Cryptococcus neoformansGene Expression during Murine Macrophage Infection

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.

Gib2, A Novel Gβ-like/RACK1 Homolog, Functions as a Gβ Subunit in cAMP Signaling and Is Essential in Cryptococcus neoformans

“…This is rather intriguing because Smg1 was previously identified as a multicopy suppressor of gpa1 mutation for melanin deficiency and is implicated in melanin formation and virulence (56,57). Results of our gene disruption studies have contradicted this proposition, because smg1 mutant strains did not exhibit any altered levels of melanin pigmentation.…”

“…Whereas the prospect of Gib2 binding to an SH3 domain would support Gib2 being a G␤-and RACK1-like protein because the mammalian RACK1 is known to negatively regulate the activity of the Src kinase through binding, we chose to examine the clone containing the oxidoreductase domain, as it represents an internal region of Smg1 (GenBank AF349949), a protein previously identified as a multicopy suppressor of gpa1 mutation. Smg1 has been implicated as one of the downstream targets of the Gpa1-cAMP signaling pathway in C. neoformans (56,57). Physical association between Gib2 and the full-length Smg1 was also confirmed (Fig.…”

Differential gene expression analysis of Paracoccidioides brasiliensis during keratinocyte infection

“…This is similar to the situation in Cryptococcus neoformans, where patterns of gene expression and adaptation were tissue specific for lung infection in mice (Hu et al, 2008) or in a rabbit model of experimental meningitis (Lee et al, 2010). Several studies have focused on the pathways of gluconeogenesis, the glyoxylate cycle and b-oxidation of fatty acids, as these pathways are specifically upregulated during contact with host immune cells in a variety of human fungal and bacterial pathogens, such as P. brasiliensis (Derengowski et al, 2008), Cryptococcus neoformans (Fan et al, 2005;Hu et al, 2008;Rude et al, 2002), Candida albicans (Barelle et al, 2006;Fradin et al, 2005;Ramírez & Lorenz, 2009), Aspergillus fumigatus (Ebel et al, 2006;Olivas et al, 2008) and Mycobacterium tuberculosis (McKinney et al, 2000;Muñoz-Elías & McKinney, 2005).…”

The fatal fungal outbreak on Vancouver Island is characterized by enhanced intracellular parasitism driven by mitochondrial regulation

“…Subsequently, the pellet was resuspended in 10 mL H 2O supplemented with 0.05% SDS (SDS) for 5 min to remove macrophage RNA. At this SDS concentration, the macrophages were lysed while cryptococci remained intact and viable (37). The remaining yeast cells were washed 3 times with ice-cold H 2O by centrifugation and resuspension.…”