Role of a VPS41 homologue in starvation response, intracellular survival and virulence of Cryptococcus neoformans

Abstract

SummaryPrevious studies have demonstrated an important role for the vacuole in the virulence of the fungus Cryptococcus and studies in yeast have implicated the vacuolar protein Vps41 in copper loading of proteins such as iron transporters. However, our studies found that a cryptococcal vps41D strain displayed wild-type growth on media containing iron and copper chelators and normal activity of the copper-containing virulence factor laccase as well as almost normal growth at 37°C and wild-type production of the virulence factor capsule. Despite these attributes, the vps41D mutant strain showed a dramatic attenuation of virulence in mice and co-incubation of mutant cells with the macrophage cell line, J774.16, resulted in a dramatic loss in viability of the vps41D mutant strain at 10 h compared with wild-type and complemented strains. Closer examination revealed that the vps41D mutant displayed a dramatic loss in viability after nutrient starvation which was traced to a failure to undergo G2 arrest, but there was no defect in the formation of autophagic or proteolytic vesicles. Our results indicate that VPS41 plays a key role in regulating starvation response in this pathogenic organism and that defects in cell cycle arrest are associated with attenuated pathogenic fitness in mammalian hosts.

Mating Pheromone in Cryptococcus neoformans Is Regulated by a Transcriptional/Degradative “Futile” Cycle

“…After digestion with MunI, the fragment was cloned into a cryptococcal expression vector, pORA-KUTAP, containing the EF1-␣ terminator under the control of a cryptococcal actin promoter (25), and introduced into ⌬vad1-FOA mutant cells (YP510) by electroporation as described previously (26), and transformants were selected on uracil-containing asparagine minimum-selective medium agar plates. For GPD-GFP constructs (strains YP104, YP108, YP111, YP114, YP119, and YP126), the indicated 5Ј-UTR and/or 3Ј-UTR of the MF␣1 gene was PCR-amplified using primers described in supplemental Table S2 and cloned into pORA-KUTAP.…”

“…Finding a role for MF␣1 3Ј-UTR would suggest a function in RNA stability affecting processes such as deadenylation, which is dependent on the 3Ј-UTR (45). To test these possibilities, we expressed MF␣1-GFP, replacing the MF␣1 3Ј-UTR with an unrelated EF1-␣ 3Ј-UTR, which has been successfully used to express proteins in C. neoformans (25). As shown in Fig.…”

Role of a CUF1/CTR4 copper regulatory axis in the virulence of Cryptococcus neoformans

“…The cryptococcal shuttle vector pORA-KUTAP was used to express a fusion protein between the CTR4 promoter region and a recently described GFP possessing C. neoformans codon usage (53). First, pORA-KUTAP containing the synthetic GFP upstream of the EF-1α terminator was digested with BglII and EcoRI to remove the ACT1 promoter; next, a PCR-amplified fragment of genomic DNA from H99 (using primers GCCGCCGGATCCCAGAT-TAAGTGAAGCAAG [CTR4-857S-Bam] and CTTGGAATTCCCCAT-GTTCATGTGGCTGTG [CTR4-1934A-RI]) was digested with compatible enzymes and ligated to produce pCTR4-GFP.…”

PI3K signaling of autophagy is required for starvation tolerance and virulenceof Cryptococcus neoformans

“…Retention of near WT growth at 37°C is in contrast to a more severe temperature-dependent growth defect of the homolog mutant strain from Sc (16). This difference could be due to redundancy in growth pathways in the basidiomycetous fungi and has also been found with other vps-class mutants from this fungus (17). To further characterize the cryptococcal Vps34 activity, a Sc vps34Δ mutant was complemented with a cDNA fragment of the cryptococcal gene expressed under a Saccharomyces constitutive promoter.…”

“…The ATG8 gene was PCR amplified from an H99 cDNA mass-excised library using primers ATG8-2238S-RI (5′-GCC-GCGAATTCTGTGAGAAGGCTGAGAAGA) and ATG8-2859A-XhoI (5′-GCCGCCCTCGAGTTAAACATAGTTGCTTTGCTTA) and 2 fragments of the ATG8 gene were ligated downstream from an ACT1 promoter of a pORA-KUTAP cryptococcal shuttle vector described previously (17), in a sense-antisense fashion on either side of a linker fragment consisting of a 500-bp PCR-amplified fragment of intron I of LAC1 generated from a LAC1 genomic clone using primers INTRON-XhoS and INTRONXhoA to produce plasmid ORA-i-ATG8. ORA-i-ATG8 was linearized and transformed into H99 Matα, ura5-19 described previously (29), and transformants selected on minimal media.…”