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Cryptococcus spp. are environmental fungi that first must adapt to the host environment before they can cause life-threatening meningitis in immunocompromised patients. Host CO 2 concentrations are 100-fold higher than the external environment and strains unable to grow at host CO 2 concentrations are not pathogenic. Using a genetic screening and transcriptional profiling approach, we report that the TOR pathway is critical for C. neoformans adaptation to host CO 2 partly through Ypk1-dependent remodeling of phosphatidylserine asymmetry at the plasma membrane. We also describe a C. neoformans ABC/PDR transporter ( PDR9 ) that is highly expressed in CO 2 -sensitive environmental strains, suppresses CO 2 -induced phosphatidylserine/phospholipid remodeling, and increases susceptibility to host concentrations of CO 2 . Interestingly, regulation of plasma membrane lipid asymmetry by the TOR-Ypk1 axis is distinct in C. neoformans compared to S. cerevisiae . Finally, host CO 2 concentrations suppress the C. neoformans pathways that respond to host temperature (Mpk1) and pH (Rim101), indicating that host adaptation requires a stringent balance among distinct stress responses. Here, Ristow et al show that the environmental fungus Cryptococcus adapts to the higher carbon dioxide levels present in human tissue through activation of the Target-of-Rapamycin stress response pathway leading to reorganization of its outer membrane lipids and host adaptation.

The environmental pathogen Cryptococcus neoformans claims over 180,000 lives each year. Survival of this basidiomycete at host CO 2 concentrations has only recently been considered an important virulence trait. Through screening gene knockout libraries constructed in a CO 2 -tolerant clinical strain, we found mutations leading to CO 2 sensitivity are enriched in pathways activated by heat stress, including calcineurin, Ras1-Cdc24, cell wall integrity, and R egulator of A ce2 and M orphogenesis (RAM). Overexpression of Cbk1, the conserved terminal kinase of the RAM pathway, partially restored defects of these mutants at host CO 2 or temperature levels. In ascomycetes such as Saccharomyces cerevisiae and Candida albicans , transcription factor Ace2 is an important target of Cbk1, activating genes responsible for cell separation. However, no Ace2 homolog or any downstream component of the RAM pathway has been identified in basidiomycetes. Through in vitro evolution and comparative genomics, we characterized mutations in suppressors of cbk1 Δ in C. neoformans that partially rescued defects in CO 2 tolerance, thermotolerance, and morphology. One suppressor is the RNA translation repressor Ssd1, which is highly conserved in ascomycetes and basidiomycetes. The other is a novel ribonuclease domain-containing protein, here named PSC1 , which is present in basidiomycetes and humans but surprisingly absent in most ascomycetes. Loss of Ssd1 in cbk1 Δ partially restored cryptococcal ability to survive and amplify in the inhalation and intravenous murine models of cryptococcosis. Our discoveries highlight the overlapping regulation of CO 2 tolerance and thermotolerance, the essential role of the RAM pathway in cryptococcal adaptation to the host condition, and the potential importance of post-transcriptional control of virulence traits in this global pathogen.

The fungal cell wall is a dynamic structure, monitoring and responding to internal and external stimuli. It provides a formidable armor to the fungus. However, in a weakened state, the cell wall also triggers host immune attack when PAMPs, including glucan, chitin, and mannoproteins, are exposed. In this work, we found that the aspartyl peptidase May1 impairs the cell wall of Cryptococcus neoformans and increases the exposure of PAMPs in the acidic environment by reducing the chitosan level. Under acidic conditions, May1 is involved in the degradation of the chitin synthase Chs3, which supplies chitin to be deacetylated to chitosan. Consistently, the severe deficiency of chitosan in acidic pH can be rescued by overexpressing CHS3 . These findings improve our understanding of cell wall remodeling and reveal a potential target to compromise the cell wall integrity in this important fungal pathogen.

