The Role of Secreted Aspartyl Proteinases in Candida albicans Keratitis

Abstract

purpose. To evaluate virulence in a murine keratitis model using Candida albicans homozygous mutants deficient in one or more secreted aspartyl proteinases encoded by SAP genes or in transcriptional factors encoded by EFG1 and CPH1 genes.

methods. Corneas of BALB/c mice were scarified and topically inoculated with 10⁶ colony-forming units of a C. albicans human isolate (SC5314), triple SAP-null mutants (SAP1-3 ^−/− and SAP4-6 ^−/−), double mutants (SAP4/5 ^−/−, SAP4/6 ^−/−, SAP5/6 ^−/−, and SAP9/10 ^−/− and EFG1 ^−/−/CPH1 ^−/−), single mutants (SAP4 ^−/−, SAP5 ^−/− and SAP6 ^−/−, EFG1 ^−/−, and CPH1 ^−/−), SAP6 rescuant, or parental controls (CAF2-1 and CAI-4). Animals were evaluated daily for up to 8 days after inoculation.

results. Wild-type C. albicans induced severe, sustained ulcerative keratitis, and the fungal strains (CAF2-1 and CAI-4) used to generate mutants had similar corneal pathogenicity. SAP1-3 ^−/−, SAP4/5 ^−/−, and SAP9/10 ^−/− mutants produced moderate keratitis similar to the virulent parental strain. SAP4-6 ^−/−, SAP4/6 ^−/−, and SAP5/6 ^−/− gave rise to significantly less severe corneal inflammation. The SAP6 ^−/− single mutant resulted in mild nonulcerative keratitis that resolved spontaneously within 5 days, and the SAP6 rescuant reestablished moderate disease severity. The EFG1 ^−/−/CPH1 ^−/− and EFG1 ^−/− mutants had reduced corneal virulence, but the CPH1 ^−/− strain resulted in persistent keratitis similar to control corneas.

conclusions. The EFG1-regulated SAP6 gene of C. albicans encodes a unique secreted aspartyl proteinase that contributes to corneal pathogenicity. The role of SAP6 during corneal infection appears to be associated with the morphogenic transformation of C. albicans yeasts into invasive filamentous forms.

Virulence factors contributing to the pathogenesis of Candida albicans infection include the secreted aspartyl proteinases (Saps). ¹ At least 10 proteins comprise the Sap family, encoded by genes SAP1 to SAP10. ²  

The role of Saps in the development and progression of candidiasis has been studied for systemic and mucosal candidal infections. In hematogenous murine and guinea pig models, specific deletions in SAP1 to SAP6 attenuate virulence. ^{3 4} In oral disease, SAP1 to SAP3 are important, whereas SAP4 to SAP6 are apparently not required. ⁵ No SAP gene appears necessary for C. albicans to colonize or infect the gastrointestinal tract. ⁶ SAP1, -2, -4, and -5 are involved in vaginitis. ^{7 8}  

The differing roles of Saps in candidal infections at various sites indicate the need for tissue-specific studies. We previously reported a mouse model of C. albicans keratitis for the study of the pathogenic effect of genetically encoded fungal virulence factors. ⁹ In this study, we sought to determine how the aspartyl proteinases of C. albicans affect corneal virulence, by using SAP gene-deficient mutants and mutants lacking the transcription factor genes EFG1 and CPH1, which regulate SAP activity and fungal filamentation. 

Materials and Methods

Fungal Strains

C. albicans mutants were derived from a highly virulent C. albicans human isolate, SC5314. ¹⁰ CAF2-1 and CAI-4 served as parental controls for selected gene disruptions of the SAP mutants and the EFG1 and CPH1 mutants, respectfully. ¹¹ Strains with single-, double-, and triple-null homozygous mutations involving SAP1, -2, -3, -4, -5, -6, -9, and -10 ^{3 4 6 12} and strains with single and combined mutations for EFG1 and CPH1 ^{13 14 15} were constructed with the Ura-blaster cassette consisting of URA3 and hisG flanking sequences. To reconstitute SAP6 expression in the SAP6-null mutant, a CIp10 plasmid containing SAP6 was integrated into the SAP6-null mutant, and the positive transformant was confirmed by Southern blot analysis. ¹⁶ All yeast strains were routinely grown in YPD (1% yeast extract, 2% peptone-tryptone, and 2% dextrose) medium at 30°C. Yeasts were harvested during exponential growth and suspended in sterile phosphate-buffered saline (PBS). The yeast strains are listed in Table 1 . 

