C. albicans Biofilm Resistance

A Multiple Mechanism Process

David Andes University of Wisconsin

Biofilms

•  3 dimensional microbial community = network of cells, adherent to each other and another surface and encapsulated within an extracellular matrix

•  Phenotypically distinct –  Growth rate = slower –  Adherent –  Matrix –  Drug resistance

Candida Biofilm Resistance

•  50% of all hospital infections are associated with medical devices. These infections invariably involve biofilm growth

•  The most important modifiable factor associated with reduced mortality in patients with candidemia is catheter removal (N=1915, Mortality 28% removed vs 41% retained)

•  Drug treatment of Candida biofilms is largely ineffective, primarily due to drug resistance

Drug (µg/ml) Biofilm-Associated

Resistance (Biofilm EC50/Plank MIC)

Amphotericin B 6-100x

Anidulafungin 8-32x

Fluconazole >1000x

Flucytosine >1000x

Baillie et al. 1998, Andes in press, Mukherjee et al. 2003

MIC=minimal inhibitory concentration EC50 (Effective concentration) is the drug concentration associated with a 50% reduction

•  Candida mechanisms considered •  Learning from prokaryotes •  Candida matrix composition and

resistance contribution •  Genetic basis for matrix associated

resistance •  Proposed Model •  Current studies

Candida Biofilm Resistance Overview

•  Pump mutants are more susceptible, but a large amount of resistance phenotype remains unexplained

Impact of Efflux Pumps

Strain Planktonic MIC

Biofilm MIC

3153a 4 >256 Δcdr1 0.25 128 Δcdr2 0.25 128 Δcdr1/Δcdr2 0.25 256 Δmdr1 0.25 64 Δcdr1/Δmdr1 0.25 256

Ramage et al JAC 2002;49:973

Uppuluri et al AAC 2008;52:1127

Role of Calcineurin Pathway

•  Pharmacologic and genetic inhibition of the pathway enhances triazole biofilm susceptibility

Impact of Growth Rate

Rate of growth controlled by glucose content of media (lower concentration = slower growth) l Planktonic cells n  o ¡ Biofilm cells

AmB, Fluc, and 5FC added at 20X planktonic MIC = no impact on biofilm viability regardless of growth rate

Ballie and Douglas AAC 1998;42:1900

EARLY LATE

Mukerjee et al AAC 2003;71:4333

Impact of Sterol Changes?

Persister Phenotype

•  Persister cells exhibited resistance to AmB •  Upregulation of glucan genes and down regulation of

sterol genes

Khot et al 2006;50:3708

Impact of Matrix - Penetration

Biofilm on filter disc

Fresh filter disc

Antifungal containing agar

Candida containing agar

Zone of inhibition

Fresh filter disc

Zone of inhibition correlated with standard curve from Antifungal spiked filter discs

Al-Fattini and Douglas AAC 2004:48:3291

Impact of Matrix - Penetration

C = biofilm diameter C0 = spiked disc diameter Each symbol represents different Candida species After 1 h the penetrated concentration exceeded the planktonic MIC many fold

Al-Fattini and Douglas AAC 2004:48:3291

Impact of Amount of Matrix

MORE MATRIX LESS MATRIX

•  Reduced susceptibility to AmB associated with increase in matrix

Al-Fattani et al J Med Microbiol 2006;55:999

• P. aeruginosa biofilm drug resistance

- Cyclic β-1,3 glucan in the periplasm sequesters antibiotic

•  rapA influence on E. coli biofilm resistance. –  Decreased mutant matrix

carbohydrate

Mah et al Nature 2003;426:306, Lynch et al. Antimicrob Agents Chemother. 2007

Bacterial Glucan-Mediated Drug Resistance

Cell Wall β-1,3 glucan Content From Biofilm and Planktonic Cells

Nett et al. Antimicrob Agents Chemother 2007;51:510

Cell Growth Condition

Biofilm Log Stationary

Mic

rogr

ams

0

200

400

600

800

1000

1200

1400

1600

Alkali soluble glucanB 1,6 glucanB 1,3 glucanB 1,3 and 1,6 glucanTotal carbohydrate

