The Lonely Guy (LOG) Homologue SiRe_0427 from the Thermophilic Archaeon Sulfolobus islandicus REY15A Is a Phosphoribohydrolase Representing a Novel Group

ABSTRACT Lonely Guy (LOG) proteins are important enzymes in cellular organisms, which catalyze the final step in the production of biologically active cytokinins via dephosphoribosylation. LOG proteins are vital enzymes in plants for the activation of cytokinin precursors, which is crucial for plant growth and development. In fungi and bacteria, LOGs are implicated in pathogenic or nonpathogenic interactions with their plant hosts. However, LOGs have also been identified in the human pathogen Mycobacterium tuberculosis, and the accumulation of cytokinin-degraded products, aldehydes, within bacterial cells is lethal to the bacterium in the presence of nitric oxide, suggesting diverse roles of LOGs in various species. In this study, we conducted biochemical and genetic analysis of a LOG homologue, SiRe_0427, from the hyperthermophilic archaeon Sulfolobus islandicus REY15A. The protein possessed the LOG motif GGGxGTxxE and exhibited phosphoribohydrolase activity on adenosine-5-monophosphate (AMP), similar to LOGs from eukaryotes and bacteria. Alanine mutants at either catalytic residues or substrate binding sites lost their activity, resembling other known LOGs. SiRe_0427 is probably a homotetramer, as revealed by size exclusion chromatography and chemical cross-linking. We found that the gene encoding SiRe_0427 could be knocked out; however, the Δsire_0427 strain exhibited no apparent difference in growth compared to the wild type, nor did it show any difference in sensitivity to UV irradiation under our laboratory growth conditions. Overall, these findings indicate that archaeal LOG homologue is active as a phosphoribohydrolase. IMPORTANCE Lonely Guy (LOG) is an essential enzyme for the final biosynthesis of cytokinins, which regulate almost every aspect of growth and development in plants. LOG protein was originally discovered 12 years ago in a strain of Oryza sativa with a distinct floral phenotype of a single stamen. Recently, the presence of LOG homologues has been reported in Mycobacterium tuberculosis, an obligate human pathogen. To date, active LOG proteins have been reported in plants, pathogenic and nonpathogenic fungi, and bacteria, but there have been no experimental reports of LOG protein from archaea. In the current work, we report the identification of a LOG homologue active on AMP from Sulfolobus islandicus REY15A, a thermophilic archaeon. The protein likely forms a tetramer in solution and represents a novel evolutionary lineage. The results presented here expand our knowledge regarding proteins with phosphoribohydrolase activities and open an avenue for studying signal transduction networks of archaea and potential applications of LOG enzymes in agriculture and industry.

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The Copper-Responsive RicR Regulon Contributes to Mycobacterium tuberculosis Virulence

mBio ◽

10.1128/mbio.00876-13 ◽

2014 ◽

Vol 5 (1) ◽

Cited By ~ 34

Author(s):

Xiaoshan Shi ◽

Richard A. Festa ◽

Thomas R. Ioerger ◽

Susan Butler-Wu ◽

James C. Sacchettini ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Microbial Growth ◽

Human Pathogen ◽

Content Type ◽

Cu Toxicity ◽

Cu Homeostasis ◽

Cu Resistance ◽

Life On Earth

ABSTRACTAs with most life on Earth, the transition metal copper (Cu) is essential for the viability of the human pathogenMycobacterium tuberculosis. However, infected hosts can also use Cu to control microbial growth. Several Cu-responsive pathways are present inM. tuberculosis, including the regulated in copper repressor (RicR) regulon, which is unique to pathogenic mycobacteria. In this work, we describe the contribution of each RicR-regulated gene to Cu resistancein vitroand to virulence in animals. We found that the deletion or disruption of individual RicR-regulated genes had no impact on virulence in mice, although several mutants had Cu hypersensitivity. In contrast, a mutant unable to activate the RicR regulon was not only highly susceptible to Cu but also attenuated in mice. Thus, these data suggest that several genes of the RicR regulon are required simultaneously to combat Cu toxicityin vivoor that this regulon is also important for resistance against Cu-independent mechanisms of host defense.IMPORTANCEMycobacterium tuberculosisis the causative agent of tuberculosis, killing millions of people every year. Therefore, understanding the biology ofM. tuberculosisis crucial for the development of new therapies to treat this devastating disease. Our studies reveal that although host-supplied Cu can suppress bacterial growth,M. tuberculosishas a unique pathway, the RicR regulon, to defend against Cu toxicity. These findings suggest that Cu homeostasis pathways in both the host and the pathogen could be exploited for the treatment of tuberculosis.

