Abstract

Candida albicans produces chlamydospores, which can be used as a diagnostic tool for species identification. It has been suggested that these chlamydospores are degenerate spores. If so, then their production might be linked to the mating loci, and clinical strains that are homozygous for the C. albicans mating locus MTL may be altered in chlamydospore formation, which could cause problems in diagnostics and species identification. In Saccharomyces cerevisiae diploid cells, the heterodimeric transcriptional repressor formed by the products of the mating genes MATa1 and MATα2 is an important regulator of sporulation. It was therefore of interest to determine if the disruptions of the MATa1 and MATα2 homologs in C. albicans, MTLa1 and MTLα2, result in inhibition of chlamydospore formation. Laboratory strains containing disruptions of either the entire MTL locus or specific genes within the locus were assayed for their ability to form chlamydospores. Clinical strains that are homozygous for one of the two MTL loci were also assayed. No change in chlamydospore formation was seen in these strains compared to the standard laboratory strain.

Candida albicans is an opportunistic pathogen that can spread to the bloodstream in neutropenic patients, leading to a potentially fatal systemic infection 3–5, although this is controversial. The closely related species Candida dubliniensis readily forms chlamydospores, which differ slightly in shape and localization from C. albicans6 and which can form on media that do not support C. albicans chlamydospore formation 7, 8. This difference has been used as a diagnostic tool to differentiate these two closely related species 9, 10. Chlamydospores are rarely detected in vivo11, 12. However, the ability to form chlamydospores in vitro has been occasionally exploited in clinical settings as a diagnostic of C. albicans and C. dubliniensis.

Chlamydospores are believed to be dormant growth forms 2, that are induced under that conditions that induce sporulation in other fungi. Therefore, it was of interest to ask if genes involved in sporulation effect chlamydospore formation. In S. cerevisiae diploids, the sporulation inhibitor, RME1, is repressed by the heterodimeric transcriptional repressor formed by the products of MATa1 and MATα2 thus allowing for sporulation 13. The C. albicans genome does not contain any open reading frames with significant homology to RME1. Nonetheless, it was of interest to determine if the disruptions of the MATa1 and MATα2 homologs in C. albicans, MTLa1 and MTLα2, result in disregulation of chlamydospore formation. This would establish a link between mating and chlamydospore formation, suggestive that chlamydospores are pseudo-spores, or non-viable spores.

Little is known about the mechanisms that lead to chlamydospore formation. At least two genes that are known to regulate hyphal formation, EFG1 and NRG1, have also been shown to regulate chlamydospore formation 14, 15 The EFG1 deletion is unable to form chlamydospores 14 and the NRG1 deletion results in chlamydospore formation on media that do not normally support chlamydospore formation 15. Conditional regulation of the essential OLE1 gene encoding a fatty acid desaturase also results in reduced hyphal and chlamydospore formation. A gene important for modification of glycosylphosphatidylinositol [GPI]-anchors on proteins, GPI7, has also been shown to inhibit hyphal formation and enhance chlamydospore formation 16. It is not clear whether these genes, which all have an effect on hyphal formation, have a direct effect on chlamydospore formation or an indirect effect resulting from their effect on hyphal formation which is a prerequisite to chlamydospore formation. Farnesol is a compound that is used by C. albicans for quorum sensing and hyphal formation. It has recently been shown that it also promotes chlamydospore formation 17.

In addition to the association between hyphae and chlamydospore formation, there are other pathways that appear to affect chlamydospore formation. The MAP kinase gene HOG1 is important for oxidative stress, conditions in which chlamydospores are formed. Therefore, it is not surprising that deletion of the HOG1 gene results in strains that are unable to produce chlamydospores 18, 19. Finally, a forward genetic screen for mutants that affect chlamydospore formation identified six genes, including ISW2, MDS2, RIM13, RIM101, SCH9 and SUV320. SUV3 is involved in mitochondrial biogenesis and thus oxidative stress. SCH9 is involved in stress resistance and nutrient sensing. ISW2 is involved in chromatin remodeling and may be related to HOG1 function. RIM13, RIM101, and MDS2 are involved in pH response, but they also regulate hyphal formation. Thus a small number of genes have been shown to have an effect on chlamydospore formation in C. albicans.

Neither a traditional mating cycle nor a spore form of Candida has ever been described. However, homologs to genes in Saccharomyces cerevisiae involved in the mating process have been described in C. albicans21, including homologs to the key mating gene transcriptional regulators MATa1, MATα1, and MATα2 [MTLa1, MTLα1, and MTLα2]22. In both S. cerevisiae and C. albicans, these mating regulators are located in a unique mating locus. In haploid S. cerevisiae, the presence of the MATa or MATα alleles at the mating type (MAT) locus determines the a or α mating type of the cell respectively. Mating can only occur between cells of complementary mating type. In wild type diploid S. cerevisiae, and in 97% of C. albicans23, one copy of each of the mating type alleles was found on sister chromosomes.