Proliferation kinetics for each strain was calculated when grown in either YPD or SD medium (0.67% Difco yeast nitrogen base without amino acids, 2% dextrose; Difco, Detroit, MI) lacking uracil supplementation (SD-ura) at 33°C. Cultures were incubated at 33°C to mimic the temperature of the cornea. Each C. albicans strain was grown overnight in 5 mL of either YPD or SD-ura liquid medium. 

The concentration was calculated using a spectrophotometer to measure the optical density (OD) at a wavelength of 600 nm and a conversion factor of one OD₆₀₀ unit equivalent to 3 × 10⁷ colony-forming units (CFU)/mL. ⁹ Three cultures were analyzed for each yeast strain. For growth in YPD, 10³ CFU of yeast was added to 5 mL YPD liquid media and incubated at 33°C with continuous shaking for 2 days. For growth in SD-ura, 10⁵ CFU was added to 5 mL SD-ura medium and incubated at 33°C with continuous shaking for 2 days. Concentrations were measured during the exponential phase of growth at 9, 24, 32, and 48 hours after inoculation (PI). The doubling time for each culture was calculated from the plotted data, and the average for each strain was reported. Statistical analyses of the results applied Student’s t-test. 

Keratitis Infection Model

Immunocompetent adult female BALB/c mice (Harlan Sprague-Dawley, Houston, TX) 6 to 8 weeks old were anesthetized, and their corneas superficially scarified as described previously. ⁹ The scarified cornea received a 5-μL inoculum of each C. albicans yeast strain that totaled 10⁶ CFU or, for the negative control, 5 μL of sterile PBS. Animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and the research protocol was approved by the Baylor College of Medicine Institutional Animal Care and Use Committee. 

Mice were monitored daily for up to 8 days PI and scored with the aid of a dissecting microscope for disease severity, as described previously. ^{9 17} A grade of 0 to 4 was assigned for three criteria (area of opacity, density of opacity, and surface regularity), to yield a possible total score of 0 to 12. A total score ≤5 was categorized as mild eye disease, 5 to 9 was considered moderate, and >9 was severe. Three to 15 mice were used for each strain. Score results were evaluated for statistical significance by the Kruskal-Wallis one-way analysis of variance (ANOVA). 

Mice used for histologic examination were killed, and the eyes were enucleated at 1 day PI. The eyes from C. albicans-infected and mock-infected mice were formalin fixed, paraffin embedded, and sectioned at a thickness of 8 μm for histologic study. ^{9 17} Sections were deparaffinized and stained with periodic acid-Schiff (PAS; Sigma-Aldrich, St. Louis, MO) or modified Grocott’s methenamine silver (GMS; Richard-Allan Scientific, Kalamazoo, MI) stains. 

Results

The in vitro growth kinetics in YPD showed that experimental fungal strains had doubling times similar to wild-type SC5314 (P > 0.9). CAF2-1 and its derivative strains were developed from SC5314 by a technique that makes strains heterozygous for the URA3 gene ¹¹ ; thus, in vitro growth kinetics was also performed in media lacking uracil (SD-ura). CAI-4 is homozygous for the URA3 gene, and growth analysis in SD-ura medium was therefore not performed for this strain. Growth in SD-ura was similar in all yeast strains when compared with wild-type (Table 2) . 

