Autophagy Regulates Fungal Virulence and Sexual Reproduction in Cryptococcus neoformans.

Autophagy (macroautophagy) is an evolutionarily conserved degradation pathway involved in bulk degradation of cytoplasmic organelles, old protein, and other macromolecules and nutrient recycling during starvation. Extensive studies on functions of autophagy-related genes have revealed that autophagy plays a role in cell differentiation and pathogenesis of pathogenic fungi. In this study, we identified and characterized 14 core autophagy machinery genes (ATGs) in C. neoformans. To understand the function of autophagy in virulence and fungal development in C. neoformans, we knocked out the 14 ATGs in both α and a mating type strain backgrounds in C. neoformans, respectively, by using biolistic transformation and in vivo homologous recombination. Fungal virulence assay showed that virulence of each atgΔ mutants was attenuated in a murine inhalation systemic-infection model, although virulence factor production was not dramatically impaired in vitro. Fungal mating assays showed that all the 14 ATGs are essential for fungal sexual reproduction as basidiospore production was blocked in bilateral mating between each atgΔ mutants. Fungal nuclei development assay showed that nuclei in the bilateral mating of each atgΔ mutants failed to undergo meiosis after fusion, indicating autophagy is essential for regulating meiosis during mating. Overall, our study showed that autophagy is essential for fungal virulence and sexual reproduction in C. neoformans, which likely represents a conserved novel virulence and sexual reproduction control mechanism that involves the autophagy-mediated proteolysis pathway.

The Cys2His2 zinc finger protein Zfp1 regulates sexual reproduction and virulence in Cryptococcus neoformans.

Cryptococcus neoformans is a ubiquitous yeast pathogen that often infects the human central nervous system (CNS) to cause meningitis in immunocompromised individuals. Although numerous signaling pathways and factors important for fungal sexual reproduction and virulence have been investigated, their precise mechanism of action remains to be further elucidated. In this study, we identified and characterized a novel zinc finger protein Zfp1 that regulates fungal sexual reproduction and virulence in C. neoformans. qRT-PCR and ZFP1 promoter regulatory activity assays revealed a ubiquitous expression pattern of ZFP1 in all stages during mating. Subcellular localization analysis indicates that Zfp1 is targeted to the cytoplasm of C. neoformans. In vitro assays of stress responses showed that zfp1Δ mutants and the ZFP1 overexpressed strains ZFP1OE are hypersensitive to SDS, but not Congo red, indicating that Zfp1 may regulate cell membrane integrity. Zfp1 is also essential for fungal sexual reproduction because basidiospore production was blocked in bilateral mating between zfp1Δ mutants or ZFP1 overexpressed strains. Fungal nuclei development assay showed that nuclei in the bilateral mating of zfp1Δ mutants or ZFP1 overexpressed strains failed to undergo meiosis after fusion, indicating Zfp1 is important for regulating meiosis during mating. Although zfp1Δ mutants showed normal growth and produced normal major virulence factors, virulence was attenuated in a murine model. Interestingly, we found that the ZFP1 overexpressed strains were avirulent in a murine systemic-infection model. Overall, our study showed that the zinc finger protein Zfp1 is essential for fungal sporulation and virulence in C. neoformans.

Total synthesis of (3R,3'R,6'R)-lutein and its stereoisomers.

(3R,3'R,6'R)-lutein (1) is a major dietary carotenoid that is abundant in most fruits and vegetables commonly consumed in the U.S. and that accumulates in the human plasma, major organs, and ocular tissues. Numerous epidemiological and experimental studies have shown that 1 has important biological activities and may play an important role in the prevention of age-related macular degeneration (AMD). While the total synthesis of 1 has been previously reported in a poor overall yield, the total synthesis of the other seven stereoisomers of lutein has not yet been accomplished. We have developed a relatively straightforward methodology for the total synthesis of 1 and three of its stereoisomers, (3R,3'S,6'S)-lutein (2), (3R,3'S,6'R)-lutein or 3'-epilutein (3), and (3R,3'R,6'S)-lutein (4) by C(15) + C(10) + C(15) Wittig coupling reactions. Employing this methodology, the other four stereoisomers of lutein that are enantiomeric to the aforementioned lutein isomers can be similarly prepared. One of the important features of this strategy is its application to the total synthesis of (13)C-labeled luteins and their metabolites with appropriate stereochemistry for metabolic studies in animals and humans. This synthesis also provides access to the C(15)-precursors of optically active carotenoids with a 3-hydroxy-epsilon end group that are otherwise difficult to synthesize.

Partial synthesis of (3R,6'R)-alpha-cryptoxanthin and (3R)-beta-cryptoxanthin from (3R,3'R,6'R)-lutein.

(3R,3'R,6'R)-Lutein (1), (3R,3'R)-zeaxanthin (2), (3R,6'R)-alpha-cryptoxanthin (3), and (3R)-beta-cryptoxanthin (4) are among dietary hydroxycarotenoids that have been identified in human serum, milk, and ocular tissues. While 1 containing 6% of 2 is commercially available, industrial production of optically active 3 and 4 has not yet been accomplished. Several processes have been developed that transform 1 into 3, 4, and minor quantities of (3R,5'RS,6'R)-3',4'-didehydro-5',6'-dihydro-beta,beta-caroten-3-ol (5) (a regioisomer of 3). In one process, lutein (1) was cleanly deoxygenated to 3 in the presence of trifluoroacetic acid (TFA) and Me3N.BH3 in CH2Cl2 at ambient temperature in nearly 90% yield. Reaction of lutein (1) with a Lewis acid (AlCl3, ZnBr2, ZnI2) and a hydride donor (Me3N.BH3, Na[BH3(OCOCF3)], NaCNBH3) in solvents such as CH2Cl2, THF, and TBME produced similar results. In a two-step process, high-temperature acid-catalyzed dehydration of 1 (propanol/water/acid, 90 degrees C) gave a mixture of anhydroluteins 6, 7, and 8 in 86% yield. In the second step, these dehydration products underwent ionic hydrogenation with TFA/Me3N.BH3 in CH2Cl2 to afford a mixture of 3 and 4 in nearly 80% yield that contained only 1% of 5.

Three-dimensional solution structures of the chromodomains of cpSRP43.

Chloroplasts contain a unique signal recognition particle (cpSRP). Unlike the cytoplasmic forms, the cpSRP lacks RNA but contains a conserved 54-kDa GTPase and a novel 43-kDa subunit (cpSRP43). Recently, three functionally distinct chromodomains (CDs) have been identified in cpSRP43. In the present study, we report the three-dimensional solution structures of the three CDs (CD1, CD2, and CD3) using a variety of triple resonance NMR experiments. The structure of CD1 consists of a triple-stranded beta-sheet segment. The C-terminal helical segment typically found in the nuclear chromodomains is absent in CD1. The secondary structural elements in CD2 and CD3 include a triple-stranded antiparallel beta-sheet and a C-terminal helix. Interestingly, the orientation of the C-terminal helix is significantly different in the structures of CD2 and CD3. Critical comparison of the structures of the chromodomains of cpSRP43 with those found in nuclear chromodomain proteins revealed that the diverse protein-protein interactions mediated by the CDs appear to stem from the differences that exist in the surface charge potentials of each CD. Results of isothermal titration calorimetry experiments confirmed that only CD2 is involved in binding to cpSRP54. The negatively charged C-terminal helix in CD2 possibly plays a crucial role in the cpSRP54-cpSRP43 interaction.