DYRK-family kinases regulate Candida albicans morphogenesis and virulence through the Ras1/PKA pathway.

IMPORTANCE: Candida albicans is an opportunistic human fungal pathogen that frequently causes life-threatening infections in immunocompromised individuals. To cause disease, the fungus employs several virulence traits, including its ability to transition between yeast and filamentous states. Previous work identified a role for the kinase Yak1 in regulating C. albicans filamentation. Here, we demonstrate that Yak1 regulates morphogenesis through the canonical cAMP/PKA pathway and that this regulation is environmentally contingent, as host-relevant concentrations of CO2 bypass the requirement of Yak1 for C. albicans morphogenesis. We show a related kinase, Pom1, is important for filamentation in the absence of Yak1 under these host-relevant conditions, as deletion of both genes blocked filamentous growth under all conditions tested. Finally, we demonstrate that Yak1 is required for filamentation in a mouse model of C. albicans dermatitis using genetic and pharmacological approaches. Overall, our results expand our understanding of how Yak1 regulates an important virulence trait in C. albicans.

Multifactor transcriptional control of alternative oxidase induction integrates diverse environmental inputs to enable fungal virulence.

Metabolic flexibility enables fungi to invade challenging host environments. In Candida albicans, a common cause of life-threatening infections in humans, an important contributor to flexibility is alternative oxidase (Aox) activity. Dramatic induction of this activity occurs under respiratory-stress conditions, which impair the classical electron transport chain (ETC). Here, we show that deletion of the inducible AOX2 gene cripples C. albicans virulence in mice by increasing immune recognition. To investigate further, we examined transcriptional regulation of AOX2 in molecular detail under host-relevant, ETC-inhibitory conditions. We found that multiple transcription factors, including Rtg1/Rtg3, Cwt1/Zcf11, and Zcf2, bind and regulate the AOX2 promoter, conferring thousand-fold levels of inducibility to AOX2 in response to distinct environmental stressors. Further dissection of this complex promoter revealed how integration of stimuli ranging from reactive species of oxygen, nitrogen, and sulfur to reduced copper availability is achieved at the transcriptional level to regulate AOX2 induction and enable pathogenesis.

Functional genomic analysis of Candida albicans protein kinases reveals modulators of morphogenesis in diverse environments.

Candida albicans is a leading cause of mycotic infection. The ability to transition between yeast and filamentous forms is critical to C. albicans virulence and complex signaling pathways regulate this process. Here, we screened a C. albicans protein kinase mutant library in six environmental conditions to identify regulators of morphogenesis. We identified the uncharacterized gene orf19.3751 as a negative regulator of filamentation and follow-up investigations implicated a role for orf19.3751 in cell cycle regulation. We also uncovered a dual role for the kinases Ire1 and protein kinase A (Tpk1 and Tpk2) in C. albicans morphogenesis, specifically as negative regulators of wrinkly colony formation on solid medium but positive regulators of filamentation in liquid medium. Further analyses suggested Ire1 modulates morphogenesis in both media states in part through the transcription factor Hac1 and in part through independent mechanisms. Overall, this work provides insights into the signaling governing morphogenesis in C. albicans.

The GARP complex is required for filamentation in Candida albicans.