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OsdR of Streptomyces coelicolor and the Dormancy Regulator DevR of Mycobacterium tuberculosis Control Overlapping Regulons

mSystems ◽

10.1128/msystems.00014-16 ◽

2016 ◽

Vol 1 (3) ◽

Cited By ~ 11

Author(s):

Mia Urem ◽

Teunke van Rossum ◽

Giselda Bucca ◽

Geri F. Moolenaar ◽

Emma Laing ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Host Defense ◽

Growth And Development ◽

Response Regulator ◽

Streptomyces Coelicolor ◽

Regulatory Element ◽

Defense System ◽

Content Type ◽

The Core ◽

Host Defense System

ABSTRACT Dormancy is a state of growth cessation that allows bacteria to escape the host defense system and antibiotic challenge. Understanding the mechanisms that control dormancy is of key importance for the treatment of latent infections, such as those from Mycobacterium tuberculosis. In mycobacteria, dormancy is controlled by the response regulator DevR, which responds to conditions of hypoxia. Here, we show that OsdR of Streptomyces coelicolor recognizes the same regulatory element and controls a regulon that consists of genes involved in the control of stress and development. Only the core regulon in the direct vicinity of dosR and osdR is conserved between M. tuberculosis and S. coelicolor, respectively. Thus, we show how the system has diverged from allowing escape from the host defense system by mycobacteria to the control of sporulation by complex multicellular streptomycetes. This provides novel insights into how bacterial growth and development are coordinated with the environmental conditions. Two-component regulatory systems allow bacteria to respond adequately to changes in their environment. In response to a given stimulus, a sensory kinase activates its cognate response regulator via reversible phosphorylation. The response regulator DevR activates a state of dormancy under hypoxia in Mycobacterium tuberculosis, allowing this pathogen to escape the host defense system. Here, we show that OsdR (SCO0204) of the soil bacterium Streptomyces coelicolor is a functional orthologue of DevR. OsdR, when activated by the sensory kinase OsdK (SCO0203), binds upstream of the DevR-controlled dormancy genes devR, hspX, and Rv3134c of M. tuberculosis. In silico analysis of the S. coelicolor genome combined with in vitro DNA binding studies identified many binding sites in the genomic region around osdR itself and upstream of stress-related genes. This binding correlated well with transcriptomic responses, with deregulation of developmental genes and genes related to stress and hypoxia in the osdR mutant. A peak in osdR transcription in the wild-type strain at the onset of aerial growth correlated with major changes in global gene expression. Taken together, our data reveal the existence of a dormancy-related regulon in streptomycetes which plays an important role in the transcriptional control of stress- and development-related genes. IMPORTANCE Dormancy is a state of growth cessation that allows bacteria to escape the host defense system and antibiotic challenge. Understanding the mechanisms that control dormancy is of key importance for the treatment of latent infections, such as those from Mycobacterium tuberculosis. In mycobacteria, dormancy is controlled by the response regulator DevR, which responds to conditions of hypoxia. Here, we show that OsdR of Streptomyces coelicolor recognizes the same regulatory element and controls a regulon that consists of genes involved in the control of stress and development. Only the core regulon in the direct vicinity of dosR and osdR is conserved between M. tuberculosis and S. coelicolor, respectively. Thus, we show how the system has diverged from allowing escape from the host defense system by mycobacteria to the control of sporulation by complex multicellular streptomycetes. This provides novel insights into how bacterial growth and development are coordinated with the environmental conditions.

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From infection niche to therapeutic target: the intracellular lifestyle of Mycobacterium tuberculosis

Microbiology ◽

10.1099/mic.0.001041 ◽

2021 ◽

Vol 167 (4) ◽

Author(s):

Leah Isobella Rankine-Wilson ◽

Tirosh Shapira ◽

Carine Sao Emani ◽

Yossef Av-Gay

Keyword(s):

Mycobacterium Tuberculosis ◽

Side Effects ◽

Type Species ◽

Life Stage ◽

Maturation Process ◽

Human Pathogen ◽

Phagosome Maturation ◽

Content Type ◽

The Past ◽

Multiple Drugs

Mycobacterium tuberculosis (Mtb) is an obligate human pathogen killing millions of people annually. Treatment for tuberculosis is lengthy and complicated, involving multiple drugs and often resulting in serious side effects and non-compliance. Mtb has developed numerous complex mechanisms enabling it to not only survive but replicate inside professional phagocytes. These mechanisms include, among others, overcoming the phagosome maturation process, inhibiting the acidification of the phagosome and inhibiting apoptosis. Within the past decade, technologies have been developed that enable a more accurate understanding of Mtb physiology within its intracellular niche, paving the way for more clinically relevant drug-development programmes. Here we review the molecular biology of Mtb pathogenesis offering a unique perspective on the use and development of therapies that target Mtb during its intracellular life stage.

