Melanin Externalization in Candida albicans Depends on Cell Wall Chitin Structures

The fungal pathogen Candida albicans produces dark-pigmented melanin after 3 to 4 days of incubation in medium containing l-3,4-dihydroxyphenylalanine (l-DOPA) as a substrate. Expression profiling of C. albicans revealed very few genes significantly up- or downregulated by growth in l-DOPA. We were unable to determine a possible role for melanin in the virulence of C. albicans. However, we showed that melanin was externalized from the fungal cells in the form of electron-dense melanosomes that were free or often loosely bound to the cell wall exterior. Melanin production was boosted by the addition of N-acetylglucosamine to the medium, indicating a possible association between melanin production and chitin synthesis. Melanin externalization was blocked in a mutant specifically disrupted in the chitin synthase-encoding gene CHS2. Melanosomes remained within the outermost cell wall layers in chs3Δ and chs2Δ chs3Δ mutants but were fully externalized in chs8Δ and chs2Δ chs8Δ mutants. All the CHS mutants synthesized dark pigment at equivalent rates from mixed membrane fractions in vitro, suggesting it was the form of chitin structure produced by the enzymes, not the enzymes themselves, that was involved in the melanin externalization process. Mutants with single and double disruptions of the chitinase genes CHT2 and CHT3 and the chitin pathway regulator ECM33 also showed impaired melanin externalization. We hypothesize that the chitin product of Chs3 forms a scaffold essential for normal externalization of melanosomes, while the Chs8 chitin product, probably produced in cell walls in greater quantity in the absence of CHS2, impedes externalization.Candida albicans is a major opportunistic fungal human pathogen that causes a wide variety of infections (9, 68). In healthy individuals C. albicans resides as a commensal within the oral cavity and gastrointestinal and urogenital tracts. However, in immunocompromised hosts, C. albicans causes infections ranging in severity from mucocutaneous infections to life-threatening disseminated diseases (9, 68). Research into the pathogenicity of C. albicans has revealed a complex mix of putative virulence factors (7, 60), perhaps reflecting the fine balance this species strikes between commensal colonization and opportunistic invasion of the human host.Melanins are biological pigments, typically dark brown or black, formed by the oxidative polymerization of phenolic compounds. They are negatively charged hydrophobic molecules with high molecular weights and are insoluble in both aqueous and organic solvents. Their insolubility makes melanins difficult to study, and no definitive structure has yet been found for them; they probably represent an amorphous mixture of polymers (35). There are various types of melanin in nature, including eumelanin and phaeomelanin (76). Two principal types of melanin are found in the fungal kingdom. The majority are 1.8-dihydroxynapthalene (DNH) melanins synthesized from acetyl-coenzyme A (CoA) via the polyketide pathway (5). DNH melanins have been found in a wide range of opportunistic fungal pathogens of humans, including dark (dematiaceous) molds, such as Cladosporium, Fonsecaea, Phialophora, and Wangiella species, and as conidial pigments in Aspergillus fumigatus and Aspergillus niger (41, 80, 87, 88). However, several other fungal pathogens, including Blastomyces dermatitidis, Coccidioides posadasii, Cryptococcus neoformans, Histoplasma capsulatum, Paracoccidioides brasiliensis, and Sporothrix schenckii, produce eumelanin (3,4-dihydroxyphenylalanine [DOPA]-melanin) through the activity of a polyphenol oxidase (laccase) and require an exogenous o-diphenolic or p-diphenolic substrate, such as l-DOPA (16, 30, 63,–65, 67, 79).The production of melanin in humans and other mammals is a function of specialized cells called melanocytes. Particles of melanin polymers, sometimes, including more than one melanin type, are built up within membrane-bound organelles called melanosomes (76), and these are actively transported along microtubules to the tips of dendritic outgrowths of melanocytes, from where they are transferred to neighboring cells (32, 81). The mechanism of intercellular transfer of