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.

Prospective Evaluation of the Fungitell® (1→3) Beta-D-Glucan Assay as a Diagnostic Tool for Invasive Fungal Disease in Pediatric Allogeneic Hematopoietic Cell Transplantation: A Report from the Children's Oncology Group.

BACKGROUND: Invasive fungal disease (IFD) is a major source of morbidity and mortality for hematopoietic cell transplant (HCT) recipients. Non-invasive biomarkers, such as the beta-D-glucan assay, may improve the diagnosis of IFD. The objective was to define the utility of surveillance testing using Fungitell® beta-D-glucan (BDG) assay in children receiving antifungal prophylaxis in the immediate post-HCT period. METHODS: Weekly surveillance blood testing with the Fungitell® BDG assay was performed during the early post-HCT period in the context of a randomized trial of children, adolescents, and young adults undergoing allogeneic HCT allocated to triazole or caspofungin prophylaxis. Positivity was defined at the manufacturer cutoff of 80 pg/ml. IFD was adjudicated using blinded central reviewers. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated for the Fungitell® BDG assay for the outcome of proven or probable IFD. RESULTS: A total of 51 patients (out of 290 patients in the parent trial) contributed blood specimens. In total, 278 specimens were evaluated. Specificity was 80.8% (95% confidence interval [CI]: 75.6%-85.3%), and NPV was over 99% (95% CI: 86.8%-99.9%). However, there were no true positive results, resulting in sensitivity of 0% (95% CI: 0.0%-84.2%) and PPV of 0% (95% CI: 0.0%-6.7%). CONCLUSIONS: Fungitell® BDG screening is of limited utility in diagnosing IFD in the post-HCT period, mainly due to high false-positive rates. Fungitell® BDG surveillance testing should not be performed in children during the early post-HCT period while receiving antifungal prophylaxis as the pretest probability for IFD is low.

Multicenter Prospective Study of Biomarkers for Diagnosis of Invasive Candidiasis in Children and Adolescents.

BACKGROUND: Diagnosis of invasive candidiasis (IC) relies on insensitive cultures; the relative utility of fungal biomarkers in children is unclear. METHODS: This multinational observational cohort study enrolled patients aged >120 days and <18 years with concern for IC from 1 January 2015 to 26 September 2019 at 25 centers. Blood collected at onset of symptoms was tested using T2Candida, Fungitell (1→3)-ß-D-glucan, Platelia Candida Antigen (Ag) Plus, and Platelia Candida Antibody (Ab) Plus assays. Operating characteristics were determined for each biomarker, and assays meeting a defined threshold considered in combination. Sterile site cultures were the reference standard. RESULTS: Five hundred participants were enrolled at 22 centers in 3 countries, and IC was diagnosed in 13 (2.6%). Thirteen additional blood specimens were collected and successfully spiked with Candida species, to achieve a 5.0% event rate. Valid T2Candida, Fungitell, Platelia Candida Ag Plus, and Platelia Candida Ab Plus assay results were available for 438, 467, 473, and 473 specimens, respectively. Operating characteristics for T2Candida were most optimal for detecting IC due to any Candida species, with results as follows: sensitivity, 80.0% (95% confidence interval, 59.3%-93.2%), specificity 97.1% (95.0%-98.5%), positive predictive value, 62.5% (43.7%-78.9%), and negative predictive value, 98.8% (97.2%-99.6%). Only T2Candida and Platelia Candida Ag Plus assays met the threshold for combination testing. Positive result for either yielded the following results: sensitivity, 86.4% (95% confidence interval, 65.1%- 97.1%); specificity, 94.7% (92.0%-96.7%); positive predictive value, 47.5% (31.5%-63.9%); and negative predictive value, 99.2% (97.7%-99.8%). CONCLUSIONS: T2Candida alone or in combination with Platelia Candida Ag Plus may be beneficial for rapid detection of Candida species in children with concern for IC. CLINICAL TRIALS REGISTRATION: NCT02220790.

Severe Acute Respiratory Syndrome Coronavirus 2 Infections Among Children in the Biospecimens from Respiratory Virus-Exposed Kids (BRAVE Kids) Study.

