Transcription factor Efg1 shows a haploinsufficiency phenotype in modulating the cell wall architecture and immunogenicity of Candida albicans

Abstract

The Candida albicans transcription factor Efg1 is known to be involved in many different cellular processes, including morphogenesis, general metabolism, and virulence. Here we show that besides its manifold roles, Efg1 also has a prominent effect on cell wall structure and composition, strongly affecting the structural glucan part. Deletion of only one allele of EFG1 already results in severe phenotypes for cell wall biogenesis, comparable to those with deletion of both alleles, indicative of a severe haploinsufficiency for EFG1. The observed defects in structural setup of the cell wall, together with previously reported alterations in expression of cell surface proteins, result in altered immunogenic properties of strains with compromised Efg1 function. This is shown by interaction studies with macrophages and primary dendritic cells. The structural changes in the cell wall carbohydrate meshwork presented here, together with the manifold changes in cell wall protein composition and metabolism reported in other studies, contribute to the altered immune response mounted by innate immune cells and to the altered virulence phenotypes observed for strains lacking EFG1.

Fig 1

(A) Drop tests for sensitivity to the cell wall-disturbing agent Congo red (CR). Plates were cultivated at 30°C and documented after 2 days. The sensitivities of post-exponential-phase (B) and exponentially growing (C) cells to Zymolyase 100T are shown. Data represent averages for three independent experiments.

Fig 2

Transcriptional analysis of EFG1. Levels of EFG1 mRNA transcripts were determined for exponential-phase and post-exponential-phase cells of strains carrying one or two copies of EFG1. Levels were normalized to the 18S rRNA transcript level. In total, three biological replicates were performed, and the results of a representative one are presented, with standard errors originating from qRT-PCR replicates. For each strain, mRNA levels were related to the absolute level of EFG1 transcripts in exponentially growing wild-type SC5314, which was set to 1.

Fig 3

Polysaccharide composition of the fungal cell wall. (A) Absolute amounts of glucose released by hydrolysis of total dry cell biomass and cytosolic contents of exponentially growing strains SC5314, CAI4 URA3, HLC17, HLC17Rev, HLC52, and HLC74. (B) Absolute polysaccharide cell wall compositions related to dry cell biomass of exponentially growing strains. All of the data are based on three independent experiments, and average values with standard deviations are presented.

Fig 4

Cell wall thicknesses and representative transmission electron microscopy images. (A) Cell wall thicknesses were estimated from over 20 independent cell measurements obtained from different microscopic images. Representative images of the cell walls of wild-type strain SC5314 (B) and the efg1/efg1 mutant HLC52 (C) present differences in thickness of the electron-translucent polysaccharide layer. Images were taken at a magnification of ×20,000 and represent identical areas.

Fig 5

β-1,3-Glucan antibody staining of the cell surface. (A) Exponentially growing and β-1,3-glucan antibody-labeled strains were examined under a microscope. All figures represent equal areas. Fluorescent images were taken with a constant exposition time to compare signal intensities. For control samples labeled only with secondary antibodies, no visible fluorescence was detectable (not shown). (B) Labeled β-1,3-glucan in exponential-phase and post-exponential-phase cells was quantified by FACS analysis. Intensity medians for three biological replicates were combined and supplemented with standard errors. Fluorescence intensity was normalized to that of exponentially grown wild-type SC5314, which was set to 1.

Fig 6

ROS release by macrophages challenged with strains SC5314, CAI4 URA3, HLC17, HLC17Rev, HLC52, and HLC74 in exponential-phase (A) or post-exponential-phase (B) growth. Data were reproduced in three independent experiments performed in quadruplicate. Average results for representative experiments are presented. Standard deviations for individual time points were within 10% of absolute values.

Fig 7

Transcriptional responses of unstimulated mDCs and mDCs in contact with the SC5314 wild-type, CAI4 URA3 revertant, and EFG1 mutant strains. mRNA levels were related to the absolute level of mDCs treated with wild-type SC5314, which was set to 1. Determinations were performed in triplicate, and average values with standard deviations are presented. Results are representative of two independent experiments. In the presence of wild-type strain SC5314, the level of IL-4 was induced by 1 order of magnitude, while the rest of the studied cytokines were induced by 2 orders of magnitude, compared to the basal levels in unstimulated mDCs.