melanosomes has not yet been established, but the export process probably involves the fusion of cell and vesicular membranes rather than secretion of naked melanin (82). In pathogenic fungi, melanins are often reported to be associated with or “in” the cell wall (35, 36, 50, 72, 79). However, there is variation between species: the melanin may be located external to the wall, e.g., in P. brasiliensis (79); within the wall itself (reviewed in reference 42); or as a layer internal to the wall and external to the cell membrane, e.g., in C. neoformans (22, 45, 85). However, mutants of C. neoformans bearing disruptions of three CDA genes involved in the biosynthesis of cell wall chitosan, or of CHS3, encoding a chitin synthase, or of CSR2, which probably regulates Chs3, all released melanin into the culture supernatant, suggesting a role for chitin or chitosan in retaining the pigment polymer in its normal intracellular location (3, 4). However, vesicles externalized from C. neoformans cells also show laccase activity (21), so the effect of chitin may be on vesicle externalization rather than on melanin itself. Internal structures compatible with mammalian melanosomes have been observed in Cladosporium carrionii (73) and in Fonsecaea pedrosoi (2, 26). Remarkably, F. pedrosoi also secretes melanin and locates the polymer within the cell wall (1, 2, 25, 27, 74).Melanization has been found to play an important role in the virulence of several human fungal pathogens, such as C. neoformans, A. fumigatus, P. brasiliensis, S. schenckii, H. capsulatum, B. dermatitidis, and C. posadasii (among recent reviews are references 29, 42, 62, 74, and 79). From these and earlier reviews of the extensive literature, melanin has been postulated to be involved in a range of virulence-associated properties, including interactions with host cells; protection against oxidative stresses, UV light, and hydrolytic enzymes; resistance to antifungal agents; iron-binding activities; and even the harnessing of ionizing radiation in contaminated soils (15). The most extensively studied fungal pathogen for the role of melanization is C. neoformans, which possesses two genes, LAC1 and LAC2, encoding melanin-synthesizing laccases (52, 69, 90). It has been known since early studies with naturally occurring albino variants of C. neoformans (39) that melanin-deficient strains are attenuated in mouse models of cryptococcosis. Deletion of both the LAC1 and LAC2 genes reduced survival of C. neoformans in macrophages (52), and a study based on otherwise isogenic LAC1⁺ and LAC1⁻ strains confirmed the importance of LAC1 in experimental virulence (66). Other genes in the regulatory pathway for LAC1 are similarly known to be essential to virulence (12, 84).C. albicans has been shown to produce melanin with DOPA as a substrate for production of the polymer (53). The cells could be treated with hot acids to produce typical melanin “ghosts,” and antibodies specific for melanin reacted with the fungal cells by immunohistochemistry with tissues from experimentally infected mice, demonstrating that C. albicans produces melanin in vivo (53). However, no candidate genes encoding laccases have yet been identified in the C. albicans genome (http://www.candidagenome.org/). In this study, we investigated the production of melanin by C. albicans and showed that its normal externalization from wild-type cells, including formation of melanosomes, can be altered to an intracellular and intrawall location by mutation of genes involved in chitin synthesis. C. albicans has four genes encoding chitin synthase enzymes. CHS1 is an essential gene under normal conditions (59), and its product is the main enzyme involved in septum formation (83). Chs3 forms the bulk of the chitin in the cell wall and the chitinous ring at sites of bud emergence (8, 51, 57), while Chs2 contributes to differential chitin levels found between yeast and hyphal forms of the fungus, and Chs8 influences the architecture of chitin microfibrils (43, 51, 55, 57, 58). We found that melanin externalization was unaffected in a chs8Δ mutant but was reduced or abrogated in chs2Δ and chs3Δ mutants. Expression profiles of melanin-producing cells grown in the presence of l-DOPA did not identify any potential laccase-synthesizing genes.     