BACKGROUND: Child with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection typically have mild symptoms that do not require medical attention, leaving a gap in our understanding of the spectrum of SARS-CoV-2-related illnesses that the viruses causes in children. METHODS: We conducted a prospective cohort study of children and adolescents (aged <21 years) with a SARS-CoV-2-infected close contact. We collected nasopharyngeal or nasal swabs at enrollment and tested for SARS-CoV-2 using a real-time polymerase chain reaction assay. RESULTS: Of 382 children, 293 (77%) were SARS-CoV-2-infected. SARS-CoV-2-infected children were more likely to be Hispanic (P < .0001), less likely to have asthma (P = .005), and more likely to have an infected sibling contact (P = .001) than uninfected children. Children aged 6-13 years were frequently asymptomatic (39%) and had respiratory symptoms less often than younger children (29% vs 48%; P = .01) or adolescents (29% vs 60%; P < .001). Compared with children aged 6-13 years, adolescents more frequently reported influenza-like (61% vs 39%; P < .001) , and gastrointestinal (27% vs 9%; P = .002), and sensory symptoms (42% vs 9%; P < .0001) and had more prolonged illnesses (median [interquartile range] duration: 7 [4-12] vs 4 [3-8] days; P = 0.01). Despite the age-related variability in symptoms, wWe found no difference in nasopharyngeal viral load by age or between symptomatic and asymptomatic children. CONCLUSIONS: Hispanic ethnicity and an infected sibling close contact are associated with increased SARS-CoV-2 infection risk among children, while asthma is associated with decreased risk. Age-related differences in clinical manifestations of SARS-CoV-2 infection must be considered when evaluating children for coronavirus disease 2019 and in developing screening strategies for schools and childcare settings.

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.

Safety, Tolerability, and Population Pharmacokinetics of Intravenous and Oral Isavuconazonium Sulfate in Pediatric Patients.

Isavuconazole, administered as the water-soluble prodrug isavuconazonium sulfate, is a new triazole agent used to treat invasive fungal infections. This phase 1 study evaluated the pharmacokinetics (PK), safety, and tolerability of isavuconazole in 46 immunocompromised pediatric patients, stratified by age (1 to <6 [intravenous (i.v.) only], 6 to <12, and 12 to <18 years), receiving 10 mg/kg body weight (maximum, 372 mg) isavuconazonium sulfate either i.v. or orally. A population PK model using weight-based allometric scaling was constructed with the pediatric i.v. and oral data plus i.v. data from a phase 1 study in adults. The best model was a 3-compartment model with combined zero-order and first-order input, with linear elimination. Stepwise covariate modeling was performed in Perl-speaks-NONMEM version 4.7.0. None of the covariates examined, including age, sex, race, and body mass index, were statistically significant for any of the PK parameters. The area under the concentration-time curve at steady state (AUCSS) was predicted for pediatric patients using 1,000 Monte Carlo simulations per age cohort for each administration route. The probability of target attainment (AUCSS range, 60 to 233 µg · h/ml) was estimated; this target range was derived from plasma drug exposures in adults receiving the recommended clinical dose. Predicted plasma drug exposures were within the target range for >80% and >76% of simulated pediatric patients following i.v. or oral administration, respectively. Intravenous and oral administration of isavuconazonium sulfate at the studied dosage of 10 mg/kg was well tolerated and resulted in exposure in pediatric patients similar to that in adults. (This study has been registered at ClinicalTrials.gov under identifier NCT03241550).

Risks and outcomes of adenovirus disease in pediatric hematopoietic stem cell transplant recipients-Comparison of current antiviral treatment options.

BACKGROUND: Adenovirus disease (ADVd) is a significant burden in pediatric hematopoietic stem cell transplant (HSCT) recipients. However, current knowledge of risk factors associated with poor clinical outcome and the effectiveness of antiviral therapy are not well understood. This study determined the relationship between transplant characteristics and risk of ADVd and also compared time to resolution of disease between pediatric patients who did and did not receive antiviral therapy. METHODS: We conducted a retrospective, single-center cohort study of pediatric patients undergoing HSCT at Duke University (2005-2016). Cases of ADVd were defined a priori using a classification tool. Cox proportional hazards (CPH) regression models were used to compare the hazard of ADVd between HSCT recipients differing by type of transplant and type of conditioning regimen. The hazard of time to resolution of ADVd by antiviral therapy (cidofovir, brincidofovir, both, or neither) was compared. RESULTS: Ninety-three of 830 subjects had ADVd post-HSCT (11.2%). Umbilical cord transplant (UCT) recipients had 2.30 (95% CI 1.57, 6.90, P = .002) higher hazard of developing ADVd compared to non-cord allogeneic transplants, and 6.30 higher (95% CI 2.70, 19.61, P < .001) hazard compared to autologous transplants. Subjects who did not receive antiviral therapy experienced earlier resolution of ADVd compared to subjects who received therapy, even after adjusting for subjects with disseminated disease (HR [95% CI]: 3.75 [1.57, 8.93], P = .003). CONCLUSIONS: Pediatric UCT recipients are at a higher risk for ADVd. Antiviral therapy was not associated with an earlier resolution of ADVd, even in patients with higher disease burden.

