ESCMID Online Lecture Library © by Author

Library

Quorum sensingLecture in Candida speciesauthor Onlineby Mariana Henriques ©Paris | January 2011

ESCMID Quorum sensing in Candida spp. Candida infections

Library Comensal Candida albicans microorganisms Opportunistic pathogens Non‐Candida albicans Lecture candida (NCAC) species Morbility and Mortality author Superficial Candida glabrata and systemicby Onlineinfections Candida dubliniensis Nosocomial © infections Candida krusei Candida parapsilosis Candida tropicalis

High costs (> $1.7ESCMID billion/ year in the USA) Quorum sensing in Candida spp. Candida infections

Candida virulence factors Library

Adhesion and biofilm formation on abiotic surfaces

Colonization, invasion and damage of hostLecture tissues author DimorphismOnline phenomenonby (from yeast to filamentous form) © Enzymes production • Secreted aspartyl proteinases (Saps) • Phospholipases and lipases • HliHaemolysins ESCMID Quorum sensing in Candida spp. Candida infections

Candida biofilm Library

Lecture

Matrix

Cells author Surface Onlineby ©

ESCMID Quorum sensing in Candida spp. Candida infections

Candida biofilm Library

C. tropicalisC. C. albicansparapsilosis C. glabrata Lecture author Onlineby ©

ESCMID Quorum sensing in Candida spp. Candida infections

Library

Lecture author Copyright © AlfredOnline B. Cunningham, Johnby E. Lennox, and Rockford J. Ross, Eds. 2001-2008 Cell signa lling © “complex system of communication based on chemical signals that governs basic cellular activities and coordinates cell actions” * ESCMID * http://en.wikipedia.org/wiki/Cell_signaling [01-12-2007] Quorum sensing in Candida spp. Candida infections

Quorum sensing

Quorum sensing was firstly identified in bacteria, namelyLibrary in Vibrio fischeri, as a mechanism of cells to respond to changes in cell population density Lecture author Onlineby ©

QSM in C. albicans Library • FlFarnesol • Tyrosol

Effect of Farnesol and Tyrosol • C. albicans Lecture • NCAC spp author DetectionOnline ofby extracellular molecules (EM) © Effect of EM • C. albicans • NCAC spp ESCMID Quorum sensing in Candida spp. Farnesol and Tyrosol

Farnesol in C. albicans Library

Lecture author

OnlineMorphogenesisby

©Biofilm formation

Virulence ESCMID Quorum sensing in Candida spp. Farnesol and Tyrosol

Tyrosol in C. albicans Library

Lecture author Onlineby © Morphogenesis Biofilm formation

ESCMID Quorum sensing in Candida spp. Farnesol and Tyrosol

Farnesol vs Tyrosol Library

Lecture author Onlineby ©

FEMS Yeast Res 9 (2009) 990–999

ESCMID Quorum sensing in Candida spp. Outline

QSM in C. albicans Library • FlFarnesol • Tyrosol

Farnesol in C. albicans

Morphology Library C. albicans Faaesolrnesol without farnesol

Lecture

Biofilm formation author Biofilm metabolic Farnesol Biofilm Biomass Onlineby activity © 24h

Mitochondrial activity ESCMIDBiomass Quorum sensing in Candida spp. Effect of Farnesol and Tyrosol

Farnesol in NCAC spp Library 0 h 24 h Growth and morphology Farnesol Tirar foh50 uM

C. glabrata C. krusei C. tropicalis Lecture author Onlineby © Growth delay Growth delay Growth delay No changes in cell viability No changes in cell viability Changes in cell viability No alteration onESCMID morphology No alteration on morphology No alteration on morphology Quorum sensing in Candida spp. Effect of Farnesol and Tyrosol

Farnesol in NCAC spp

3 h 48 h Biofilm formation Library Farnesol Farnesol Tirar foh50 uM C. dublinienssis C. parapsilosis C. tropicalis

Lecture author Onlineby ©

ESCMID Quorum sensing in Candida spp. Effect of Farnesol and Tyrosol

Tyrosol in C. albicans 3 h 48 h Library Tyrosol Tyrosol

Mitochondrial activity Biomass cells dd Lecture Adhere author Onlineby ofilms ii © Mature b ESCMID Quorum sensing in Candida spp. Effect of Farnesol and Tyrosol

Tyrosol in NCAC spp Library C. dubliniensis C. parapsilosis C. tropicalis ed cells rr Lecture Adhe author Onlineby iofilms © Mature b ESCMID Quorum sensing in Candida spp. Outline

QSM in C. albicans Library • FlFarnesol • Tyrosol

Cross-effect of supernatants

Morphology Library

* * * * * * icans *

bb * * * * Lecture C. al author * Online* by *

liniensis * bb © * * * C. du

ESCMID * Martins et al., Eukariotic cell 6:12, 2429 P<0.05 Quorum sensing in Candida spp. Detection of extracellular molecules

Purification of cell signalling molecules Library Farnesol as an example*

Lecture author Onlineby © Large sample volume Solvent extraction

Complexity ESCMID * Hornby et al. (2001) App Env Microbiol Time consuming Quorum sensing in Candida spp. Detection of extracellular molecules

Library

HS – SPME

LectureSupernatant filtration Storage at 4 °C

authorCarbowax divinylbenzen Supernatant Onlineby e 20 ml sample © 4 ggaC NaCl 40°C ± 0.1°C 15 min 45 min

Sheme adapted from Vas and Vékey (2004) J Mass Spectrom ESCMID Martins et al., Eukariotic cell 6:12, 2429 Quorum sensing in Candida spp. Detection of extracellular molecules