Manipulation of genes in the Candida mating type-like (MTL) locus can result in an unorthodox sexual cycle 24–26. This cycle consists of the fusion of two diploid cells that have complementary homozygous MTL alleles, resulting in a tetraploid zygote. The zygote can then randomly shed chromosomes to form a stable diploid daughter cell 24. Although much of this mating cycle is nontraditional, it does appear that the genes in the C. albicans MTL locus maintain a critical regulatory role in mating, analogous to the S. cerevisiae MAT locus genes 22. Both mating locus alleles are present in the majority of C. albicans clinical isolates, rendering these strains unable to mate. However, recent data from our lab has indicated that strains homozygous for the MTLa locus or for the MTLα locus are found in over 20% of azole resistant clinical isolates from patients with active candidiasis undergoing azole treatment 27.

Chlamydospore formation was assessed on several MTL mutant strains to test the hypothesis that MTL genes regulate chlamydospore formation in the same manner as spore formation is regulated in related fungi. Those strains include clinical strains that are homozygous at the MTL locus, and laboratory strains that have gene disruptions in the mating locus. It is important to know if there is any connection between the mating locus and chlamydospore formation, given the increased frequency of MTL homozygosity in azole-resistant clinically important isolates and the role of chlamydospore formation as a diagnostic assay.

Strains with gene disruptions of the MTLa locus, the MTLα locus, the MTLa1 gene, and both MTLα1 and MTLα2 genes were generously provided by Christina Hull [Duke University] and were constructed in the laboratory of Alexander Johnson [University of California, San Francisco] 25. Further disruptions of MTLα1 [MTLK1], OBPα [MTLK2], and both MTLα1 and MTLα2 [MTLK3] in strain CHY484 were created using standard methodologies [Rustad TR, White TC, unpublished observations, 2005].

Clinical homozygous MTL strains were isolated from collections of C. albicans clinical isolates, as previously described 27. Strains CAI4 28, the parent strain to the gene-disrupted isolates, as well as 3153A and SC5314 were used as controls. Strains were stored in YEPD (10 g yeast extract, 20 g Bacto peptone, 20 g dextrose per liter) with 10% glycerol at −80° C. Active cultures were subcultured weekly on YEPD plates [YEPD with 20 g l−1 bactoagar]. Chlamydospores were induced using the Dalmau method 5. Briefly, cells were scratched into the surface of a cornmeal agar plate containing Tween 80, covered with a sterile cover slip and incubated at room temperature. The cover slip created a gradient of hypoxia that included conditions ideal for chlamydospore formation.

To determine if the genes in the MTL locus regulate the formation of chlamydospores, a collection of clinical and laboratory strains with disruptions at the MTL locus (Table 1) were assessed for their ability to form chlamydospores. MTL heterozygous strains were used as positive controls (CAI4, 3153A, and SC5314). A set of matched clinical isolates from a serial set of isolates wherein MTL heterozygosity was lost in conjunction with the development of azole resistance [FH1 and FH5] 29, and in a second set where isolates before and after the acquisition of azole resistance are MTL heterozygous [2–76 and 12–99] 30, were tested for chlamydospore formation. Strains with disruptions of individual MTL genes [CHY247, CHY257, CHY420, CHY439] and disruptions of the entire MTL locus [CHY484 and CHY486] were assayed to determine the effect of deregulation of MTLa1p/MTLα2p controlled genes on chlamydospore formation. Further disruptions were performed on genes within the MTLα allele in Δmtla background to look for a phenotype masked by genes on the complementary MTL locus (MTLK1, MTLK2, MTLK3) [Rustad TR, White TC, unpublished observations, 2005]. Since laboratory strains are not always perfect predictors of behavior in clinical isolates, six MTLa homozygous isolates and six MTLα homozygous clinical isolates were also assayed (Table 1). All strains in Table 1 formed chlamydospores that appeared to have the same morphology and density as wild type strains [data not shown]. No significant difference was seen between the parent MTL heterozygous strain CAI4 and the gene disrupted strains in frequency or morphology of chlamydospores.

