Ctr2 links copper homeostasis to polysaccharide capsule formation and phagocytosis inhibition in the human fungal pathogen Cryptococcus neoformans

Abstract

Cryptococcus neoformans is a human opportunistic fungal pathogen responsible for approximately 1/3 of HIV/AIDS deaths worldwide. This budding yeast expresses a polysaccharide capsule necessary for virulence. Capsule production inhibits phagocytosis by macrophages. Here we describe results that link copper homeostasis to capsule production and the inhibition of phagocytosis. Specifically, using Agrobacterium-mediated insertional mutagenesis, we identified an insertion in the promoter region of the putative copper transporter-encoding gene CTR2 that results in reduced expression of CTR2 and increased phagocytosis by murine RAW264.7 macrophages. The mutant also displayed sensitivity to copper starvation and defects in polysaccharide capsule production and melanization. These defects were all reversed by genetic correction of the promoter insertion by homologous targeting. Several melanization-defective mutants identified previously, those in the RIM20, RIM101, and VPS25 genes, also display sensitivity to copper starvation, reduced capsule production and increased phagocytosis. Together these results indicate a previously undescribed link between copper homeostasis to polysaccharide capsule production and phagocytosis inhibition in Cryptococcus neoformans.

Figure 1. C. neoformans mutant strains demonstrate increased rates of unopsonized phagocytosis.

(A) Mutant C. neoformans strains show increased rates of phagocytosis by RAW264.7 macrophages. The indicated C. neoformans strains were co-incubated with RAW264.7 macrophages for 24 hours. The macrophages were then washed three times with PBS to remove unphagocytosed yeast, and the percentage of macrophages with associated yeast was assayed. At least 200 macrophages were counted per strain, and each strain was performed in triplicate. Error bars denote SD. (B) RT-qPCR of CNAG_07701 transcript in wild type, the mutant strain 1F8, and the strain 1F8 + pCNAG_07701 (1F8 + p), where the mutated promoter of CNAG_07701 has been replaced with an intact copy. Error bars denote SD from strains grown in duplicate.

Figure 2. Disruption and complementation of promoter of CNAG_07701.

(A) Schematic of A. tumefaciens-mediated disruption of the promoter of CNAG_07701 in the strain 1F8. (B) Schematic of complementation of 1F8 using the full promoter of CNAG_07701, as in the strain 1F8 + pCNAG_07701. A construct containing the promoter of CNAG_07701 flanked by a G418-resistance cassette was transformed into the strain 1F8, and transformants were screened for acquisition of G418-resistance and loss of nourseothricin (NAT)-resistance.

Figure 3. C. neoformans CNAG_07701 shows homology to copper transporters from other species.

(A) Schematic of CNAG_07701. Dark grey boxes indicate presence of three MXXM motifs (residues 1–10). Light grey box indicates predicted transmembrane domain (residues 15–37). (B) ClustalW diagram comparing the sequence of CNAG_07701 to copper transporters in Aspergillus fumigatus, Histoplasma capsulatum, Pleurotus ostreatus, Homo sapiens, and Saccharomyces cerevisiae. Asterisks (*) denote locations of MXXM motif in CNAG_07701.

Figure 4. Growth and melanization defects of C. neoformans mutant strains.

(A) Growth on YNB, YNB plates containing 3.2 mM BCS (a copper chelator, for copper-limiting conditions), and LIM (limited iron medium). The indicated strains were spotted from overnight culture in five-fold serial dilution. LIM plate, right panel, the images shown are all from the same plate on the same day – strains were re-arranged in image post-processing to reflect YNB and YNB + BCS plate arrangement. (B) Melanization of indicated strains. Overnight cultures were spotted on L-DOPA melanin-inducing plates.

Figure 5. Capsule production in C. neoformans mutant strains.

(A) Cells were grown in capsule-inducing conditions, and capsule and cell diameters were measured, and their ratio calculated, for at least thirty cells per strain. Error bars denote SD. (B) Cells from capsule-inducing conditions were stained by India ink (DIC) and anti-capsular polysaccharide antibody (FITC). Scale bar denotes 5 µm.

Figure 6. A model of the genetic basis of copper homeostasis in C. neoformans.

Copper is required for iron uptake, through the actions of the oxidoreductase Cfo1. Copper uptake and copper-dependent growth depend on CTR2, CUF1, RIM20, RIM101, and VPS25. There is a requirement for copper for capsule biosynthesis and attachment, and for inhibition of phagocytosis, although it remains unclear if the two phenotypes are linked.