Keratitis scores for wild-type (SC5314) and parental control (CAF2-1 and CAI-4) corneas showed severe and persistent keratitis and were statistically similar at all days for each set of experiments. We used multiplex mutants SAP1-3 ^−/−, SAP4-6 ^−/−, and SAP9/10 ^−/− to determine which SAP group, if any, contributes to corneal virulence (Fig. 1) . Whereas the severity of experimental keratitis induced by SAP1-3 ^−/− or by the SAP9/10 ^−/− strains was similar to the control, SAP4-6 ^−/− showed partial attenuation. Examination of the dual mutants for this subgroup showed moderately severe keratitis for SAP4/5 ^−/−, whereas SAP4/6 ^−/− and SAP5/6 ^−/− produced moderate disease at 1 day PI that resolved by 5 days PI (Fig. 2) . 

All three single mutants—SAP4 ^−/−, SAP5 ^−/−, and SAP6 ^−/−—produced moderate keratitis at 1 day PI (Fig. 3) . Initially, SAP4 ^−/− and SAP5 ^−/− caused moderate disease for 4 days, and then the severity slowly decreased. However, SAP6 ^−/− showed less corneal infiltration, similar to the double mutants SAP4/6 ^−/− and SAP5/6 ^−/−, with inflammation resolving by 5 days PI (Fig. 4) . The use of a mutant having the SAP6 gene reintroduced into the SAP6 ^−/− mutant strain resulted in disease severity similar to wild-type virulence (Fig. 3) . 

Genes encoding transcription factors EFG1 and CPH1 were also examined in the mouse keratitis model (Fig. 5) . A mutant strain with both EFG1 and CPH1 deleted produced no detectable keratitis. The single mutant lacking EFG1 also produced a mild infiltrate that cleared by 3 days PI. The single mutant deficient in CPH1 produced moderate ulcerative keratitis. 

Histologic examination of infected eyes showed extensive hyphal invasion into the mid stroma within 1 day PI for strain SC5314 (Fig. 6A)and for parental control strains CAF2-1 and CAI-4. A few budding yeasts were seen in SAP6 ^−/−-infected animals, but infection was patchy and limited to the superficial cornea (Fig. 6B) . The SAP6 rescuant produced mild to moderate pseudohyphae invading the anterior stroma (Fig. 6C) . Neutrophilic infiltration and edematous corneal stroma were prominent in corneas infected with strain SC5314 and were present after inoculation with the SAP6 rescuant, but minimal inflammation occurred in corneas exposed to the SAP6 ^−/− strain. Mock-infected eyes did not show signs of infection or inflammation but did acquire some microscopic changes from the initial scarification procedure. 

Discussion

Fungi such as C. albicans are commonly part of the normal microflora of the outer eye and occasionally cause corneal infection. ^{18 19 20} The immune status and physiological integrity of the ocular surface are key factors in preventing fungal keratitis, ^{9 21} but pathogenic fungi have the ability to overcome host defenses and to invade the cornea. ²² The processes by which C. albicans and other opportunistic fungi transition from commensal saprophytes into invasive pathogens involve a network of virulence traits, ^{23 24} including hydrolytic enzymes. ²⁵ C. albicans produces at least 10 secreted Saps encoded by corresponding genes on five chromosomes. ¹  

Knowledge of the metabolic pathways involved in the production and activity of Saps has recently emerged. Genetic transcription of these enzymes is regulated by transcriptional activators such as Efg1p (enhanced filamentous growth protein). ²⁶ After activation, the aspartyl proteinases Sap1p to Sap8p are secreted extracellularly, whereas Sap9p and Sap10p are anchored in the fungal membrane. ²⁷ Saps may act on proteins of the extracellular matrix such as collagen and fibronectin and can upregulate proinflammatory cytokines within the host’s tissues. Among Candida’s extracellular aspartyl isoenzymes, Sap1p, -2p, and -3p are more active in an acidic environment, appear involved in C. albicans epithelial adherence, and can be inhibited by lysozyme. ^{28 29} The subgroup of Sap4p, -5p, and -6p retains activity at neutral pH, and their genes are primarily expressed during filamentation. ³⁰  

The construction of fungal strains through targeted gene disruption allows exploration of the role of Saps at various sites of infection in animal models. ^{3 4} Experiments using null mutants indicate that SAP1, -2, or -3 contribute to oral, cutaneous, and vaginal infections. ^{5 31 32} In contrast, SAP4, -5, and/or -6 influence the virulence of experimental disseminated candidiasis and candidal peritonitis. ^{3 4 16 33 34} Our study suggests that SAP6 is also involved in the pathogenesis of C. albicans keratitis. 

