We used a library of insertional mutants and a genetic screen to identify C. neoformans genes involved in inhibition or evasion of phagocytosis by macrophages. Through this screen, we identified the copper transporter Ctr2, which has homology to known copper transporters in other yeast. As expected, a mutant with reduced CTR2 expression shows sensitivity to growth in copper-limited conditions. Unexpectedly, it also shows defects in capsule formation. We determined that this is not an isolated relationship – strains with knockouts in RIM20, RIM101 and VPS25 are also sensitive to growth in copper-limited conditions and show decreased capsule production. We believe this points to a new, previously uncharacterized links between copper uptake and capsule synthesis, and between copper uptake and anti-phagocytic behavior ().
It remains uncertain if the reduced capsule production is directly responsible for the reduction in anti-phagocytic behavior. It is striking that the strains 1F8, rim20
display more phagocytosis than cap10
strains, despite generating more capsule, suggesting that the relationship between capsule and phagocytosis inhibition is not a linear one. This may suggest copper-dependent synthesis of an inhibitory factor, or a copper-dependent modification of the capsule.
This study is the first to link sensitivity to growth on copper-limited conditions with capsule production in C. neoformans
. It is interesting that the ESCRT-II component Vps25 emerged as important for capsule formation and copper uptake, as it is important for vesicular trafficking to the vacuole. Previous studies have shown that the copper transporters Ctr2 (S. cerevisiae
) and Ctr6 (S. pombe
) are localized to the vacuole, which may serve as a site for copper storage and mobilization 
. In S. cerevisiae
, a recent screen identified the strain vps25
as well as strains mutated in other members of the ESCRT-II and ESCRT-III complexes as sensitive to copper overload, confirming a role for Vps25 and the ESCRT machinery in copper homeostasis 
. Additionally, studies have suggested that capsule formation occurs within the cell and is transported by vesicles, as evidenced by anti-GXM antibody staining of vesicles in a mutant defective for secretion 
. It is therefore possible that the defects of the vps25
strain in both capsule synthesis and copper homeostasis may be attributable to a requirement in both for proper vesicle formation.
Cft1 is a homolog of the S. cerevisiae
high affinity iron transporter Ftr1. Ftr1 complexes with the multicopper ferroxidase Fet3, which utilizes copper to oxidize Fe2+
for transport by Ftr1 into the cell. Therefore, depletion of copper is known to render cells defective in iron uptake, although there is no known reciprocal requirement for iron in copper uptake in yeast 
. As copper uptake is presumed to be normal in C. neoformans cft1
cells, it is therefore expected that the cft1
strain would not display a phagocytosis phenotype.
It is currently unknown what factors regulate CTR2
transcriptional expression. Previous studies have shown that CTR4
is regulated by Cuf1 and Rim101 
; it remains to be seen if CTR2
is similarly regulated. However, O'Meara et al 
performed microarrays comparing gene expression in a rim101
strain to wild type and did not identify CTR2
in their analysis, suggesting that its expression is Rim101-independent. In our hands, the strain rim101Δ
displayed some surprising phenotypes that were unexpected based on the studies performed by O'Meara et al. Specifically, we observed no growth defect in LIM and increased levels of phagocytosis. O'Meara et al observed a mild growth defect for strain rim101Δ
when grown in liquid LIM culture, but noted that the mutant did eventually reach saturation phase. Our LIM assay utilized cultures at saturation in LIM prior to spotting onto LIM plates, and it is possible that in those conditions there are Rim101-independent adaptations to growth in limited iron that allow for robust growth on LIM plates. In addition, while we saw increased levels of phagocytic uptake of rim20Δ
strains, O'Meara et al reported no change in the phagocytic index of the rim101Δ
strain when compared to wild type. This is likely due to their use of opsonization (personal communication), as we have observed robust uptake of wild type and mutant strains when opsonized with anti-capsular antibodies that masks subtler phagocytosis phenotypes (data not shown). We were able to confirm their observation that rim20Δ
cells do not display capsule by India ink staining when cultured in DMEM, at 37°C with 5% CO2
. This is in contrast with our earlier findings 
, where we reported no capsule phenotypes for these strains or the strain vps25Δ
. This is due to a difference in the conditions used to induce capsule: in our previous studies, capsule-induction was performed in 10% Saboraud dextrose medium, and in these conditions, rim20Δ
strains generate capsule on levels comparable to wild type (Figure S2
). This suggests that there are different signaling pathways that feed into capsule production, and that Rim101 is required for capsule attachment in one condition but not the other. This is to our knowledge the first evidence for transcriptional regulation of different pathways for capsule synthesis in response to different stimuli.
This study links for the first time capsule biogenesis and phagocytosis inhibition to copper homeostasis in C. neoformans. Future studies are required to determine the exact mechanisms through which copper affects these two processes.