Biofilm Planktonic2 um

A

1 um

B

Biofilm Planktonic2 um

A

2 um

A

1 um

B

1 um

B

Biofilm  

200  nm  

260  nm  

Planktonic  

120  nm  

200  nm  

Candida Biofilm Matrix

Extracellular Matrix Composition Carbohydrate 41% Phosphorus 0.4% Glucose 15.9% Protein 5.2% Hexosamine 3.4%

Ballie and Douglas JAC 2000;46:397

In  vitro  Supernatant  

In  vitro  Matrix  

In  vivo  Serum  

Beta-­‐1,3  glucan  (pg/ml)  

0  

50  

100  

150  

200  

250  

300  Biofilm    

Non-­‐Biofilm  

*  

*  *  

Candida Matrix Composition

•  Increased Beta 1,3 glucan, compared to planktonic cells both in vivo and in vitro

Nett Antimicrob Agents Chemother. 2007 ;51:510

Biofilm:Antifungal Association

- Using radio-labeled fluconazole, the majority of fluconazole associating with the intact biofilm was found in the biofilm matrix component

Nett et al. J Infect Dis. 2010

0500

1000150020002500300035004000

H3

Fluc

onaz

ole

(CPM

)

Inta

ctB

iofil

m

Bio

film

Mat

rix

Cel

l Wal

l

Cyt

opla

sm

-  In vitro combination assays found disruption of glucan by β 1,3 glucanase treatment rendered biofilms more susceptible to fluconazole,

-  Disruption of glucan by β 1,3 glucanase decreased the amount of radio-labeled fluconazole associating with the intact biofilm and the biofilm matrix *p<0.05

Impact of Glucan Modification on Fluconazole Susceptibility In vitro

Nett et al. Antimicrob Agents Chemother. 2007, Nett et al. J Infect Dis. 2010

50%

100%

0 125

Fluconazole (µg/ml)

Bio

film

Gro

wth

(%)

Control

B 1,3 glucanase

C. albicans Biofilm Gene Expression

114 > 1.5 fold at 24 h 111 > 1.5 fold at 12 h 82 same genes

Nett et al J Infect Dis 2009

ORF Gene Putative Function 19.2495 FKS1 β-1,3 glucan synthase 19.2929 GSL2 Glucan synthase

19.5058 SMI1 β-1,3 glucan synthesis 19.3829 PHR1 β-1,3 glucanosyltransferase 19.4565 BGL2 β-1,3 glucosyltransferase 19.2990 XOG1 β-1,3 glucanase 19.2237 SPR1 β-1,3 glucosidase 19.6276 GDB1 Glucanotransferase 19.1719 SGA1 Glucosidase 19.4668 SCW11 β-1,3 glucosidase 19.7434 GLG2 Glucosyltransferase

Role of FKS1 in C. albicans Biofilm Resistance to Fluconazole In vitro

Nett et al. J Infect Dis. 202;171:2010

- Modulation of FKS1 impacts biofilm resistance to fluconazole *p<0.05

Bio

film

Gro

wth

50%

100%

0 15 7.5 1

Doxycycline (ng/ml) Inhibition of FKS1

Control

Fluconazole 62.5 µg/ml

* *

* * 50%

100%

Reference FKS1/fks1∆ FKS1-S645F

Control Fluconazole 15.6 µg/ml Fluconazole 250 µg/ml

Fluconazole Dose (mg/kg/12 h)!3! 10! 30!

Cha

nge

Log1

0 C

FU/!

Kid

neys!

-2"

-1"

0"

1"

Reference strain!

FKS1/fks1∆!

Nett et al J Infect Dis 202;171:2010

Log1

0 C

FU/C

athe

ter!

Reference"

FKS1/fks1∆"

4"

5"

6"

7"

NaCl !Control"

Fluconazole!250 μg/ml"

*!

