Tuning sterol extraction kinetics yields a renal-sparing polyene antifungal.

Decades of previous efforts to develop renal-sparing polyene antifungals were misguided by the classic membrane permeabilization model1. Recently, the clinically vital but also highly renal-toxic small-molecule natural product amphotericin B was instead found to kill fungi primarily by forming extramembraneous sponge-like aggregates that extract ergosterol from lipid bilayers2-6. Here we show that rapid and selective extraction of fungal ergosterol can yield potent and renal-sparing polyene antifungals. Cholesterol extraction was found to drive the toxicity of amphotericin B to human renal cells. Our examination of high-resolution structures of amphotericin B sponges in sterol-free and sterol-bound states guided us to a promising structural derivative that does not bind cholesterol and is thus renal sparing. This derivative was also less potent because it extracts ergosterol more slowly. Selective acceleration of ergosterol extraction with a second structural modification yielded a new polyene, AM-2-19, that is renal sparing in mice and primary human renal cells, potent against hundreds of pathogenic fungal strains, resistance evasive following serial passage in vitro and highly efficacious in animal models of invasive fungal infections. Thus, rational tuning of the dynamics of interactions between small molecules may lead to better treatments for fungal infections that still kill millions of people annually7,8 and potentially other resistance-evasive antimicrobials, including those that have recently been shown to operate through supramolecular structures that target specific lipids9.

Enhanced fungal specificity and in vivo therapeutic efficacy of a C-22-modified FK520 analog against C. neoformans.

IMPORTANCE: Fungal infections cause significant morbidity and mortality globally. The therapeutic armamentarium against these infections is limited, and the development of antifungal drugs has been hindered by the evolutionary conservation between fungi and the human host. With rising resistance to the current antifungal arsenal and an increasing at-risk population, there is an urgent need for the development of new antifungal compounds. The FK520 analogs described in this study display potent antifungal activity as a novel class of antifungals centered on modifying an existing orally active FDA-approved therapy. This research advances the development of much-needed newer antifungal treatment options with novel mechanisms of action.

Enhanced fungal specificity and in vivo therapeutic efficacy of a C-22 modified FK520 analog against C. neoformans.

Fungal infections are of mounting global concern, and the current limited treatment arsenal poses challenges when treating such infections. In particular, infections by Cryptococcus neoformans are associated with high mortality, emphasizing the need for novel therapeutic options. Calcineurin is a protein phosphatase that mediates fungal stress responses, and calcineurin inhibition by the natural product FK506 blocks C. neoformans growth at 37°C. Calcineurin is also required for pathogenesis. However, because calcineurin is conserved in humans, and inhibition with FK506 results in immunosuppression, the use of FK506 as an anti-infective agent is precluded. We previously elucidated the structures of multiple fungal calcineurin-FK506-FKBP12 complexes and implicated the C-22 position on FK506 as a key point for differential modification of ligand inhibition of the mammalian versus fungal target proteins. Through in vitro antifungal and immunosuppressive testing of FK520 (a natural analog of FK506) derivatives, we identified JH-FK-08 as a lead candidate for further antifungal development. JH-FK-08 exhibited significantly reduced immunosuppressive activity and both reduced fungal burden and prolonged survival of infected animals. JH-FK-08 exhibited additive activity in combination with fluconazole in vivo . These findings further advance calcineurin inhibition as an antifungal therapeutic approach. Importance: Fungal infections cause significant morbidity and mortality globally. The therapeutic armamentarium against these infections is limited and development of antifungal drugs has been hindered by the evolutionary conservation between fungi and the human host. With rising resistance to the current antifungal arsenal and an increasing at-risk population, there is an urgent need for the development of new antifungal compounds. The FK520 analogs described in this study display potent antifungal activity as a novel class of antifungals centered on modifying an existing orally-active FDA approved therapy. This research advances the development of much needed newer antifungal treatment options with novel mechanisms of action.

Calcineurin Inhibitor CN585 Exhibits Off-Target Effects in the Human Fungal Pathogen Aspergillus fumigatus.

