Research Article: Amino acid permeases in Cryptococcus neoformans are required for high temperature growth and virulence; and are regulated by Ras signaling

Date Published: January 25, 2019

Publisher: Public Library of Science

Author(s): Crislaine Lambiase Calvete, Kevin Felipe Martho, Gabrielle Felizardo, Alexandre Paes, João Miguel Nunes, Camila Oliveira Ferreira, Marcelo A. Vallim, Renata C. Pascon, Yong-Sun Bahn.


Cryptococcosis is an Invasive Fungal Infection (IFI) caused by Cryptococcus neoformans, mainly in immunocompromised patients. Therapeutic failure due to pathogen drug resistance, treatment inconstancy and few antifungal options is a problem. The study of amino acid biosynthesis and uptake represents an opportunity to explore possible development of novel antifungals. C. neoformans has 10 amino acids permeases, two of them (Aap3 and Aap7) not expressed at the conditions tested, and five were studied previously (Aap2, Aap4, Aap5, Mup1 and Mup3). Our previous results showed that Aap4 and Aap5 are major permeases with overlapping functions. The aap4Δ/aap5Δ double mutant fails to grow in amino acids as sole nitrogen source and is avirulent in animal model. Here, we deleted the remaining amino acid permeases (AAP1, AAP6, AAP8) that showed gene expression modulation by nutritional condition and created a double mutant (aap1Δ/aap2Δ). We studied the virulence attributes of these mutants and explored the regulatory mechanism behind amino acid uptake in C. neoformans. The aap1Δ/aap2Δ strain had reduced growth at 37°C in L-amino acids, reduced capsule production and was hypovirulent in the Galleria mellonella animal model. Our data, along with previous studies, (i) complement the analysis for all 10 amino acid permeases mutants, (ii) corroborate the idea that these transporters behave as global permeases, (iii) are required during heat and nutritional stress, and (iv) are important for virulence. Our study also indicates a new possible link between Ras1 signaling and amino acids uptake.

Partial Text

Cryptococcus neoformans is a cosmopolitan yeast with the ability to infect humans and animals. Cryptococcosis is the disease caused by this pathogen, which is a systemic infection, often fatal in immunodeficient population. The disease progression leads to invasion of the Central Nervous System, causing cryptococcal meningitis, which is the second main cause of death after tuberculosis [1–5]. In order to invade the host, C. neoformans expresses several phenotypic features that guarantee successful colonization. In this context, metabolic versatility is an important feature, allowing the pathogen to use several carbon and nitrogen sources, as well as other nutritional elements, such as iron, phosphate, sulfur, amino acids etc., which are important to survival, but they may not be abundant in the animal host [6,7]. Also, the production of a polysaccharide capsule is required to overcome the immune system, and help the yeast to escape phagocytosis by the alveolar macrophages [8]. The ability to cope with stress factors is also an essential characteristic for survival, such as resistance to high temperature, oxidative and osmotic stress [9]. Other virulence factors are considered important, such as, production of urease and phospholipase [10,11]. Failure to express these traits results in avirulence or hypovirulence in animal model as G. mellonella and murine [12–19]. The ability to sporulate through the sexual cycle and haploid fruiting is also very important since the production of spores is linked to the dissemination of C. neoformans and its inhalation is the most common form of contamination [20].

C. neoformans is an opportunistic pathogen that survives in the human body. This adaptation is due to several virulence factors that favor the pathogen establishment in the host [15]. Adaptation to nutritional challenges may be crucial to survival and virulence [7].

Amino acid uptake is an important feature for survival during nutritional (nitrogen and carbon) and heat stress. Failure to assimilate amino acids leads to reduced capsule synthesis and virulence in animal model. In this paper we have found that, besides being important for virulence, amino acid permeases are, at least partially, regulated by the Ras pathway. This is the first report in literature to associate amino acid uptake regulation and a signaling pathway in C. neoformans.




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