Candida albicans is an opportunistic human fungal pathogen that is able to cause both mucosal and systemic infections. It is also a frequent human commensal, where it is typically found inhabiting multiple niches including the gastrointestinal tract. One of the most remarkable features of C. albicans biology is its ability to undergo heritable and reversible switching between different phenotypic states, a phenomenon known as phenotypic switching. This is best exemplified by the white-opaque switch, in which cells undergo epigenetic transitions between two alternative cellular states. Here, we describe assays to quantify the frequency of switching between states, as well as methods to help identify cells in different phenotypic states. We also describe the use of environmental cues that can induce switching into either the white or opaque state. Finally, we introduce the use of mNeonGreen and mScarlet fluorescent proteins that have been optimized for use in C. albicans and which outperform commonly used fluorescent proteins for both fluorescence microscopy and flow cytometry. © 2019 by John Wiley & Sons, Inc.

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Peptide-based pheromones are used throughout the fungal kingdom for coordinating sexual responses between mating partners. Here, we address the properties and function of Bar1, an aspartyl protease that acts as a "barrier" and antagonist to pheromone signaling in multiple species. Candida albicans Bar1 was purified and shown to exhibit preferential cleavage of native α pheromone over pheromones from related fungal species. This result establishes that protease substrate specificity coevolved along with changes in its pheromone target. Pheromone cleavage by Bar1 occurred between residues Thr-5 and Asn-6 in the middle of the tridecapeptide sequence. Surprisingly, proteolytic activity was independent of the amino acid residues present at the scissile bond and instead relied on residues at the C terminus of α pheromone. Unlike most aspartyl proteases, Bar1 also exhibited a near-neutral pH optimum and was resistant to the class-wide inhibitor pepstatin A. In addition, genetic analysis was performed on C. albicans BAR1 and demonstrated that the protease not only regulates endogenous pheromone signaling but also can limit interspecies pheromone signaling. We discuss these findings and propose that the unusual substrate specificity of Bar1 is a consequence of its coevolution with the α pheromone receptor Ste2 for their shared peptide target.

Importance

Pheromones are important for intraspecies communication across the tree of life. In the fungal kingdom, extracellular proteases play a key role in antagonizing pheromone signaling in multiple species. This study examines the properties and function of Candida albicans Bar1, an aspartyl protease that cleaves and thereby inactivates α pheromone. We demonstrate that Bar1 plays important roles in regulating both intra- and interspecies pheromone signaling. The fungal protease shows preferential activity on the endogenous pheromone, but, surprisingly, cleavage activity is dependent on amino acid residues distal to the scissile bond. We propose that the unusual substrate specificity of Bar1 is a direct result of coevolution with Ste2, the receptor for α pheromone, for recognition of the same peptide target. The novel specificity of Bar1 reveals the complex forces shaping the evolution of mating pathways in fungi and uncovers a protease with potentially important applications in the biotechnology industry.

Background: Quality of life (QOL) in hidradenitis suppurativa (HS) patients is negatively impacted by physical and psychosocial problems. The aim of this study was to investigate the frequency and severity of HS-specific symptoms and to correlate these with disease severity. Methods We analyzed medical record data from 145 patients seen in an academic HS specialty clinic between August 2009 to March 2018. Results Hurley stage III patients had significantly higher mean Dermatology Life Quality Index (DLQI) scores (20.2) compared to patients with Hurley stage I (11.3) and II (13.9), (P<0.001 and P=0.001, respectively). More than 75% of patients reported physical symptoms of drainage, irritation, pain, itching, bleeding, and odor. There were associated psychosocial problems of embarrassment and self-consciousness. Symptom severity was most strongly correlated with disease severity for odor (correlation coefficient 0.4, P<0.001), difficulty moving arms (0.323, P<0.001), negative impact on job/school (0.303, P<0.001), and negative impact on relationships (0.298, P<0.001). Conclusion Our results highlight the significant burden of HS and the need for a more comprehensive, HS-specific evaluation tool to better assess the QOL of this patient population. Limitations A small cohort in a single academic center.

