Pseudohyphal growth of Cryptococcus neoformans is a reversible dimorphic transition in response to ammonium that requires Amt1 and Amt2 ammonium permeases

Abstract

Cryptococcus neoformans is a human-pathogenic basidiomycete that commonly infects HIV/AIDS patients to cause meningoencephalitis (7, 19). C. neoformans grows as a budding yeast during vegetative growth or as hyphae during sexual reproduction. Pseudohyphal growth of C. neoformans has been observed rarely during murine and human infections but frequently during coculture with amoeba; however, the genetics underlying pseudohyphal growth are largely unknown. Our studies found that C. neoformans displays pseudohyphal growth under nitrogen-limiting conditions, especially when a small amount of ammonium is available as a sole nitrogen source. Pseudohyphal growth was observed with Cryptococcus neoformans serotypes A and D and Cryptococcus gattii. C. neoformans pseudohyphae bud to produce yeast cells and normal smooth hemispherical colonies when transferred to complete media, indicating that pseudohyphal growth is a conditional developmental stage. Subsequent analysis revealed that two ammonium permeases encoded by the AMT1 and AMT2 genes are required for pseudohyphal growth. Both amt1 and amt2 mutants are capable of forming pseudohyphae; however, amt1 amt2 double mutants do not form pseudohyphae. Interestingly, C. gattii pseudohypha formation is irreversible and involves a RAM pathway mutation that drives pseudohyphal development. We also found that pseudohyphal growth is related to the invasive growth into the medium. These results demonstrate that pseudohyphal growth is a common reversible growth pattern in C. neoformans but a mutational genetic event in C. gattii and provide new insights into understanding pseudohyphal growth of Cryptococcus.

Fig 1

Pseudohypha formation of an αAAα diploid in response to nitrogen limitation. (A) The αAAα diploid strain MMRL1368 produced pseudohyphae on the edge of colonies when glucose, galactose, or sucrose was used as the fermentable carbon source. However, when maltose was used as the carbon source, the strain did not form pseudohyphae. (B) Differential interference contrast (DIC) image of pseudohyphae of MMRL1368 displays branched pseudohyphae without the clamp connections observed during hypha formation. (C) Scanning electron microscopic images show details of pseudohyphae. Scale = 5 μm.

Fig 2

Pseudohyphal growth in serotype A and serotype D haploids. Both mating types of both serotype A (H99α and KN99a) and serotype D (JEC21α and JEC20a) form pseudohyphae in ¼ YNB media. Scale = 10 μm.

Fig 3

Daughter cells bud from pseudohyphae. When pseudohyphae formed on ¼ YNB media were transferred onto rich YPD media, yeast daughter cells budded from the apex of the pseudohyphae. Numbers represent minutes of growth. Scale = 5 μm.

Fig 4

Requirement of both ammonium permeases to form pseudohyphae in C. neoformans. (A) Wild-type cells and amt1 and amt2 single mutants were able to exhibit pseudohyphal growth on ¼ YNB media; however, amt1 amt2 double mutants were not able to undergo pseudohyphal growth. Scale = 10 μm. (B) Wild-type (WT), amt1, amt2, and amt1 amt2 progeny were obtained from a cross between congenic strains JR11 (amt1 amt2) and KN99a (wild type). Wild-type, amt1, and amt2 progeny exhibited pseudohyphal growth on ¼ YNB media, showing rough-edged, wrinkled colonies and invasive growth into the agar, whereas the amt1 amt2 double mutant formed smooth colonies with no pseudohyphae and less invasive growth into the agar. Scale = 5 μm.

Fig 5

Pseudohyphal growth of C. gattii. (A) The C. gattii 97/433 strain exhibited pseudohyphal growth on filamentation agar. (B) Microdissected pseudohyphae of C. gattii cells continued to grow as pseudohyphae, unlike those of C. neoformans, indicating that inheritable genetic change(s) had occurred that promoted pseudohyphal growth. Scale = 10 μm. (C) The mutants always displayed pseudohyphal growth, even on rich YPD media, whereas wild-type cells grew as yeast. When washed, the pseudohyphae showed more invasive growth into the agar than wild-type cells on YPD media. The pseudohypha-forming mutants also exhibited temperature-sensitive growth at 37°C and were unable to proliferate.