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A flucytosine-responsive Mbp1/Swi4-like protein, Mbs1, plays pleiotropic roles in antifungal drug resistance, stress response, and virulence of Cryptococcus neoformans.

ABSTRACT: Cryptococcosis, caused by the basidiomycetous fungus Cryptococcus neoformans, is responsible for more than 600,000 deaths annually in AIDS patients. Flucytosine is one of the most commonly used antifungal drugs for its treatment, but its resistance and regulatory mechanisms have never been investigated at the genome scale in C. neoformans. In the present study, we performed comparative transcriptome analysis by employing two-component system mutants (tco1? and tco2?) exhibiting opposing flucytosine susceptibility. As a result, a total of 177 flucytosine-responsive genes were identified, and many of them were found to be regulated by Tco1 or Tco2. Among these, we discovered an APSES-like transcription factor, Mbs1 (Mbp1- and Swi4-like protein 1). Expression analysis revealed that MBS1 was regulated in response to flucytosine in a Tco2/Hog1-dependent manner. Supporting this, C. neoformans with the deletion of MBS1 exhibited increased susceptibility to flucytosine. Intriguingly, Mbs1 played pleiotropic roles in diverse cellular processes of C. neoformans. Mbs1 positively regulated ergosterol biosynthesis and thereby affected polyene and azole drug susceptibility. Mbs1 was also involved in genotoxic and oxidative stress responses. Furthermore, Mbs1 promoted production of melanin and capsule and thereby was required for full virulence of C. neoformans. In conclusion, Mbs1 is considered to be a novel antifungal therapeutic target for treatment of cryptococcosis.

SUBMITTER: Song MH

PROVIDER: S-EPMC3255937 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature

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A flucytosine-responsive Mbp1/Swi4-like protein, Mbs1, plays pleiotropic roles in antifungal drug resistance, stress response, and virulence of Cryptococcus neoformans.

Song Min-Hee MH Lee Jang-Won JW Kim Min Su MS Yoon Ja-Kyung JK White Theodore C TC Floyd Anna A Heitman Joseph J Strain Anna K AK Nielsen Judith N JN Nielsen Kirsten K Bahn Yong-Sun YS

Eukaryotic cell 20111111 1

Cryptococcosis, caused by the basidiomycetous fungus Cryptococcus neoformans, is responsible for more than 600,000 deaths annually in AIDS patients. Flucytosine is one of the most commonly used antifungal drugs for its treatment, but its resistance and regulatory mechanisms have never been investigated at the genome scale in C. neoformans. In the present study, we performed comparative transcriptome analysis by employing two-component system mutants (tco1Δ and tco2Δ) exhibiting opposing flucytos ...[more]

PMID: 22080454

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Project description:The target of rapamycin (TOR) pathway is an evolutionarily conserved signal transduction system that is activated by varying nutrient and environmental signals and governs a plethora of eukaryotic biological processes, but its role in Cryptococcus neoformans remains elusive. In this study, we investigated the TOR pathway by functionally characterizing two Tor-like kinases, Tor1 and Tlk1, in C. neoformans. We successfully deleted TLK1, but not TOR1. TLK1 deletion did not result in any evident in vitro phenotypes, suggesting that Tlk1 is mainly dispensable for the growth of C. neoformans. We further demonstrated that Tor1, but not Tlk1, is essential and the target of rapamycin by constructing and analyzing conditionally regulated strains and sporulation analysis of heterozygous mutants in the diploid strain background. To analyze the functions of Tor1 in more detail, we constructed constitutive TOR1 overexpression strains. Tor1 negatively regulated thermotolerance and the DNA damage response, which are two important virulence factors of C. neoformans. We also found that TOR1 overexpression reduced Mpk1 phosphorylation, which is required for cell wall integrity and thermoresistance, and Rad53 phosphorylation, which governs the DNA damage response pathway. Tor1 is localized to the cytoplasm but enriched in the vacuole membrane. Phosphoproteomics and transcriptomics revealed that Tor1 regulates a variety of biological processes, including metabolic processes, cytoskeleton organization, ribosome biogenesis, autophagy, and stress response. TOR inhibition by rapamycin caused actin depolarization in a Tor1-dependent manner. Finally, screening rapamycin-sensitive and -resistant kinase and transcription factor mutants revealed that the TOR pathway may crosstalk with a number of signaling pathways, including the Hog1 pathway. In conclusion, our study demonstrates that a single Tor1 kinase plays a variety of roles in the fungal meningitis pathogen C. neoformans.

Project description:Amoeboid predators, such as amoebae, are proposed to select for survival traits in soil microbes such as Cryptococcus neoformans; these traits can also function in animal virulence by defeating phagocytic immune cells, such as macrophages. Consistent with this notion, incubation of various fungal species with amoebae enhanced their virulence, but the mechanisms involved are unknown. In this study, we exposed three strains of C. neoformans (1 clinical and 2 environmental) to predation by Acanthamoeba castellanii for prolonged times and then analyzed surviving colonies phenotypically and genetically. Surviving colonies comprised cells that expressed either pseudohyphal or yeast phenotypes, which demonstrated variable expression of traits associated with virulence, such as capsule size, urease production, and melanization. Phenotypic changes were associated with aneuploidy and DNA sequence mutations in some amoeba-passaged isolates, but not in others. Mutations in the gene encoding the oligopeptide transporter (CNAG_03013; OPT1) were observed among amoeba-passaged isolates from each of the three strains. Isolates derived from environmental strains gained the capacity for enhanced macrophage toxicity after amoeba selection and carried mutations on the CNAG_00570 gene encoding Pkr1 (AMP-dependent protein kinase regulator) but manifested reduced virulence in mice because they elicited more effective fungal-clearing immune responses. Our results indicate that C. neoformans survival under constant amoeba predation involves the generation of strains expressing pleiotropic phenotypic and genetic changes. Given the myriad potential predators in soils, the diversity observed among amoeba-selected strains suggests a bet-hedging strategy whereby variant diversity increases the likelihood that some will survive predation.IMPORTANCECryptococcus neoformans is a ubiquitous environmental fungus that is also a leading cause of fatal fungal infection in humans, especially among immunocompromised patients. A major question in the field is how an environmental yeast such as C. neoformans becomes a human pathogen when it has no need for an animal host in its life cycle. Previous studies showed that C. neoformans increases its pathogenicity after interacting with its environmental predator amoebae. Amoebae, like macrophages, are phagocytic cells that are considered an environmental training ground for pathogens to resist macrophages, but the mechanism by which C. neoformans changes its virulence through interactions with protozoa is unknown. Our study indicates that fungal survival in the face of amoeba predation is associated with the emergence of pleiotropic phenotypic and genomic changes that increase the chance of fungal survival, with this diversity suggesting a bet-hedging strategy to ensure that some forms survive.

Dataset Information

A flucytosine-responsive Mbp1/Swi4-like protein, Mbs1, plays pleiotropic roles in antifungal drug resistance, stress response, and virulence of Cryptococcus neoformans.

Publications

A flucytosine-responsive Mbp1/Swi4-like protein, Mbs1, plays pleiotropic roles in antifungal drug resistance, stress response, and virulence of Cryptococcus neoformans.

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