SAP1-3 triple-null mutants and SAP4 and SAP5 single-null mutants produced moderately severe keratitis, but SAP6-altered strains did not establish persistent infection in the murine cornea and failed to invade or to trigger inflammation. Reintroduction of the SAP6 gene into the fungal genome reconstituted corneal pathogenicity. 

We also studied strains deficient in transcription factors that influence genes with a potential role in fungal virulence. Efg1p is a transcriptional factor that promotes the transition from yeast to hyphae and that independently affects the production of Sap6p. ^{14 16} Similar to findings in other studies linking EFG1-regulated candidal morphogenesis with tissue invasiveness, ³⁵ we found that EFG1-altered mutants did not establish infection in the traumatized cornea. Corneal infection by C. albicans probably involves parallel processes regulated by EFG1, the production of fungal hyphae and the expression of SAP6. 

The importance of Sap6p and Efg1p in candidiasis is probably related to the transition of C. albicans from blastoconidia to hyphae. Sap6p is associated with filamentation, ²⁸ and its expression is modified in a strain lacking EFG1 that cannot readily form hyphae. ¹³ C. albicans undergoes dimorphic transformation from yeasts to filamentous forms during the early stages of fungal keratitis, ²⁴ and this morphogenic conversion is closely associated with the production of Sap6p. 

The initial events of C. albicans keratitis may be similar to the molecular processes involved in invasive candidiasis. In a murine model of parenchymal infection after intraperitoneal injection, ¹⁶ fungal strains lacking SAP6 expression had reduced ability to invade the liver, spleen, and pancreas. ¹⁶ We found that these SAP6 mutants also had curtailed virulence when applied to the scarified cornea by topical ophthalmic exposure. Together with our previous work on the pathogenesis of experimental keratomycosis, ^{9 22 23 24} a representative scheme of the early events of C. albicans keratitis is materializing (Fig. 7) . 

Modeling experimental posttraumatic keratomycosis provides an opportunity to elucidate how microbial virulence genes influence the severity of fungal keratitis, but our study is subject to some limitations. We used fungal mutants with an altered URA3 genetic marker that could have a lower level of pathogenicity, ³⁶ although these auxotrophic strains form germ tubes and proliferate similarly to a wild-type isolate. ³⁷ Also, fungal growth was examined in vitro but not in corneal tissue, because previous studies have shown that quantitative fungal recovery during C. albicans keratitis does not correlate well with clinical severity or with histopathologic appearance. ^{17 38} Further work in ophthalmic mycology is needed to apply sensitive measures of fungal growth in ocular tissue, since fungal proliferation in vivo could affect the extent and outcome of experimental keratomycosis. SAP expression could also be studied during corneal infection using quantitative reverse transcription-polymerase chain reaction, in vivo expression technology, or immunoelectron microscopy. ^{1 26} Additional study is also needed to elucidate the role of Saps during corneal invasion by C. albicans and the interaction with the ocular inflammatory response. 

Our results are consistent with those in previous studies on systemic models of candidiasis but suggest that the pathogenesis of C. albicans keratitis differs from that of other mucocutaneous infections. The finding that SAP6 is associated with fungal invasion after corneal trauma indicates that this aspartyl proteinase plays a key role in keratomycosis and may offer a new target in antifungal chemotherapy. 

 Supported by National Eye Institute Grants T32-EY07001 (BEJ) and P30-EY02520, the Research to Prevent Blindness, Inc., the Lions Eye Bank Foundation, and the Sid W. Richardson Foundation.

 Submitted for publication January 30, 2007; revised March 23, 2007; accepted May 25, 2007.

 Disclosure: B.E. Jackson, None; K.R. Wilhelmus, None; B. Hube, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Table 1.

