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Characterization of the Complete Uric Acid Degradation Pathway in the Fungal Pathogen Cryptococcus neoformans

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I Russel Lee
I Russel Lee
I Russel Lee
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Liting Yang
Liting Yang
Liting Yang
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Gaseene Sebetso at Botswana Harvard AIDS Institute
Gaseene Sebetso
Rebecca Elspeth Allen at Royal Brisbane Hospital
Rebecca Elspeth Allen
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Abstract and Figures

Degradation of purines to uric acid is generally conserved among organisms, however, the end product of uric acid degradation varies from species to species depending on the presence of active catabolic enzymes. In humans, most higher primates and birds, the urate oxidase gene is non-functional and hence uric acid is not further broken down. Uric acid in human blood plasma serves as an antioxidant and an immune enhancer; conversely, excessive amounts cause the common affliction gout. In contrast, uric acid is completely degraded to ammonia in most fungi. Currently, relatively little is known about uric acid catabolism in the fungal pathogen Cryptococcus neoformans even though this yeast is commonly isolated from uric acid-rich pigeon guano. In addition, uric acid utilization enhances the production of the cryptococcal virulence factors capsule and urease, and may potentially modulate the host immune response during infection. Based on these important observations, we employed both Agrobacterium-mediated insertional mutagenesis and bioinformatics to predict all the uric acid catabolic enzyme-encoding genes in the H99 genome. The candidate C. neoformans uric acid catabolic genes identified were named: URO1 (urate oxidase), URO2 (HIU hydrolase), URO3 (OHCU decarboxylase), DAL1 (allantoinase), DAL2,3,3 (allantoicase-ureidoglycolate hydrolase fusion protein), and URE1 (urease). All six ORFs were then deleted via homologous recombination; assaying of the deletion mutants' ability to assimilate uric acid and its pathway intermediates as the sole nitrogen source validated their enzymatic functions. While Uro1, Uro2, Uro3, Dal1 and Dal2,3,3 were demonstrated to be dispensable for virulence, the significance of using a modified animal model system of cryptococcosis for improved mimicking of human pathogenicity is discussed.
Uro1, Uro2, Uro3, Dal1, Dal2,3,3 and Ure1 are not required for C. neoformans initiation of mating. Filamentation assays on V8, MS and pigeon guano (PG) medium showed that filament formation appeared indistinguishable between the crosses of H99 6 KN99a and each of the uric acid mutants 6 KN99a. (A) Periphery of each individual colony. (B) Under higher magnification (6400). doi:10.1371/journal.pone.0064292.g004
Uro1, Uro2, Uro3, Dal1, Dal2,3,3 and Ure1 are not required for C. neoformans initiation of mating. Filamentation assays on V8, MS and pigeon guano (PG) medium showed that filament formation appeared indistinguishable between the crosses of H99 6 KN99a and each of the uric acid mutants 6 KN99a. (A) Periphery of each individual colony. (B) Under higher magnification (6400). doi:10.1371/journal.pone.0064292.g004
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While degradation of purines to uric acid is generally conserved among organisms, the end product of uric acid catabolism varies among taxa often due to the loss of functional catabolic enzymes in the pathway.
While degradation of purines to uric acid is generally conserved among organisms, the end product of uric acid catabolism varies among taxa often due to the loss of functional catabolic enzymes in the pathway.
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While the metabolic pathway of purine degradation is present in bacteria, fungi, plants and animals, there is hidden complexity between species. In addition to species in which only part of the pathway is present, these variations include at least three examples of convergent evolution (conversion of xanthine to uric acid, allantoin to allantoate, and urea to ammonia), offshoots of the pathway that are only present in some species (allantoin racemase, ureidoglycine production and hydrolysis) as well as points at which spontaneous conversion occur without the need for enzyme activity (dashed arrows). Steps predicted to be absent from C. neoformans are opaque.
While the metabolic pathway of purine degradation is present in bacteria, fungi, plants and animals, there is hidden complexity between species. In addition to species in which only part of the pathway is present, these variations include at least three examples of convergent evolution (conversion of xanthine to uric acid, allantoin to allantoate, and urea to ammonia), offshoots of the pathway that are only present in some species (allantoin racemase, ureidoglycine production and hydrolysis) as well as points at which spontaneous conversion occur without the need for enzyme activity (dashed arrows). Steps predicted to be absent from C. neoformans are opaque.