Candida albicans is an opportunistic fungal pathogen that causes superficial infections in immunocompetent individuals, as well as life-threatening systemic disease in immunocompromised patients. A key virulence trait of this pathogen is its ability to transition between yeast and filamentous morphologies. A functional genomic screen to identify novel regulators of filamentation previously revealed VPS53 as being important for morphogenesis. Vps53 belongs to the Golgi-associated retrograde protein (GARP) complex, which mediates retrograde trafficking from the endosome to the trans-Golgi network. Here, we explored the role of the entire GARP complex in regulating morphogenesis. Deletion of any of the four genes encoding GARP complex subunits severely impaired filamentation in response to diverse filament-inducing cues, including upon internalization by macrophages. Genetic pathway analysis revealed that while hyperactivation of protein kinase A (PKA) signaling is insufficient to drive filamentation in GARP complex mutants, these strains are capable of filamentation upon overexpression of transcriptional activators or upon deletion of transcriptional repressors of hyphal morphogenesis. Finally, compromise of the GARP complex induced lipotoxicity, and pharmacological inhibition of sphingolipid biosynthesis phenocopied genetic compromise of the GARP complex by impairing filamentation. Together, this work identifies the GARP complex as an important mediator of filamentation in response to multiple inducing cues, maps genetic circuitry important for filamentation upon compromise of GARP function, and supports a model whereby GARP deficiency impairs lipid homeostasis, which is important for supporting filamentous growth in C. albicans.

Trial of labor after two cesarean sections: A retrospective case-control study.

AIM: The objective of this study was to compare neonatal and maternal outcomes among women with two previous cesarean deliveries who undergo trial of labor after two cesarean section (TOLA2C) versus elective repeat cesarean delivery (ERCD). Our primary outcome was neonatal intensive care unit (NICU) admission. Secondary outcomes included APGAR score <7 at 5 min, TOLA2C success rate, uterine rupture, postpartum hemorrhage, maternal blood transfusion, maternal bowel and bladder injury, immediate postpartum infection, and maternal mortality. METHODS: This retrospective cohort study was undertaken at a community medical center from January 1, 2008 to December 31, 2018. Inclusion criteria were women with a vertex singleton gestation at term and a history of two prior cesarean sections. Exclusion criteria included a previous successful TOLA2C, prior classical uterine incision or abdominal myomectomy, placenta previa or invasive placentation, multiple gestation, nonvertex presentation, history of uterine rupture or known fetal anomaly. Maternal and neonatal outcomes were assessed using Fisher exact test and Wilcoxon rank sum test. RESULTS: A total of 793 patients fulfilled study criteria. There were no differences in neonatal intensive care unit admissions or 5-min APGAR scores <7 between the two groups. Sixty-eight percent of women who underwent TOLAC (N = 82) had a successful vaginal delivery. The uterine rupture rate was 1.16% (N = 1) in the TOLA2C group with no case of uterine rupture in the ERCD group. No difference in maternal morbidity was noted between the two groups. No maternal or neonatal mortalities occurred in either group. CONCLUSIONS: There was no difference in maternal or neonatal morbidity among patients in our study population with two previous cesarean sections who opted for TOLA2C versus ERCD.

Targeting fungal membrane homeostasis with imidazopyrazoindoles impairs azole resistance and biofilm formation.

Fungal infections cause more than 1.5 million deaths annually. With an increase in immune-deficient susceptible populations and the emergence of antifungal drug resistance, there is an urgent need for novel strategies to combat these life-threatening infections. Here, we use a combinatorial screening approach to identify an imidazopyrazoindole, NPD827, that synergizes with fluconazole against azole-sensitive and -resistant isolates of Candida albicans. NPD827 interacts with sterols, resulting in profound effects on fungal membrane homeostasis and induction of membrane-associated stress responses. The compound impairs virulence in a Caenorhabditis elegans model of candidiasis, blocks C. albicans filamentation in vitro, and prevents biofilm formation in a rat model of catheter infection by C. albicans. Collectively, this work identifies an imidazopyrazoindole scaffold with a non-protein-targeted mode of action that re-sensitizes the leading human fungal pathogen, C. albicans, to azole antifungals.

Mitochondrial perturbation reduces susceptibility to xenobiotics through altered efflux in Candida albicans.