 

Experimental C. albicans Strains

Strain	Genetic Construct	Genotype	Reference
SC5314	Wild-type	Wild-type	10
CAF2-1	Parental control	ura3::λimm434	11
		URA3
M119	SAP1–3 null mutant	ura3::λimm434 sap1::hisG sap2::hisG sap3::hisG	6
		ura3::λimm434 sap1::hisG sap2::hisG sap3::hisG-URA3-hisG
DSY459	SAP4–6 null mutant	ura3::λimm434 sap6::hisG sap4::hisG sap5::hisG	4
		ura3::λimm434 sap6::hisG sap4::hisG sap5::hisG-URA3-hisG
M127	SAP9,10 null mutant	ura3::λimm434 sap9::hisG sap10::hisG	12
		ura3::λimm434 sap9::hisG sap10::hisG-URA3-hisG
M28	SAP4/5 null mutant	ura3::λimm434 sap4::hisG sap5::hisG	16
		ura3::λimm434 sap4::hisG sap5::hisG-URA3-hisG
M29	SAP4/6 null mutant	ura3::λimm434 sap6::hisG sap4::hisG	4
		ura3::λimm434 sap6::hisG sap4::hisG-URA3-hisG
M30	SAP5/6 null mutant	ura3::λimm434 sap6::hisG sap5::hisG	16
		ura3::λimm434 sap6::hisG sap5::hisG-URA3-hisG
M25	SAP4 null mutant	ura3::λimm434 sap4::hisG	4
		ura3::λimm434 sap4::hisG-URA3-hisG
M26	SAP5 null mutant	ura3::λimm434 sap5::hisG	4
		ura3::λimm434 sap5::hisG-URA3-hisG
M27	SAP6 null mutant	ura3::λimm434 sap6::hisG	4
		ura3::λimm434 sap6::hisG-URA3-hisG
M1066	SAP6 rescuant	ura3::λimm434 sap6::hisG	16
		ura3::λimm434 sap6::hisG-URA3-hisG pCIp10-SAP6 (pAF3)
CAI-4	Parental control	ura3::λimm434	11
		ura3::λimm434
HLC52	EFG1 null mutant	ura3::λimm434 efg1::hisG	14
		ura3::λimm434 efg1::hisG-URA3-hisG
JKC19	CPH1 null mutant	ura3::λimm434 cph1::hisG	15
		ura3::λimm434 cph1::hisG-URA3-hisG
HLC54	EFG1/CPH1 null mutant	ura3::λimm434 cph1::hisG efg1::hisG	14
		ura3::λimm434 cph1::hisG efg1::hisG-URA3-hisG

Table 2.

 

In Vitro Growth of C. albicans Strains in Rich and Minimum Media

Yeast Strain	Doubling Times (h)
		YPD	SD-ura
SC5314	1.7 ± 0.01	2.1 ± 0.1
CAF2-1	1.6 ± 0.03	2.1 ± 0.1
SAP1-3 −/−	1.6 ± 0.02	1.8 ± 0.1
SAP4-6 −/−	1.5 ± 0.03	2.0 ± 0.1
SAP9/10 −/−	1.6 ± 0.02	1.8 ± 0.1
SAP4/6 −/−	1.7 ± 0.03	1.5 ± 0.1
SAP5/6 −/−	1.7 ± 0.03	2.0 ± 0.1
SAP5/6 −/−	1.7 ± 0.03	2.0 ± 0.1
SAP4 −/−	1.5 ± 0.02	2.1 ± 0.1
SAP5 −/−	1.5 ± 0.02	2.0 ± 0.1
SAP6 −/−	1.4 ± 0.04	2.5 ± 0.1
SAP6 rescuant	1.6 ± 0.02	2.2 ± 0.1
CAI-4	1.9 ± 0.07	ND
EFG1 −/−	1.7 ± 0.05	2.4 ± 0.3
CPH1 −/−	1.6 ± 0.04	2.1 ± 0.1
EFG1 −/−/CPH1 −/−	1.9 ± 0.03	2.6 ± 0.1

 Data are mean ± SD. YPD, yeast peptone dextrose medium; SD-ura, synthetic dextrose without uracil medium. ND, not determined.

