Eukaryot Cell 4(8): 1420-1433

<em>Cryptococcus neoformans</em> Gene Expression during Murine Macrophage Infection<sup><a href="#fn1" rid="fn1" class=" fn">†</a></sup>

Departments of Molecular Genetics and Microbiology,^Medicine,^{Pharmacology and Cancer Biology,}^{Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710}⁴

^{Corresponding author. Mailing address: Department of Molecular Genetics and Microbiology, 322 CARL Building, Research Dr., Duke University Medical Center, Durham, NC 27710. Phone: (919) 684-2824. Fax: (919) 684-5458. E-mail:}ude.ekud@100mtieh.

^{Present address: Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06620.}

^{Present address: Département Chimie-biologie, Université du Québec à Trois-Rivières, 3351 Boul. des Forges, Trois-Rivières, Québec G9A 5H7, Canada.}

Received 2005 May 5; Accepted 2005 Jun 3.

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.

Abstract

The basidiomycetous fungus Cryptococcus neoformans is an opportunistic pathogen that is common in AIDS patients and the major cause of fungal meningitis worldwide (9). C. neoformans is a free-living organism commonly found in many environmental niches, including soil, trees, and bird guano. It is prototrophic for amino acids, sugars, and lipids and can utilize a variety of carbon sources for growth (9). C. neoformans has a bipolar mating system with a and α mating types and can undergo sexual reproduction or monokaryotic fruiting to produce basidiospores, which are thought to be the infectious propagule (28, 59).

Several features of C. neoformans have been characterized as critical for its infectivity. These include the ability to grow at host body temperature (37°C) (44), formation of a polysaccharide capsule (10), production of melanin (89), and secretion of degradative enzymes such as urease (17), phospholipase B (16), and proteases (13). Expression of these virulence factors has been shown to be regulated by several signal transduction pathways (37, 46). The cyclic AMP-protein kinase A (cAMP/PKA) pathway controls capsule formation, melanin production, mating, and virulence (46). This pathway is activated by environmental stimuli, such as glucose, through an as-yet-unknown G-protein-coupled receptor and the Gα protein, Gpa1 (3, 4, 25, 26). Both the Ras (Ras1 and Ras2) and calcineurin pathways regulate the ability of this pathogenic organism to grow at 37°C (1, 61, 90). A mitogen-activated protein kinase cascade regulates filamentous growth and morphological differentiation in the mating process in response to activation by pheromones (14, 20, 32, 74, 90).

C. neoformans infection is likely to begin when cryptococcal cells, either in the yeast or the spore form, are inhaled. One of the first lines of innate defense in the host is the alveolar macrophage within the lung. Although C. neoformans can be efficiently phagocytosed and killed by macrophages, it can also inhibit and evade phagocytosis with its polysaccharide capsule. However, following phagocytosis, this yeast is able to survive the exposure to antifungal compounds produced by the activated macrophage and adapts to the intracellular environment of the phagosome (29). While residing within the macrophage, C. neoformans may then be able to shield itself from other innate defense mechanisms, such as complement, antibodies, serum, and alveolar immune factors, or even antifungal drugs (9, 29, 30). It has also been proposed that residency with macrophages provides a route for C. neoformans to enter the bloodstream and lymphatic system, enabling dissemination from the lung to distant organs, including its propensity to invade the central nervous system (9, 29, 30). However, how C. neoformans adapts to the intracellular environment of the macrophage and what signal transduction pathways control its intracellular growth and maintenance are largely unknown.

The completion of the C. neoformans genome has enabled global gene expression profiling with microarray technology (50; http://cneo.genetics.duke.edu/index.html). We previously developed a genomic microarray comprising >6,000 PCR-amplified genomic DNA fragments. This microarray has been employed to identify the gene expression profile in response to growth at high temperature (42) and genes controlled by the Gpa1 virulence cascade (64). Herein we report and analyze the transcriptional profile of C. neoformans during intracellular growth within murine macrophages.

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Acknowledgments

We thank Arturo Casadevall for kindly providing the 18B7 monoclonal antibody against C. neoformans polysaccharide, Emily Wenink for technical assistance, Steve Giles for advice on macrophage cell culture, Alex Idnurm for assistance with Agrobacterium-mediated transformation of C. neoformans, Raphael Valdivia for advice on immunofluorescent staining, John Perfect and Andrew Alspaugh for critical reading of the manuscript, and Andrew Alspaugh for sharing unpublished data.

This work was supported by NIAID R01 grants AI50113 and AI50438 to J. Heitman and by NIAID P01 program project grant AI44975 to the Duke University Mycology Research Unit. Joseph Heitman is a Burroughs Wellcome Scholar in Molecular Pathogenic Mycology and an Investigator of the Howard Hughes Medical Institute.

Acknowledgments

Footnotes

^{Supplemental material for this article may be found at}http://ec.asm.org/.

Footnotes

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