Calcineurin (CN) is an attractive antifungal target as it is critical for growth, stress response, drug resistance, and virulence in fungal pathogens. The immunosuppressive drugs, tacrolimus (FK506) and cyclosporin A (CsA), are fungistatic and specifically inhibit CN through binding to their respective immunophilins, FK506-binding protein (FKBP12), and cyclophilin (CypA). We are focused on CN structure-based approaches for the development of non-immunosuppressive FK506 analogs as antifungal therapeutics. Here, we examined the effect of the novel CN inhibitor, CN585, on the growth of the human pathogen Aspergillus fumigatus, the most common cause of invasive aspergillosis. Unexpectedly, in contrast to FK506, CN585 exhibited off-target effect on A. fumigatus wild-type and the azole- and echinocandin-resistant strains. Unlike with FK506 and CsA, the A. fumigatus CN, FKBP12, CypA mutants (ΔcnaA, Δfkbp12, ΔcypA) and various FK506-resistant mutants were all sensitive to CN585. Furthermore, in contrast to FK506 the cytosolic to nuclear translocation of the CN-dependent transcription factor (CrzA-GFP) was not inhibited by CN585. Molecular docking of CN585 onto human and A. fumigatus CN complexes revealed differential potential binding sites between human CN versus A. fumigatus CN. Our results indicate CN585 may be a non-specific inhibitor of CN with a yet undefined antifungal mechanism of activity.

Structure-Guided Synthesis of FK506 and FK520 Analogs with Increased Selectivity Exhibit In Vivo Therapeutic Efficacy against Cryptococcus.

Calcineurin is an essential virulence factor that is conserved across human fungal pathogens, including Cryptococcus neoformans, Aspergillus fumigatus, and Candida albicans. Although an excellent target for antifungal drug development, the serine-threonine phosphatase activity of calcineurin is conserved in mammals, and inhibition of this activity results in immunosuppression. FK506 (tacrolimus) is a naturally produced macrocyclic compound that inhibits calcineurin by binding to the immunophilin FKBP12. Previously, our fungal calcineurin-FK506-FKBP12 structure-based approaches identified a nonconserved region of FKBP12 that can be exploited for fungus-specific targeting. These studies led to the design of an FK506 analog, APX879, modified at the C-22 position, which was less immunosuppressive yet maintained antifungal activity. We now report high-resolution protein crystal structures of fungal FKBP12 and a human truncated calcineurin-FKBP12 bound to a natural FK506 analog, FK520 (ascomycin). Based on information from these structures and the success of APX879, we synthesized and screened a novel panel of C-22-modified compounds derived from both FK506 and FK520. One compound, JH-FK-05, demonstrates broad-spectrum antifungal activity in vitro and is nonimmunosuppressive in vivo. In murine models of pulmonary and disseminated C. neoformans infection, JH-FK-05 treatment significantly reduced fungal burden and extended animal survival alone and in combination with fluconazole. Furthermore, molecular dynamic simulations performed with JH-FK-05 binding to fungal and human FKBP12 identified additional residues outside the C-22 and C-21 positions that could be modified to generate novel FK506 analogs with improved antifungal activity. IMPORTANCE Due to rising rates of antifungal drug resistance and a limited armamentarium of antifungal treatments, there is a paramount need for novel antifungal drugs to treat systemic fungal infections. Calcineurin has been established as an essential and conserved virulence factor in several fungi, making it an attractive antifungal target. However, due to the immunosuppressive action of calcineurin inhibitors, they have not been successfully utilized clinically for antifungal treatment in humans. Recent availability of crystal structures of fungal calcineurin-bound inhibitor complexes has enabled the structure-guided design of FK506 analogs and led to a breakthrough in the development of a compound with increased fungal specificity. The development of a calcineurin inhibitor with reduced immunosuppressive activity and maintained therapeutic antifungal activity would add a significant tool to the treatment options for these invasive fungal infections with exceedingly high rates of mortality.

Protein Kinase A Regulates Autophagy-Associated Proteins Impacting Growth and Virulence of Aspergillus fumigatus.

