Significant variation of filamentation phenotypes in clinical Candida albicans strains

Abstract

Introduction: Candida albicans is an opportunistic human pathogen that typically resides as part of the microbiome in the gastrointestinal and genitourinary tracts of a large portion of the human population. This fungus lacks a true sexual cycle and evolves in a largely clonal pattern. The ability to cause disease is consistent across the species as strains causing systemic infections appear across the known C. albicans intra-species clades.

Methods: In this work, strains collected from patients with systemic C. albicans infections isolated at the Nebraska Medicine clinical laboratory were typed by MLST analysis. Since the ability to form filaments has been linked to pathogenesis in C. albicans, these clinical strains, as well as a previously genotyped set of clinical strains, were tested for their ability to filament across a variety of inducing conditions.

Results: Genotyping of the clinical strains demonstrated that the strains isolated at one of the major medical centers in our region were as diverse as strains collected across the United States. We demonstrated that clinical strains exhibit a variety of filamentation patterns across differing inducing conditions. The only consistent pattern observed in the entire set of clinical strains tested was an almost universal inability to filament in standard solid inducing conditions used throughout the C. albicans field. A different solid filamentation assay that produces more robust filamentation profiles from clinical strains is proposed in this study, although not all strains expected to filament in vivo were filamentous in this assay.

Discussion: Our data supports growing evidence that broad phenotypic diversity exists between the C. albicans type strain and clinical strains, suggesting that the type strain poorly represents filamentation patterns observed in most clinical isolates. These data further highlight the need to use diverse clinical strains in pathogenesis assays.

Keywords: Candida albicans; clinical strains; filamentation; hyphal morphogenesis; phylogeny.

Figure 1

UPGMA dendrogram consisting of the 20 clinical isolates and 224 reference strains of C. albicans. C. albicans clades are indicated by the brackets. Arrows indicate location of the clinical isolates from the UNMC collection genotyped in this study. Black arrows are within known clades while the red arrow designates a singleton strain.

Figure 2

Filamentation phenotypes of the clinical isolates. Cells of the indicated strains were tested for filamentation in standard solid and liquid filamentation assays in FBS (F), Lee’s (L), RPMI (R), or spider (S) media. Cells that filamented like the type strain were given a score of 4 (yellow) while strains that did not filament were given a score of 0 (blue). Strains with scores below 2.5 were considered defective in filamentation. Strains labeled “Y” in the “Sys” column were isolated from systemic infections, while those labeled “N” were isolated from other infections or healthy patients. Strains labeled “UNKN” in the “SYS” column had an unknown origin. Clade identification of each strain is also indicated.

Figure 3

Additional filamentation observations in the clinical strains. (A) Clinical strains were generally defective in filamentation on solid media. The number of assays with filamentation scores above 2.5 in liquid (blue) or solid (orange) filamentation conditions was calculated for each strain. Absence of a bar indicates that the strain filamented in none of the conditions. (B) Cells that filament well in solid media are often hyperfilamentous. P37039 and B527-15 cells were grown on solid YPD media for 4-5 days. Images were taken at the edge of the colonies at 4X on an Evos microscope. The white bar represents 400 µm. (C) Clinical C. albicans strains clearly filament more robustly in liquid media. The number of strains with a filamentation score above 2.5 was calculated for each filamentation condition. Letters correspond to the letters used for filamentation assays in Figure 2 .

Figure 4

Comparison of standard solid filamentation assays and a shortened solid filamentation assay. (A) Respective images from standard and shortened solid filamentation assays for strain B444-12. The strain was grown in the indicated filamentation induction media for 4-5 days (standard) or for 3 hours (short) at 37˚C and imaged through the bottom of the agar plate on an Evos microscope at 4X (standard) or 10X (short). Bars in the images represent 100 µm for each figure set (short or standard). (B) A heat map of filamentation scores in standard and shortened solid filamentation assays. Cells of the indicated strains were tested for filamentation in standard and shortened solid filamentation assays in FBS (F), Lee’s (L), RPMI (R), or spider (S) media. Cells that filamented like the type strain were given a score of 4 (yellow) while strains that were afilamentous were given a score of 0 (blue). Strains with scores below 2.5 were considered defective in filamentation. (C) Some clinical strains can filament in solid media, but only in the shortened assay. The number of assays with filamentation scores above 2.5 in standard (blue) or shortened (orange) solid filamentation conditions was calculated for each strain. Absence of a bar indicates that the strain filamented in none of the conditions. (D) B527-15 filaments in the standard solid filamentation assay, but few cells were filamentous in the shortened assay. Respective images from standard and shortened solid filamentation assays for strain B527-15. The strain was grown in the indicated filamentation for 4-5 days (standard) or for 3 hours (short) and imaged on an Evos microscope at 4X (standard) or 10X (short). Bars in the images represent 100 µm for each figure set (short or standard).