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(A) Based on the enzymatic activity of homologs in other systems, inactivation of each respective catabolic enzyme (in red) is predicted to disrupt the ability of C. neoformans to utilize one or more pathway intermediates as the sole nitrogen source. (B) Ten-fold spot dilution assays on YNB supplemented with 10 mM uric acid or its derivatives revealed that the uro1Δ, uro2Δ, uro3Δ, dal1Δ, dal2,3,3Δ and ure1Δ mutants showed a correlation with the anticipated nitrogen utilization abilities.
(A) Based on the enzymatic activity of homologs in other systems, inactivation of each respective catabolic enzyme (in red) is predicted to disrupt the ability of C. neoformans to utilize one or more pathway intermediates as the sole nitrogen source. (B) Ten-fold spot dilution assays on YNB supplemented with 10 mM uric acid or its derivatives revealed that the uro1Δ, uro2Δ, uro3Δ, dal1Δ, dal2,3,3Δ and ure1Δ mutants showed a correlation with the anticipated nitrogen utilization abilities.
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Filamentation assays on V8, MS and pigeon guano (PG) medium showed that filament formation appeared indistinguishable between the crosses of H99 × KN99a and each of the uric acid mutants × KN99a. (A) Periphery of each individual colony. (B) Under higher magnification (×400).
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Filamentation assays on V8, MS and pigeon guano (PG) medium showed that filament formation appeared indistinguishable between the crosses of H99 × KN99a and each of the uric acid mutants × KN99a. (A) Periphery of each individual colony. (B) Under higher magnification (×400).
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Characterization of the Complete Uric Acid Degradation Pathway in the Fungal Pathogen Cryptococcus neoforma
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Supplementary resources (12)

Table S1
Data
May 2013
I. Russel Lee · Liting Yang · Gaseene Sebetso · Rebecca Elspeth Allen · James A. Fraser
Figure S8
Data
May 2013
I. Russel Lee · Liting Yang · Gaseene Sebetso · Rebecca Elspeth Allen · James A. Fraser
Figure S6
Data
May 2013
I. Russel Lee · Liting Yang · Gaseene Sebetso · Rebecca Elspeth Allen · James A. Fraser
Table S4
Data
May 2013
I. Russel Lee · Liting Yang · Gaseene Sebetso · Rebecca Elspeth Allen · James A. Fraser
Table S2
Data
May 2013
I. Russel Lee · Liting Yang · Gaseene Sebetso · Rebecca Elspeth Allen · James A. Fraser
Figure S5
Data
May 2013
I. Russel Lee · Liting Yang · Gaseene Sebetso · Rebecca Elspeth Allen · James A. Fraser
Figure S1
Data
May 2013
I. Russel Lee · Liting Yang · Gaseene Sebetso · Rebecca Elspeth Allen · James A. Fraser
Table S3
Data
May 2013
I. Russel Lee · Liting Yang · Gaseene Sebetso · Rebecca Elspeth Allen · James A. Fraser
Figure S7
Data
May 2013
I. Russel Lee · Liting Yang · Gaseene Sebetso · Rebecca Elspeth Allen · James A. Fraser
Figure S4
Data
May 2013
I. Russel Lee · Liting Yang · Gaseene Sebetso · Rebecca Elspeth Allen · James A. Fraser
Figure S3
Data
May 2013
I. Russel Lee · Liting Yang · Gaseene Sebetso · Rebecca Elspeth Allen · James A. Fraser
Figure S2
Data
May 2013
I. Russel Lee · Liting Yang · Gaseene Sebetso · Rebecca Elspeth Allen · James A. Fraser
... Three enzymes in the uricolytic pathway were downregulated in quasi-aposymbionts ( Figure 5). Urate oxidase was initially supposed to be the only enzyme responsible for the conversion of uric acid into allantoin in many species, including B. germanica, [29,84]. However, two genes sharing a common history of loss or gain events with urate oxidase were identified through phylogenetic genome comparison [85]. ...
