Morphogenic and genetic differences between Candida albicans strains are associated with keratomycosis virulence

Fungal strains

C. albicans strains included SC5314, a human clinical isolate recovered from a patient with generalized candidiasis [19,20], and VE175, a human corneal isolate obtained from a patient with candidal keratitis [21]. After reviewing the genomic differences between SC5314 and VE175, a mutant strain DSY459 deficient in secreted aspartyl proteinase (SAP) SAP4, SAP5, and SAP6 genes (Δsap4-6) was obtained from Sanglard and associates who derived this triple-null homozygote from SC5314 using a Ura-blaster cassette [22].

In vitro growth kinetics

Yeast strains were grown in 1% yeast-extract, 2% peptone, and 2% dextrose (YPD) liquid medium at 30 °C, harvested during exponential growth, and suspended in sterile phosphate-buffered saline (PBS). Optical density (OD) was measured with an Ultraspec 2000 spectrophotometer (Pharmacia Biotech, Princeton, NJ) at a wavelength of 600 nm (OD₆₀₀). A conversion factor of one OD₆₀₀ unit equivalent to 3×10⁷ colony-forming units (CFU)/ml was used to determine fungal concentration [23]. Triplicate samples of 3×10⁵ CFU for each strain were inoculated into 25 ml M199 liquid media (Invitrogen, Grand Island, NY) at pH 6.0, pH 7.3, and pH 8.0 and incubated at 27 °C with continuous shaking. C. albicans concentrations were determined spectrophotometrically at 1.5, 3, 4.5, 6, 9, 12, 15, 21, 27, 35, and 48 h post inoculation (pi).

Filamentation assay

C. albicans strains were grown overnight on Sabouraud dextrose agar (Difco, Detroit, MI) for 3 days at 25 ºC. Yeasts were harvested, diluted in sterile PBS to yield 1 CFU/μl, inoculated onto M199 agar plates containing Earle’s salts and glutamine but lacking sodium bicarbonate that were buffered with 2 M Tris-HCl to yield media pH values of 7.3 and 8.0, and incubated at 37 ºC. Colonies were observed daily by inverted microscopy for 10 days.

Genomic microarray

C. albicans strains SC5314 and VE175 were separately inoculated into 10 ml of YPD media and incubated in a room-temperature (22 ºC) shaker until the late exponential growth phase. Ten replicate 1 ml fungal suspensions in 50 ml M199 media at pH 8.0 were held at 37 °C in a 180 rpm incubator, and fungi were harvested after 4 h [24]. Hot acidic phenol extraction was performed to prepare yeast ribonucleic acid (RNA) [25]. The RNA concentration was measured with a ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE), followed by gel electrophoresis to confirm purity and integrity.

Twenty µg of total RNA were labeled by an aminoallyl-derivative protocol using the Micromax RNA labeling kit (PerkinElmer, Waltham, MA). Samples were mixed and hybridized to a glass-slide microarray in the C. albicans Array Ready Oligo Set (Operon Biotechnology, Huntsville, AL) composed of 8,117 70-mer oligonucleotides representing 6,655 genes from Assembly 19 of the C. albicans genome [26], plus specific and non-specific controls, resulting in slightly more than the predicted 6,354 protein-coding genes due to the inclusion of some tRNA genes and spurious genes predicted in earlier assemblies [27]. The array was printed in duplicate on Codelink II (GE Healthcare, Princeton, NJ) slides (Microarrays, Nashville, TN). Labeled samples were mixed and hybridized to the arrays. Two technical replicates and two biological replicates were processed for each sample for a total of eight spots for each gene. Arrays were scanned using an Axon 4000B scanner (Molecular Devices, Sunnywale, CA), and data were analyzed using GenePix Pro 6.0 software (Molecular Devices, Downingtown, PA) as previously described [28]. Inconsistent or saturated spots were filtered, and spot values were calculated by subtracting the background intensity from the median pixel intensity of each spot. Values were then normalized to equalize median intensities between the two channels across the entire array. From the eight replicate spots, geometric means of the ratios and p-values were estimated. Genes having an expression ratio of at least 2 fold difference and p <0.05 between the two strains were grouped into functional categories using information curated by the CandidaGenome Database [29].

