Time course of global gene expression alterations in Candida albicans during infection of HeLa cells

Abstract

Candida albicans (C. albicans) is an opportunistic fungus that quickly adapts to various microniches. It causes candidiasis, a common fungal infection for which the pathogenic mechanism has not been elucidated yet. To explore the pathogenic mechanism of candidiasis we used several methods, including microscopic observation of morphological changes of HeLa cells and fungus, analysis of differentially expressed genes using gene chips, and a series of biological and bioinformatic analyses to explore genes that are possibly involved in the pathogenesis of C. albicans. During the C. albicans infection, significant morphological changes of the fungus were observed, and the HeLa cells were gradually destroyed. The gene chip experiments showed upregulated expression of 120 genes and downregulated expression of 178 genes. Further analysis showed that some genes may play an important role in the pathogenesis of C. albicans. Overall, morphological variation and adaptive gene expression within a particular microniche may exert important effects during C. albicans infections.

FIGURE 1

Attachment and hyphal formation of C. albicans during infection of HeLa cells. (A) In uninfected (normal) HeLa cells, we could clearly see the cell membranes and nuclei. (B) After 1 hour, the fungus attached to the surface of HeLa cells and transformed from its yeast to hyphal form. (C) After 3 hours, the hyphae adhered extensively to the cell surface so that the cell-cell interactions could not be clearly observed, and the cell surface became loose. (D-E) In the invasion phase (3-12 hours), the hyphae were abundant, the shape of the epithelial cells changed, the membranes were ruptured and the cell content overflowed. (F) In the late phase (12–24 hours), the epithelial cells were completely destroyed and only a large number of C. albicans hyphal forms was observed.

FIGURE 2

Twelve significant differential expression tendencies (profiles 2, 3, 5, 6, 11, 12, 30, 38, 39, 41, 56, and 65). Eighty theoretical expression tendencies were observed, but only 12 expression trends were considered significant. The most significant expression trend was observed in the profile 3, where gene expression decreased initially but after that increased slightly at the last time point.

FIGURE 3

Heat map of genes detected by quantitative reverse transcription PCR (RT-qPCR). The heat map illustrates that the 15 selected differentially expressed genes were in accordance with the results of gene chip and differential gene expression tendency analyses. The rows represent genes and the columns represent replicates of each time points. Blue indicates down-regulation, red up-regulation, and white indicates the gene expression of Candida albicans before infection. The average and standard deviation (STD) can be seen in Supplemental Figure 1.

SUPPLEMENTAL FIGURE 1

Validation of mRNA expression. As shown in the bar chart, the mRNA expression was in accordance with the gene chip results and differential expression tendencies.

FIGURE 4

Series tests of cluster (STC) - Gene Ontology (GO) analysis of the third significant expression tendency (profile 3). The STC-GO analysis showed that the genes from profile 3 were associated with a variety of biological functions, including hyphal growth, pseudohyphal growth, budding cell bud growth, cell-matrix adhesion, biofilm formation, mRNA polyadenylation, and response to osmotic stress among others. When the P-value was <0.05, distribution map of significant functions was performed with the negative logarithm of P value; the abscissa was -LogP, and the ordinate was significant GO. In this figure, the closer it was to the top, the more significant the GO function was.

SUPPLEMENTAL FIGURE 2

Series tests of cluster (STC) - Gene Ontology (GO) analysis of 12 significant gene expression tendencies. The STC-GO analysis showed associations between different genes and biological functions, apart from the biological functions presented in the profile 3.

FIGURE 5

Analysis of signaling pathways. Overall, 17 significant signaling pathways with differentially expressed genes were observed, and these included glycerophospholipid metabolism, amino sugar and nucleotide sugar metabolism, biosynthesis of secondary metabolites, and alpha-linolenic acid metabolism.

FIGURE 6

Gene Co-expression Network (GCN) analysis of genes from 12 significant differential expression trends. The GCN contained 557 genes and the results varied in relation to the infection time points. The nodes represent genes and the line segments represent the interactions between genes. The solid lines represent positively correlated genes, which means that the products of one or more genes can promote the expression of other genes. The dashed lines represent negatively correlated genes, meaning mutual inhibition between genes and their products. The colors represent the value of k-core, and k-cores of the same value reflect a similarity and indicate a correlation in gene functions. The complexity of gene relationships increases with k-core value rank. The identically colored nodes are from the same subgraph.

FIGURE 7

(A) The co-expression network with excluded genes for which names were not assigned. Some genes corresponded to multiple probes, some probes did not have a gene name; for a more intuitive analysis of a correlation between genes, we excluded the genes for which names were not assigned and reconstructed the co-expression network. This network contained 134 genes, where PDA1 and ZRT2 genes appeared to have important roles in C. albicans. (B) Gene Co-expression Network (GCN) analysis of genes from the intersection set. According to the GCN, CHT3, SHM2, PDA1, SDH41, ADE13, LAT1, IDP1, SER33, VMA2, HEM12, MUQ1, and HXK1 genes may have an important role in C. albicans. (C) Gene Co-expression Network (GCN) analysis of genes from the union set. PDA1, ZRT2, CHS8, KEL1, ALG7, IPF168, HOG1, LAT1, DCW1, and HXK1 genes were in the center of the GCN, indicating their possible important roles in C. albicans.