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Cryptococcus neoformans and Cryptococcus gattii are two species complexes in the large fungal genus Cryptococcus and are responsible for potentially lethal disseminated infections. These two complexes share several phenotypic traits, such as production of the protective compound melanin. In C. neoformans , the pigment associates with key cellular constituents that are essential for melanin deposition within the cell wall. Consequently, melanization is modulated by changes in cell-wall composition or ultrastructure. However, whether similar factors influence melanization in C. gattii is unknown. Herein, we used transmission EM, biochemical assays, and solid-state NMR spectroscopy of representative isolates and "leaky melanin" mutant strains from each species complex to examine the compositional and structural factors governing cell-wall pigment deposition in C. neoformans and C. gattii. The principal findings were the following. 1) C. gattii R265 had an exceptionally high chitosan content compared with C. neoformans H99; a rich chitosan composition promoted homogeneous melanin distribution throughout the cell wall but did not increase the propensity of pigment deposition. 2) Strains from both species manifesting the leaky melanin phenotype had reduced chitosan content, which was compensated for by the production of lipids and other nonpolysaccharide constituents that depended on the species or mutation. 3) Changes in the relative rigidity of cell-wall chitin were associated with aberrant pigment retention, implicating cell-wall flexibility as an independent variable in cryptococcal melanin assembly. Overall, our results indicate that cell-wall composition and molecular architecture are critical factors for the anchoring and arrangement of melanin pigments in both C. neoformans and C. gattii species complexes., (© 2020 Chrissian et al.)

Cryptococcus neoformans is a fungal pathogen that causes chronic meningitis in 10% of patients with AIDS. Genetic and biochemical studies were conducted to determine whether myristoyl-CoA:protein N-myristoyltransferase (Nmt) is a target for development of a new class of fungicidal drugs. A single copy of a conditional lethal C. neoformans NMT allele was introduced into the fungal genome by homologous recombination. The allele (nmt487D) produces temperature-sensitive myristic acid auxotrophy. This phenotype is due, in part, to under-myristoylation of a cellular ADP ribosylation factor (Arf) and can be rescued by forced expression of human Nmt. Two isogenic strains with identical growth kinetics at 35 degreesC were used to test the biological effects of an Nmt inhibitor. CPA8 contained a single copy of wild type C. neoformans NMT. HMC1 contained nmt487D plus 10 copies of human NMT. Since a single copy of nmt487D will not support growth at 35 degreesC, survival of HMC1 depends upon its human Nmt. ALYASKLS-NH2, an inhibitor derived from an Arf, was fully depeptidized: p-[(2-methyl-1-imidazol-1-yl)butyl]phenyl-acetyl was used to represent the GLYA tetrapeptide, whereas SKLS was replaced with a chiral tyrosinol scaffold. Kinetic studies revealed Ki (app) values of 1.8 +/- 1 and 9 +/- 2.4 microM for purified fungal and human Nmts, respectively. The minimal inhibitory concentration of the compound was 2-fold lower for CPA8 compared with HMC1. A single dose of 100 microM produced a 5-fold greater inhibition of protein synthesis in CPA8 versus HMC1. The strain specificity of these responses indicates that the fungicidal effect was Nmt-dependent. These two strains may be useful for screening chemical libraries for Nmt-based fungicidal compounds with relatively little activity against the human enzyme.