Biofilm Catheter Disseminated – No Biofilm

Planktonic – In vitro Strain MIC (mg/l)

WT 0.5 FKS1 -/+ 0.5

Role of FKS1 in C. albicans Biofilm Resistance In vivo and for Planktonic Cells

Reference   FKS1/&s1∆   TDH3-­‐FKS1  

Impact of FKS1 Modulation on C. albicans Biofilm β-Glucan Production and Drug Sequestration

Ne5  et  al.  J  Infect  Dis  202;171;:2010    

Reference  FKS1/&s1∆  TDH3-­‐FKS1  

Glucan  (pg)/serum

 (ml)  

In  vitro-­‐    Matrix  

In  vitro-­‐  Supernatant  

Glucan  (pg)/Biofilm  cells  

(106)  

0  

200  

400  

500  

1000  

*  

In  vivo-­‐  Serum  

*  

*  *  

*  

[H3 ]  Flucona

zole  CPM

/biofi

lm  weight  (m

g)  

0  

20000  

40000  

60000  

80000  

100000  

Intact  Biofilm   Matrix  

*  

*  

*  

*  

*  *  

0  

20000  

40000  

60000  

80000  

100000  

*  

*  

*  

*  

*  *  

Reference  FKS1/&s1∆  FKS1-­‐S645F  TDH3-­‐FKS1  

- Modulation of FKS1 influenced biofilm matrix glucan production in vitro and in vivo *p<0.05

- FKS1 modulation impacted association of radio-labeled fluconazole with biofilm matrix *p<0.05

- Less matrix was visualized in the FKS1/fks1∆ biofilm and more was seen in the TDH3-FKS1 biofilm growing on a rat venous catheters

scanning  electron  micrographs  obtained  at  5,000x  measurement  bars  represent  1  µM  

Biofilm Glucan Matrix and the Cell Wall Integrity Pathway?

S. cerevisae (Candida)

Increased Biofilm

Susceptibility?

Reduced Matrix Glucan?

Pkc1 N N

Bck1 N N

Mkk2 N N

Slt2 (Mkc1) N N

Knr4 (Smi1) Y Y

Rlm1 Y Y

Fks1 Y Y

Interaction of Glucan matrix and CWI Pathway, but different triggers and upstream control

Fks1p

Smi1p

Rlm1p

Fluconazole Resistance and SMI1 in an in vitro Candida biofilm model

•  Deletion of smi1 modulates susceptibility to a drugs from a variety of classes, including fluconazole, amphotericin B and anidulafungin •  Planktonic susceptibility to these drugs was not impacted. •  Fluconazole binding efficiency of the smi1 biofilm and matrix was decreased

Fluconazole (µg/ml)

Perc

ent R

educ

tion

25%

50%

75%

0 250 125

* *

Reference smi1∆/smi1∆ smi1∆/smi1∆ + pSMI1

CP

M/N

orm

aliz

ed B

iofil

m (1

0exp

)

0

20

40

60

80

100

Reference smi1/smi1

Intact Biofilm Matrix

SMI1 Impact on Biofilm Formation and Matrix

200

400

600

800

1000

Biofilm Matrix

Reference

SMI1/smi1∆

smi1∆ /smi1∆

Β 1

,3 g

luca

n (p

g/m

l)

Regulation of Glucan Synthesis by SMI1

•  Disruption of SMI1 diminishes transcription of FKS1 •  Overexpression of FKS1 in an smi1Δ/smi1Δ background restores resistance to antifungal

Transcript

Nor

mal

ized

Exp

ress

ion

Rat

io

A

SMI1 FKS1

0.2

0.4

0.6

0.8

1

1.2 Reference

SMI1/smi1∆

smi1∆/smi1∆

B

Fluconazole (µg/ml)

Perc

ent r

educ

tion

10%

20%

30%

40%

50%

0 125 500

smi1∆/smi1∆

smi1∆/smi1∆ +TDH3-FKS1

**

Impact of FKS1 Modulation on C. albicans Biofilm Cell Wall and Susceptibility to

Stress Inducing Agents FKS1/fks1∆ TDH3-FKS1 Reference

TET-FKS1 +Doxycyline

TET-FKS1 - Doxycycline FKS1-S645F

Strain

Growth Response to Cell Stress (MIC or EC50)

37oC 42oC NaCl (M)