Table 1

Strains used in this study

Type of strain Strain MTL genotype Auxotrophy Reference 
Wild type/ Control isolates 3153A MTLa/ MTLα wt 31 
 SC5314 MTLa/ MTLα wt 28 
 CAI4 MTLa/ MTLα Ura- 28 
     
Matched clinical isolates FH1 MTLa/ MTLα wt 29 
 FH5 MTLα/ MTLα wt 29 
 2–76 MTLa/ MTLα wt 30 
 12–99 MTLa/ MTLα wt 30 
     
MTL gene disruptions CHY 247 mtla1/ MTLα wt 22, 25 
 CHY 257 mtla1/ MTLα Ura- 22, 25 
 CHY 420 MTLa1/ mtlα1, mtlα2 wt 22, 25 
 CHY 439 MTLa1/ mtlα1, mtlα2 Ura- 22, 25 
     
MTL locus disruptions CHY 484 mtla/ MTLα Ade- 22, 25 
 CHY 4841 mtla/ MTLα Ura-Ade- This study 
 CHY 486 MTLa/ mtlα Ade- 22, 25 
 CHY 4861 MTLa/ mtlα Ura-Ade- This study 
     
MTL double disruptions MTLK 1 mtla/ mtlα1 Ade- This study 
 MTLK 2 mtla/ obpα Ade- This study 
 MTLK 3 mtla/ mtlα1, mtlα2 wt This study 
     
Clinical MTLhom isolates S14 MTLa/ MTLa wt 27 
 S15 MTLa/ MTLa wt 27 
 P24 MTLa/ MTLa wt 27 
 P32 MTLa/ MTLa wt 27 
 P39 MTLa/ MTLa wt 27 
 P43 MTLa/ MTLa wt 27 
 S5 MTLα/ MTLα wt 27 
 S37 MTLα/ MTLα wt 27 
 P41 MTLα/ MTLα wt 27 
 P42 MTLα/ MTLα wt 27 
 P47 MTLα/ MTLα wt 27 
 A556 MTLα/ MTLα wt 27 
Type of strain Strain MTL genotype Auxotrophy Reference 
Wild type/ Control isolates 3153A MTLa/ MTLα wt 31 
 SC5314 MTLa/ MTLα wt 28 
 CAI4 MTLa/ MTLα Ura- 28 
     
Matched clinical isolates FH1 MTLa/ MTLα wt 29 
 FH5 MTLα/ MTLα wt 29 
 2–76 MTLa/ MTLα wt 30 
 12–99 MTLa/ MTLα wt 30 
     
MTL gene disruptions CHY 247 mtla1/ MTLα wt 22, 25 
 CHY 257 mtla1/ MTLα Ura- 22, 25 
 CHY 420 MTLa1/ mtlα1, mtlα2 wt 22, 25 
 CHY 439 MTLa1/ mtlα1, mtlα2 Ura- 22, 25 
     
MTL locus disruptions CHY 484 mtla/ MTLα Ade- 22, 25 
 CHY 4841 mtla/ MTLα Ura-Ade- This study 
 CHY 486 MTLa/ mtlα Ade- 22, 25 
 CHY 4861 MTLa/ mtlα Ura-Ade- This study 
     
MTL double disruptions MTLK 1 mtla/ mtlα1 Ade- This study 
 MTLK 2 mtla/ obpα Ade- This study 
 MTLK 3 mtla/ mtlα1, mtlα2 wt This study 
     
Clinical MTLhom isolates S14 MTLa/ MTLa wt 27 
 S15 MTLa/ MTLa wt 27 
 P24 MTLa/ MTLa wt 27 
 P32 MTLa/ MTLa wt 27 
 P39 MTLa/ MTLa wt 27 
 P43 MTLa/ MTLa wt 27 
 S5 MTLα/ MTLα wt 27 
 S37 MTLα/ MTLα wt 27 
 P41 MTLα/ MTLα wt 27 
 P42 MTLα/ MTLα wt 27 
 P47 MTLα/ MTLα wt 27 
 A556 MTLα/ MTLα wt 27 

There are several explanations for the apparent lack of effect on chlamydospore formation of disruptions of the MTL locus. It is possible that the change in the rate of chlamydospore formation was too subtle for this assay to detect. Quantitative analysis of chlamydospore formation is not possible due to the large variability in numbers of chlamydospores across the array of hypoxia created in the Dalmau method, and a subtle change may be difficult to observe. Alternatively, the techniques used to induce chlamydospore formation in this assay may bypass the regulatory effect of the mating locus genes may have in nature.

Although there is evidence suggesting C. albicans can undergo a form of mating, there remains little evidence that this occurs frequently in clinical settings 27. It is possible that the genes responsible for chlamydospore formation have evolved into a new function, and in doing so became uncoupled from the regulation of the mating locus genes. Finally, it is entirely possible that chlamydospore formation is completely unrelated to spore formation, and therefore has never been regulated by the mating locus genes.

This study suggests that there is no relationship between the formation of chlamydospores and the C. albicans MTL. Clinical diagnosis of C. albicans by chlamydospore formation is therefore unaffected by the homozygous mating locus found in a significant portion of azole resistant clinical isolates.

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