Forward genetics in Candida albicans that reveals the Arp2/3 complex is required for hyphal formation, but not endocytosis

Abstract

SummaryCandida albicans is a diploid fungal pathogen lacking a defined complete sexual cycle, and thus has been refractory to standard forward genetic analysis. Instead, transcription profiling and reverse genetic strategies based on Saccharomyces cerevisiae have typically been used to link genes to functions. To overcome restrictions inherent in such indirect approaches, we have investigated a forward genetic mutagenesis strategy based on the UAU1 technology. We screened 4700 random insertion mutants for defects in hyphal development and linked two new genes (ARP2 and VPS52) to hyphal growth. Deleting ARP2 abolished hyphal formation, generated round and swollen yeast phase cells, disrupted cortical actin patches and blocked virulence in mice. The mutants also showed a global lack of induction of hyphae-specific genes upon the yeast-to-hyphae switch. Surprisingly, both arp2D/D and arp2D/Darp3D/D mutants were still able to endocytose FM4-64 and Lucifer Yellow, although as shown by time-lapse movies internalization of FM4-64 was somewhat delayed in mutant cells. Thus the non-essential role of the Arp2/3 complex discovered by forward genetic screening in C. albicans showed that uptake of membrane components from the plasma membrane to vacuolar structures is not dependent on this actin nucleating machinery.

Functional Genomic Analysis of Candida albicans Adherence Reveals a Key Role for the Arp2/3 Complex in Cell Wall Remodelling and Biofilm Formation

“…Other members of this complex play important roles in C . albicans virulence, morphogenesis and cell wall organization [17]; however, this complex has not been previously implicated in cellular adhesion or biofilm development. To determine whether transcriptional repression of other components of the Arp2/3 complex causes similar phenotypes to those observed in the tetO-ARC18/arc18Δ strain, we performed equivalent adherence, biofilm, cell surface hydrophobicity and fluorescence staining assays on non-barcoded conditional expression strains for ARC15 , ARC19 , ARC35 , and ARC40 .…”

“…albicans strains lacking ARP2 or ARP3 have dramatic actin cytoskeleton defects and delayed rates of endocytosis [17]. A common feature of cells with endocytic defects and depolarized actin patches is hyperactivity of cell wall stress pathways [19].…”

“…In C . albicans , Rho1-mediated endocytosis has not been characterized, but is a strong candidate for the compensatory endocytosis observed in the absence of Arp2/3 activity [17]. To test this, we constructed strains where RHO1 expression is under the control of a doxycycline-repressible promoter in both a wild-type strain and the arc40Δ/Δ mutant.…”

“…Although previous work established that the Arp2/3 complex is not essential for endocytosis in C . albicans [17], the compensatory pathway that acts in the absence of Arp2/3 complex function remained enigmatic. Our results suggest that endocytosis through Rho1 signaling is the elusive pathway through which endocytosis occurs in the absence of Arp2/3 function in C .…”

The Ccr4–Not complex

“…Furthermore in Candida albicans cells that lack Not4 fail to form hyphae (Krueger et al, 2004). The other subunits of the complex such as Not5 (Cheng et al, 2003a), Not1 (Epp et al, 2010;Uhl et al, 2003) and both Ccr4 and Caf1 (Dagley et al, 2011) have also been implicated in filamentous growth in this yeast.…”