Neocentromeres Form Efficiently at Multiple Possible Loci in Candida albicans

Centromeres are critically important for chromosome stability and integrity. Most eukaryotes have regional centromeres that include long tracts of repetitive DNA packaged into pericentric heterochromatin. Neocentromeres, new sites of functional kinetochore assembly, can form at ectopic loci because no DNA sequence is strictly required for assembly of a functional kinetochore. In humans, neocentromeres often arise in cells with gross chromosome rearrangements that rescue an acentric chromosome. Here, we studied the properties of centromeres in Candida albicans, the most prevalent fungal pathogen of humans, which has small regional centromeres that lack pericentric heterochromatin. We functionally delimited centromere DNA on Chromosome 5 (CEN5) and then replaced the entire region with the counter-selectable URA3 gene or other marker genes. All of the resulting cen5Δ::URA3 transformants stably retained both copies of Chr5, indicating that a functional neocentromere had assembled efficiently on the homolog lacking CEN5 DNA. Strains selected to maintain only the cen5Δ::URA3 homolog and no wild-type Chr5 homolog also grew well, indicating that neocentromere function is independent of the presence of any wild-type CEN5 DNA. Two classes of neocentromere (neoCEN) strains were distinguishable: “proximal neoCEN” and “distal neoCEN” strains. Neocentromeres in the distal neoCEN strains formed at loci about 200–450 kb from cen5Δ::URA3 on either chromosome arm, as detected by massively parallel sequencing of DNA isolated by CENP-ACse4p chromatin immunoprecipitation (ChIP). In the proximal neoCEN strains, the neocentromeres formed directly adjacent to cen5Δ::URA3 and moved onto the URA3 DNA, resulting in silencing of its expression. Functional neocentromeres form efficiently at several possible loci that share properties of low gene density and flanking repeated DNA sequences. Subsequently, neocentromeres can move locally, which can be detected by silencing of an adjacent URA3 gene, or can relocate to entirely different regions of the chromosome. The ability to select for neocentromere formation and movement in C. albicans permits mechanistic analysis of the assembly and maintenance of a regional centromere

Deletion of Cryptococcus neoformans AIF Ortholog Promotes Chromosome Aneuploidy and Fluconazole-Resistance in a Metacaspase-Independent Manner

Apoptosis is a form of programmed cell death critical for development and homeostasis in multicellular organisms. Apoptosis-like cell death (ALCD) has been described in several fungi, including the opportunistic human pathogen Cryptococcus neoformans. In addition, capsular polysaccharides of C. neoformans are known to induce apoptosis in host immune cells, thereby contributing to its virulence. Our goals were to characterize the apoptotic signaling cascade in C. neoformans as well as its unique features compared to the host machinery to exploit the endogenous fungal apoptotic pathways as a novel antifungal strategy in the future. The dissection of apoptotic pathways revealed that apoptosis-inducing factor (Aif1) and metacaspases (Mca1 and Mca2) are independently required for ALCD in C. neoformans. We show that the apoptotic pathways are required for cell fusion and sporulation during mating, indicating that apoptosis may occur during sexual development. Previous studies showed that antifungal drugs induce ALCD in fungi and that C. neoformans adapts to high concentrations of the antifungal fluconazole (FLC) by acquisition of aneuploidy, especially duplication of chromosome 1 (Chr1). Disruption of aif1, but not the metacaspases, stimulates the emergence of aneuploid subpopulations with Chr1 disomy that are resistant to fluconazole (FLCR) in vitro and in vivo. FLCR isolates in the aif1 background are stable in the absence of the drug, while those in the wild-type background readily revert to FLC sensitivity. We propose that apoptosis orchestrated by Aif1 might eliminate aneuploid cells from the population and defects in this pathway contribute to the selection of aneuploid FLCR subpopulations during treatment. Aneuploid clinical isolates with disomies for chromosomes other than Chr1 exhibit reduced AIF1 expression, suggesting that inactivation of Aif1 might be a novel aneuploidy-tolerating mechanism in fungi that facilitates the selection of antifungal drug resistance

Low Dosage of Histone H4 Leads to Growth Defects and Morphological Changes in Candida albicans

Chromatin function depends on adequate histone stoichiometry. Alterations in histone dosage affect transcription and chromosome segregation, leading to growth defects and aneuploidies. In the fungal pathogen Candida albicans, aneuploidy formation is associated with antifungal resistance and pathogenesis. Histone modifying enzymes and chromatin remodeling proteins are also required for pathogenesis. However, little is known about the mechanisms that generate aneuploidies or about the epigenetic mechanisms that shape the response of C. albicans to the host environment. Here, we determined the impact of histone H4 deficit in the growth and colony morphology of C. albicans. We found that C. albicans requires at least two of the four alleles that code for histone H4 (HHF1 and HHF22) to grow normally. Strains with only one histone H4 allele show a severe growth defect and unstable colony morphology, and produce faster-growing, morphologically stable suppressors. Segmental or whole chromosomal trisomies that increased wild-type histone H4 copy number were the preferred mechanism of suppression. This is the first study of a core nucleosomal histone in C. albicans, and constitutes the prelude to future, more detailed research on the function of histone H4 in this important fungal pathogen

Diploids in the Cryptococcus neoformans Serotype A Population Homozygous for the α Mating Type Originate via Unisexual Mating

