Eca1, a Sarcoplasmic/Endoplasmic Reticulum Ca²⁺-ATPase, Is Involved in Stress Tolerance and Virulence inCryptococcus neoformans

Abstract

The basidiomycetous fungal pathogen Cryptococcus neoformans is adapted to survive challenges in the soil and environment and within the unique setting of the mammalian host. A C. neoformans mutant was isolated with enhanced virulence in a soil amoeba model that nevertheless exhibits dramatically reduced growth at mammalian body temperature (37°C). This mutant phenotype results from an insertion in the ECA1 gene, which encodes a sarcoplasmic/endoplasmic reticulum (ER) Ca 2؉ -ATPase (SERCA)-type calcium pump. Infection in murine macrophages, amoebae (Acanthamoeba castellanii), nematodes (Caenorhabditis elegans), and wax moth (Galleria mellonella) larvae revealed that the eca1 mutants are virulent or hypervirulent at permissive growth temperatures but attenuated at 37°C. Deletion mutants lacking the entire ECA1 gene were also hypersensitive to the calcineurin inhibitors cyclosporin and FK506 and to ER and osmotic stresses. An eca1⌬ cna1⌬ mutant lacking both Eca1 and the calcineurin catalytic subunit was more sensitive to high temperature and ER stresses than the single mutants and exhibited reduced survival in C. elegans and attenuated virulence towards wax moth larvae at temperatures that permit normal growth in vitro. Eca1 is likely involved in maintaining ER function, thus contributing to stress tolerance and virulence acting in parallel with Ca 2؉

Regulatory Circuitry Governing Fungal Development, Drug Resistance, and Disease

“…In C. neoformans, ECA1 encodes the SERCA ATPase. eca1 mutants are virulent or hypervirulent at permissive growth temperatures in a murine macrophage model, in the wax moth G. mellonella, and in the nematode Caenorhabditis elegans but are attenuated in virulence at 37°C (172). Furthermore, eca1 mutants exhibit hypersensitivity to calcineurin inhibition and to osmotic stresses, with eca1 cna1 double mutants showing an elevated sensitivity to high temperatures and ER stresses compared to either single mutant.…”

Survival Defects of Cryptococcus neoformans Mutants Exposed to Human Cerebrospinal Fluid Result in Attenuated Virulence in an Experimental Model of Meningitis

“…To assess whether the CSF survival defects resulted in an inability to respond to the host's innate immune response, we performed the C. elegans killing assay on our 13 CSF survival-defective mutant strains (36). Lacking an adaptive immune response and possessing tractable genetics, C. elegans has emerged as an efficient way to screen for candidate genes that mediate virulence through interactions with innate immunity (15,36,37). This model allows investigators to distinguish generally "sick" strains and appreciate possible differences in mutant susceptibility to host responses.…”

Identification of ENA1 as a Virulence Gene of the Human Pathogenic Fungus Cryptococcus neoformans through Signature-Tagged Insertional Mutagenesis

“…However, with the exception of the recent partial systematic mutagenesis screen of C. neoformans (32), almost all investigations of genes required for virulence in this fungus have focused on candidate genes or used in vitro screening approaches for known virulence traits like melanin and capsule production and the ability to grow at mammalian body temperature. Screens of insertion mutants generated by biolistic transformation in nonmammalian alternative hosts, the nematode Caenorhabditis elegans and the amoeba Acanthamoeba castellani, have led to the identification of three genes: KIN1 and ROM2, which are required for killing of the host, fertility, and viability (41,57), and ECA1, whose role in virulence depends on the host used (16). Importantly, both kin1 and rom2 deletion mutants are attenuated for virulence in murine inhalation models while eca1 mutants are temperature sensitive and, as such, are expected to be unable to survive in the mammalian host.…”

“…However, a search of the C. neoformans genome reveals only a single copy of the ENA1 gene, with the next best BLAST match to the S. cerevisiae Ena1 gene being the C. neoformans Eca1 gene. Eca1 is a putative sarcoplasmic-endoplasmic reticulum calcium ATPase-like Ca 2ϩ P-type ATPase that was identified in an amoeba virulence screen, and the mutant strains are predicted to be attenuated for virulence in mammalian models due to temperature sensitivity (16).…”