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The Influence of the Thyroid Function on the Metabolic Rate of Prothrombin, Factor VII, and Factor X in the Rat

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647959 ◽

1976 ◽

Vol 35 (03) ◽

pp. 607-619 ◽

Cited By ~ 8

Author(s):

Allan T. van Oosterom ◽

Herman Mattie ◽

Wim Th Hermens ◽

Jan J. Veltkamp

Keyword(s):

Metabolic Rate ◽

Thyroid Function ◽

Coagulation Factors ◽

Biologically Active ◽

Factor Vii ◽

Synthesis Rate ◽

Apparent Difference ◽

Vitamin K1 ◽

Factor X ◽

Significant Difference

SummaryThe influence of the thyroid function on the metabolic rate of prothrombin, factor VII, and X was studied in the rat. Disappearance rates of the three coagulation factors were measured after synthesis had been blocked with appropriate doses of warfarin, and reappearance rates were assessed upon induction of synthesis by high doses of vitamin K1 injected into rats displaying coumarin induced hypocoagulability.No statistically significant difference in the disappearance and production rates of any of the factors could be found between normal euthyroid rats and thyroxin-treated hypothyroid rats proven to be euthyroid. The differences between the two euthyroid groups and the hypothyroid group were highly significant, however: hypothyroidism results in an approximately 50% decrease of the metabolic rates of the three coagulation factors under study.The reappearance of the three factors, under euthyroid as well as hypothyroid conditions, showed a biphasic pattern: in the first two hours after vitamin K1 administration to warfarin treated rats, a rapid reappearance was observed, to the same extent for all three factors, in hypo- as well as euthyroid rats. This finding suggests that in vitamin K1 deficiency an intracellular accumulation of precursor proteins (PIVKAs) occurs, which after rapid conversion into biologically active coagulation factors by vitamin K1 are shed into circulation.The subsequent phase of reappearance is much slower and reflects the synthesis rate of coagulation enzymes. It is characteristic for each factor and clearly slower in hypothyroid rats than in euthyroid rats. From this an influence of thyroid function on the synthesis rate of the protein moiety of coagulation factors can be inferred.An apparent difference between disappearance and reappearance rate of the coagulation factors in the plasma, particularly pronounced for factors VII and X in euthyroid rats, could theoretically be explained as the consequence of the model used for derivation of these rates.

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Mutations in fbiD (Rv2983) as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01948-20 ◽

2020 ◽

Vol 65 (1) ◽

pp. e01948-20

Author(s):

Dalin Rifat ◽

Si-Yang Li ◽

Thomas Ioerger ◽

Keshav Shah ◽

Jean-Philippe Lanoix ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Clinical Evidence ◽

Clinical Samples ◽

Loss Of Function ◽

Wild Type ◽

Content Type ◽

Solid Media ◽

High Level ◽

Level Resistance

ABSTRACTThe nitroimidazole prodrugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10−5 CFU. Whole-genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, of which 91% occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance, namely, fbiC (56%), fbiA (15%), ddn (12%), fgd (4%), and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983 (fbiD), a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

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Regulation of Cell Division in Bacteria by Monitoring Genome Integrity and DNA Replication Status

Journal of Bacteriology ◽

10.1128/jb.00408-19 ◽

2019 ◽

Vol 202 (2) ◽

Cited By ~ 5

Author(s):