Comparative Transcriptomics of Fat Bodies between Symbiotic and Quasi-Aposymbiotic Adult Females of Blattella germanica with Emphasis on the Metabolic Integration with Its Endosymbiont Blattabacterium and Its Immune System
Article
Full-text available
  • Apr 2024
  • INT J MOL SCI
We explored the metabolic integration of Blattella germanica and its obligate endosymbiont Blattabacterium cuenoti by the transcriptomic analysis of the fat body of quasi-aposymbiotic cockroaches, where the endosymbionts were almost entirely removed with rifampicin. Fat bodies from quasi-aposymbiotic insects displayed large differences in gene expression compared to controls. In quasi-aposymbionts, the metabolism of phenylalanine and tyrosine involved in cuticle sclerotization and pigmentation increased drastically to compensate for the deficiency in the biosynthesis of these amino acids by the endosymbionts. On the other hand, the uricolytic pathway and the biosynthesis of uric acid were severely decreased, probably because the reduced population of endosymbionts was unable to metabolize urea to ammonia. Metabolite transporters that could be involved in the endosymbiosis process were identified. Immune system and antimicrobial peptide (AMP) gene expression was also reduced in quasi-aposymbionts, genes encoding peptidoglycan-recognition proteins, which may provide clues for the maintenance of the symbiotic relationship, as well as three AMP genes whose involvement in the symbiotic relationship will require additional analysis. Finally, a search for AMP-like factors that could be involved in controlling the endosymbiont identified two orphan genes encoding proteins smaller than 200 amino acids underexpressed in quasi-aposymbionts, suggesting a role in the host–endosymbiont relationship.
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... Birds could be a suitable animal model for investigating human HUA and gout because the final product of purine metabolism is urate in birds (but allantoin in non-primate mammals such as rats and mice) owing to the lacking of uricase in birds, which is also the case in humans. Furthermore, birds are prone to accumulating urate, and feed formulation (in analogy with the human diet) is a critical factor contributing to the development of bird gout, for example, chicken articular or visceral gout [24][25][26]. HUA models have been described in different avian species such as broiler chickens [21], red-tailed hawks [27], and quails [28]. A high-protein diet with and without high-calcium supplementation, purine-rich feed, and sodium bicarbonate in drinking water were used to establish the models in these birds. ...
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... This deletion has been demonstrated to attenuate the virulence in an animal model [65], the PKA2 gene which regulates the mating, haploid fruiting, and virulence in serotype D strains of Cryptococcus [66]. The URO1 gene, known as dispensable for virulence [67], and the SKI2 gene, whose mutant exhibited a lower virulence and susceptibility to anti-ribosomal drugs [68] (Table S3). These results describe that C. neoformans VNI present changes in specific genes that could not only favor its survival and adaptation but also possibly modify the clinical and therapeutic outcomes. ...
A Landscape of the Genomic Structure of Cryptococcus neoformans in Colombian Isolates
Article
Full-text available
  • Jan 2023
  • J. Fungi
Cryptococcus neoformans species complexes are recognized as environmental fungi responsible for lethal meningoencephalitis in immunocompromised individuals. Despite the vast knowledge about the epidemiology and genetic diversity of this fungus in different regions of the world, more studies are necessary to comprehend the genomic profiles across South America, including Colombia, considered to be the second country with the highest number of Cryptococcosis. Here, we sequenced and analyzed the genomic architecture of 29 Colombian C. neoformans isolates and evaluated the phylogenetic relationship of these strains with publicly available C. neoformans genomes. The phylogenomic analysis showed that 97% of the isolates belonged to the VNI molecular type and the presence of sub-lineages and sub-clades. We evidenced a karyotype without changes, a low number of genes with copy number variations, and a moderate number of single-nucleotide polymorphisms (SNPs). Additionally, a difference in the number of SNPs between the sub-lineages/sub-clades was observed; some were involved in crucial fungi biological processes. Our study demonstrated the intraspecific divergence of C. neoformans in Colombia. These findings provide evidence that Colombian C. neoformans isolates do not probably require significant structural changes as adaptation mechanisms to the host. To the best of our knowledge, this is the first study to report the whole genome sequence of Colombian C. neoformans isolates.
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... IPR017595) respectively. The HiuH and Uao proteins are responsible for the second (hydrolysis of HIU to OHCU) and third (the decarboxylation of OHCU to (S)-allantoin) steps of uric acid processing, respectively (Lee et al. 2013 were found, including four plasmids with the hpxO gene and five plasmids without hpxO (Table 1 and S4). Among those plasmids with the hpxO gene, pA297-3 and pA1429c ...
