Characterization of genetic determinants that modulate Candida albicans filamentation in the presence of bacteria

Abstract

In the human body, fungi and bacteria share many niches where the close contact of these organisms maintains a balance among the microbial population. However, when this microbial balance is disrupted, as with antibiotic treatment, other bacteria or fungi can grow uninhibited. C. albicans is the most common opportunistic fungal pathogen affecting humans and can uniquely control its morphogenesis between yeast, pseudohyphal, and hyphal forms. Numerous studies have shown that C. albicans interactions with bacteria can impact its ability to undergo morphogenesis; however, the genetics that govern this morphological control via these bacterial interactions are still relatively unknown. To aid in the understanding of the cross-kingdom interactions of C. albicans with bacteria and the impact on morphology we utilized a haploinsufficiency based C. albicans mutant screen to test for the ability of C. albicans to produce hyphae in the presence of three bacterial species (Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus). Of the 18,144 mutant strains tested, 295 mutants produced hyphae in the presence of all three bacterial species. The 295 mutants identified 132 points of insertion, which included identified/predicted genes, major repeat sequences, and a number of non-coding/unannotated transcripts. One gene, CDR4, displayed increased expression when co-cultured with S. aureus, but not E. coli or P. aeruginosa. Our data demonstrates the ability to use a large scale library screen to identify genes involved in Candida-bacterial interactions and provides the foundation for comprehending the genetic pathways relating to bacterial control of C. albicans morphogenesis.

Figure 1. Distribution of mutants that filament in the presence of the three bacterial species tested.

Overall 836 mutants were identified that filamented in the presence of the three bacterial species tested. Ec:E. coli; Pa: P. aeruginosa; Sa: S. aureus.

Figure 2. Representative colony morphologies.

(A) SC5314 wild-type non-filamentous growth on YPD agar (30° C); (B) SC5314 wild-type filamentation on M199 agar (37° C); (C) SC5314 wild-type filamentation inhibition in the presence of P. aeruginosa on M199 agar (37° C); (D) 4X magnification of A; (E) 4X magnification of B; (F) 4X magnification of C; (G), (H), (I) Representative examples of library mutant filamentation in the presence of bacteria due to haploinsufficiency on M199 agar (37° C) (4X magnification).

Figure 3. Representative photos of observed phenotypes of SC5314, library transposon candidates, and heterozygous and homozygous deletion strains.

YPD growth control (30° C), 40x magnification; M199 filamentation control (37° C), 100x magnification; E. coli interactions with C. albicans strains, 100x magnification; P. aeruginosa interactions with C. albicans strains, 100x magnification; S. aureus interactions with C. albicans strains, 100x magnification.

Figure 4. Phenotypes observed when SC5314, library transposon candidates, heterozygous and homozygous deletion strains grown in liquid culture (37° C) with bacteria or spent media.

A) C. albicans strains with M199 filamentation control and coculture with E. coli, P. aeruginosa, and S. aureus. Magnification 400x; B) C. albicans strains with M199 filamentation control and culture in spent media E. coli, P. aeruginosa, and S. aureus. Magnification 400x.

Figure 5. C. albicans CDR4 transcript levels grown in the presence of bacteria.

C. albicans SC5314, E. coli, P. aeruginosa, and S. aureus were grown to mid log phase in liquid culture, mixed together in equal amounts, co-incubated at 37° C, and aliquots were taken at 0, 10, 20, 30, and 60 minutes post addition. RNA was isolated and expression of CDR4 was measured by reverse transcription. ENO1 was used as a loading control and reference gene for expression comparisons. Graphical representation of CDR4 expression over time for SC5314 co-incubated with E. coli, P. aeruginosa, or S. aureus. Data is representative of three independent experiments with mean value and standard deviation bars shown. The asterisks indicate a statistically significant difference (P<0.05) in mean intensity of test conditions over the control.