Reverse transcription (RT) of RNA and RT-polymerase chain reaction (RT-PCR)

Total RNA was separately isolated from C. albicans strains SC5314 and VE175 following cultivation for 4 h at pH 7.3 and pH 8.0, respectively. First-strand cDNA was synthesized from 1 μg total RNA with Ready-To-Go You-Prime First-Strand Beads (GE Healthcare) and random hexamers (Applied Biosystems, Foster City, CA). RT-PCR was performed using PuReTaq Ready-To-Go PCR beads (GE Healthcare). Primers for SAP4 (P1: GTC AAC GCT GGT GTC CTC TT; P2: GCA GGA ACG GAA ATC TTG AG, 197 bp), SAP5 (P2: CCG AAT TCC TTT TCC AAA CA; P2: TGG AGC CAT GGA GAT TTT CT, 158 bp), and SAP6 (P1: GTC AAC GCT GGT GTC CTC TT; P2: TTC ACG AAC ACG AAT TTC ACA; 270 bp) were synthesized (Sigma, St. Louis, MO), with ACT1 (TGC TGA ACG TAT GCA AAA GG; P2: TGA ACA ATG GAT GGA CCA GA; 186 bp) as the housekeeping gene. Semiquantitative RT-PCR was established by terminating reactions at intervals of 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, and 40 cycles for each primer pair to ensure that PCR products were within the linear portion of the amplification curve. All products were separated by 2% agarose gel electrophoresis and visualized with 0.5 mg/ml ethidium bromide. Fidelity of RT-PCR products was confirmed by comparing cDNA bands and by sequencing PCR products.

Ex vivo human cornea model

Thirty-six human corneas were obtained from the Lions Eye Bank of Texas, Houston, TX after informed consent for research use was obtained from the decedent donors’ next-of-kin. Donor corneas were maintained at 4 ºC in Optisol-GS (Bausch & Lomb, Irvine, CA) before being transferred to modified supplemented hormonal epithelial medium (SHEM), consisting of equal volumes of Dulbecco’s modified Eagle’s medium and Ham’s F12 medium that contained 5 ng/ml epidermal growth factor, 5 μg/ml insulin, 5 μg/ml transferrin, 5 μg/ml sodium selenite, 0.5 μg/ml hydrocortisone, 30 ng/ml cholera toxin A, 0.5% dimethylsulfoxide, 50 μg/ml gentamicin, and 5% fetal bovine serum, and was buffered with 2 M Tris-HCL to pH 7.3 or 8.0. Corneas were superficially scarified using a 22 gauge needle to produce a 15x15 cross-hatch pattern, similar to a protocol previously described for an experimental fungal keratitis model [14]. A Horizon artificial anterior chamber (Refractive Technologies, Cleveland, OH) fixated the corneal button during superficial scarification, followed by topical application of 10 μl of 1×10⁵ CFU C. albicans per cornea. Inoculated corneas were put epithelial side up into a 6 well culture dish (Corning, Corning, NY) so that the sclera was immersed in modified SHEM and the central cornea vaulted upward. Tissues were incubated at 34 °C in 5% CO₂ with 95% humidity, and culture medium was changed daily. After 24 and 72 h, corneas were embedded in OCT compound (Sakura Finetec, Torrance, CA) and frozen at -80 °C for subsequent histopathologic processing.

Hyphal penetration

Frozen sections (10 µm) were cut, and three sections were examined for each cornea at 10 μm intervals for 100 µm from the corneal mid-point. Sections were stained with periodic acid-Schiff (PAS) reagent (Sigma-Aldrich, St. Louis, MO). Images were captured of an entire limbus-to-limbus section from each cornea with a DS-Fil digital camera (Nikon, Tokyo, Japan) attached to a Nikon Y-FL microscope. We measured the overall hyphal depth with 10X magnification at 5 different points at equidistant intervals. The entire corneal thickness was then estimated at 5X magnification at the same positions for each section using the NIS-Element 3.0 image analysis system (Nikon). The overall average depth of penetration was calculated as the percentage of corneal thickness. The maximal percentage of hyphal penetration was also estimated from measurements taken at regions of each corneal section demonstrating the greatest depth of corneal penetration. To assure that points of maximum hyphal penetration were selected, results from the three largest hyphal-depth percentages were averaged from 5 measurements taken for each histological section and calculated as percentage of corneal thickness. Results were compared by the Student t-test.

