cAMP signaling in Aspergillus fumigatus is involved in the regulation of the virulence gene pksP and in defense against killing by macrophages

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Abstract
Aspergillus fumigatus is an important pathogen of immunocompromised hosts, causing pneumonia and invasive disseminated disease and resulting in high mortality. In order to determine the importance of the cAMP signaling pathway for virulence, three genes encoding putative elements of the pathway have been cloned and characterized: the adenylate cyclase gene acyA, and gpaA and gpaB, both of which encode α subunits of heterotrimeric G proteins. The acyA and gpaB genes were each deleted in A. fumigatus. Both mutants showed reduced conidiation, with the ΔacyA mutant producing very few conidia. The growth rate of the ΔacyA mutant was also reduced, in contrast to that of the ΔgpaB mutant. Addition of 10 mM dibutyryl-cAMP to the culture medium completely restored the wild-type phenotype in both mutant strains. To study the influence of GPAB on the expression of the gene pksP, which encodes a virulence factor that is involved in pathogenicity, a pksPp- lacZ gene fusion was generated and integrated as a single copy at the pyrG gene locus of both the parental strain and the ΔgpaB mutant strain. The ΔgpaB mutant showed reduced expression of the pksPp-lacZ reporter gene relative to that in the parental strain. In mycelia of both the parental strain and the ΔgpaB mutant pksPp-lacZ expression was increased when isobutyl-methyl-xanthine, an inhibitor of intracellular phosphodiesterases, was added to the medium. The survival rate of conidia after ingestion by human monocyte-derived macrophages was also determined. The killing rate for conidia from ΔacyA and ΔgpaB strains was significantly higher than that for wild-type conidia. Taken together, these findings suggest that cAMP triggers a system that protects A. fumigatus from the effects of immune effector cells of the host.