methods
o One of the most broadly used chemoinformatics approacheso Quantitative models that correlate the variation in biological activity with the
variation in molecular structureo Applied to closely related sequence variantso QSARs /artificial neural networks (ANNs): accelerate the discovery
and design of AMPs and applied to a larger peptides with high sequence diversity. o Successfully used for as antiviral/anti-HIV inhibitory peptideso Various commercial programs for QSAR: MOE, ACD labs and Tsar
Artificial neural networks
o Artificial neural networks: mathematical modeling algorithms
o Reliable and efficient way for in silico identification of novel AMPs
o Successfully applied for prediction of the antifungal activities
o ANN analysis can be performed with Tsar or Neural Network Toolbox
Structure-based drug design methods: Fuzzy logic modeling
o Fuzzy logic modeling : analysis of AMPs to distinguish active from inactive peptides with impressive accuracy.
o Fuzzy calculation can be performed using Gait-CAD
Molecular simulations and dynamics
o Demonstrate the inhibition activity and elucidate the mode of action of AMPs ( effect on bacterial membranes, formation of transmembrane ion-permeable pores and detergent-like effect)
o Use of live bacterial strains (availability and handling)
o Current area of research involves simulating bacterial membrane and AMP interactions with phospholipid bilayers
Membrane externe:Bicouche de phospholipides
Peptidoglycane
Membrane interne:Bicouche de phospholipides Membrane interne:
Bicouche de phospholipides
Couche épaisse de peptidoglycane
Acide lipoteichoique(LTA)
Acide teichoique(TA)
Molecular simulations and dynamics
Listeria monocytogenesEscherichia coli
Molecular simulations and dynamics
https://www.youtube.com/watch?v=c2utqcDdX_w
Concluding remarks
o Urgent need for new antimicrobials
o Experimental approaches
o The recent development in genomics and bioinformatics tools combined to existing structural and functional data offer a new opportunity to develop novel peptide candidates as antimicrobial molecules.
o Bioinformatics approaches: Rapid, reproducible, low cost, safe, high yields
o Amina Sghairi
o Mohamed Kacem Ben Fradj
o Jeannette Ben Hamida
o Eric Biron
oMuriel Subirade
o Sylvie Rebuffat
Acknowledgments