Abstract
Metabolic diversity is an important factor during microbial adaptation to different environments. Among metabolic processes, amino acid biosynthesis has been demonstrated to be relevant for survival for many microbial pathogens, whereas the association between pathogenesis and amino acid uptake and recycling are less well-established. Cryptococcus neoformans is an opportunistic fungal pathogen with many habitats. As a result, it faces frequent metabolic shifts and challenges during its life cycle. Here we studied the C. neoformans tryptophan biosynthetic pathway and found that the pathway is essential. RNAi indicated that interruptions in the biosynthetic pathway render strains inviable. However, auxotroph complementation can be partially achieved by tryptophan uptake when a non preferred nitrogen source and lower growth temperature are applied, suggesting that amino acid permeases may be the target of nitrogen catabolism repression (NCR). We used bioinformatics to search for amino acid permeases in the C. neoformans and found eight potential global permeases (AAP1 to AAP8). The transcriptional profile of them revealed that they are subjected to regulatory mechanisms which are known to respond to nutritional status in other fungi, such as (i) quality of nitrogen (Nitrogen Catabolism Repression, NCR) and carbon sources (Carbon Catabolism Repression, CCR), (ii) amino acid availability in the extracellular environment (SPS-sensing) and (iii) nutritional deprivation (Global Amino Acid Control, GAAC). This study shows that C. neoformans has fewer amino acid permeases than other model yeasts, and that these proteins may be subjected to complex regulatory mechanisms. Our data suggest that the C. neoformans tryptophan biosynthetic pathway is an excellent pharmacological target. Furthermore, inhibitors of this pathway cause Cryptococcus growth arrest in vitro.
Highlights
Many microbial pathogens are subjected to rapidly changing environmental conditions and require robust metabolic strategies to survive and succeed during these changes [1,2,3]
The amino acid sequence of these proteins were used as queries to search for homologues in C. neoformans and C. gattii genomes deposited at the Broad Institute and NCBI to gain knowledge about genome organization of this pathway in Cryptococcus
Cryptococcus tryptophan synthase encoded by the TRP5 gene has the same configuration as S. cerevisiae and other fungi, that is, the A and B subunits are encoded by the same gene, and both enzymatic functions are confined to the same protein
Summary
Many microbial pathogens are subjected to rapidly changing environmental conditions and require robust metabolic strategies to survive and succeed during these changes [1,2,3]. Grubii) and C. gattii species—face this type of challenge during their life cycle [4]. Yeasts from the Cryptococcus genus—mainly C. neoformans Both species can be found in vegetables, fruits, trees, soil, decaying wood, and bird guano. They are adapted to the animal and human environment, encountering extreme nutritional and physical-chemical alterations when entering the host [5,6,7]. Grubii) is the most clinically prevalent species, and is the main cause of fungal meningitis in immune suppressed populations, especially patients with HIV/AIDS [5, 10, 11]. In 2009, Park and collaborators evaluated the global burden of cryptococcal meningitis based on the population studied as part of the UN’s 2007 Programme on HIV/AIDS and found that there is an estimated 957,900 new cases per year worldwide (and 624,700 deaths), most of which are in sub-Saharan Africa [12]
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