Analysis of pCl107 a large plasmid carried by an ST25 Acinetobacter baumannii strain reveals a complex evolutionary history and links to multiple antibiotic resistance and metabolic pathways
Article
Full-text available
  • Nov 2022
Acinetobacter baumannii has successfully spread during the last decades as one of the main critically important pathogens. However, many aspects including plasmids, are still under-investigated. Here, we report the complete sequence of an Acinetobacter baumannii strain, belonging to the ST25IP (Institut Pasteur) sequence type and recovered in 2012 in Lebanon, using a combination of Illumina MiSeq and Oxford Nanopore sequencing and a hybrid assembly approach. This strain (Cl107) carries a 198 kb plasmid called pCl107 that encodes the MPFI conjugative transfer system. The plasmid carries the aacA1, aacC2, sul2, strAB, and tetA(B) antibiotic resistance genes. pCl107 region encompassing the sul2, strAB, tetA(B) is closely related to AbGRI1 chromosomal resistance islands, which are widespread in A. baumannii strains belonging to Global Clone 2. The resistance region found in pCl107 is one of the missing links in the evolutionary history of the AbGRI1 islands. pCl107 also contains a BREX Type 1 region and represents one of the two main evolution patterns observed in BREX clusters found in plasmids related to pCl107. pCl107 also harbours a ptx phosphonate metabolism module, which plays an ancestral structure compared to other large plasmids in ST25 strains. While the uric acid metabolic module found in pCl107 is incomplete, we identified possible ancestors from plasmids and chromosomes of Acinetobacter spp. Our analyses indicate a complex evolutionary history of plasmids related to pCl107 with many links to multiple antibiotic resistance and metabolic pathways.
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... Like XO, the next enzyme uricase utilizes molecular oxygen and produces H 2 O 2 as a side product (Figure 3). The animalpathogenic C. neoformans, which is commonly isolated from uric acid rich pigeon guano, may catabolise uric acid into ammonia (Lee et al., 2013). Unicellular yeasts such as S. cerevisiae and Ascomycota filamentous fungi in turn are rich in uric acid (Hafez et al., 2017). ...
Basidiomycota Fungi and ROS: Genomic Perspective on Key Enzymes Involved in Generation and Mitigation of Reactive Oxygen Species
Article
Full-text available
  • Mar 2022
Our review includes a genomic survey of a multitude of reactive oxygen species (ROS) related intra-and extracellular enzymes and proteins among fungi of Basidiomycota, following their taxonomic classification within the systematic classes and orders, and focusing on different fungal lifestyles (saprobic, symbiotic, pathogenic). Intra-and extracellular ROS metabolism-involved enzymes (49 different protein families, summing 4170 protein models) were searched as protein encoding genes among 63 genomes selected according to current taxonomy. Extracellular and intracellular ROS metabolism and mechanisms in Basidiomycota are illustrated in detail. In brief, it may be concluded that differences between the set of extracellular enzymes activated by ROS, especially by H 2 O 2 , and involved in generation of H 2 O 2 , follow the differences in fungal lifestyles. The wood and plant biomass degrading white-rot fungi and the litter-decomposing species of Agaricomycetes contain the highest counts for genes encoding various extracellular peroxidases, mono-and peroxygenases, and oxidases. These findings further confirm the necessity of the multigene families of various extracellular oxidoreductases for efficient and complete degradation of wood lignocelluloses by fungi. High variations in the sizes of the extracellular ROS-involved gene families were found, however, among species with mycorrhizal symbiotic lifestyle. In addition, there are some differences among the sets of intracellular thiol-mediation involving proteins, and existence of enzyme mechanisms for quenching of intracellular H 2 O 2 and ROS. In animal-and plant-pathogenic species, extracellular ROS enzymes are absent or rare. In these fungi, intracellular peroxidases are seemingly in minor role than in the independent saprobic, filamentous species of Basidiomycota. Noteworthy is that our genomic survey and review of the literature point to that there are differences both in generation of extracellular ROS as well as in mechanisms of response to oxidative stress and mitigation of ROS between fungi of Basidiomycota and Ascomycota.
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... These crystals, being insoluble in tissue fluids and having sharp ends, cause physical damage to the tissues leading to a cascading inflammatory reaction However, other mammals, such as rats, mice, and rabbits, contain uricase and can metabolize urate to allantoin, which is soluble in water and easy to excrete (Lee et al., 2013). Chickens affected by renal damage can continue to be productive until less than one third of their normal kidney mass remains functional. ...