Cellular recycling via autophagy-associated proteins is a key catabolic pathway critical to invasive fungal pathogen growth and virulence in the nutrient-limited host environment. Protein kinase A (PKA) is vital for the growth and virulence of numerous fungal pathogens. However, the underlying basis for its regulation of pathogenesis remains poorly understood in any species. Our Aspergillus fumigatus PKA-dependent whole proteome and phosphoproteome studies employing advanced mass spectroscopic approaches identified numerous previously undefined PKA-regulated proteins in catabolic pathways. Here, we demonstrate reciprocal inhibition of autophagy and PKA activity, and identify 16 autophagy-associated proteins as likely novel PKA-regulated effectors. We characterize the novel PKA-phosphoregulated sorting nexin Atg20, and demonstrate its importance for growth, cell wall stress response, and virulence of A. fumigatus in a murine infection model. Additionally, we identify physical and functional interaction of Atg20 with previously characterized sorting nexin Atg24. Furthermore, we demonstrate the importance of additional uncharacterized PKA-regulated putative autophagy-associated proteins to hyphal growth. Our data presented here indicate that PKA regulates the autophagy pathway much more extensively than previously known, including targeting of novel effector proteins with fungal-specific functions important for invasive disease.

Leveraging Fungal and Human Calcineurin-Inhibitor Structures, Biophysical Data, and Dynamics To Design Selective and Nonimmunosuppressive FK506 Analogs.

Calcineurin is a critical enzyme in fungal pathogenesis and antifungal drug tolerance and, therefore, an attractive antifungal target. Current clinically accessible calcineurin inhibitors, such as FK506, are immunosuppressive to humans, so exploiting calcineurin inhibition as an antifungal strategy necessitates fungal specificity in order to avoid inhibiting the human pathway. Harnessing fungal calcineurin-inhibitor crystal structures, we recently developed a less immunosuppressive FK506 analog, APX879, with broad-spectrum antifungal activity and demonstrable efficacy in a murine model of invasive fungal infection. Our overarching goal is to better understand, at a molecular level, the interaction determinants of the human and fungal FK506-binding proteins (FKBP12) required for calcineurin inhibition in order to guide the design of fungus-selective, nonimmunosuppressive FK506 analogs. To this end, we characterized high-resolution structures of the Mucor circinelloides FKBP12 bound to FK506 and of the Aspergillus fumigatus, M. circinelloides, and human FKBP12 proteins bound to the FK506 analog APX879, which exhibits enhanced selectivity for fungal pathogens. Combining structural, genetic, and biophysical methodologies with molecular dynamics simulations, we identify critical variations in these structurally similar FKBP12-ligand complexes. The work presented here, aimed at the rational design of more effective calcineurin inhibitors, indeed suggests that modifications to the APX879 scaffold centered around the C15, C16, C18, C36, and C37 positions provide the potential to significantly enhance fungal selectivity. IMPORTANCE Invasive fungal infections are a leading cause of death in the immunocompromised patient population. The rise in drug resistance to current antifungals highlights the urgent need to develop more efficacious and highly selective agents. Numerous investigations of major fungal pathogens have confirmed the critical role of the calcineurin pathway for fungal virulence, making it an attractive target for antifungal development. Although FK506 inhibits calcineurin, it is immunosuppressive in humans and cannot be used as an antifungal. By combining structural, genetic, biophysical, and in silico methodologies, we pinpoint regions of the FK506 scaffold and a less immunosuppressive analog, APX879, centered around the C15 to C18 and C36 to C37 positions that could be altered with selective extensions and/or deletions to enhance fungal selectivity. This work represents a significant advancement toward realizing calcineurin as a viable target for antifungal drug discovery.

In Vitro Activity of APX2041, a New Gwt1 Inhibitor, and In Vivo Efficacy of the Prodrug APX2104 against Aspergillus fumigatus.

Invasive aspergillosis (IA) due to Aspergillus fumigatus is a deadly infection for which new antifungal therapies are needed. Here, we demonstrate the efficacy of a Gwt1 inhibitor, APX2041, and its prodrug, APX2104, against A. fumigatus. The wild-type, azole-resistant, and echinocandin-resistant A. fumigatus strains were equally susceptible to APX2041 in vitro. APX2104 treatment in vivo significantly prolonged survival of neutropenic mice challenged with the wild-type and azole-resistant strains, revealing APX2104 as a potentially promising therapeutic against IA.

The Protein Kinase A-Dependent Phosphoproteome of the Human Pathogen Aspergillus fumigatus Reveals Diverse Virulence-Associated Kinase Targets.

