Transcriptional regulators Cph1p and Efg1p mediate activation of the Candida albicans virulence gene SAP5 during infection

Abstract

The opportunistic fungal pathogen Candida albicans can cause superficial as well as systemic infections. Successful adaptation to the different host niches encountered during infection requires coordinated expression of various virulence traits, including the switch between yeast and hyphal growth forms and secretion of aspartic proteinases. Using an in vivo expression technology that is based on genetic recombination as a reporter of gene activation during experimental candidiasis in mice, we investigated whether two signal transduction pathways controlling hyphal growth, a mitogen-activated protein kinase cascade ending in the transcriptional activator Cph1p and a cyclic AMP-dependent regulatory pathway that involves the transcription factor Efg1p, also control expression of the SAP5 gene, which encodes one of the secreted aspartic proteinases and is induced by host signals soon after infection. Our results show that both transcriptional regulators are important for SAP5 activation in vivo. SAP5 expression was reduced in a cph1 mutant, although filamentous growth in infected tissue was not detectably impaired. SAP5 expression was also reduced, but not eliminated, in an efg1 null mutant, although this strain grew exclusively in the yeast form in infected tissue, demonstrating that in contrast to in vitro conditions, SAP5 activation during infection does not depend on growth of C. albicans in the hyphal form. In a cph1 efg1 double mutant, however, SAP5 expression in infected mice was almost completely eliminated, suggesting that the two signal transduction pathways are important for SAP5 expression in vivo. The avirulence of the cph1 efg1 mutant seemed to be caused not only by the inability to form hyphae but also by a loss of expression of additional virulence genes in the host.

FIG. 1.

Integration of the deletable FRT-MPA^r-FRT cassette into one of the ACT1 alleles of strains JKC18 (cph1/cph1), HLC67 (efg1/efg1), and HLC69 (cph1/cph1 efg1/efg1). (A) Integration scheme. The ACT1 coding region is represented by the open arrow, and the MPA^r marker is represented by the grey arrow. The 34-bp FRT site is not drawn to scale. The probe used for verification of correct integration by Southern hybridization is represented by the black bar, and the diagnostic BglII sites are shown. Bg, BglII; ScI, SacI. (B) Southern hybridization of BglII-digested genomic DNA of parent strains and their transformants carrying the FRT-MPA^r-FRT cassette with an ACT1-specific probe. The positions of the fragments are indicated on the right, and molecular sizes (in kilobases) are indicated on the left. Lane 1, CAI4; lane 2, CFI1; lane 3, JKC18; lane 4, CFI2; lane 5, HLC67; lane 6, CFI3; lane 7, HLC69; lane 8, CFI4.

FIG. 2.

Integration of the P_SAP5-ecaFLP fusion into one of the SAP5 alleles of strains CFI2 (cph1/cph1), CFI3 (efg1/efg1), and CFI4 (cph1/cph1 efg1/efg1). (A) Integration scheme. The coding regions of the SAP5 and ecaFLP genes are represented by the open and cross-hatched arrows, respectively. The SAP5 promoter (P_SAP5) is indicated by the solid arrow, the ACT1 transcription termination sequence (ACT1T) is indicated by the solid circle, and the URA3 selection marker is indicated by the grey arrow. The probe used for verification of correct integration by Southern hybridization is represented by the black bar, and the diagnostic BglII sites are shown. The BglII site in parentheses is not present in the SAP5-2 allele of strain CAI4 and its derivatives. Bg, BglII; ScI, SacI; X, XbaI. (B) Southern hybridization of BglII-digested genomic DNA of parent strains and their transformants carrying the ecaFLP reporter gene with the SAP5 promoter fragment as the probe. The positions of the wild-type SAP5 alleles (boldface) and the cross-hybridizing SAP4 and SAP6 fragments are indicated on the left. The molecular sizes (in kilobases) of the wild-type SAP5 fragments and the fragments corresponding to the reporter gene fusions are indicated on the right. Lane 1, CAI4; lane 2, CFI1; lane 3, S5FI2A; lane 4, S5FI2B; lane 5, JKC18; lane 6, CFI2; lane 7, C2S5F1A; lane 8, C2S5F1B; lane 9, HLC67; lane 10, CFI3; lane 11, C3S5F1A; lane 12, C3S5F1B; lane 13, HLC69; lane 14, CFI4; lane 15, C4S5F1A; lane 16, C4S5F1B.

FIG. 3.

Integration of the P_SAP2-ecaFLP fusion into one of the SAP2 alleles of strains CFI2 (cph1/cph1), CFI3 (efg1/efg1), and CFI4 (cph1/cph1 efg1/efg1). (A) Integration scheme. The coding regions of the SAP2 and ecaFLP genes are represented by the open and cross-hatched arrows, respectively. The SAP2 promoter (P_SAP2-1) is indicated by the solid arrow, the ACT1 transcription termination sequence (ACT1T) is indicated by the solid circle, and the URA3 selection marker is indicated by the grey arrow. The probe used for verification of correct integration by Southern hybridization is represented by the black bar, and the diagnostic ClaI sites are shown. The ClaI site in parentheses is not present in the SAP2-2 allele of strain CAI4 and its derivatives. C, ClaI; ScI, SacI; X, XbaI. (B) Southern hybridization of ClaI-digested genomic DNA of parent strains and their transformants carrying the ecaFLP reporter gene with the SAP2 promoter fragment as the probe. The positions of the wild-type SAP2 alleles are indicated on the left, and the molecular sizes (in kilobases) of the wild-type SAP2 fragments and the fragment corresponding to the reporter gene fusion are indicated on the right. Lane 1, CAI4; lane 2, CFI1; lane 3, S2FI5B; lane 4, JKC18; lane 5, CFI2; lane 6, C2S2F1A; lane 7, C2S2F1B; lane 8, HLC67; lane 9, CFI3; lane 10, C3S2F1D; lane 11, C3S2F1E; lane 12, HLC69; lane 13, CFI4; lane 14, C4S2F1B; lane 15, C4S2F1C.

FIG. 4.

Microscopic appearance of reporter strains S5FI1A (wild type) (A), C2S5F1A (cph1) (B), C3S5F1A (efg1) (C), and C4S5F1A (cph1 efg1) (D) 24 h after infection. Peritoneal infection by the efg1 single mutant and the cph1 efg1 double mutant was characterized by inflammatory infiltrates composed of yeast cells surrounded by inflammatory cells which were attached to the liver surface (C and D), whereas the wild-type strain and the cph1 mutant invaded the liver (A and B).

FIG. 5.

Expression of the SAP5 gene in wild-type (WT) and cph1, efg1, and cph1 efg1 mutant strains at various stages of an intraperitoneal infection. In vivo SAP5 induction was monitored by determining the percentage of MPA-sensitive cells recovered by peritoneal lavage after 30 min and recovered from the liver after 4, 24, and 48 h. The results are results from two independent experiments in which one or two mice were infected per strain and time point; each bar represents one animal. The light grey bars show the results obtained with strains S5FI2A, C2S5F1A, C3S5F1A, and C4S5F1A, and the dark grey bars show the results obtained with strains S5FI2B, C2S5F1B, C3S5F1B, and C4S5F1B. a, mutant strains were significantly different from the wild type (P < 0.005, as determined by Student’s t test); b, cph1 efg1 mutants were significantly different from the cph1 single mutant; c, cph1 efg1 mutants were significantly different from the efg1 single mutant.