Splitting the BLOSUM Score into Numbers of Biological Significance

Mathematical tools developed in the context of Shannon information theory were used to analyze the meaning of the BLOSUM score, which was split into three components termed as the BLOSUM spectrum (or BLOSpectrum). These relate respectively to the sequence convergence (the stochastic similarity of the two protein sequences), to the background frequency divergence (typicality of the amino acid probability distribution in each sequence), and to the target frequency divergence (compliance of the amino acid variations between the two sequences to the protein model implicit in the BLOCKS database). This treatment sharpens the protein sequence comparison, providing a rationale for the biological significance of the obtained score, and helps to identify weakly related sequences. Moreover, the BLOSpectrum can guide the choice of the most appropriate scoring matrix, tailoring it to the evolutionary divergence associated with the two sequences, or indicate if a compositionally adjusted matrix could perform better.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]     

Isocitrate Dehydrogenase Is Important for Nitrosative Stress Resistance in Cryptococcus neoformans,but Oxidative Stress Resistance Is Not Dependent on Glucose-6-Phosphate Dehydrogenase

The opportunistic intracellular fungal pathogen Cryptococcus neoformans depends on many antioxidant and denitrosylating proteins and pathways for virulence in the immunocompromised host. These include the glutathione and thioredoxin pathways, thiol peroxidase, cytochrome c peroxidase, and flavohemoglobin denitrosylase. All of these ultimately depend on NADPH for either catalytic activity or maintenance of a reduced, functional form. The need for NADPH during oxidative stress is well established in many systems, but a role in resistance to nitrosative stress has not been as well characterized. In this study we investigated the roles of two sources of NADPH, glucose-6-phosphate dehydrogenase (Zwf1) and NADP⁺-dependent isocitrate dehydrogenase (Idp1), in production of NADPH and resistance to oxidative and nitrosative stress. Deletion of ZWF1 in C. neoformans did not result in an oxidative stress sensitivity phenotype or changes in the amount of NADPH produced during oxidative stress compared to those for the wild type. Deletion of IDP1 resulted in greater sensitivity to nitrosative stress than to oxidative stress. The amount of NADPH increased 2-fold over that in the wild type during nitrosative stress, and yet the idp1Δ strain accumulated more mitochondrial damage than the wild type during nitrosative stress. This is the first report of the importance of Idp1 and NADPH for nitrosative stress resistance.The alveolar macrophage can produce microbicidal amounts of toxic reactive oxygen species (ROS) and reactive nitrogen species (RNS) following phagocytosis (27, 53). Despite this, the opportunistic fungal pathogen Cryptococcus neoformans is able to inhabit and replicate within phagocytes of the mammalian host and to exit these cells unharmed (1, 2, 40). The intracellular pathogenicity of C. neoformans is most likely facilitated by stress resistance mechanisms, including a number of antioxidant proteins and pathways involved in the detoxification of ROS and RNS. Specifically, these include the synthesis of mannitol, a free radical scavenger (9, 20); the small protein flavohemoglobin denitrosylase (Fhb1), which is essential for resistance of C. neoformans to nitrosative stress (10, 14, 32); and the glutathione and thioredoxin antioxidant systems, which are both important for stress resistance and virulence (42, 43, 45).Even with different mechanisms of catalysis and/or cellular localization, one thing that these stress resistance proteins and pathways have in common is the requirement for NADPH as a cofactor. NADPH is used as an electron donor either in recycling of oxidized, inactive enzymes to reduced, active forms or directly in catalytic activity. For example, Fhb1 binds NADPH during its catalytic activity and uses it directly as an electron donor for the reduction of NO· to NO₃ (21). Catalases, which are highly conserved antioxidants that dismute H₂O₂ to molecular oxygen and water, consist of four units each with a molecule of NADPH bound in the core (18, 36, 59). The tripeptide glutathione (GSH) is oxidized to glutathione disulfide (GSSG), a homodimer held