Protein kinase A (PKA) signaling plays a critical role in the growth and development of all eukaryotic microbes. However, few direct targets have been characterized in any organism. The fungus Aspergillus fumigatus is a leading infectious cause of death in immunocompromised patients, but the specific molecular mechanisms responsible for its pathogenesis are poorly understood. We used this important pathogen as a platform for a comprehensive and multifaceted interrogation of both the PKA-dependent whole proteome and phosphoproteome in order to elucidate the mechanisms through which PKA signaling regulates invasive microbial disease. Employing advanced quantitative whole-proteomic and phosphoproteomic approaches with two complementary phosphopeptide enrichment strategies, coupled to an independent PKA interactome analysis, we defined distinct PKA-regulated pathways and identified novel direct PKA targets contributing to pathogenesis. We discovered three previously uncharacterized virulence-associated PKA effectors, including an autophagy-related protein, Atg24; a CCAAT-binding transcriptional regulator, HapB; and a CCR4-NOT complex-associated ubiquitin ligase, Not4. Targeted mutagenesis, combined with in vitro kinase assays, multiple murine infection models, structural modeling, and molecular dynamics simulations, was employed to characterize the roles of these new PKA targets in growth, environmental and antimicrobial stress responses, and pathogenesis in a mammalian system. We also elucidated the molecular mechanisms of PKA regulation for these effectors by defining the functionality of phosphorylation at specific PKA target sites. We have comprehensively characterized the PKA-dependent phosphoproteome and validated PKA targets as direct regulators of infectious disease for the first time in any pathogen, providing new insights into PKA signaling and control over microbial pathogenesis.IMPORTANCE PKA is essential for the virulence of eukaryotic human pathogens. Understanding PKA signaling mechanisms is therefore fundamental to deciphering pathogenesis and developing novel therapies. Despite its ubiquitous necessity, specific PKA effectors underlying microbial disease remain unknown. To address this fundamental knowledge gap, we examined the whole-proteomic and phosphoproteomic impacts of PKA on the deadly fungal pathogen Aspergillus fumigatus to uncover novel PKA targets controlling growth and virulence. We also defined the functional consequences of specific posttranslational modifications of these target proteins to characterize the molecular mechanisms of pathogenic effector regulation by PKA. This study constitutes the most comprehensive analysis of the PKA-dependent phosphoproteome of any human pathogen and proposes new and complex roles played by PKA signaling networks in governing infectious disease.

Functional heterogeneity of alveolar macrophage population based on expression of CXCL2.

Alveolar macrophages (AMs) are the major lung-resident macrophages and have contradictory functions. AMs maintain tolerance and tissue homeostasis, but they also initiate strong inflammatory responses. However, such opposing roles within the AM population were not known to be simultaneously generated and coexist. Here, we uncovered heterogeneous AM subpopulations generated in response to two distinct pulmonary fungal infections, Cryptococcus neoformans and Aspergillus fumigatus Some AMs are bona fide sentinel cells that produce chemoattractant CXCL2, which also serves as a marker for AM heterogeneity, in the context of pulmonary fungal infections. However, other AMs do not produce CXCL2 and other pro-inflammatory molecules. Instead, they highly produce anti-inflammatory molecules, including interleukin-10 (IL-10) and complement component 1q (C1q). These two AM subpopulations have distinct metabolic profiles and phagocytic capacities. We report that polarization of pro-inflammatory and anti-inflammatory AM subpopulations is regulated at both epigenetic and transcriptional levels and that these AM subpopulations are generally highly plastic. Our studies have uncovered the role of C1q expression in programming and sustaining anti-inflammatory AMs. Our finding of the AM heterogeneity upon fungal infections suggests a possible pharmacological intervention target to treat fungal infections by tipping the balance of AM subpopulations.

Aspergillus fumigatus Cyp51A and Cyp51B Proteins Are Compensatory in Function and Localize Differentially in Response to Antifungals and Cell Wall Inhibitors.

