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, 4 (8), 1420-33

Cryptococcus Neoformans Gene Expression During Murine Macrophage Infection

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Cryptococcus Neoformans Gene Expression During Murine Macrophage Infection

Weihua Fan et al. Eukaryot Cell.

Abstract

The fungal pathogen Cryptococcus neoformans survives phagocytosis by macrophages and proliferates within, ultimately establishing latent infection as a facultative intracellular pathogen that can escape macrophage control to cause disseminated disease. This process is hypothesized to be important for C. neoformans pathogenesis; however, it is poorly understood how C. neoformans adapts to and overcomes the hostile intracellular environment of the macrophage. Using DNA microarray technology, we have investigated the transcriptional response of C. neoformans to phagocytosis by murine macrophages. The expression profiles of several genes were verified using quantitative reverse transcription-PCR and a green fluorescent protein reporter strain. Multiple membrane transporters for hexoses, amino acids, and iron were up-regulated, as well as genes involved in responses to oxidative stress. Genes involved in autophagy, peroxisome function, and lipid metabolism were also induced. Interestingly, almost the entire mating type locus displayed increased expression 24 h after internalization, suggesting an intrinsic connection between infection and the MAT locus. Genes in the Gpa1-cyclic AMP-protein kinase A pathway were also up-regulated. Both gpa1 and pka1 mutants were found to be compromised in macrophage infection, confirming the important role of this virulence pathway. A large proportion of the repressed genes are involved in ribosome-related functions, rRNA processing, and translation initiation/elongation, implicating a reduction in translation as a central response to phagocytosis. In summary, this gene expression profile allows us to interpret the adaptation of C. neoformans to the intracellular infection process and informs the search for genes encoding novel virulence attributes.

Figures

FIG. 1.
FIG. 1.
Activation of MATα locus transcription during phagocytosis. Log-transformed microarray data of a random set of genes and of genes spanning the MATα locus are plotted here, as well as log-transformed data from real-time RT-PCR analysis of selected genes in the MATα locus. The lines at 1 and −1 indicate the threshold of significance (twofold increase or decrease). A diagram of the serotype A MATα locus indicates the position of genes in this region. The RT-PCR was performed three times in duplicate with unamplified total RNA as template. The expression value of each gene was determined by the threshold cycle method, and the value of ACT1 (encoding actin) was used to normalize the expression of genes of interest. For each gene, the relative induction was determined by comparing its normalized expression values in the macrophage-ingested sample with the medium control. The average values from six replicates are shown here.
FIG. 2.
FIG. 2.
Expression and functional analysis of phagocytosis-induced genes. A. Quantitative real-time RT-PCR analysis of the CZC1, CGL1, and CPP2 genes. RT-PCR was performed three times in duplicate at each time point with unamplified total RNA as template. The expression value of each gene was determined by the threshold cycle method, and the value of ACT1 (encoding actin) was used to normalize the expression of genes of interest. For each gene, the relative induction was determined by comparing its normalized expression in the macrophage-ingested sample with the medium control. The average inductions deduced from RT-PCR and from microarray are shown here. B. Growth of the deletion mutants in macrophages. Cells of two independent mutant isolates for CZC1 (czc1-3 and czc1-4) or CPP2 (cpp2-11 and cpp2-18) and the fhb1 mutant and its complemented strain (FHB1) (21) were inoculated into macrophages at an MOI of 2:1, and cryptococcal cells were reisolated after 24 h of incubation. Growth was measured by plating reisolated cells and determining CFU. Open columns, growth in macrophages; closed columns, growth in culture medium under conditions for macrophage culture. The experiments were repeated three times, and error bars represent the standard deviation of the mean.
FIG. 3.
FIG. 3.
Growth of C. neoformans mutants in macrophages. Cryptococcal cells of the gpa1, pka1, lac1, or fhb1 mutants were inoculated with macrophages at an MOI of 2:1, and cryptococcal cells were reisolated after 24 h of incubation. Growth was measured by plating the reisolated cells and counting CFU. A. Growth in macrophages. Open columns, initial inoculum; solid columns, growth after 24-h incubation in macrophages. B. Growth in macrophage culture medium alone at 37°C and 5% CO2.
FIG. 4.
FIG. 4.
Expression of the LAC1pro-GFP reporter. The LAC1pro-GFP reporter strain was constructed in the H99 background, and its expression was monitored by fluorescence microscopy. A. Induction of the LAC1pro-GFP reporter under melanin-inducing conditions. Cryptococcus cells of the H99 or the LAC1pro-GFP strain were grown in asparagine medium with 0.1% glucose at 30°C for 4 h before examination by fluorescence microscopy. Upper panels are images from light microscopy; lower panels are fluorescent images. B. Re-repression of the LAC1pro-GFP reporter by glucose. The LAC1pro-GFP reporter strain was incubated in asparagine medium with 0.1% glucose for 4 h before shifting to YPD with 2% glucose with continued incubation for 2 or 4 h. Upper panels, light images; lower panels, fluorescent images. Arrows indicate a bud lacking GFP. C. Expression of the LAC1pro-GFP reporter gene in macrophages. H99 or the LAC1pro-GFP reporter strain was inoculated into macrophages and incubated for either 4 or 16 h before examination by fluorescence microscopy. Upper panels, light images; lower panels, fluorescence images. “Extracellular” indicates H99 cells not ingested by macrophages.

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