Myristoyl-CoA:protein N-myristoyltransferase (Nmt) transfers myristate from CoA to the N-terminal Gly residue of cellular proteins in an ordered reaction mechanism that first involves binding of myristoyl-CoA to the apoenzyme. The gene encoding Saccharomyces cerevisiae Nmt1p (NMT1) is essential for vegetative growth. Candida albicans, Cryptococcus neoformans var. neoformans, and Histoplasma capsulatum are the principal causes of systemic fungal infections in immunocompromised humans. Metabolic labeling studies indicate that they synthesize a small set of cellular N-myristoylproteins during exponential growth on rich media, the most prominent of which co-migrate with two essential functionally interchangeable S. cerevisiae N-myristoylproteins, ADP ribosylation factor-1 (Arf1p) and Arf2p. NMT and ARF genes have been recovered from C. neoformans and H. capsulatum using the polymerase chain reaction. They are single copy genes, interrupted by multiple introns. C. neoformans and H. capsulatum Nmts have approximately 50% amino acid sequence identity with the orthologous S. cerevisiae, C. albicans, and Homo sapiens N-myristoyltransferases, whereas C. neoformans and H. capsulatum Arfs are approximately 80% identical with C. albicans Arf and S. cerevisiae Arf1p and Arf2p. Functional studies of C. neoformans and C. albicans Nmts conducted in Escherichia coli reveal that (i) both efficiently acylate S. cerevisiae Arf2p; (ii) C. neoformans Arf is a substrate for C. neoformans Nmt; and (iii) substitution of an Asp for a Gly located 5 residues from the C terminus of these two enzymes causes marked temperature-dependent reductions in their catalytic efficiency, just as it does with S. cerevisiae and H. sapiens Nmts. Wild type C. neoformans, C. albicans, and H. sapiens NMTs can fully complement the lethal phenotype of a S. cerevisiae nmt1 null allele at 24 and 37 degrees C when the GAL1-10 promoter controlling their expression is induced by galactose. Only the C. albicans enzyme is able to do so when the promoter is repressed with glucose. This complementation profile likely arises, at least in part, from differences in the protein substrate specificities of the orthologous Nmts. A Gly-->Asp mutation in S. cerevisiae, C. neoformans, C. albicans, and H. sapiens Nmts produces temperature-sensitive growth arrest in isogenic S. cerevisiae strains with a nmt1 null allele. Growth of strains producing the mutant C. albicans or H. sapiens, but not the C. neoformans, enzyme can be rescued by myristate at the non-permissive temperature (37 degrees C) even in the presence of cerulenin, an inhibitor of fatty acid synthetase.(ABSTRACT TRUNCATED AT 400 WORDS)

Candida albicans and Cryptococcus neoformans are major causes of systemic fungal infections, particularly in patients with acquired immunodeficiency syndrome. Metabolic labeling studies revealed that these organisms synthesize a small number of N-myristoylproteins, the most prominent being 20-kDa ADP-ribosylation factors (Arfs). C. albicans Arf has approximately 80% identity with the essential Arf1 and Arf2 proteins of Saccharomyces cerevisiae. [3H]Myristic acid analogs with oxygen for -CH2- substitutions at C4, C6, C11, and C13 are incorporated into cellular N-myristoylproteins, phospholipids, and neutral lipids produced by these three yeasts during exponential growth at 30 degrees C in complex media. Analog- and organism-specific differences in the efficiency of labeling of proteins and lipid classes were observed. The effects of oxatetradecanoic acids with oxygen for -CH2- substitutions at C3-C13 on C. neoformans, C. albicans, and S. cerevisiae were assessed during mid-log phase growth at 30 degrees C. A single dose of 3-oxa-, 4-oxa-, 5-oxa- or 6-oxatetradecanoic acid (O3-O6, final concentration = 300 microM) was able to inhibit growth of C. neoformans in the order O4 greater than O5 greater than O3 approximately O6. The other compounds were inactive. 4-Oxatetradecanoic acid was fungicidal, producing a 10,000-fold reduction in viable cell number 1 h after administration and continued suppression of cell growth for 7 h. A clear dose response was observed over a concentration range of 100-300 microM. 4-Oxatridecanoic acid was 100-fold less potent in reducing cell viability than 4-oxatetradecanoic acid but more potent than 5-oxatridecanoic acid. O4 produced approximately 10-100-fold reductions in the viability of C. albicans and S. cerevisiae at 300-500 microM, respectively, whereas O5 and O6 were less active. Since N-myristoylation of the Pr55gag polyprotein precursor produced by human immunodeficiency virus I (HIV-I) is essential for its assembly, we also assessed the antiviral effects of 4-oxatetradecanoic acid. O4 is able to produce a 50% reduction in the replication of HIV-I in acutely infected human T-lymphocyte cell lines at a concentration of 18 microM. Together, these data suggest that (i) the position of the oxygen for methylene substitution is a critical determinant of the fungicidal activity of O4 and (ii) NMT may be an attractive therapeutic target for treating opportunistic fungal infections in patients infected with HIV-I.