Congenic strains have been utilized in numerous model organisms to determine the genetic underpinning of various phenotypic traits. Congenic strains are usually derived after 10 backcrosses to a recipient parent, at which point they are 99.95% genetically identical to the parental strain. In recent decades, congenic pairs have provided an invaluable tool for genetics and molecular biology research in the Cryptococcus neoformans species complex. Here, we summarize the history of Cryptococcus congenic pairs and their application in Cryptococcus research on topics including the impact of the mating type locus on unisexual reproduction, virulence, tissue tropism, uniparental mitochondrial inheritance, and the genetic underpinning of other various traits. We also discuss the limitations of these approaches and other biological questions, which could be explored by employing congenic pairs.

Cryptococcus neoformans is a ubiquitous soil fungus and airborne pathogen that causes over 180,000 deaths each year. Cryptococcus must adapt to host CO 2 levels to cause disease, but the genetic basis for this adaptation is unknown. We utilized quantitative trait loci mapping with 374 progeny from a cross between a CO 2 -tolerant clinical isolate and a CO 2 -sensitive environmental isolate to identify genetic regions regulating CO 2 tolerance. To identify specific quantitative trait genes, we applied fine mapping through bulk segregant analysis of near-isogenic progeny with distinct tolerance levels to CO 2 . We found that virulence among near-isogenic strains in a murine model of cryptococcosis correlated with CO 2 tolerance. Moreover, we discovered that sensitive strains may adapt in vivo to become more CO 2 tolerant and more virulent. These findings highlight the underappreciated role of CO 2 tolerance and its importance in the ability of an opportunistic environmental pathogen to cause disease. Quantitative trait loci mapping reveals that tolerance to host CO 2 is critical for virulence of the human fungal pathogen Cryptococcus neoformans .

Across lower eukaryotes, the transcription factor Crz1 is dephosphorylated by calcineurin, which facilitates Crz1 translocation to the nucleus to regulate gene expression. In the fungal pathogen Cryptococcus neoformans, calcineurin–Crz1 signaling maintains calcium homeostasis, thermotolerance, cell wall integrity, and morphogenesis. How Crz1 distinguishes different stressors and differentially regulates cellular responses is poorly understood. Through monitoring Crz1 subcellular localization over time, we found that Crz1 transiently localizes to granules after exposure to high temperature or calcium. These granules also host the phosphatase calcineurin and Pub1, a ribonucleoprotein stress granule marker, suggesting a role of stress granules in modulating calcineurin–Crz1 signaling. Additionally, we constructed and analyzed an array of Crz1 truncation mutants. We identified the intrinsically disordered regions in Crz1 contribute to proper stress granule localization, nuclear localization, and function. Our results provide the groundwork for further determination of the mechanisms behind the complex regulation of Crz1.

Repeated exposure to cocaine increases dendritic spine density on nucleus accumbens (NAc) neurons. Here the authors show that the small GTPase Rac1 is necessary and sufficient for cocaine-induced behavior and spine changes in NAc neurons, adding support for a causal role for structural plasticity in cocaine-induced behavior. Repeated cocaine administration increases the dendritic arborization of nucleus accumbens neurons, but the underlying signaling events remain unknown. Here we show that repeated exposure to cocaine negatively regulates the active form of Rac1, a small GTPase that controls actin remodeling in other systems. Further, we show, using viral-mediated gene transfer, that overexpression of a dominant negative mutant of Rac1 or local knockout of Rac1 is sufficient to increase the density of immature dendritic spines on nucleus accumbens neurons, whereas overexpression of a constitutively active Rac1 or light activation of a photoactivatable form of Rac1 blocks the ability of repeated cocaine exposure to produce this effect. Downregulation of Rac1 activity likewise promotes behavioral responses to cocaine exposure, with activation of Rac1 producing the opposite effect. These findings establish that Rac1 signaling mediates structural and behavioral plasticity in response to cocaine exposure.