Triazole antifungals are the primary therapeutic option against invasive aspergillosis. However, resistance to azoles has increased dramatically over the last decade. Azole resistance is known to primarily occur due to point mutations in the azole target protein Cyp51A, one of two paralogous 14-α sterol demethylases found in Aspergillus fumigatus Despite the importance of Cyp51A, little is known about the function of its paralog, Cyp51B, and the behavior of these proteins within the cell or their functional interrelationship. In this study, we addressed two important aspects of the Cyp51 proteins: (i) we characterized their localization patterns under normal growth versus stress conditions, and (ii) we determined how the proteins compensate for each other's absence and respond to azole treatment. Both the Cyp51A and Cyp51B proteins were found to localize in distinct endoplasmic reticulum (ER) domains, including the perinuclear ER and the peripheral ER. Occasionally, the Cyp51 proteins concentrated in the peripheral ER network of tubules along the hyphal septa and at the hyphal tips. Exposure to voriconazole, caspofungin, and Congo red led to significant increases in fluorescence intensity in these alternative localization sites, indicative of Cyp51 protein translocation in response to cell wall stress. Furthermore, deletion of either Cyp51 paralog increased susceptibility to voriconazole, though a greater effect was observed following deletion of cyp51A, indicating a compensatory response to stress conditions.

The class V myosin interactome of the human pathogen Aspergillus fumigatus reveals novel interactions with COPII vesicle transport proteins.

The human fungal pathogen Aspergillus fumigatus causes life-threatening invasive aspergillosis in immunocompromised individuals. Adaptation to the host environment is integral to survival of A. fumigatus and requires the coordination of short- and long-distance vesicular transport to move essential components throughout the fungus. We previously reported the importance of MyoE, the only class V myosin, for hyphal growth and virulence of A. fumigatus. Class V myosins are actin-based, cargo-carrying motor proteins that contain unique binding sites for specific cargo. Specific cargo carried by myosin V has not been identified in any fungus, and previous studies have only identified single components that interact with class V myosins. Here we utilized a mass spectrometry-based whole proteomic approach to identify MyoE interacting proteins in A. fumigatus for the first time. Several proteins previously shown to interact with myosin V through physical and genetic approaches were confirmed, validating our proteomic analysis. Importantly, we identified novel MyoE-interacting proteins, including members of the cytoskeleton network, cell wall synthesis, calcium signaling and a group of coat protein complex II (COPII) proteins involved in the endoplasmic reticulum (ER) to Golgi transport. Furthermore, we analyzed the localization patterns of the COPII proteins, UsoA (Uso1), SrgE (Sec31), and SrgF (Sec23), which suggested a potential role for MyoE in ER to Golgi trafficking.

FKBP12 dimerization mutations effect FK506 binding and differentially alter calcineurin inhibition in the human pathogen Aspergillus fumigatus.

The 12-kDa FK506-binding protein (FKBP12) is the target of the commonly used immunosuppressive drug FK506. The FKBP12-FK506 complex binds to calcineurin and inhibits its activity, leading to immunosuppression and preventing organ transplant rejection. Our recent characterization of crystal structures of FKBP12 proteins in pathogenic fungi revealed the involvement of the 80's loop residue (Pro90) in the active site pocket in self-substrate interaction providing novel evidence on FKBP12 dimerization in vivo. The 40's loop residues have also been shown to be involved in reversible dimerization of FKBP12 in the mammalian and yeast systems. To understand how FKBP12 dimerization affects FK506 binding and influences calcineurin function, we generated Aspergillus fumigatus FKBP12 mutations in the 40's and 50's loop (F37 M/L; W60V). Interestingly, the mutants exhibited variable FK506 susceptibility in vivo indicating differing dimer strengths. In comparison to the 80's loop P90G and V91C mutants, the F37 M/L and W60V mutants exhibited greater FK506 resistance, with the F37M mutation showing complete loss in calcineurin binding in vivo. Molecular dynamics and pulling simulations for each dimeric FKBP12 protein revealed a two-fold increase in dimer strength and significantly higher number of contacts for the F37M, F37L, and W60V mutations, further confirming their varying degree of impact on FK506 binding and calcineurin inhibition in vivo.

Harnessing calcineurin-FK506-FKBP12 crystal structures from invasive fungal pathogens to develop antifungal agents.