The ubiquitous environmental human pathogen Cryptococcus neoformans is traditionally considered a haploid fungus with a bipolar mating system. In nature, the α mating type is overwhelmingly predominant over a. How genetic diversity is generated and maintained by this heterothallic fungus in a largely unisexual α population is unclear. Recently it was discovered that C. neoformans can undergo same-sex mating under laboratory conditions generating both diploid intermediates and haploid recombinant progeny. Same-sex mating (α-α) also occurs in nature as evidenced by the existence of natural diploid αADα hybrids that arose by fusion between two α cells of different serotypes (A and D). How significantly this novel sexual style contributes to genetic diversity of the Cryptococcus population was unknown. In this study, ∼500 natural C. neoformans isolates were tested for ploidy and close to 8% were found to be diploid by fluorescence flow cytometry analysis. The majority of these diploids were serotype A isolates with two copies of the α MAT locus allele. Among those, several are intra-varietal allodiploid hybrids produced by fusion of two genetically distinct α cells through same-sex mating. The majority, however, are autodiploids that harbor two seemingly identical copies of the genome and arose via either endoreplication or clonal mating. The diploids identified were isolated from different geographic locations and varied genotypically and phenotypically, indicating independent non-clonal origins. The present study demonstrates that unisexual mating produces diploid isolates of C. neoformans in nature, giving rise to populations of hybrids and mixed ploidy. Our findings underscore the importance of same-sex mating in shaping the current population structure of this important human pathogenic fungus, with implications for mechanisms of selfing and inbreeding in other microbial pathogens

Acquisition of aneuploidy provides increased fitness during the evolution of antifungal drug resistance.

The evolution of drug resistance is an important process that affects clinical outcomes. Resistance to fluconazole, the most widely used antifungal, is often associated with acquired aneuploidy. Here we provide a longitudinal study of the prevalence and dynamics of gross chromosomal rearrangements, including aneuploidy, in the presence and absence of fluconazole during a well-controlled in vitro evolution experiment using Candida albicans, the most prevalent human fungal pathogen. While no aneuploidy was detected in any of the no-drug control populations, in all fluconazole-treated populations analyzed an isochromosome 5L [i(5L)] appeared soon after drug exposure. This isochromosome was associated with increased fitness in the presence of drug and, over time, became fixed in independent populations. In two separate cases, larger supernumerary chromosomes composed of i(5L) attached to an intact chromosome or chromosome fragment formed during exposure to the drug. Other aneuploidies, particularly trisomies of the smaller chromosomes (Chr3-7), appeared throughout the evolution experiment, and the accumulation of multiple aneuploid chromosomes per cell coincided with the highest resistance to fluconazole. Unlike the case in many other organisms, some isolates carrying i(5L) exhibited improved fitness in the presence, as well as in the absence, of fluconazole. The early appearance of aneuploidy is consistent with a model in which C. albicans becomes more permissive of chromosome rearrangements and segregation defects in the presence of fluconazole

Growth defect and phenotypic instability of mutants containing a single allele of histone H4.

<p>A small colony of a histone H4 mutant (DAY1072) was re-streaked on YPD medium and incubated 4 days at 30°C (A), close-up picture (B). Re-isolated small (C) and large (C, E) colonies of <i>hhf22-hht2Δ/hhf22-hht2Δ HHF1/hhf1Δ</i> (DAY1072), and small (D) and large (D, F) colonies of <i>HHF22-HHT2/hhf22-hht2Δ hhf1Δ/hhf1Δ</i> (DAY1074 and DAY1079) incubated on YPD for 48 hrs at 30°C.</p

Genomic organization of the histone H3 and H4 loci in <i>Candida albicans</i>.

<p>The black boxes indicate the extent of the deleted regions in the mutants, which are replaced by the auxotrophic markers <i>ARG4</i>, <i>URA3</i>, <i>URA3-dpl200</i> or the <i>dpl200</i> loop-out. <i>HHF1</i> is deleted from nucleotide +114 to the STOP codon (65% of the gene) and the <i>HHF22-HHT2</i> cluster is deleted from the STOP codon of <i>HHF22</i> to nucleotide +329 of <i>HHT2</i> (80% of the <i>HHT2</i> gene). orf19.1060 is a possible spurious ORF. In red, the regions recognized by the probes D and G used for determining <i>HHF1</i> or <i>HHF22</i> copy dosage by Southern Blot are shown. H: HindIII; N: NcoI.</p

Quantitative Southern blot of histone H4 alleles.

<p>DNA samples were obtained from 30°C overnight cultures of small and large colonies isolated from (A) <i>hhf22-hht2Δ/Δ HHF1/hhf1Δ</i> (DAY1072) and (B and C) <i>HHF22-HHT2/hhf22-hht2Δ hhf1Δ/Δ</i> (DAY1074, DAY1075, DAY1076, DAY1078 and DAY1079). Due to the formation of suppressors, the proportion of small to large colonies was verified for each overnight culture by colony count on YPD to ensure that the DNA extraction was representative of a small or a large colony population. Overnight cultures from small colonies with a minimum of 80% of small colonies were used to prepare DNA for the Southern blot. Representative colonies from these plates corresponding to different ratios of mutated to wild-type histone H4 ratio are shown (D). Probes D and G were used to detect the alleles: <i>hhf1::URA3</i> and <i>HHF1</i>, and <i>hhf22::ARG4</i> and <i>HHF22</i>, respectively. <i>HHF22-HHT2/hhf22-hht2Δ HHF1/hhf1Δ</i> (DAY1070) was used in (B) as a control for a 1:1 mutated to wild-type <i>HHF22</i> ratio.</p

Strains used in this study.

<p>Strains used in this study.</p

Amino acid sequence alignment of histone H3 and H4 genes.

<p>Comparison of the amino acid sequences of histone H3 (A) and histone H4 (B) alleles from <i>C. albicans</i> and <i>S. cerevisiae</i> using ClustalW.</p