Peter E. Burby ◽

Lyle A. Simmons

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Cell Division ◽

Dna Replication ◽

Cell Cycle Progression ◽

Genome Instability ◽

Sos Response ◽

Bacterial Cells ◽

Cycle Progression ◽

Content Type

ABSTRACT All organisms regulate cell cycle progression by coordinating cell division with DNA replication status. In eukaryotes, DNA damage or problems with replication fork progression induce the DNA damage response (DDR), causing cyclin-dependent kinases to remain active, preventing further cell cycle progression until replication and repair are complete. In bacteria, cell division is coordinated with chromosome segregation, preventing cell division ring formation over the nucleoid in a process termed nucleoid occlusion. In addition to nucleoid occlusion, bacteria induce the SOS response after replication forks encounter DNA damage or impediments that slow or block their progression. During SOS induction, Escherichia coli expresses a cytoplasmic protein, SulA, that inhibits cell division by directly binding FtsZ. After the SOS response is turned off, SulA is degraded by Lon protease, allowing for cell division to resume. Recently, it has become clear that SulA is restricted to bacteria closely related to E. coli and that most bacteria enforce the DNA damage checkpoint by expressing a small integral membrane protein. Resumption of cell division is then mediated by membrane-bound proteases that cleave the cell division inhibitor. Further, many bacterial cells have mechanisms to inhibit cell division that are regulated independently from the canonical LexA-mediated SOS response. In this review, we discuss several pathways used by bacteria to prevent cell division from occurring when genome instability is detected or before the chromosome has been fully replicated and segregated.

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Spo0A Suppresses sin Locus Expression in Clostridioides difficile

mSphere ◽

10.1128/msphere.00963-20 ◽

2020 ◽

Vol 5 (6) ◽

Author(s):

Babita Adhikari Dhungel ◽

Revathi Govind

Keyword(s):

Diarrheal Disease ◽

Toxin Production ◽

The United States ◽

Upstream Region ◽

Pcr Analysis ◽

Bacterial Cells ◽

Master Regulator ◽

Content Type ◽

Clostridioides Difficile

ABSTRACT Clostridioides difficile is the leading cause of nosocomial infection and is the causative agent of antibiotic-associated diarrhea. The severity of the disease is directly associated with toxin production, and spores are responsible for the transmission and persistence of the organism. Previously, we characterized sin locus regulators SinR and SinR′ (we renamed it SinI), where SinR is the regulator of toxin production and sporulation. The SinI regulator acts as its antagonist. In Bacillus subtilis, Spo0A, the master regulator of sporulation, controls SinR by regulating the expression of its antagonist, sinI. However, the role of Spo0A in the expression of sinR and sinI in C. difficile had not yet been reported. In this study, we tested spo0A mutants in three different C. difficile strains, R20291, UK1, and JIR8094, to understand the role of Spo0A in sin locus expression. Western blot analysis revealed that spo0A mutants had increased SinR levels. Quantitative reverse transcription-PCR (qRT-PCR) analysis of its expression further supported these data. By carrying out genetic and biochemical assays, we show that Spo0A can bind to the upstream region of this locus to regulates its expression. This study provides vital information that Spo0A regulates the sin locus, which controls critical pathogenic traits such as sporulation, toxin production, and motility in C. difficile. IMPORTANCE Clostridioides difficile is the leading cause of antibiotic-associated diarrheal disease in the United States. During infection, C. difficile spores germinate, and the vegetative bacterial cells produce toxins that damage host tissue. In C. difficile, the sin locus is known to regulate both sporulation and toxin production. In this study, we show that Spo0A, the master regulator of sporulation, controls sin locus expression. Results from our study suggest that Spo0A directly regulates the expression of this locus by binding to its upstream DNA region. This observation adds new detail to the gene regulatory network that connects sporulation and toxin production in this pathogen.

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Characterisation of Persistent Anti-Xa Activity Following Administration of the Low Molecular Weight Heparin Enoxaparin Sodium (Clexane)

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648852 ◽

1994 ◽

Vol 72 (02) ◽

pp. 275-280 ◽

Cited By ~ 12

Author(s):

David Brieger ◽

Joan Dawes

Keyword(s):

Molecular Weight ◽

Enoxaparin Sodium ◽

Antithrombin Iii ◽

Anticoagulant Activity ◽

Active Material ◽

Biologically Active ◽

Size Exclusion ◽

Low Molecular Weight ◽

Exclusion Chromatography ◽

Liver And Kidney

SummaryIt is widely reported that persistent anti-Xa activity follows administration of low molecular weight heparins. To identify the effectors of this activity we have injected 125I-labelled Enoxaparin sodium into rabbits and subsequently analysed the circulating radiolabelled material and anti-Xa activity by affinity and size exclusion chromatography. Antithrombin III-binding material derived from the injected drug was responsible for all the anti-Xa amidolytic activity. At early times after injection additional anticoagulant activity which was largely attributable to tissue factor pathway inhibitor was measured by the Heptest clotting assay after removal of glycosaminoglycans from plasma samples. Small radiolabelled fragments, including penta/hexasaccharide with affinity for antithrombin III, were detectable in the circulation 1 week later, and sulphated oligosaccharides persisted for 3-4 weeks. Significant quantities of radiolabel remained in the liver and kidney several weeks post-injection; these organs may sequester some of the injected drug and give rise to circulating biologically active material by degradation and secretion of catabolic products into the plasma.