Candida albicans can stochastically switch between two phenotypes, white and opaque. Opaque cells are the sexually competent form of C. albicans and therefore undergo efficient polarized growth and mating in the presence of pheromone. In contrast, white cells cannot mate, but are induced - under a specialized set of conditions - to form biofilms in response to pheromone. In this work, we compare the genetic regulation of such "pheromone-stimulated" biofilms with that of "conventional" C. albicans biofilms. In particular, we examined a network of six transcriptional regulators (Bcr1, Brg1, Efg1, Tec1, Ndt80, and Rob1) that mediate conventional biofilm formation for their potential roles in pheromone-stimulated biofilm formation. We show that four of the six transcription factors (Bcr1, Brg1, Rob1, and Tec1) promote formation of both conventional and pheromone-stimulated biofilms, indicating they play general roles in cell cohesion and biofilm development. In addition, we identify the master transcriptional regulator of pheromone-stimulated biofilms as C. albicans Cph1, ortholog of Saccharomyces cerevisiae Ste12. Cph1 regulates mating in C. albicans opaque cells, and here we show that Cph1 is also essential for pheromone-stimulated biofilm formation in white cells. In contrast, Cph1 is dispensable for the formation of conventional biofilms. The regulation of pheromone- stimulated biofilm formation was further investigated by transcriptional profiling and genetic analyses. These studies identified 196 genes that are induced by pheromone signaling during biofilm formation. One of these genes, HGC1, is shown to be required for both conventional and pheromone-stimulated biofilm formation. Taken together, these observations compare and contrast the regulation of conventional and pheromone-stimulated biofilm formation in C. albicans, and demonstrate that Cph1 is required for the latter, but not the former.

©2015. American Geophysical Union. All Rights Reserved. Campaign GPS data collected from 2002 to 2014 result in 41 new site velocities from the San Bernardino Mountains and vicinity. We combined these velocities with 93 continuous GPS velocities and 216 published velocities to obtain a velocity profile across the Pacific-North America plate boundary through the San Bernardino Mountains. We modeled the plate boundary-parallel, horizontal deformation with 5-14 parallel and one obliquely oriented screw dislocations within an elastic half-space. Our rate for the San Bernardino strand of the San Andreas Fault (6.5±3.6mm/yr) is consistent with recently published latest Quaternary rates at the 95% confidence level and is slower than our rate for the San Jacinto Fault (14.1±2.9mm/yr). Our modeled rate for all faults of the Eastern California Shear Zone (ECSZ) combined (15.7±2.9mm/yr) is faster than the summed latest Quaternary rates for these faults, even when an estimate of permanent, off-fault deformation is included. The rate discrepancy is concentrated on faults near the 1992 Landers and 1999 Hector Mine earthquakes; the geodetic and geologic rates agree within uncertainties for other faults within the ECSZ. Coupled with the observation that postearthquake deformation is faster than the pre-1992 deformation, this suggests that the ECSZ geodetic-geologic rate discrepancy is directly related to the timing and location of these earthquakes and is likely the result of viscoelastic deformation in the mantle that varies over the timescale of an earthquake cycle, rather than a redistribution of plate boundary slip at a timescale of multiple earthquake cycles or longer.

The human fungal pathogen Candida albicans can reversibly switch between two cell types named "white" and "opaque," each of which is stable through many cell divisions. These two cell types differ in their ability to mate, their metabolic preferences and their interactions with the mammalian innate immune system. A highly interconnected network of eight transcriptional regulators has been shown to control switching between these two cell types. To identify additional regulators of the switch, we systematically and quantitatively measured white-opaque switching rates of 196 strains, each deleted for a specific transcriptional regulator. We identified 19 new regulators with at least a 10-fold effect on switching rates and an additional 14 new regulators with more subtle effects. To investigate how these regulators affect switching rates, we examined several criteria, including the binding of the eight known regulators of switching to the control region of each new regulatory gene, differential expression of the newly found genes between cell types, and the growth rate of each mutant strain. This study highlights the complexity of the transcriptional network that regulates the white-opaque switch and the extent to which switching is linked to a variety of metabolic processes, including respiration and carbon utilization. In addition to revealing specific insights, the information reported here provides a foundation to understand the highly complex coupling of white-opaque switching to cellular physiology.