together by a disulfide bridge, during its oxidative state. GSSG can be reduced back to GSH by glutathione reductase, an enzyme that requires NADPH for electrons used in reduction. Similarly, glutathione peroxidase and thiol peroxidase ultimately depend on NADPH for recycling from an oxidized, inactive form back to a reduced, active form (57).NADPH is classically recognized as being produced by the highly conserved, cytosolic pentose phosphate pathway. This pathway has been shown to be important for reductive biochemistry during oxidative stress in many organisms. The pentose phosphate pathway is an essential factor in maintaining health of erythrocytes, cells that, due to their biological function, have considerable risk for oxidative damage. Humans deficient in the pathway have hemolytic anemia, as their erythrocytes are unable to maintain sufficient pools of reduced glutathione (68). Also, the pressure of oxidative stress can stimulate the pentose phosphate pathway. This has been shown in human lymphocytes (56); in the rat adrenal gland, liver, and pancreas (15, 16); and in bacteria (63).In fungi, the pentose phosphate pathway has been implicated in both oxidative stress resistance and adaptation to oxidative stress. In the model yeast Saccharomyces cerevisiae, NADPH-generating systems, including the pentose phosphate pathway, are critical for the ability of this organism to resist and adapt to high levels of oxidative stress (35, 47). It has also been shown that the cytosolic copper/zinc superoxide dismutase and the pentose phosphate pathway have overlapping roles in protecting S. cerevisiae from oxidative stress and that both systems are critical for maintaining the intracellular redox state (62). Furthermore, fungi may rely on the pentose phosphate pathway for more than reducing oxidative stress. Aspergillus nidulans requires a functional pentose phosphate pathway for nitrogen metabolism. Four A. nidulans mutants with independent defects in the pentose phosphate pathway were unable to grow on nitrite, nitrate, or various carbon sources, including 1% glucose, d-xylose, or d-glucoronate (28).The pathway has two phases, the oxidative phase and the nonoxidative phase. The oxidative phase consists of two successive oxidations and results in the production of NADPH. The first enzyme in the oxidative phase of the pentose phosphate pathway is glucose-6-phosphate dehydrogenase (Zwf). Zwf catalyzes the oxidation of glucose-6-phosphate to 6-phosphogluconate and is highly specific for NADP⁺ as a cofactor (49, 67). There is abundant evidence supporting the role of Zwf in oxidative stress resistance. In addition to Zwf deficiency causing hemolytic anemia, Zwf has been also been implicated in maintenance of DNA repair systems during oxidative stress, as some cancers and aging disorders have also been linked to Zwf deficiency (30). For instance, Chinese hamster ovarian cells that are Zwf null have enhanced radiation sensitivity and a reduced ability to repair double-strand breaks due to the inactivation of Ku, a heterodimer DNA repair protein. In this case, the inactivation of Ku is the result of overoxidation of key cysteine residues on the protein due to the lack of sufficient reduced GSH (3). In the model yeast Saccharomyces cerevisiae, deletion of ZWF1 results in sensitivity to oxidative stress. ZWF1 is also important for the adaptive response to oxidative stress in S. cerevisiae. ZWF1-null mutants and wild-type cells were pretreated with 0.2 mM H₂O₂ and then challenged with 2 mM H₂O₂. While a large increase in tolerance to the high level of H₂O₂ was observed in the wild-type cells pretreated with 0.2 mM H₂O₂, the zwf1Δ strain was unable to tolerate the higher concentration (33). In Candida albicans, another pathogenic fungus, ZWF1 is upregulated during oxidative stress (38).Another source of NADPH is NADP⁺-dependent isocitrate dehydrogenase (Idp) (55), a ubiquitous enzyme that in systems ranging from humans to yeasts to plants has been found in the cytosol, peroxisomes, or mitochondria (12, 19, 70). Although this enzyme can be targeted to mitochondria, it is distinct from the NAD⁺-dependent isocitrate dehydrogenase (Idh) that functions in the mitochondria as part of the Krebs cycle. However, similarly to Idh, Idp catalyzes the decarboxylation of isocitrate to α-ketoglutarate (29). This reaction can be performed in the mitochondria, in the cytosol, or in peroxisomes using