Candida albicans is a leading human fungal pathogen, which can cause superficial infections or life-threatening systemic disease in immunocompromised individuals. The ability to transition between yeast and filamentous forms is a major virulence trait of C. albicans, and a key regulator of this morphogenetic transition is the molecular chaperone Hsp90. To explore the mechanisms governing C. albicans morphogenesis in response to Hsp90 inhibition, we performed a functional genomic screen using the gene replacement and conditional expression collection to identify mutants that are defective in filamentation in response to the Hsp90 inhibitor, geldanamycin. We found that transcriptional repression of genes involved in mitochondrial function blocked filamentous growth in response to the concentration of the Hsp90 inhibitor used in the screen, and this was attributable to increased resistance to the compound. Further exploration revealed that perturbation of mitochondrial function reduced susceptibility to two structurally distinct Hsp90 inhibitors, geldanamycin and radicicol, such that filamentous growth was restored in the mitochondrial mutants by increasing the compound concentration. Deletion of two representative mitochondrial genes, MSU1 and SHY1, enhanced cellular efflux and reduced susceptibility to diverse intracellularly acting compounds. Additionally, screening a C. albicans efflux pump gene deletion library implicated Yor1 in the efflux of geldanamycin and Cdr1, in the efflux of radicicol. Deletion of these transporter genes restored sensitivity to Hsp90 inhibitors in MSU1 and SHY1 homozygous deletion mutants, thereby enabling filamentation. Taken together, our findings suggest that mitochondrial dysregulation elevates cellular efflux and consequently reduces susceptibility to xenobiotics in C. albicans.

Functional connections between cell cycle and proteostasis in the regulation of Candida albicans morphogenesis.

Morphological plasticity is a key virulence trait for many fungal pathogens. For the opportunistic fungal pathogen Candida albicans, transitions among yeast, pseudohyphal, and hyphal forms are critical for virulence, because the morphotypes play distinct roles in the infection process. C. albicans morphogenesis is induced in response to many host-relevant conditions and is regulated by complex signaling pathways and cellular processes. Perturbation of either cell-cycle progression or protein homeostasis induces C. albicans filamentation, demonstrating that these processes play a key role in morphogenetic control. Regulators such as cyclin-dependent kinases, checkpoint proteins, the proteasome, the heat shock protein Hsp90, and the heat shock transcription factor Hsf1 all influence morphogenesis, often through interconnected effects on the cell cycle and proteostasis. This review highlights the major cell-cycle and proteostasis regulators that modulate morphogenesis and discusses how these two processes intersect to regulate this key virulence trait.

The Proteasome Governs Fungal Morphogenesis via Functional Connections with Hsp90 and cAMP-Protein Kinase A Signaling.

Protein homeostasis is critical for proliferation and viability of all organisms. For Candida albicans, protein homeostasis also modulates the transition between yeast and filamentous forms, which is critical for virulence. A key regulator of morphogenesis is the molecular chaperone Hsp90, which mediates proteostasis under physiological and stress conditions. Hsp90 regulates morphogenesis by repressing cyclic AMP-protein kinase A (cAMP-PKA) signaling, such that inhibition of Hsp90 causes filamentation in the absence of an inducing cue. We explored the effect of perturbation of another facet of protein homeostasis and discovered that morphogenesis is also regulated by the proteasome, a large 33-subunit protein complex consisting of a 20S catalytic core and two 19S regulatory particles, which controls degradation of intracellular proteins. We identified a conserved role of the proteasome in morphogenesis as pharmacological inhibition of the proteasome induced filamentation of C. albicans and the related species Candida dubliniensis, Candida tropicalis, Candida krusei, and Candida parapsilosis For C. albicans, genetic depletion of any of 29 subunits of the 19S or 20S particle induced filamentation. Filaments induced by inhibition of either the proteasome or Hsp90 have shared structural characteristics, such as aberrant nuclear content, and shared genetic dependencies, such as intact cAMP-PKA signaling. Consistent with a functional connection between these facets of protein homeostasis that modulate morphogenesis, we observed that proteasome inhibition results in an accumulation of ubiquitinated proteins that overwhelm Hsp90 function, relieving Hsp90-mediated repression of morphogenesis. Together, our findings provide a mechanism whereby interconnected facets of proteostasis regulate C. albicans morphogenesis.IMPORTANCE Fungi cause life-threatening infections and pose a serious threat to human health as there are very few effective antifungal drugs. Candida albicans is a major human fungal pathogen and cause of morbidity and mortality in immunocompromised individuals. A key trait that enables C. albicans virulence is its ability to transition between yeast and filamentous forms. Understanding the mechanisms regulating this virulence trait can facilitate the development of much-needed, novel therapeutic strategies. A key regulator of morphogenesis is the molecular chaperone Hsp90, which is crucial for proteostasis. Here, we expanded our understanding of how proteostasis regulates fungal morphogenesis and identified the proteasome as a repressor of filamentation in C. albicans and related species. Our work suggests that proteasome inhibition overwhelms Hsp90 function, thereby inducing morphogenesis. This work provides a foundation for understanding the role of the proteasome in fungal virulence and offers potential for targeting the proteasome to disarm fungal pathogens.

