Transcriptional response of Candida albicans upon internalization by macrophages

Abstract

The opportunistic fungal pathogen Candida albicans is both a benign gut commensal and a frequently fatal systemic pathogen. The interaction of C. albicans with the host's innate immune system is the primary factor in this balance; defects in innate immunity predispose the patient to disseminated candidiasis. Because of the central importance of phagocytic cells in defense against fungal infections, we have investigated the response of C. albicans to phagocytosis by mammalian macrophages using genomic transcript profiling. This analysis reveals a dramatic reprogramming of transcription in C. albicans that occurs in two successive steps. In the early phase cells shift to a starvation mode, including gluconeogenic growth, activation of fatty acid degradation, and downregulation of translation. In a later phase, as hyphal growth enables C. albicans to escape from the macrophage, cells quickly resume glycolytic growth. In addition, there is a substantial nonmetabolic response imbedded in the early phase, including machinery for DNA damage repair, oxidative stress responses, peptide uptake systems, and arginine biosynthesis. Further, a surprising percentage of the genes that respond specifically to macrophage contact have no known homologs, suggesting that the organism has undergone substantial evolutionary adaptations to the commensal or pathogen lifestyle. This transcriptional reprogramming is almost wholly absent in the related, but nonpathogenic, yeast Saccharomyces cerevisiae, suggesting that these large-scale and coordinated changes contribute significantly to the ability of this organism to survive and cause disease in vivo.

FIG. 1.

In vitro C. albicans-macrophage interaction system. C. albicans strain SC5314 was incubated in RPMI plus 10% FBS at 37°C with or without macrophage line J774A. Germ tube formation, which occurs with roughly the same kinetics with or without macrophages, allows escape from the macrophage during the 6-h experiment.

FIG. 2.

Complete induction of alternative carbon metabolism upon phagocytosis. The pathways of β-oxidation, the glyoxylate cycle, gluconeogenesis, and glycolysis are shown, with the C. albicans gene names. Induction ratios (i.e., with macrophages/without macrophages) at 1 h are given, with genes in boldface regulated at least threefold. The dashed arrow shows the net reaction of these pathways: the conversion of fatty acids to glucose.

FIG. 3.

Widespread repression of translation by phagocytosis. Hierarchical clustering of expression data for genes related to translation during the entire 6-h time course. (A) All 54 ribosomal proteins included on our array. (B) All of the translation initiation factors (n = 23), translation elongation factors (n = 11), and cytoplasmic tRNA synthetases (n = 20) found on our array. Gene names are from S. cerevisiae homologs. Red represents induced genes, green represents repressed genes, and black indicates no change.

FIG. 4.

Schematic for additional array experiments. (A) Time course of the interaction between the Δcph1 Δefg1 strain (HLC54) and macrophages. Because this strain is nonfilamentous, it cannot escape the macrophage. (B) Schematic for the in vitro starvation experiments. Cells pregrown in minimal medium with abundant carbon and nitrogen were shifted to medium lacking either nitrogen or carbon (or both). A ratio was calculated for each gene in each condition compared to condition 1 (medium with carbon and nitrogen).

FIG. 5.

Similarities between starvation and phagocytosis. Data from the macrophage experiments in wild-type and Δcph1 Δefg1 strains were clustered with the starvation conditions (no nitrogen and no carbon, no carbon, or acetate as a carbon source, with a 1-, 2-, and 3-h time point for each condition). (A) Cluster of genes upregulated by macrophages and by starvation. Genes related to carbon metabolism are indicated by a black line; nutrient transporters are indicated by a green line. Of the 66 genes in this cluster, 19 fit one of these categories. (B) Downregulated cluster. Ribosomal proteins are indicated by black lines, tRNA synthases are indicated in blue, translational factors are indicated in orange, and glycolytic genes are indicated in purple. Of the 145 genes, 52 fit one of these categories.

FIG. 6.

Macrophage-signature expression cluster. Data from the wild-type and Δcph1 Δefg1 strains with macrophages and the wild-type strain in various starvation conditions were clustered. This cluster contains 227 genes that are upregulated by macrophage contact but not by starvation. All 19 conditions shown were clustered together; the separation of the three experiments is for clarity only.

FIG. 7.

Summary of C. albicans phagocytic responses. Phagocytosis induces C. albicans to adopt a slower growth program and to induce stress responses and alternate carbon metabolism compared to cells in medium alone. The morphogenetic response then allows escape, and these adaptations are reversed.