Guideline for Antibacterial Prophylaxis Administration in Pediatric Cancer and Hematopoietic Stem Cell Transplantation.

BACKGROUND: Bacteremia and other invasive bacterial infections are common among children with cancer receiving intensive chemotherapy and in pediatric recipients of hematopoietic stem cell transplantation (HSCT). Systemic antibacterial prophylaxis is one approach that can be used to reduce the risk of these infections. Our purpose was to develop a clinical practice guideline (CPG) for systemic antibacterial prophylaxis administration in pediatric patients with cancer and those undergoing HSCT. METHODS: An international and multidisciplinary panel was convened with representation from pediatric hematology/oncology and HSCT, pediatric infectious diseases (including antibiotic stewardship), nursing, pharmacy, a patient advocate, and a CPG methodologist. The panel used the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) approach to generate recommendations based on the results of a systematic review of the literature. RESULTS: The systematic review identified 114 eligible randomized trials of antibiotic prophylaxis. The panel made a weak recommendation for systemic antibacterial prophylaxis for children receiving intensive chemotherapy for acute myeloid leukemia and relapsed acute lymphoblastic leukemia (ALL). Weak recommendations against the routine use of systemic antibacterial prophylaxis were made for children undergoing induction chemotherapy for ALL, autologous HSCT and allogeneic HSCT. A strong recommendation against its routine use was made for children whose therapy is not expected to result in prolonged severe neutropenia. If used, prophylaxis with levofloxacin was recommended during severe neutropenia. CONCLUSIONS: We present a CPG for systemic antibacterial prophylaxis administration in pediatric cancer and HSCT patients. Future research should evaluate the long-term effectiveness and adverse effects of prophylaxis.

Revision and Update of the Consensus Definitions of Invasive Fungal Disease From the European Organization for Research and Treatment of Cancer and the Mycoses Study Group Education and Research Consortium.

BACKGROUND: Invasive fungal diseases (IFDs) remain important causes of morbidity and mortality. The consensus definitions of the Infectious Diseases Group of the European Organization for Research and Treatment of Cancer and the Mycoses Study Group have been of immense value to researchers who conduct clinical trials of antifungals, assess diagnostic tests, and undertake epidemiologic studies. However, their utility has not extended beyond patients with cancer or recipients of stem cell or solid organ transplants. With newer diagnostic techniques available, it was clear that an update of these definitions was essential. METHODS: To achieve this, 10 working groups looked closely at imaging, laboratory diagnosis, and special populations at risk of IFD. A final version of the manuscript was agreed upon after the groups' findings were presented at a scientific symposium and after a 3-month period for public comment. There were several rounds of discussion before a final version of the manuscript was approved. RESULTS: There is no change in the classifications of "proven," "probable," and "possible" IFD, although the definition of "probable" has been expanded and the scope of the category "possible" has been diminished. The category of proven IFD can apply to any patient, regardless of whether the patient is immunocompromised. The probable and possible categories are proposed for immunocompromised patients only, except for endemic mycoses. CONCLUSIONS: These updated definitions of IFDs should prove applicable in clinical, diagnostic, and epidemiologic research of a broader range of patients at high-risk.

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.

A multicenter study to define the epidemiology and outcomes of Clostridioides difficile infection in pediatric hematopoietic cell and solid organ transplant recipients.

Hematopoietic cell transplant (HCT) and solid organ transplant (SOT) recipients are at increased risk for Clostridioides difficile infection (CDI). We conducted a multicenter retrospective study to describe the incidence of CDI in children transplanted between January 2010 and June 2013. Nested case-control substudies, matched 1:1 by transplant type, institution, patient age, and time of year (quartile) of transplant, identified CDI risk factors. Cohorts included 1496 HCT and 1090 SOT recipients. Among HCT recipients, 355 CDI episodes were diagnosed in 265 recipients (18.2%). Nested case-control study identified prior history of CDI (odds ratio [OR] 2.6, 95% confidence interval [CI] 1.5-4.7), proton pump inhibitors (PPIs; OR 2.1, 95% CI 1.3-3.4), and exposure to third- (OR 2.4, 95% CI 1.4-4.2) or fourth-generation (OR 2.1, 95% CI 1.2-3.7) cephalosporins as risk factors. Notably, fluoroquinolone exposure appeared protective (OR 0.6, 95% CI 0.3-0.9). Ninety-two episodes of CDI were diagnosed among 79 SOT recipients (7.3%), and exposure to PPIs (OR 2.4, 95% CI 1.1-5.4) and third-generation cephalosporin therapy (OR 3.9, 95% CI 1.4-10.5) were identified as risk factors. Strategies to decrease PPI use and changes in the class of prophylactic antibiotics may impact CDI incidence and warrant further study.

