Brain glucose induces tolerance of Cryptococcus neoformans to amphotericin B during meningitis.

Antibiotic tolerance is the ability of a susceptible population to survive high doses of cidal drugs and has been shown to compromise therapeutic outcomes in bacterial infections. In comparison, whether fungicide tolerance can be induced by host-derived factors during fungal diseases remains largely unknown. Here, through a systematic evaluation of metabolite-drug-fungal interactions in the leading fungal meningitis pathogen, Cryptococcus neoformans, we found that brain glucose induces fungal tolerance to amphotericin B (AmB) in mouse brain tissue and patient cerebrospinal fluid via the fungal glucose repression activator Mig1. Mig1-mediated tolerance limits treatment efficacy for cryptococcal meningitis in mice via inhibiting the synthesis of ergosterol, the target of AmB, and promoting the production of inositolphosphorylceramide, which competes with AmB for ergosterol. Furthermore, AmB combined with an inhibitor of fungal-specific inositolphosphorylceramide synthase, aureobasidin A, shows better efficacy against cryptococcal meningitis in mice than do clinically recommended therapies.

Regulatory basis for reproductive flexibility in a meningitis-causing fungal pathogen.

Pathogenic fungi of the genus Cryptococcus can undergo two sexual cycles, involving either bisexual diploidization (after fusion of haploid cells of different mating type) or unisexual diploidization (by autodiploidization of a single cell). Here, we construct a gene-deletion library for 111 transcription factor genes in Cryptococcus deneoformans, and explore the roles of these regulatory networks in the two reproductive modes. We show that transcription factors crucial for bisexual syngamy induce the expression of known mating determinants as well as other conserved genes of unknown function. Deletion of one of these genes, which we term FMP1, leads to defects in bisexual reproduction in C. deneoformans, its sister species Cryptococcus neoformans, and the ascomycete Neurospora crassa. Furthermore, we show that a recently evolved regulatory cascade mediates pre-meiotic unisexual autodiploidization, supporting that this reproductive process is a recent evolutionary innovation. Our findings indicate that genetic circuits with different evolutionary ages govern hallmark events distinguishing unisexual and bisexual reproduction in Cryptococcus.

Translation Efficiency and Degradation of ER-Associated mRNAs Modulated by ER-Anchored poly(A)-Specific Ribonuclease (PARN).

Translation is spatiotemporally regulated and endoplasmic reticulum (ER)-associated mRNAs are generally in efficient translation. It is unclear whether the ER-associated mRNAs are deadenylated or degraded on the ER surface in situ or in the cytosol. Here, we showed that ER possessed active deadenylases, particularly the poly(A)-specific ribonuclease (PARN), in common cell lines and mouse tissues. Consistently, purified recombinant PARN exhibited a strong ability to insert into the Langmuir monolayer and liposome. ER-anchored PARN was found to be able to reshape the poly(A) length profile of the ER-associated RNAs by suppressing long poly(A) tails without significantly influencing the cytosolic RNAs. The shortening of long poly(A) tails did not affect global translation efficiency, which suggests that the non-specific action of PARN towards long poly(A) tails was beyond the scope of translation regulation on the ER surface. Transcriptome sequencing analysis indicated that the ER-anchored PARN trigged the degradation of a small subset of ER-enriched transcripts. The ER-anchored PARN modulated the translation of its targets by redistributing ribosomes to heavy polysomes, which suggests that PARN might play a role in dynamic ribosome reallocation. During DNA damage response, MK2 phosphorylated PARN-Ser557 to modulate PARN translocation from the ER to cytosol. The ER-anchored PARN modulated DNA damage response and thereby cell viability by promoting the decay of ER-associated MDM2 transcripts with low ribosome occupancy. These findings revealed that highly regulated communication between mRNA degradation rate and translation efficiency is present on the ER surface in situ and PARN might contribute to this communication by modulating the dynamic ribosome reallocation between transcripts with low and high ribosome occupancies.

Cryptococcus neoformans sexual reproduction is controlled by a quorum sensing peptide.

Bacterial quorum sensing is a well-characterized communication system that governs a large variety of collective behaviours. By comparison, quorum sensing regulation in eukaryotic microbes remains poorly understood, especially its functional role in eukaryote-specific behaviours, such as sexual reproduction. Cryptococcus neoformans is a prevalent fungal pathogen that has two defined sexual cycles (bisexual and unisexual) and is a model organism for studying sexual reproduction in fungi. Here, we show that the quorum sensing peptide Qsp1 serves as an important signalling molecule for both forms of sexual reproduction. Qsp1 orchestrates various differentiation and molecular processes, including meiosis, the hallmark of sexual reproduction. It activates bisexual mating, at least in part through the control of pheromone, a signal necessary for bisexual activation. Notably, Qsp1 also plays a major role in the intercellular regulation of unisexual initiation and coordination, in which pheromone is not strictly required. Through a multi-layered genetic screening approach, we identified the atypical zinc finger regulator Cqs2 as an important component of the Qsp1 signalling cascade during both bisexual and unisexual reproduction. The absence of Cqs2 eliminates the Qsp1-stimulated mating response. Together, these findings extend the range of behaviours governed by quorum sensing to sexual development and meiosis.

A deadenylase assay by size-exclusion chromatography.

The shortening of the 3'-end poly(A) tail, also called deadenylation, is crucial to the regulation of mRNA processing, transportation, translation and degradation. The deadenylation process is achieved by deadenylases, which specifically catalyze the removal of the poly(A) tail at the 3'-end of eukaryotic mRNAs and release 5'-AMP as the product. To achieve their physiological functions, all deadenylases have numerous binding partners that may regulate their catalytic properties or recruit them into various protein complexes. To study the effects of various partners, it is important to develop new deadenylase assay that can be applied either in vivo or in vitro. In this research, we developed the deadenylase assay by the size-exclusion chromatography (SEC) method. The SEC analysis indicated that the poly(A) or oligo(A) substrate and the product AMP could be successfully separated and quantified. The enzymatic parameters of deadenylase could be obtained by quantifying the AMP generation. When using the commercial poly(A) as the substrate, a biphasic catalytic process was observed, which might correlate to the two distinct states of poly(A) in the commercial samples. Different lots of commercial poly(A) had dissimilar size distributions and were dissimilar in response to the degradation of deadenylase. The deadenylation pattern, processive or distributive, could also be investigated using the SEC assay by monitoring the status of the substrate and the generation kinetics of AMP and A2. The SEC assay was applicable to both simple samples using the purified enzyme and complex enzyme reaction conditions such as using protein mixtures or crude cell extracts as samples. The influence of solutes with absorption at 254 nm could be successfully eliminated by constructing the different SEC profiles.