Library GC-MS

Lecture author Farnesol Onlineby E- nerolidol © 2-phenylethanol

1-dodecanol ESCMID Isoamylalcohol Quorum sensing in Candida spp. Detection of extracellular molecules

Identification of Candida extracellular molecules Library

Isoamyl alcohol 2-Phenylethanol 1-Dodecanol E-Nerolidol E,E-Farnesol C. albicans +++++Lecture C. dubliniensis +++++ C. parapsilosis +++author − + C. tropicalis +++− + Onlineby ©

ESCMID Martins et al., J Basic Microbiol, 50(1): S89-S97, 2010 Quorum sensing in Candida spp. Outline

QSM in C. albicans Library • FlFarnesol • Tyrosol

Morphology Library

C. albicans C. dubliniensis Control 2-Phenylethanol % hyphae % pseudohyphae

inhibition inhibition ans cc Isoamyl alcohol 46 µM 68.4 ± 12.3 70.7Lecture ± 15.6

2-Phenylethanol 5µM 71.4 ± 15.7 72.9 ± 17.0 C. albi sis 1-Dodecanol 2 nM 57. 8 ± 14. 3 54. 1 ± 12author. 1 nn E-Nerolidol 1.5 nM 51.1 Online± 9.3by 68.3 ± 5.0 E,E-Farnesol 1.5 nM 63.5 ± 10.8 57.8 ± 4.8 © C. dublinie

ESCMID Martins et al., Eukariotic cell 6:12, 2429 Quorum sensing in Candida spp. Effect of extracellular molecules

3 h 48 h Biofilm formation Library EM EM

Mitochondrial activity Biomass Lecture C. albicans C. dubliniensis C. parapsilosis C. tropicalis Isoamyl alcohol 23 mM ↓ 26 % ↓ 46 % author↓ 52 % ↓ 52 % ↑ 34 % 46 µM ≈ ↓ 43 % 2-Phenylethanol 500 µM ↓ 19 % 5µM Online≈≈≈by ↓ 10 % 1-Dodecanol 2 µM © ↑ 46 % 2nM ≈ ≈ ↓ 33 % E-Nerolidol 1.5 µM ↓ 26 % ↓ 13 % ↓ 23 % 1.5 nM ≈≈↓ 30 % ↓ 13 % ESCMID Martins et al., J Basic Microbiol, 50(1): S89-S97, 2010 Quorum sensing in Candida spp. Effect of extracellular molecules

3 h 48 h Biofilm formation Library EM EM

Mitochondrial activity Biomass Lecture C. albicans C. dubliniensis C. parapsilosis C. tropicalis Isoamyl alcohol 23 mM ≈ ≈ 46 µM author ≈ ≈ 2-Phenylethanol 500 µM 5µM Online≈≈≈by ≈ 1-Dodecanol 2µM © ≈ ≈ 2nM ≈ ≈ E-Nerolidol 1.5 µM ↓ 26 % ↓ 25 % 1.5 nM ≈≈↓ 30 % ↓ 25 % ESCMID Martins et al., J Basic Microbiol, 50(1): S89-S97, 2010 Quorum sensing in Candida spp. Effect of extracellular molecules

EM cocktail

Morphology Library

C. albicans C. dubliniensis

* Lecture author Onlineby © 1- C. albicans planktonic 2- C. albicans biofilm * 3- C. dubliniensis P<0.05 planktonic 4- C. dubliniensis biofilm ESCMID Martins et al., Eukariotic cell 6:12, 2429 Quorum sensing in Candida spp. Conclusions

QSM in C. albicans •Farnesol • Tyrosol Library Effect of Farnesol and Tyrosol • C. albicans • NCAC spp

DiDetection of extracellul ar mollleculLecturees (EM)Tyrosol

ESCMID Quorum sensing in Candida spp. Conclusions

QSM in C. albicans Tyrosol •Farnesol • Tyrosol LibraryFarnesol Effect of Farnesol and Tyrosol • C. albicans • NCAC spp

DiDetection of extracellul ar mollleculLecturees (EM)

Effect of EM • C. albicans author • NCAC spp Farnesol - Effect on Tyrosol - Effect on growthOnline and onby biofilms is biofilms © dddtependent on dependent on the species the species

ESCMID Quorum sensing in Candida spp. Conclusions

QSM in C. albicans Tyrosol •Farnesol • Tyrosol LibraryFarnesol Effect of Farnesol and Tyrosol Farnesol Tyrosol • C. albicans • NCAC spp

DiDetection of extracellul ar mollleculLecturees (EM)

Isoamylalcohol ESCMID Quorum sensing in Candida spp. Conclusions

QSM in C. albicans Tyrosol •Farnesol • Tyrosol LibraryFarnesol Effect of Farnesol and Tyrosol Farnesol Tyrosol • C. albicans • NCAC spp

E- nerolidol 2-phenylethanol DiDetection of extracellul ar molllecules (EM) 1-dodecanol Lecture Isoamylalcohol

Effect of EM • C. albicans author • NCAC spp

ESCMIDDependent on the species and on the alcohol Quorum sensing in Candida spp. Conclusions

QSM in C. albicans Tyrosol •Farnesol • Tyrosol LibraryFarnesol Effect of Farnesol and Tyrosol Farnesol Tyrosol • C. albicans • NCAC spp

E- nerolidol 2-phenylethanol DiDetection of extracellul ar molllecules (EM) 1-dodecanol Lecture Isoamylalcohol

Effect of EM • C. albicans author • NCAC spp Onlineby © The classification of these alcohols as quorum sensing molecules is still a controversy ESCMID Quorum sensing in Candida spp Acknowledgments

Library

University of Minho, Braga, Portugal

ESCMID