Candida albicans Strain-Dependent Virulence and
Rim13p-Mediated Filamentation in
Bradley M. Mitchell,1,2Tzu G. Wu,1Beth E. Jackson,1and Kirk R. Wilhelmus1
PURPOSE. To compare the virulence of wild-type Candida al-
bicans strains in a murine model of corneal candidiasis and to
investigate the role of fungal filamentation in disease progres-
METHODS. Scarified corneas of immunocompetent or cyclo-
phosphamide-treated BALB/c mice were topically inoculated
with one of three human isolates of C. albicans, a homozygous
mutant of the pH-dependent filamentation gene rim13 or a
mutant reference strain control. Mock-inoculated eyes served
as negative controls. Corneal disease was categorized daily for
8 days with quantitative fungal culturing of eyes at 6 hours, 1
day, 4 days, and 8 days after infection and histopathologic
examination at 1 day and 4 days after infection.
RESULTS. Corneal disease severity differed significantly among
wild-type strains (P ? 0.02). The rim13–/–mutant Tn7-rim13
was fully attenuated, whereas the mutant control DAY286 was
fully virulent. Pretreatment of mice with cyclophosphamide
increased susceptibility to wild-type C. albicans and partially
rescued the attenuated phenotype of the genetically deficient
rim13–/–fungal mutant. All strains replicated with similar ki-
netics in vitro, and wild-type strains had similar clearance from
infected eyes. Histopathologic findings correlated with disease
CONCLUSIONS. Wild-type strains of C. albicans that differ signif-
icantly in ocular pathogenicity correlate with the ability of
yeast to produce pseudohyphae and hyphae and to invade
corneal tissue. Full attenuation of the fungal rim13–/–mutant is
the first direct demonstration of a hyphal morphogenesis-re-
lated gene as a specific virulence factor for C. albicans during
corneal infection. (Invest Ophthalmol Vis Sci. 2007;48:
Candida albicans is found on the ocular surface of up to one
third of otherwise healthy persons.1–4Although this fungal
species is typically commensal, severe ocular disease develops
under certain conditions. How this shift to pathogenic invasion
occurs is still unclear, but the transition is finely balanced and
genetically regulated.5Fungal mechanisms crucial for mucosal
and systemic infection by C. albicans include adherence, ger-
mination, production of extracellular proteinases and phos-
pseudohyphae and hyphae.6–9Previous work has suggested
that the conversion to filamentous forms contributes to ocular
virulence of C. albicans.10,11
Environmental cues such as pH12,13influence filamentation
growth of Candida, Saccharomyces, and Aspergillus through
a conserved alkaline-response pathway.14,15Mutant strains un-
able to grow at a given pH in vitro are less capable of infecting
selective sites in vivo.16,17This system, called the RIM101
pathway or the PacC pathway, depending on fungal genus,18is
governed by the zinc finger–containing transcription factor
Rim101p/PacC.15,19–21In acidic conditions, Rim101p is full-
length and inactive, whereas in neutral to alkaline conditions
its C-terminal portion is cleaved, resulting in activation of the
protein that regulates gene expression.22–24Several gene prod-
ucts are required for this proteolytic processing, including
Rim8p/PalF, Rim9p/PalI, Rim13p/PalB, Rim20p/PalA, and
Rim21p/PalH.24,25Rim13p/PalB is a calpain-like protease that
cleaves the C-terminal glutamate-aspartate-rich domain of
Rim101p during activation.18
Because of the complex life cycle of fungi, investigating the
pathogenesis of oculomycosis requires in vivo models that
allow high reproducibility and sensitive quantitation.10,11,26–28
This study compared the degrees of virulence of three different
C. albicans wild-type strains in mice and evaluated the contri-
bution of the RIM101 signal transduction pathway.15,20,21Our
findings provide direct evidence for the importance of filamen-
tation in keratomycosis and demonstrate that a morphogenesis-
related fungal gene is a specific virulence factor for C. albicans
during corneal infection.
MATERIALS AND METHODS
Three wild-type strains of C. albicans originally isolated from human
infection, a Tn7 transposon mutant, and a mutant control reference
strain were evaluated. Strain VE175 is a corneal isolate that has been
used to induce experimental keratomycosis in rabbits.29Strain B311
(ATCC 32354; American Type Culture Collection, Rockville, MD) is an
isolate that is highly virulent in mice when injected intravenously30and
that causes ocular disease in mice after corneal inoculation.11Strain
SC5314 has been used extensively for genetic studies of C. albicans
and experimentally causes corneal disease in rabbits.29,31Strain Tn7-
rim13 is a homozygous mutant with a transposon insertion at position
239 of the rim13 coding sequence18that was transformed into strain
BWP17,15a derivative of strain SC5314, as part of a homozygous
insertion mutant library.32The mutant control reference strain
DAY286 has a Ura?Arg?His?genotype and was created by the trans-
formation of strain BWP17.15
All yeast strains were grown on Sabouraud dextrose agar (Difco,
Detroit, MI) for 3 days at 25°C. For corneal inoculation, the yeasts were
harvested and diluted in sterile phosphate-buffered saline (PBS) to yield
Cullen Eye Institute, Department of Ophthalmology, and the2Depart-
ment of Molecular Virology and Microbiology, Baylor College of Med-
icine, Houston, Texas.
Supported by a clinical investigator award (EY00377), cooperative
agreement (EY09696), and core grant (EY02520) from the National Eye
Institute; a senior scientific investigator award and grant from Research
to Prevent Blindness, Inc.; the J. S. Abercrombie Foundation; the Retina
Research Foundation; and the Sid W. Richardson Foundation.
Submitted for publication July 12, 2006; revised September 5,
2006; accepted December 1, 2006.
Disclosure: B.M. Mitchell, None; T.G. Wu, None; B.E. Jackson,
None; K.R. Wilhelmus, None
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be marked “advertise-
ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Corresponding author: Kirk R. Wilhelmus, Department of Oph-
thalmology, NC-205, Baylor College of Medicine, 6565 Fannin, Hous-
ton, TX 77030; email@example.com.
1Sid W. Richardson Ocular Microbiology Laboratory,
Investigative Ophthalmology & Visual Science, February 2007, Vol. 48, No. 2
Copyright © Association for Research in Vision and Ophthalmology
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