Comprehensive analysis of epigenetic regulation demonstrates a series of complex, interacting chromatin mechanisms by which chronic exposure to opiates downregulates Bdnf gene transcription in the ventral tegmental area. This regulatory cascade is also shown to play a role in controlling opiate-induced behavioral plasticity. Brain-derived neurotrophic factor (BDNF) has a crucial role in modulating neural and behavioral plasticity to drugs of abuse. We found a persistent downregulation of exon-specific Bdnf expression in the ventral tegmental area (VTA) in response to chronic opiate exposure, which was mediated by specific epigenetic modifications at the corresponding Bdnf gene promoters. Exposure to chronic morphine increased stalling of RNA polymerase II at these Bdnf promoters in VTA and altered permissive and repressive histone modifications and occupancy of their regulatory proteins at the specific promoters. Furthermore, we found that morphine suppressed binding of phospho-CREB (cAMP response element binding protein) to Bdnf promoters in VTA, which resulted from enrichment of trimethylated H3K27 at the promoters, and that decreased NURR1 (nuclear receptor related-1) expression also contributed to Bdnf repression and associated behavioral plasticity to morphine. Our findings suggest previously unknown epigenetic mechanisms of morphine-induced molecular and behavioral neuroadaptations.

The family Hantaviridae within the Bunyavirales order comprises tri-segmented negative sense RNA viruses, many of which are rodent-borne emerging pathogens associated with fatal human disease. In contrast, hantavirus infection of corresponding rodent hosts results in inapparent or latent infections, which can be recapitulated in cultured cells that become persistently infected. In this study, we used Tula virus (TULV) to investigate the location of hantavirus replication during early, peak and persistent phases of infection, over a 30-day time course. Using immunofluorescent (IF) microscopy, we showed that the TULV nucleocapsid protein (NP) is distributed within both punctate and filamentous structures, with the latter increasing in size as the infection progresses. Transmission electron microscopy of TULV-infected cell sections revealed these filamentous structures comprised aligned clusters of filament bundles. The filamentous NP-associated structures increasingly co-localized with the Golgi and with the stress granule marker TIA-1 over the infection time course, suggesting a redistribution of these cellular organelles. The analysis of the intracellular distribution of TULV RNAs using fluorescent in-situ hybridization revealed that both genomic and mRNAs co-localized with Golgi-associated filamentous compartments that were positive for TIA. These results show that TULV induces a dramatic reorganization of the intracellular environment, including the establishment of TULV RNA synthesis factories in re-modelled Golgi compartments.

The environmental pathogen Cryptococcus neoformans claims over 180,000 lives each year. Survival of this basidiomycete at host CO.sub.2 concentrations has only recently been considered an important virulence trait. Through screening gene knockout libraries constructed in a CO.sub.2-tolerant clinical strain, we found mutations leading to CO.sub.2 sensitivity are enriched in pathways activated by heat stress, including calcineurin, Ras1-Cdc24, cell wall integrity, and Regulator of Ace2 and Morphogenesis (RAM). Overexpression of Cbk1, the conserved terminal kinase of the RAM pathway, partially restored defects of these mutants at host CO.sub.2 or temperature levels. In ascomycetes such as Saccharomyces cerevisiae and Candida albicans, transcription factor Ace2 is an important target of Cbk1, activating genes responsible for cell separation. However, no Ace2 homolog or any downstream component of the RAM pathway has been identified in basidiomycetes. Through in vitro evolution and comparative genomics, we characterized mutations in suppressors of cbk1[DELTA] in C. neoformans that partially rescued defects in CO.sub.2 tolerance, thermotolerance, and morphology. One suppressor is the RNA translation repressor Ssd1, which is highly conserved in ascomycetes and basidiomycetes. The other is a novel ribonuclease domain-containing protein, here named PSC1, which is present in basidiomycetes and humans but surprisingly absent in most ascomycetes. Loss of Ssd1 in cbk1[DELTA] partially restored cryptococcal ability to survive and amplify in the inhalation and intravenous murine models of cryptococcosis. Our discoveries highlight the overlapping regulation of CO.sub.2 tolerance and thermotolerance, the essential role of the RAM pathway in cryptococcal adaptation to the host condition, and the potential importance of post-transcriptional control of virulence traits in this global pathogen.