Calcineurin is important for fungal virulence and a potential antifungal target, but compounds targeting calcineurin, such as FK506, are immunosuppressive. Here we report the crystal structures of calcineurin catalytic (CnA) and regulatory (CnB) subunits complexed with FK506 and the FK506-binding protein (FKBP12) from human fungal pathogens (Aspergillus fumigatus, Candida albicans, Cryptococcus neoformans and Coccidioides immitis). Fungal calcineurin complexes are similar to the mammalian complex, but comparison of fungal and human FKBP12 (hFKBP12) reveals conformational differences in the 40s and 80s loops. NMR analysis, molecular dynamic simulations, and mutations of the A. fumigatus CnA/CnB-FK506-FKBP12-complex identify a Phe88 residue, not conserved in hFKBP12, as critical for binding and inhibition of fungal calcineurin. These differences enable us to develop a less immunosuppressive FK506 analog, APX879, with an acetohydrazine substitution of the C22-carbonyl of FK506. APX879 exhibits reduced immunosuppressive activity and retains broad-spectrum antifungal activity and efficacy in a murine model of invasive fungal infection.

Calcineurin-dependent dephosphorylation of the transcription factor CrzA at specific sites controls conidiation, stress tolerance, and virulence of Aspergillus fumigatus.

Calcium signaling through calcineurin and its major transcription factor (TF), CrzA, is integral to hyphal growth, stress response and virulence of the pathogenic fungus Aspergillus fumigatus, the leading etiology of invasive aspergillosis. Dephosphorylation of CrzA by calcineurin activates the TF, but the specific phosphorylation sites and their roles in the activation/inactivation mechanism are unknown. Mass spectroscopic analysis identified 20 phosphorylation sites, the majority of which were specific to filamentous fungi and distributed throughout the CrzA protein, with particular concentration in a serine-rich region N-terminal to the conserved DNA-binding domain (DBD). Site-directed mutagenesis of phosphorylated residues revealed that CrzA activity during calcium stimulation can only be suppressed by a high degree of phosphorylation in multiple regions of the protein. Our findings further suggest that this regulation is not solely accomplished through control of CrzA nuclear import. Additionally, we demonstrate the importance of the CrzA phosphorylation state in regulating growth, conidiation, calcium and cell wall stress tolerance, and virulence. Finally, we identify two previously undescribed nuclear localization sequences in the DBD. These findings provide novel insight into the phosphoregulation of CrzA which may be exploited to selectively target A. fumigatus.

Kin1 kinase localizes at the hyphal septum and is dephosphorylated by calcineurin but is dispensable for septation and virulence in the human pathogen Aspergillus fumigatus.

Studies in yeasts have implicated the importance of Kin1 protein kinase, a member of the eukaryotic PAR1/MARK/MELK family, in polarized growth, cell division and septation through coordinated activity with the phosphatase, calcineurin. Kin1 is also required for virulence of the fungal pathogens Cryptococcus neoformans and Fusarium graminearum. Here we show that kin1 deletion in the human fungal pathogen Aspergillus fumigatus does not affect hyphal growth and septation but results in differential susceptibility to antifungals targeting the cell wall and cell membrane. The Δkin1 strain remained virulent in a Galleria mellonella model of invasive aspergillosis. Expression of Kin1 tagged to GFP or RFP showed its stable localization at the septum. Co-localization experiments revealed calcineurin (CnaA) localization on either side of Kin1 at the septum suggesting possible interaction. Bimolecular fluorescence complementation assay confirmed the interaction of Kin1 with CnaA at the hyphal tips and septa in the presence of the antifungal caspofungin. Furthermore, phosphoproteomic analyses for the first time revealed Kin1 as a substrate of calcineurin providing novel insight into Kin1 regulation through calcineurin-mediated dephosphorylation mechanism.

Scanning Quadrupole Data-Independent Acquisition, Part A: Qualitative and Quantitative Characterization.

A novel data-independent acquisition (DIA) method incorporating a scanning quadrupole in front of a collision cell and orthogonal acceleration time-of-flight mass analyzer is described. The method has been characterized for the qualitative and quantitative label-free proteomic analysis of complex biological samples. The principle of the scanning quadrupole DIA method is discussed, and analytical instrument characteristics, such as the quadrupole transmission width, scan/integration time, and chromatographic separation, have been optimized in relation to sample complexity for a number of different model proteomes of varying complexity and dynamic range including human plasma, cell lines, and bacteria. In addition, the technological merits over existing DIA approaches are described and contrasted. The qualitative and semiquantitative performance of the method is illustrated for the analysis of relatively simple protein digest mixtures and a well-characterized human cell line sample using untargeted and targeted search strategies. Finally, the results from a human cell line were compared against publicly available data that used similar chromatographic conditions but were acquired with DDA technology and alternative mass analyzer systems. Qualitative comparison showed excellent concordance of results with >90% overlap of the detected proteins.