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Regulation of uric acid and glyoxylate metabolism by UgmR protein in Pseudomonas aeruginosa
Article
  • Jun 2022
The opportunistic pathogen Pseudomonas aeruginosa has evolved several systems to adapt to complex environments. The GntR family proteins play important roles in the regulation of metabolic processes and bacterial pathogenesis. In this study, we uncovered that the gene clusters of PA1513‐PA1518 and PA1498‐PA1502 in P. aeruginosa are required for uric acid and glyoxylate metabolism, respectively. We also identified a GntR family regulator UgmR that is involved in regulation of uric acid and glyoxylate metabolism. Promoter activity measurement and biochemical assays revealed that the UgmR directly represses the transcriptional activity of PA1513‐PA1518 and PA1498‐PA1502, and this inhibition was relieved by the addition of uric acid. Importantly, further experiments showed that UgmR also participates in the glyoxylate shunt. Collectively, these findings contribute to a better understanding of the UgmR factor involved in uric acid and glyoxylate metabolism, which provide insights into the complex metabolic pathways in P. aeruginosa. This article is protected by copyright. All rights reserved.
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Structural and biochemical insights into a hyperthermostable urate oxidase from Thermobispora bispora for hyperuricemia and gout therapy
Article
  • Aug 2021
  • INT J BIOL MACROMOL
Microbial urate oxidase has emerged as a potential source of therapeutic properties for hyperuricemia in arthritic gout and renal disease. The thermostability and long-term thermal tolerance of the enzyme need to be established to prolong its therapeutic effects. Here, we present the biochemical and structural aspects of a hyperthermostable urate oxidase (TbUox) from the thermophilic microorganism Thermobispora bispora. Enzymatic characterization of TbUox revealed that it was active over a wide range of temperatures, from 30 to 70 °C, with optimal activity at 65 °C and pH 8.0, which suggests its applicability under physiological conditions. Moreover, TbUox exhibits high thermostability from 10 to 65 °C, with Tm of 70.3 °C and near-neutral pH stability from pH 7.0 to 8.0 and high thermal tolerance. The crystal structures of TbUox revealed a distinct feature of the C-terminal loop extensions that may help with protein stability via inter-subunit interactions. In addition, the high thermal tolerance of TbUox may be contributed by the extensive inter-subunit contacts via salt bridges, hydrogen bonds, and hydrophobic interactions. The findings in this study provide a molecular basis for the thermophilic TbUox urate oxidase for application in hyperuricemia and gout therapy.
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Screening of Chemical Libraries for New Antifungal Drugs against Aspergillus fumigatus Reveals Sphingolipids Are Involved in the Mechanism of Action of Miltefosine
Article
Full-text available
  • Aug 2021
The filamentous fungus Aspergillus fumigatus causes a group of diseases named aspergillosis, and their development occurs after the inhalation of conidia dispersed in the environment. Very few classes of antifungal drugs are available for aspergillosis treatment, e.g., azoles, but the emergence of global resistance to azoles in A. fumigatus clinical isolates has increased over recent decades.
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Urea Is a Product of Ureidoglycolate Degradation in Chickpea. Purification and Characterization of the Ureidoglycolate Urea-Lyase
Article
Full-text available
  • Mar 2001
  • PLANT PHYSIOL
ABSTRACT: A ureidoglycolate-degrading activity was analyzed in different organs of chickpea (Cicer arietinum). Activity was detected in all the tissues analyzed, but highest levels of specific activity were found in pods, from which it has been purified and characterized. This is the first ureidoglycolate-degrading activity that has been purified to homogeneity from any photosynthetic organism. Only one ureidoglycolate-degrading activity was found during the purification. The enzyme was purified 1,500-fold, and specific activity for the pure enzyme was 8.6 units mg(-1), which corresponds with a turnover number of 1,600 min(-1). The native enzyme has a molecular mass of 180 kD and consists of six identical or similar-sized subunits of 31 kD each. The enzyme exhibited hyperbolic, Michaelian kinetics for (-) ureidoglycolate with K(m) values of 6 and 10 microM in the presence or absence of Mn(2+), respectively. Optimum pH was between 7 and 8 and maximum activity was found at temperatures above 70 degrees C, the enzyme being extremely stable and resistant to heat denaturation. The activity was inhibited by EDTA and enhanced by several bivalent cations, thus suggesting that the enzyme is a metalloprotein. This enzyme has been characterized as a ureidoglycolate urea-lyase (EC 4.3.2.3), which catalyzes the degradation of (-) ureidoglycolate to glyoxylate and urea. This is the first time that such an activity is detected in plant tissues. A possible function for this activity and its implications in the context of nitrogen mobilization in legume plants is also discussed.
