A glucan synthase FKS1 homolog in cryptococcus neoformans is single copy and encodes an essential function.
Cryptococcal meningitis is a fungal infection, caused by Cryptococcus neoformans, which is prevalent in immunocompromised patient populations. Treatment failures of this disease are emerging in the clinic, usually associated with long-term treatment with existing antifungal agents. The fungal cell wall is an attractive target for drug therapy because the syntheses of cell wall glucan and chitin are processes that are absent in mammalian cells. Echinocandins comprise a class of lipopeptide compounds known to inhibit 1,3-beta-glucan synthesis, and at least two compounds belonging to this class are currently in clinical trials as therapy for life-threatening fungal infections. Studies of Saccharomyces cerevisiae and Candida albicans mutants identify the membrane-spanning subunit of glucan synthase, encoded by the FKS genes, as the molecular target of echinocandins. In vitro, the echinocandins show potent antifungal activity against Candida and Aspergillus species but are much less potent against C. neoformans. In order to examine why C. neoformans cells are less susceptible to echinocandin treatment, we have cloned a homolog of S. cerevisiae FKS1 from C. neoformans. We have developed a generalized method to evaluate the essentiality of genes in Cryptococcus and applied it to the FKS1 gene. The method relies on homologous integrative transformation with a plasmid that can integrate in two orientations, only one of which will disrupt the target gene function. The results of this analysis suggest that the C. neoformans FKS1 gene is essential for viability. The C. neoformans FKS1 sequence is closely related to the FKS1 sequences from other fungal species and appears to be single copy in C. neoformans. Furthermore, amino acid residues known to be critical for echinocandin susceptibility in Saccharomyces are conserved in the C. neoformans FKS1 sequence.
Thompson
J R
JR
Infectious Diseases, Merck Research Laboratories, Rahway, New Jersey 07065, USA. john_thompson@merck.com
Douglas
C M
CM
Li
W
W
Jue
C K
CK
Pramanik
B
B
Yuan
X
X
Rude
T H
TH
Toffaletti
D L
DL
Perfect
J R
JR
Kurtz
M
M
D000595:Amino Acid Sequence; D002473:Cell Wall / Q000378:metabolism; D003001:Cloning, Molecular; D016384:Consensus Sequence; D003455:Cryptococcus neoformans / Q000201:enzymology* / Q000235:genetics* / Q000302:isolation & purification; D017931:DNA Primers; D004271:DNA, Fungal / Q000302:isolation & purification; D054714:Echinocandins; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005656:Fungal Proteins / Q000096:biosynthesis / Q000235:genetics*; D020043:Genes, Essential; D005800:Genes, Fungal; D005964:Glucosyltransferases / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics*; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D004271:DNA, Fungal; D054714:Echinocandins; D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D008565:Membrane Proteins; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D005964:Glucosyltransferases; C020570:1,3-beta-glucan synthase; C091618:FKS1 protein, S cerevisiae