isocitrate formed from citrate exported across the mitochondrial membrane. This allows for the production of NADPH in cellular compartments without reliance of active transport of NADPH across membranes (11). It is important to have reductive power produced directly within organelles for protection from exogenous as well as endogenous stressor. For example, NADPH is consumed in peroxisomes by enzymes such as catalase and uric acid oxidase, that counteract the ROS produced during breakdown of lipids (4, 5, 31). Mitochondria particularly require reductive capability, as these organelles are susceptible to endogenous ROS produced during cellular respiration and also to exogenous RNS (52). The proteins that make up the electron transport chain are prone to damage by nitric oxide, peroxynitrite, and S-nitrosothiols (6). Nitric oxide and peroxynitrite have been shown to cause irreversible damage to cytochrome c reductase, NADH dehydrogenase, and the succinate-ubiquinone complex; the common mechanism of damage is sequestration of iron/sulfur centers of the proteins (54, 69). Thus, without a means of detoxification, the mitochondrial membrane loses potential and the ability to continue respiration, leading to death of the stressed cell. In C. neoformans, some antioxidant enzymes that are located at the mitochondria and dependent on NADPH for function include catalases, superoxide dismutases, cytochrome c peroxidase, and flavohemoglobin denitrosylase (7, 24, 25, 26). These enzymes are important for stress resistance or virulence of C. neoformans due to their role in high-temperature growth (24, 25) or nitrosative stress resistance (10, 14, 26).In humans, there is one IDP gene that results in mitochondrial and peroxisomal products (22). In S. cerevisiae, there are three IDP genes, which encode mitochondrial (IDP1), cytosolic (IDP2), and peroxisomal (IDP3) forms of the protein. Deletion of both ZWF1 and any one of the IDP genes in S. cerevisiae results in sensitivity to oxidative stress, likely due to a substantial decrease in NADPH produced in these double deletion mutants (41). In C. neoformans there is one predicted IDP gene (IDP1). Microarray data have indicated that this gene is upregulated 2.5-fold during nitrosative stress and thus may have a role in resistance to this stressor (44).Since so many factors essential for stress resistance in C. neoformans utilize NADPH, we hypothesize that the sources of this cofactor are likewise critical for stress resistance. Although Zwf1 is important for adaptation to oxidative stress in the fungi S. cerevisiae and C. albicans, we had previously found that C. neoformans is unable to adapt to oxidative stress (S. M. Brown and J. K. Lodge, unpublished data), and thus we had reason to suspect that the role of Zwf1 in C. neoformans may be different than that in other organisms. The role of Idp1 in stress resistance, especially in resistance to nitrosative stress, is relatively unknown. In this study we used biochemical and genetic approaches to compare the roles of Zwf1 and Idp1 in resistance to oxidative and nitrosative stress in C. neoformans. We found that the Zwf1 is dispensable for viability, for resistance to oxidative and nitrosative stress, and for NADPH production. In contrast, we found that Idp1 is important for resistance to nitrosative stress, specifically for maintaining healthy mitochondria during exposure to nitrosative stress.     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Algorithms for Finding Small Attractors in Boolean Networks

A Boolean network is a model used to study the interactions between different genes in genetic regulatory networks. In this paper, we present several algorithms using gene ordering and feedback vertex sets to identify singleton attractors and small attractors in Boolean networks. We analyze the average case time complexities of some of the proposed algorithms. For instance, it is shown that the outdegree-based ordering algorithm for finding singleton attractors works in time for , which is much faster than the naive time algorithm, where is the number of genes and is the maximum indegree. We performed extensive computational experiments on these algorithms, which resulted in good agreement with theoretical results. In contrast, we give a simple and complete proof for showing that finding an attractor with the shortest period is NP-hard.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]