Scanning Quadrupole Data-Independent Acquisition, Part B: Application to the Analysis of the Calcineurin-Interacting Proteins during Treatment of Aspergillus fumigatus with Azole and Echinocandin Antifungal Drugs.

Calcineurin is a critical cell-signaling protein that orchestrates growth, stress response, virulence, and antifungal drug resistance in several fungal pathogens. Blocking calcineurin signaling increases the efficacy of several currently available antifungals and suppresses drug resistance. We demonstrate the application of a novel scanning quadrupole DIA method for the analysis of changes in the proteins coimmunoprecipitated with calcineurin during therapeutic antifungal drug treatments of the deadly human fungal pathogen Aspergillus fumigatus. Our experimental design afforded an assessment of the precision of the method as demonstrated by peptide- and protein-centric analysis from eight replicates of the study pool QC samples. Two distinct classes of clinically relevant antifungal drugs that are guideline recommended for the treatment of invasive "aspergillosis" caused by Aspergillus fumigatus, the azoles (voriconazole) and the echinocandins (caspofungin and micafungin), which specifically target the fungal plasma membrane and the fungal cell wall, respectively, were chosen to distinguish variations occurring in the proteins coimmunoprecipitated with calcineurin. Novel potential interactors were identified in response to the different drug treatments that are indicative of the possible role for calcineurin in regulating these effectors. Notably, treatment with voriconazole showed increased immunoprecipitation of key proteins involved in membrane ergosterol biosynthesis with calcineurin. In contrast, echinocandin (caspofungin or micafungin) treatments caused increased immunoprecipitation of proteins involved in cell-wall biosynthesis and septation. Furthermore, abundant coimmunoprecipitation of ribosomal proteins with calcineurin occurred exclusively in echinocandins treatment, indicating reprogramming of cellular growth mechanisms during different antifungal drug treatments. While variations in the observed calcineurin immunoprecipitated proteins may also be due to changes in their expression levels under different drug treatments, this study suggests an important role for calcineurin-dependent cellular mechanisms in response to antifungal treatment of A. fumigatus that warrants future studies.

The tail domain of the Aspergillus fumigatus class V myosin MyoE orchestrates septal localization and hyphal growth.

Myosins are critical motor proteins that contribute to the secretory pathway, polarized growth, and cytokinesis. The globular tail domains of class V myosins have been shown to be important for cargo binding and actin cable organization. Additionally, phosphorylation plays a role in class V myosin cargo choice. Our previous studies on the class V myosin MyoE in the fungal pathogen Aspergillus fumigatus confirmed its requirement for normal morphology and virulence. However, the domains and molecular mechanisms governing the functions of MyoE remain unknown. Here, by analyzing tail mutants, we demonstrate that the tail is required for radial growth, conidiation, septation frequency and MyoE's location at the septum. Furthermore, MyoE is phosphorylated at multiple residues in vivo; however, alanine substitution mutants revealed that no single phosphorylated residue was critical. Importantly, in the absence of the phosphatase calcineurin, an additional residue was phosphorylated in its tail domain. Mutation of this tail residue led to mislocalization of MyoE from the septa. This work reveals the importance of the MyoE tail domain and its phosphorylation/dephosphorylation in the growth and morphology of A. fumigatus.

Phosphorylation of Aspergillus fumigatus PkaR impacts growth and cell wall integrity through novel mechanisms.

Protein kinase A (PKA) signaling is essential for growth and virulence of the fungal pathogen Aspergillus fumigatus. Little is known concerning the regulation of this pathway in filamentous fungi. Employing liquid chromatography-tandem mass spectroscopy, we identified novel phosphorylation sites on the regulatory subunit PkaR, distinct from those previously identified in mammals and yeasts, and demonstrated the importance of two phosphorylation clusters for hyphal growth and cell wall-stress response. We also identified key differences in the regulation of PKA subcellular localization in A. fumigatus compared with other species. This is the first analysis of the phosphoregulation of a PKA regulatory subunit in a filamentous fungus and uncovers critical mechanistic differences between PKA regulation in filamentous fungi compared with mammals and yeast species, suggesting divergent targeting opportunities.