1. Dissociation of C. albicans Biofilms by Anti-amyloid Compounds
Desmond N. Jackson1, Lin Yang1,2, Edward J. Kennelly2,3, and Peter N. Lipke1,2
1Biology Department, Brooklyn College CUNY, Brooklyn, NY; 2The Graduate Center CUNY, New York, NY;
3Department of Biological Sciences, Lehman College CUNY, Bronx, NY
Fungal biofilms represent an increasing strain on resources in the clinic, where fungal contamination of
indwelling catheters has become pervasive. Along with this increase, an alarming amount of fungi are
becoming drug resistant. This situation highlights the need for investment in the development of new anti-
fungal chemotherapy regimens to help combat invasive fungal disease caused by biofilms. Since most current
anti-fungal drugs target some aspect of the cells’ genetic machinery, we propose that other compartments in
addition to genetics be exploited in the development of new biofilm eradication strategies. Here we present
preliminary evidence in support of the hypothesis that protein-protein interactions of amyloid forming adhesins
in the extracellular compartment of C. albicans biofilms provide a promising target for intervention.
Treatment of mature C. albicans biofilms with thioflavin-T or xanthochymol resulted in significant decrease in
biofilm viability as measured by XTT assay. Confocal microscopy analysis revealed a dramatic reduction in
hyphae in the presence of anti-amyloid compounds used in these experiments. Further, there are synergistic
interactions between the anti-amyloid xanthochymol and fluconazole against fungal biofilms. Based on these
experiments we propose that the extracellular compartment of fungal biofilms containing amyloid forming
adhesins represents a credible target for intervention against established fungal biofilms.
 Biofilm growth and encapsulation in protective ECM is pervasive, resulting in
increased cost for treatment and eradication
 Candida albicans growing as biofilms shows increased resistance to antifungal
drugs.
 Candida albicans drug resistance is multifactorial
 New targets for chemotherapeutic intervention need to be developed.
 Role of amyloid forming adhesins in biofilm drug resistance remains unexplored.
Methods
Relativeabsorbance
Figure 2: C. albicans biofilms were treated with or without Thioflavin
T(30μm). XTT assay was performed immediately on addition (0hr) or 24
hours after addition.
μM Thioflavin T inhibits formation of new biofilm
Figure 1: C. albicans biofilms are thioflavin T, positive. Mature biofilms
were stained with 300nM ThT prior to epi-fluoresence imaging. Blue
fluorescence observed in Figure 1 is indicative of the presence of
amyloid-like interactions in the biofilm.
Mature Candida albicans biofilms contain
amyloid like deposits
Results
Fig. 3. Anti-amyloid peptide was
applied to mature biofilms for 24hrs.
Biofilms were washed then stained
with syto-9/PI for 30 min, followed by
CLSM. 3D projections of biofilms are
depicted in the figure.
Reconstructions were done using
Imagej.
An anti-amyloid peptide targets hyphae
Xanthochymol & Fluconazole
Control Fluconazole
Xanthochymol
Figure 5: Mature C. albicans biofilms were treated with or without xanthochymol (100μg/ml) and fluconazole for 24hrs. XTT assay
and confocal microscopy were performed. 3D projections were reconstructed using Imagej.
Xanthochymol is a potential anti-amyloid compound
Figure 4: C. albicans cells was assayed in
the absence (left) or presence (right) of
xanthochymol. 45 min aggregaton assays
with BSA-coated magnetic beads (brown).
Formation of large aggregates is dependent
on formation of cell surface amyloid
nanodomains.
Figure 6: Mature C. albicans biofilms were treated with or without rifamycin (120μM) and caspofungin (.0325 μg/ml.) for 24hrs. After treatment, the
health of the biofilm was assayed by XTT and CLSM. 3D reconstruction was done using Imagej.
Attachment phase 90
min in PBS
24 hour Biofilm growth in RPMI
–MOPS pH 7.2, 370C
2 fold dilution and addition of test
compounds and antifungals
XTT assay, absorbance
at 492nM
Confocal
Microscopy Syto-9/PI
staining
Our screens yielded two putative anti-amyloid compounds.
 Enhanced activity of fluconazole by xanthochymol against C. albicans biofilms.
 Hyphal toxicity of putative anti-amyloid xanthochymol.
 Inhibition of biofilm growth by the anti-amyloid thioflavin T.
 Rifamycin, known to be anti-amyloid, enhanced activity of caspofungin.
Conclusions
Introduction
V326N
Control
Xanthochymol is synergistic with fluconazole
The anti-amyloid compound rifamycin enhances activity of caspofungin
Control
Rifamycin + caspofungin
Rifamycin
Caspofungin
Acknowledgements
Supported by R01 GM098616