Systematic screens of a Candida albicans homozygous deletion library decouple morphogenetic switching and pathogenicity

Abstract

Candida albicans is the most common cause of serious fungal disease in humans. Creation of isogenic null mutants of this diploid organism, which requires sequential gene targeting, allows dissection of virulence mechanisms. Published analyses of such mutants show a near-perfect correlation between C. albicans pathogenicity and the ability to undergo a yeast-to-hypha morphological switch in vitro. However, most studies have used mutants constructed with a marker that is itself a virulence determinant and therefore complicates their interpretation. Using alternative markers, we created approximately 3,000 homozygous deletion strains affecting 674 genes, or roughly 11% of the C. albicans genome. Screening for infectivity in a mouse model and for morphological switching and cell proliferation in vitro, we identified 115 infectivity-attenuated mutants, of which nearly half demonstrated normal morphological switching and proliferation. Analysis of such mutants revealed that virulence requires the glycolipid glucosylceramide. To our knowledge, this is the first C. albicans small molecule that has been found to be required specifically for virulence.

Figure 1. C. albicans mutants and screens

A) Based on the published literature, Venn diagram depicting virtually complete overlap between C. albicans mutants with virulence defects and mutants with abnormal morphology (see Methods). Significance was calculated using the hypergeometric distribution. B) Construction of the homozygous gene disruption library by homologous recombination. One allele of each target gene was replaced by C. dubliniensis HIS1, and the second allele was replaced by C. maltosa LEU2. Red rectangles indicate the oligonucleotide barcodes. Successful knockout strains were defined as those with PCR-verified junctions at the 5′ and 3′ ends of the selectable markers, as well as absence of the target ORF. C) In vivo and in vitro screens of the C. albicans mutant library. As described in Methods, the 674 mutants were screened for infectivity in the mouse, for colony morphology on solid Spider medium at 30°C, and for proliferation in liquid SC at 37°C.

Figure 2. Infectivity screen

A) Schematic of the Infectivity screen. Pools of up to 48 mutants and wild type were used to infect BALB/c mice. Samples of C. albicans from the infecting inoculum and recovered from mouse kidneys were plated on Sabouraud agar. Genomic DNA was recovered and the relative abundance of each strain in the inoculum (I) and the recovered pool (R) was determined by real-time PCR (qPCR), using primers specific to the DNA barcodes. B) Histograms depicting log₂(R/I) values for C. albicans mutants (in orange), the wild type comparator (in blue), and the overlay between the two groups.

Figure 3. Colony morphology screen

A) C. albicans colony morphology. As shown in the schematic on the left, C. albicans colonies are bipartite: a central region (C) comprises yeast, pseudohyphae, and true hyphae, and a peripheral region (P) comprises mainly pseudohyphae and hyphae. On the right are examples of C. albicans colonies that illustrate the spectrum of morphologies observed in the screen. Wild type morphology is designated as 0, and aberrant morphology is scored from 1 to 3 (increased relative to wild type) and −1 to −3 (decreased relative to wild type) for both C and P. The Morphology score (M score) of each mutant is calculated as the sum of the absolute values of C and P. B) Schematic of the colony morphology screen. One isolate of each of the 674 mutants and wild type was initially plated for single colonies on Spider medium. Mutants exhibiting morphology different from wild type underwent testing of one to three additional isolates (depending on availability) to confirm consistency of the phenotype. C) Histogram of Infectivity defects among mutants with different M scores. *Denotes significant association with infectivity defect (using the hypergeometric distribution); p=0.0077 for M score of 2, p=4.8x10⁻⁶ for M score of 3, p=4.8x10⁻⁶ for M score of 4, p=0.10 for M score of 5, p=0.027 for M score of 6.

Figure 4. Proliferation screen and venn diagram

A) Histogram of infectivity defects among mutants with different doubling times. * Denotes significant association with infectivity defect (using the hypergeometric distribution); p=0.11 for 0.5 s.d.>DT_WT, p=0.13 for 1 s.d.>DT_WT, p=0.15 for 1.5 s.d.>DT_WT, p=0.034 for 2 s.d.>DT_WT, p=0.40 for 2.6 s.d.>DT_WT,, and p= 3.3x10⁻⁵ for >3 s.d.>DT_WT. B) Venn diagram illustrating the overlap of mutants from the three screens. Mutants with slow growth or inconsistent growth on Spider medium could not be scored for Morphogenesis and are not represented in the figure; these included 4 mutants with infectivity-specific defects, 8 with combined infectivity and proliferation defects, and 1 with a proliferation-specific defect.

Figure 5. Characterization of mutants affecting glucosylceramide biosynthesis

A) Schematic of the predicted glucosylceramide synthesis pathway and expected molecular masses. The position of the glucosyltransferase reaction mediated by Hsx1 has not been determined and may occur as depicted or elsewhere in the pathway. Note that the masses given are for the sodium salts of each molecule. B) TLC plate with glucosylceramide (GC) from soybean (lanes 1 and 2) and lipids extracted from equal masses of wild type (lane 3), hsx11 (lane 4), orf19.260 (lane 5), orf19.4831 (lane 6), and het1 (lane 7). The markers indicate the position of glucosylceramide, visualized with anthrone reagent. C,D,E,F,G, and H) MALDI-TOF spectra for a soybean glucosylceramide standard and lipid extracted from wild type, hsx11, orf19.260, orf19.4831, and het1, respectively.

Figure 6. Virulence analysis of mutants affecting glucosylceramide biosynthesis

A and B) The glucosylceramide pathway is required for virulence. As described in the text, BALB/c mice were infected with wild type C. albicans or one of the glucosylceramide pathway mutants, and time to illness was monitored. *denotes a significant difference from wild type by the logrank test; p=0.0020 for hsx11, p<0.0001 for orf19.260, p>0.05 for orf19.4831, and p=0.032 for het1. C,D,E, and F) Wild type genes restore competitive fitness to the glucosylceramide pathway mutants. Two-strain infectivity assays were performed comparing each mutant to a gene addback strain in 5 BALB/c mice. Shown are the R/I values of each strain in individual mice (black dots), the median R/I value (black line), and the p-value of the observed differences.