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 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.

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.

Reduction in Mortality after Umbilical Cord Blood Transplantation in Children Over a 20-Year Period (1995-2014).

Infections and graft-versus-host disease (GVHD) have historically resulted in high mortality among children undergoing umbilical cord blood transplantation (UCBT). However, recent advances in clinical practice have likely improved outcomes of these patients. We conducted a retrospective cohort study of children (<18years of age) undergoing UCBT at Duke University between January 1, 1995 and December 31, 2014. We compared 2-year all-cause and cause-specific mortality during 3 time periods based on year of transplantation (1995 to 2001, 2002 to 2007, and 2008 to 2014). We used multivariable Cox regression to identify demographic and UCBT characteristics that were associated with all-cause mortality, transplantation-related mortality, and death from invasive aspergillosis after adjustment for time period. During the 20-year study period 824 children underwent UCBT. Two-year all-cause mortality declined from 48% in 1995 to 2001 to 30% in 2008 to 2014 (P = .0002). White race and nonmalignant UCBT indications were associated with lower mortality. Black children tended to have a higher risk of death for which GVHD (18% versus 11%; P = .06) or graft failure (9% versus 3%; P = .01) were contributory than white children. Comparing 2008 to 2014 with 1995 to 2001, more than half (59%) of the reduced mortality was attributable to a reduction in infectious mortality, with 45% specifically related to reduced mortality from invasive aspergillosis. Antifungal prophylaxis with voriconazole was associated with lower mortality from invasive aspergillosis than low-dose amphotericin B lipid complex (hazard ratio, .09; 95% confidence interval, .01 to .76). With the decline in mortality from invasive aspergillosis, adenovirus and cytomegalovirus have become the most frequentinfectious causes of death in children after UCBT. Advances in clinical practice over the past 20years improved survival of children after UCBT. Reduced mortality from infections, particularly invasive aspergillosis, accounted for the largest improvement in survival and was associated with use of voriconazole for antifungal prophylaxis.

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.

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.

Caspofungin-Mediated Growth Inhibition and Paradoxical Growth in Aspergillus fumigatus Involve Fungicidal Hyphal Tip Lysis Coupled with Regenerative Intrahyphal Growth and Dynamic Changes in ß-1,3-Glucan Synthase Localization.

Caspofungin targets cell wall ß-1,3-glucan synthesis and is the international consensus guideline-recommended salvage therapy for invasive aspergillosis. Although caspofungin is inhibitory at low concentrations, it exhibits a paradoxical effect (reversal of growth inhibition) at high concentrations by an undetermined mechanism. Treatment with caspofungin at either the growth-inhibitory concentration (0.5 µg/ml) or paradoxical growth-inducing concentration (4 µg/ml) for 24 h caused similar abnormalities, including wider, hyperbranched hyphae, increased septation, and repeated hyphal tip lysis, followed by regenerative intrahyphal growth. By 48 h, only hyphae at the colony periphery treated with the high caspofungin concentration displayed paradoxical growth. A similar high concentration of caspofungin also induced the paradoxical growth of Aspergillus fumigatus during human A549 alveolar cell invasion. Localization of the ß-1,3-glucan synthase complex (Fks1 and Rho1) revealed significant differences between cells exposed to the growth-inhibitory and paradoxical growth-inducing concentrations of caspofungin. At both concentrations, Fks1 initially mislocalized from the hyphal tips to vacuoles. However, only continuous exposure to 4 µg/ml of caspofungin for 48 h led to recovery of the normal hyphal morphology with renewed localization of Fks1 to hyphal tips. Rho1 remained at the hyphal tip after treatment with both caspofungin concentrations but was required for paradoxical growth. Farnesol blocked paradoxical growth and relocalized Fks1 and Rho1 to vacuoles. Our results highlight the importance of regenerative intrahyphal growth as a rapid adaptation to the fungicidal lytic effects of caspofungin on hyphal tips and the dynamic localization of Fks1 as part of the mechanism for the caspofungin-mediated paradoxical response in A. fumigatus.