In vitro comparison of C. albicans strains

Strains SC5314 and VE175 demonstrated similar lag, log-growth, and plateau phases. The doubling time of both C. albicans strains use the same at pH 6.0 (1.94±0.04 h for SC5314 and 1.92±0.04 h for VE175) and similar at pH 7.3 (2.21±0.04 h for SC5314 and 1.94±0.01 h for VE175) but showed that SC5314 grew slightly faster (2.27±0.03 h) than VE175 (2.72±0.05 h) at alkaline pH (p=0.02). Mock-inoculated control cultures remained negative for growth throughout 24 h of observation. On M199 agar, numerous filaments surrounded SC5314 colonies 24 h after inoculation and continued growing during the following 10 days. Few filaments emerged from VE175 colonies, and while dissimilarity was present at physiologic pH, the difference with in vitro filamentation was more apparent at pH 8.0.

Genomic comparison of C. albicans strains

Among 6,655 genes detected by microarray, 601 (9.03%) genes in SC5314 were significantly (p<0.05) and differently (≥2 fold change) regulated compared to VE175. Of these, 69 named upregulated genes and 116 named downregulated genes were categorized into functional groups (Table 1), including 44 genes involved in hyphal filamentation and virulence (Figure 1). mRNA expression of SAP4, SAP5, and SAP6 was further examined by RT-PCR (Figure 2). No difference of ACT1 expression occurred between SC5314 and VE175 at either pH 7.3 or pH 8.0. mRNA expression of SAP4 was not detected in either strain at either pH. SAP5 and SAP6 mRNA were expressed higher in SC5314 than VE175 at both pH conditions.

Corneal virulence

One day after inoculation of strains SC5314 and VE175 onto human corneas incubated at pH 7.3, fungal hyphae were present in the anterior corneal stroma. By the third day SC5314 invaded toward the central stroma (Figure 3A). VE175, however, failed to continue hyphal invasion between 1 and 3 days pi and remained limited to the anterior cornea. At pH 8.0, hyphal penetration into corneal tissue was consistently greater for SC5314 than VE175. The overall and maximal penetration percentage are shown in Table 2 and Table 3. Initially, few conidia of VE175 were present on the corneal surface and no hyphal formation was observed (Figure 3B). Pseudohyphae subsequently formed with VE175 but did not penetrate to the same extent as SC5314.

Δsap4-6 deletion mutant

Fungal mutant DSY459 grew well at pH 6.0 and pH 7.3 and slightly more slowly at pH 8.0. Moderate filamentation was found in vitro at pH 7.3 and pH 8.0 (Figure 4). In explanted corneas at 1 day pi (Figure 4), the overall penetration was 5.2%±4.7% at pH 7.3 and 1.0%±2.1% at pH 8.0 (p=0.24), and the maximal penetration was 5.6%±8.5% at pH 7.3 and 2.7% ± 4.1% at pH 8.0 (p=0.38). At day 3 pi, the overall penetration was 11.1%±3.0% at pH 7.3 and 7.2%±2.1% at pH 8.0 (p=0.14), and maximal penetration was 23.8%±5.1% at pH 7.3 and 16.0%±11.3% at pH 8.0 (p=0.049). Compared to SC5314, the maximal penetration of DSY459 was significantly less at pH 7.3 at day 1 (p<0.001) and at day 3 (p<0.001) and was significantly less at pH 8.0 at day 1 (p<0.001) and at day 3 (p=0.003), but there was no difference at the overall hyphal penetration between these two strains at both pH conditions at day 1 and day 3.