H2O2 (mM)

Congo Red

(µg/ml)

EtOH (%)

SDS (%)

Planktonic Reference ++ ++ 2 16 0.6 3.1 0.03

FKS1/fks1∆ + + 2 8 0.6 1.5 0.03

Biofilm Reference + ND >2 125 62.5 25 0.035

FKS1/fks1∆ + ND >2 125 62.5 25 0.035

Nett et al J Infect Dis 202;171:2010

Interaction of AmB with C. albicans Biofilms and Glucan

Vediyappan et al AAC 2010;54:2096

AmB bound to biofilm matrix

AmB bound to glucan

Impact of FKS1 Modulation on C. albicans Biofilm Drug Resistance to Amphotericin B

Amphotericin B (µg/ml) 0

Bio

film

Gro

wth

2 1 0.5 0.25 0.13 0.06

*

* *

0%

25%

50%

75%

100% *

*

Reference

FKS1/fks1∆ TDH3-FKS1

- The TDH3-FKS1 strain was more resistant to amphotericin B *p<0.05 - The FKS1/fks1∆ biofilm was more susceptible to amphotericin B *p<0.05 - Planktonic MICs were not significantly different among the strains

- Similar impact on two additional drug classes, flucytosine and anidulafungin

Nett et al Antimicrob Agents Chemother. 2010

chitin chitin

XX

chitinchitinchitin chitinchitin

XX

Fks1pPlasma MembraneMatrix GlucanFluconazoleBiofilm Cell Wall

Fks1pPlasma MembraneMatrix GlucanFluconazoleBiofilm Cell Wall

Hypothesized Genetic Control of C. albicans Biofilm Resistance

Antifungal

?

Current Studies

•  We are near completion of refined matrix composition analysis

•  We are exploring further components of the glucan matrix pathway to identify control points and potential drug targets

•  We have identified and are refining analysis of genes important for matrix delivery or deposition

Acknowledgements Lab Members Jacque Bohrmueller Mike Cain Kyler Crawford Alex Lepak Karen Marchillo Jeniel Nett Kelly Ross Hiram Sanchez Heather Taff Robert Zarnowski

Collaborators Joe Heitman Bruce Klein Aaron Mitchell Mahmoud Ghannoum Leah Cowen Geraldine Butler Sandy Johnson Clarissa Nobile

Funding Sources NIH, VA, Mycoses Study Group Astellas, Pfizer, Merck

Impact of FKS1 Modulation on C. albicans Biofilm Drug Resistance to Flucytosine

Flucytosine (µg/ml)

0%

25%

50%

75%

100%

0 8 4 2 1 0.5 0.25

*

* * * *

*

-The FKS1/fks1∆ biofilm and the TET-FKS1 biofilm under doxycycline repression of FKS1 were more susceptible to flucytosine -Planktonic MICs were not significantly different among the strains

- Similar impact on two additional drug classes, amphotericin and anidulafungin

Flucytosine (µg/ml)

0

25%

50%

75%

100%

0 1 2 4 8

No Doxycycline

Doxycycline 7.5 ng/ml

Bio

film

Gro

wth

Reference FKS1/fks1∆ TDH3-FKS1

Nett et al Antimicrob Agents Chemother. 2010

Anidulafungin (µg/ml)

Impact of FKS1 Modulation on C. albicans Biofilm Drug Resistance to Anidulafungin

Bio

film

Gro

wth

0%

25%

50%

75%

100%

0 0.12 0.06 0.03 0.02 0.01 0.004

*

*

Anidulafungin (µg/ml) 0 0.002 0.004 0.01 0.02

0

25%

50%

75%

100%

-The FKS1/fks1∆ biofilm and the TET-FKS1 strain under doxycycline repression of FKS1 were more susceptible to anidulafungin *p<0.05 -The TDH3-FKS1 biofilm was significantly more resistant to anidulafungin *p<0.05 Planktonic MICs were not significantly different for each strain

Reference FKS1/fks1∆ TDH3-FKS1

No Doxycycline

Doxycycline 7.5 ng/ml

Nett et al Antimicrob Agents Chemother. 2010