Figure 1.

 

Keratitis severity produced by C. albicans SAP mutants. Each yeast strain was inoculated onto scarified corneas of BALB/c mice at day 0 and monitored for 4 days. The mean for each strain is reported with error bars representing the SD. SC5314 is a wild-type yeast strain. CAF2-1 is the mutant control for the SAP mutants. All mock-inoculated eyes had a 0 score for each time point. Mutant strains SAP1-3 ^−/− and SAP9/10 ^−/− produced keratitis scores similar to control scores, whereas corneas inoculated with SAP4-6 ^−/− began to resolve.

Figure 2.

 

Mean keratitis scores for C. albicans SAP double mutants. Each yeast strain was inoculated onto scarified corneas of BALB/c mice at day 0 and monitored for 8 days. Wild-type, parental control, and SAP4/5 ^−/− produced severe ulcerative keratitis, whereas both double mutants containing the SAP6 deletion had attenuated keratitis severity.

Figure 3.

 

Mean keratitis scores for C. albicans SAP single mutants. Corneas were scarified and inoculated on day 0. Both SAP4 ^−/− and SAP5 ^−/− resulted in moderately severe keratitis that slowly diminished, whereas the corneal infiltrate produced by SAP6 ^−/− resolved within 5 days.

Figure 4.

 

Mouse corneas inoculated with C. albicans. Mouse eyes were scarified and inoculated with 10⁶ colony-forming units of CAF2-1 parental control or SAP6 ^−/− mutant. Slit-lamp photographs were taken at 1 day (A, B) and 4 days (C, D) PI, with a slit-lamp camera: (A) moderate keratitis with CAF2-1 control at 1 day PI; (B) moderate keratitis with SAP6 ^−/− mutant at 1 day PI; (C) Persistent keratitis with CAF2-1 infection at 4 days PI; (D) mostly resolved disease with SAP6 ^−/− at 4 days PI.

Figure 5.

 

Mean keratitis scores for C. albicans EFG1 and CPH1 single and double mutants. Infection with a mutant lacking EFG1 resulted in mild corneal haze that resolved within 3 days, whereas CPH1 ^−/− produced moderate keratitis that persisted through day 8. A mutant deficient in both EFG1 and CPH1 genes appeared avirulent.

Figure 6.

 

Histopathology of murine keratomycosis induced by C. albicans strains SC5314, SAP6 ^−/−, and SAP6 rescuant. Mouse corneas were infected with 10⁶ colony-forming units of C. albicans, enucleated 1 day PI, sectioned, and stained with Grocott’s methenamine silver. (A) In eyes infected with wild-type SC5314, fungal hyphae invaded the anterior corneal stroma, and neutrophils infiltrated the cornea and anterior chamber. (B) SAP6 ^−/− mutant-infected eyes showed very little yeast within the superficial epithelium. (C) Infection with a C. albicans strain in which the SAP6 gene had been reintroduced into the SAP6 ^−/− mutant resulted in budding yeast and some pseudohyphae with anterior segment inflammatory infiltration. Original magnification, ×100.

Figure 7.

 

Proposed pathways of selected fungal virulence factors involved in C. albicans keratitis. Adapted from Naglik et al. ²⁶ and based on findings of Jackson et al. ²³ and Mitchell et al. ²⁴

The authors thank Dominique Sanglard, Institut de Microbiologie, University Hospital Lausanne, Lausanne, Switzerland, for constructing the CAF2-1, M119, DSY459, M25, M26, M27, M28, M29, M30, and M1066 strains; Gerald R. Fink, Whitehead Institute for Biomedical Research, Cambridge, MA, for providing CAI-4, HLC52, JKC19, and HLC54 strains; and Bradley M. Mitchell, Sid W. Richardson Ocular Microbiology Laboratory, Baylor College of Medicine, who helped design our research protocol.