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Antibacterial Peptides Produced by Alcalase from Cowpea Seed Proteins

Antibiotics ◽

10.3390/antibiotics10070870 ◽

2021 ◽

Vol 10 (7) ◽

pp. 870

Author(s):

Ali Osman ◽

Gamal Enan ◽

Abdul-Raouf Al-Mohammadi ◽

Seham Abdel-Shafi ◽

Samar Abdel-Hameid ◽

...

Keyword(s):

Size Exclusion Chromatography ◽

Seed Proteins ◽

Negative Ions ◽

Size Exclusion ◽

Ionization Mass ◽

Bacterial Cells ◽

Antibacterial Efficiency ◽

Exclusion Chromatography ◽

Molecular Masses ◽

Cowpea Seed

Cowpea seed protein hydrolysates (CPH) were output from cowpea seeds applying alcalase® from Bacillus licheniformis. CPH with an elevated level of hydrolysis was fractionated by size exclusion chromatography (SEC). Both CPH and SEC-portions showed to contain antimicrobial peptides (AMPs) as they inhibited both Gram-positive bacteria, such as Listeria monocytogenes LMG10470 (L. monocytogenes), Listeria innocua. LMG11387 (L. innocua), Staphylococcus aureus ATCC25923 (S.aureus), and Streptococcus pyogenes ATCC19615 (St.pyogenes), and Gram-negative bacteria, such as Klebsiella pnemoniae ATCC43816 (K. pnemoniae), Pseudomonas aeroginosa ATCC26853 (P. aeroginosa), Escherichia coli ATCC25468) (E.coli) and Salmonella typhimurium ATCC14028 (S. typhimurium).The data exhibited that both CPH and size exclusion chromatography-fraction 1 (SEC-F1) showed high antibacterial efficiency versus almost all the assessed bacteria. The MIC of the AMPs within SEC-F1 and CPHs were (25 µg/mL) against P. aeruginosa, E.coli and St. pyogenes. However, higher MICsof approximately 100–150 µg/mL showed for both CPHs and SEC-F1 against both S. aureus and L. innocua; it was 50 µg/mL of CPH against S.aureus. The Electro-spray-ionization-mass-spectrometry (ESI-MS) of fraction (1) revealed 10 dipeptides with a molecular masses arranged from 184 Da to 364 Da and one Penta peptide with a molecular mass of approximately 659 Da inthe case of positive ions. While the negative ions showed 4 dipeptides with the molecular masses that arranged from 330 Da to 373 Da. Transmission electron microscope (TEM) demonstrated that the SEC-F1 induced changes in the bacterial cells affected. Thus, the results suggested that the hydrolysis of cowpea seed proteins by Alcalase is an uncomplicated appliance to intensify its antibacterial efficiency.

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Melanization in Cryptococcus neoformans Requires Complex Regulation

mBio ◽

10.1128/mbio.03313-19 ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 3

Author(s):

Radames J. B. Cordero ◽

Emma Camacho ◽

Arturo Casadevall

Keyword(s):

Cryptococcus Neoformans ◽

Defense Mechanisms ◽

Environmental Stressors ◽

Antimicrobial Compounds ◽

Human Pathogen ◽

Content Type ◽

Signaling Mechanisms ◽

Complex Regulation ◽

Multiple Levels ◽

Melanin Binding

ABSTRACT The fungal human pathogen Cryptococcus neoformans undergoes melanization in response to nutrient starvation and exposure to exogenous melanin precursors. Melanization protects the fungus against host defense mechanisms such as oxidative damage and other environmental stressors (e.g., heat/cold stress, antimicrobial compounds, ionizing radiation). Conversely, the melanization process generates cytotoxic intermediates, and melanized cells are potentially susceptible to overheating and to certain melanin-binding drugs. Despite the importance of melanin in C. neoformans biology, the signaling mechanisms regulating its synthesis are poorly understood. The recent report by D. Lee, E.-H. Jang, M. Lee, S.-W. Kim, et al. [mBio 10(5):e02267-19, 2019, https://doi.org/10.1128/mBio.02267-19] provides new insights into how C. neoformans regulates melanization. The authors identified a core melanin regulatory network consisting of transcription factors and kinases required for melanization under low-nutrient conditions. The redundant and epistatic connections of this melanin-regulating network demonstrate that C. neoformans melanization is complex and carefully regulated at multiple levels. Such complex regulation reflects the multiple functions of melanin in C. neoformans biology.

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