An Adjuvant-Based Approach Enables the Use of Dominant HYG and KAN Selectable Markers in Candida albicans

Abstract

Candida albicans is a pathobiont fungus that can colonize multiple niches in the human body but is also a frequent cause of both mucosal and systemic disease. Despite its clinical importance, a paucity of dominant selectable markers has hindered the development of tools for genetic manipulation of the species. One factor limiting the utilization of dominant selectable markers is that C. albicans is inherently more resistant to antibiotics used for selection in other species. Here, we showed that the inclusion of suitable adjuvants can enable the use of two aminoglycoside antibiotics, hygromycin B and G418, for positive selection in C. albicans. Combining these antibiotics with an adjuvant, such as quinine or molybdate, substantially suppressed the background growth of C. albicans, thereby enabling transformants expressing CaHygB or CaKan markers to be readily identified. We verified that these adjuvants were not mutagenic to C. albicans and that CaHygB and CaKan markers were orthogonal to the existing marker NAT1/SAT1, and so provide complementary tools for the genetic manipulation of C. albicans strains. Our study also established that adjuvant-based approaches can enable the use of selectable markers that would otherwise be limited by high background growth from susceptible cells. IMPORTANCE Only a single dominant selectable marker has been widely adopted for use in the opportunistic fungal pathogen Candida albicans. This is in stark contrast to model fungi where a repertoire of dominant markers is readily available. A limiting factor for C. albicans has been the high levels of background growth obtained with multiple antibiotics, thereby limiting their use for distinguishing cells that carry an antibiotic-resistance gene from those that do not. Here, we demonstrated that the inclusion of adjuvants can reduce background growth and enable the robust use of both CaHygB and CaKan markers for genetic selection in C. albicans.

Keywords: antibiotic selection; dominant marker; drug resistance; fungal pathogen; selectable marker.

FIG 1

Quinine (A) and molybdate (B) act in tandem with hygromycin to inhibit C. albicans growth. 96-well checkerboard assays were performed in YPD containing the indicated drug concentrations. Plates were incubated at 30°C with orbital shaking, and OD₆₀₀ was recorded for each well every 15 min for 24 h. The relative growth of each well was normalized to the growth of the well with the highest OD.

FIG 2

Quinine acts in combination with hygromycin for the selection of a CaHygB marker. (A) Transformation with a linearized construct carrying the CaHygB gene was carried out and plated on YPD containing different combinations of hygromycin and quinine as indicated. Plates were incubated at 30°C for 72 h. (B) The parental SC5314 C. albicans strain and seven colonies from the plate containing 0.6 mg/mL hygromycin + 1.75 mg/mL quinine were subcultured on plates containing different combinations of hygromycin and quinine as indicted. Plates were incubated at 30°C for 24 h. (C) PCR check for successful integration of the CaHygB gene into the C. albicans genome. All seven transformant colonies were positive, confirming the successful integration of CaHygB. The parental SC5314 strain was used as a negative control, and a strain carrying pSFS2A-SAT1 similarly integrated at the MAL2 promoter was used as a positive control.

FIG 3

Quinine (A) and molybdate (B) act in tandem with G418 to inhibit C. albicans growth. The 96-well checkerboard assays were performed in YPD containing the indicated drug concentrations. Plates were incubated at 30°C with orbital shaking, and OD₆₀₀ was recorded for each well every 15 min for 24 h. The relative growth of each well was normalized to the growth of the well with the highest optical density (OD).

FIG 4

Quinine and molybdate act in combination with G418 for the selection of a CaKan marker. (A) Transformation of a plasmid carrying the CaKan gene was performed and transformants were cultured on YPD medium containing different combinations of G418, quinine, and molybdate as indicated. Plates were incubated at 30°C for 72 h. (B) The parental SC5314 strain and seven transformant colonies (taken from the plate containing 2 mg/mL G418 + 2 mg/mL quinine) were cultured on plates containing G418 and quinine as indicated. Plates were incubated at 30°C for 24 h. (C) In a control SC5314 strain, seven transformants from G418/molybdate selection were subcultured on plates containing different G418 and molybdate concentrations as indicated. Plates were incubated at 30°C for 24 h. (D) PCR analysis of seven transformants from G418/quinine and seven colonies from G418/molybdate were tested and all 14 contained the CaKan cassette correctly integrated at the MAL2 locus. The parental SC5314 strain was used as a negative control and a strain containing a pSFS2A-SAT1 construct similarly integrated at the MAL2 promoter was used as a positive control.

FIG 5

SAT1, CaHygB, and CaKan are three orthogonal selection markers in C. albicans. C. albicans strains carrying the SAT1 nourseothricin marker, the CaHygB hygromycin marker, the CaKan G418 marker, or no selection marker were grown on YPD, YPD + 0.2 mg/mL NAT, YPD + 0.6 mg/mL hygromycin (+1.75 mg/mL quinine), or YPD + 2 mg/mL G418 (+2 mg/mL quinine). Only cells carrying the corresponding selection marker grew on the appropriate antibiotic. Plates were incubated at 30°C for 24 h.

FIG 6

Quinine and molybdate are not significantly mutagenic to C. albicans. A C. albicans strain heterozygous for URA3 was incubated on plates containing different concentrations of adjuvants as indicated. Colonies were resuspended after 72 h of incubation at 30°C and plated to 5-FOA and YPD plates. Mutagenicity was calculated by dividing the number of colonies on 5-FOA plates by the number of colonies on YPD plates multiplied by 10,000. Each condition includes 4 replicates. The significance test was performed with Welch’s t test with a significance value of 0.05.