Pseudogymnoascus destructans is the causative agent of white-nose syndrome, a disease that has caused the deaths of millions of bats in North America. This psychrophilic fungus proliferates at low temperatures and targets hibernating bats, resulting in their premature arousal from stupor with catastrophic consequences. Despite the impact of white-nose syndrome, little is known about the fungus itself or how it infects its mammalian host. P. destructans is not amenable to genetic manipulation, and therefore understanding the proteins involved in infection requires alternative approaches. Here, we identify hydrolytic enzymes secreted by P. destructans, and use a novel and unbiased substrate profiling technique to define active peptidases. These experiments revealed that endopeptidases are the major proteolytic activities secreted by P. destructans, and that collagen, the major structural protein in mammals, is actively degraded by the secretome. A serine endopeptidase, hereby-named Destructin-1, was subsequently identified, and a recombinant form overexpressed and purified. Biochemical analysis of Destructin-1 showed that it mediated collagen degradation, and a potent inhibitor of peptidase activity was identified. Treatment of P. destructans-conditioned media with this antagonist blocked collagen degradation and facilitated the detection of additional secreted proteolytic activities, including aminopeptidases and carboxypeptidases. These results provide molecular insights into the secretome of P. destructans, and identify serine endopeptidases that have the clear potential to facilitate tissue invasion and pathogenesis in the mammalian host.

Cell identity in eukaryotes is controlled by transcriptional regulatory networks that define cell-type-specific gene expression. In the opportunistic fungal pathogen Candida albicans, transcriptional regulatory networks regulate epigenetic switching between two alternative cell states, 'white' and 'opaque', that exhibit distinct host interactions. In the present study, we reveal that the transcription factors (TFs) regulating cell identity contain prion-like domains (PrLDs) that enable liquid-liquid demixing and the formation of phase-separated condensates. Multiple white-opaque TFs can co-assemble into complex condensates as observed on single DNA molecules. Moreover, heterotypic interactions between PrLDs support the assembly of multifactorial condensates at a synthetic locus within live eukaryotic cells. Mutation of the Wor1 TF revealed that substitution of acidic residues in the PrLD blocked its ability to phase separate and co-recruit other TFs in live cells, as well as its function in C. albicans cell fate determination. Together, these studies reveal that PrLDs support the assembly of TF complexes that control fungal cell identity and highlight parallels with the 'super-enhancers' that regulate mammalian cell fate.

Candida albicans is a commensal fungus that colonizes the human oral cavity and gastrointestinal tract but also causes mucosal as well as invasive disease. The expression of virulence traits in C. albicans clinical isolates is heterogeneous and the genetic basis of this heterogeneity is of high interest. The C. albicans reference strain SC5314 is highly invasive and expresses robust filamentation and biofilm formation relative to many other clinical isolates. Here, we show that SC5314 derivatives are heterozygous for the transcription factor Rob1 and contain an allele with a rare gain-of-function SNP that drives filamentation, biofilm formation, and virulence in a model of oropharyngeal candidiasis. These findings explain, in part, the outlier phenotype of the reference strain and highlight the role heterozygosity plays in the strain-to-strain variation of diploid fungal pathogens.

The Amazon Dense Global Navigational Satellite System (GNSS) Meteorological Network ((ADGMN) provides high spatiotemporal resolution, all-weather precipitable water vapor for studying the evolution of continental tropical and sea-breeze convective regimes of Amazonia. The ADGMN campaign consisted of two experiments: a 6-week campaign in and around Belem, which coincided with the Cloud Processes of the Main Precipitation Systems in Brazil: A Contribution to Cloud-Resolving Modeling and to the Global Precipitation Measurement (CHUVA) and a 1-yr campaign in and around Manaus. The Belem network was composed of 15 GNSS/meteorological stations that provided high-frequency (5 min) PWV data as well as surface meteorological variables For the 6-week duration of the Belem experiment, days were categorized as convective (22 days) or nonconvective (19 days) based solely on a minimum cloud-top temperature of 240 K or below over the central portion of the network and a report of precipitation at at least one site during the afternoon or evening. The Manaus network commenced in April 2011 with 12 GNSS meteorological stations. Local circulations in Manaus driven by anthropogenic deforestation have, in particular, received attention.