Meiotic viral attenuation through an ancestral apoptotic pathway.

The programmed release of apoptogenic proteins from mitochondria is a core event of apoptosis, although ancestral roles of this phenomenon are not known. In mammals, one such apoptogenic protein is Endonuclease G (EndoG), a conserved mitochondrial nuclease that fragments the DNA of dying cells. In this work, we show that budding yeast executes meiotically programmed mitochondrial release of an EndoG homolog, Nuc1, during sporulation. In contrast to EndoG's ostensible pro-death function during apoptosis, Nuc1 mitochondrial release is pro-survival, attenuating the cytosolic L-A and Killer double-stranded RNA mycoviruses and protecting meiotic progeny from the catastrophic consequences of their derepression. The protective viral attenuation role of this pathway illuminates a primordial role for mitochondrial release of EndoG, and perhaps of apoptosis itself.

Functional divergence of a global regulatory complex governing fungal filamentation.

Morphogenetic transitions are prevalent in the fungal kingdom. For a leading human fungal pathogen, Candida albicans, the capacity to transition between yeast and filaments is key for virulence. For the model yeast Saccharomyces cerevisiae, filamentation enables nutrient acquisition. A recent functional genomic screen in S. cerevisiae identified Mfg1 as a regulator of morphogenesis that acts in complex with Flo8 and Mss11 to mediate transcriptional responses crucial for filamentation. In C. albicans, Mfg1 also interacts physically with Flo8 and Mss11 and is critical for filamentation in response to diverse cues, but the mechanisms through which it regulates morphogenesis remained elusive. Here, we explored the consequences of perturbation of Mfg1, Flo8, and Mss11 on C. albicans morphogenesis, and identified functional divergence of complex members. We observed that C. albicans Mss11 was dispensable for filamentation, and that overexpression of FLO8 caused constitutive filamentation even in the absence of Mfg1. Harnessing transcriptional profiling and chromatin immunoprecipitation coupled to microarray analysis, we identified divergence between transcriptional targets of Flo8 and Mfg1 in C. albicans. We also established that Flo8 and Mfg1 cooperatively bind to promoters of key regulators of filamentation, including TEC1, for which overexpression was sufficient to restore filamentation in the absence of Flo8 or Mfg1. To further explore the circuitry through which Mfg1 regulates morphogenesis, we employed a novel strategy to select for mutations that restore filamentation in the absence of Mfg1. Whole genome sequencing of filamentation-competent mutants revealed chromosome 6 amplification as a conserved adaptive mechanism. A key determinant of the chromosome 6 amplification is FLO8, as deletion of one allele blocked morphogenesis, and chromosome 6 was not amplified in evolved lineages for which FLO8 was re-located to a different chromosome. Thus, this work highlights rewiring of key morphogenetic regulators over evolutionary time and aneuploidy as an adaptive mechanism driving fungal morphogenesis.