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Urea Is a Product of Ureidoglycolate Degradation in Chickpea. Purification and Characterization of the Ureidoglycolate Urea-Lyase
Article
  • Feb 2001
  • PLANT PHYSIOL
A ureidoglycolate-degrading activity was analyzed in different organs of chickpea (Cicer arietinum). Activity was detected in all the tissues analyzed, but highest levels of specific activity were found in pods, from which it has been purified and characterized. This is the first ureidoglycolate-degrading activity that has been purified to homogeneity from any photosynthetic organism. Only one ureidoglycolate-degrading activity was found during the purification. The enzyme was purified 1,500-fold, and specific activity for the pure enzyme was 8.6 units mg 21 , which corresponds with a turnover number of 1,600 min 21 . The native enzyme has a molecular mass of 180 kD and consists of six identical or similar-sized subunits of 31 kD each. The enzyme exhibited hyperbolic, Michaelian kinetics for (2) ureidoglycolate with Km values of 6 and 10 mm in the presence or absence of Mn 21 , respectively. Optimum pH was between 7 and 8 and maximum activity was found at temperatures above 70°C, the enzyme being extremely stable and resistant to heat denaturation. The activity was inhibited by EDTA and enhanced by several bivalent cations, thus suggesting that the enzyme is a metalloprotein. This enzyme has been characterized as a ureidoglycolate urea-lyase (EC 4.3.2.3), which catalyzes the degradation of (2) ureidoglycolate to glyoxylate and urea. This is the first time that such an activity is detected in plant tissues. A possible function for this activity and its implications in the context of nitrogen mobilization in legume plants is also discussed.
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Refocusing on a Re-emergent Disease: The Current Global Burden of Cryptococcal Meningitis among Persons Living With HIV/ AIDS
Conference Paper
  • Oct 2008
Background: Cryptococcal meningitis (CM) is one of the most important HIV-related opportunistic infections, especially in the developing world, where overall mortality is very high. In order to help develop global strategies for prevention and treatment, it is important to estimate the burden of CM. Methods: We searched the English literature for studies reporting an estimate of CM among HIV populations. The median CM incidence of each UNAIDS geographic region was multiplied by the HIV population to estimate the region-specific CM cases. The range of cases in each region was calculated as ± one standard deviation from the regional estimate, using a median coefficient of variation across the regions. To estimate deaths, we assumed the 3-month case-fatality rate in high-income regions to be 9%, 55% in low- and middle-income regions, and 70% in Sub-Saharan Africa, based on available literature and expert opinion. Results: Of over 9,000 abstracts reviewed, 19 studies met the search criteria; yearly incidence ranged from 0.04%-12% among persons with HIV. Sub-Saharan Africa had the highest estimate (median incidence 3.2%, 720,000 cases, range, 144,000 - 1.3 million), followed by South/ Southeast Asia (median incidence 3.0%, 120,000 cases, range, 24,000 - 216,000). Median incidence was lowest in Western and Central Europe and Oceania (<0.1% each). Globally, approximately 957,900 cases (range, 371,700 - 1,544,000) of CM occur each year, resulting in 624,700 deaths (range, 125,000 - 1,124,900) by 3 months after infection. Conclusions: This study, the first attempt to estimate the global burden of CM, confirms the high burden of CM, particularly in Sub-Saharan Africa and South/ Southeast Asia. Further work is needed to better define and track the epidemiology of this infection, in order to prioritize prevention, diagnosis, and treatment strategies.
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The complex Arom locus of Aspergillus nidulans
Article
  • Mar 1987
The physical positions of the DNA sequences encoding the five consecutive enzyme activities required to metabolise 3-deoxy-D-arabino-heptulosonic acid-7-phosphate to 5-enolpyruvyl-shikimate-3phosphate, which are encoded by the A. nidulans Arom polypeptide have been determined. Subfragments of the Arom locus encoding EPSP synthase and 3-dehydroquinase have been expressed in appropriate E. coli aro mutants. The DNA sequence of the A. nidulans Arom locus has been shown to have homology with the corresponding unlinked E. coli aro loci strongly suggesting (1) divergent evolution from common ancestral sequences and (11) that the complex A. nidulans Arom locus arose by multiple gene fusion. The DNA and protein sequence of the two 3-dehydroquinase isoenzymes of A. nidulans share no homology, strongly indicating separate phylogenetic origins and their convergent evolution. The 5' and 3' non-translated DNA sequence of the A. nidulans Arom locus is presented along with the presumed sites for transcription initiation and polyadenylation determined by S1 nuclease protection experiments.
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