Comparative Lipidomics of Azole Sensitive and Resistant Clinical Isolates of Candida albicans Reveals Unexpected Diversity in Molecular Lipid Imprints

Although transcriptome and proteome approaches have been applied to determine the regulatory circuitry behind multidrug resistance (MDR) in Candida, its lipidome remains poorly characterized. Lipids do acclimatize to the development of MDR in Candida, but exactly how the acclimatization is achieved is poorly understood. In the present study, we have used a high-throughput mass spectrometry-based shotgun approach and analyzed the lipidome of genetically matched clinical azole-sensitive (AS) and -resistant (AR) isolates of C. albicans. By comparing the lipid profiling of matched isolates, we have identified major classes of lipids and determined more than 200 individual molecular lipid species among these major classes. The lipidome analysis has been statistically validated by principal component analysis. Although each AR isolate was similar with regard to displaying a high MIC to drugs, they had a distinct lipid imprint. There were some significant commonalities in the lipid profiles of these pairs, including molecular lipid species ranging from monounsaturated to polyunsaturated fatty acid-containing phosphoglycerides. Consistent fluctuation in phosphatidyl serine, mannosylinositolphosphorylceramides, and sterol esters levels indicated their compensatory role in maintaining lipid homeostasis among most AR isolates. Notably, overexpression of either CaCdr1p or CaMdr1p efflux pump proteins led to a different lipidomic response among AR isolates. This study clearly establishes the versatility of lipid metabolism in handling azole stress among various matched AR isolates. This comprehensive lipidomic approach will serve as a resource for assessing strategies aimed at disrupting the functions of Candida lipids, particularly the functional interactions between lipids and MDR determinants

Comparative Lipidomics in Clinical Isolates of Candida albicans Reveal Crosstalk between Mitochondria, Cell Wall Integrity and Azole Resistance

Prolonged usage of antifungal azoles which target enzymes involved in lipid biosynthesis invariably leads to the development of multi-drug resistance (MDR) in Candida albicans. We had earlier shown that membrane lipids and their fluidity are closely linked to the MDR phenomenon. In one of our recent studies involving comparative lipidomics between azole susceptible (AS) and azole resistant (AR) matched pair clinical isolates of C. albicans, we could not see consistent differences in the lipid profiles of AS and AR strains because they came from different patients and so in this study, we have used genetically related variant recovered from the same patient collected over a period of 2-years. During this time, the levels of fluconazole (FLC) resistance of the strain increased by over 200-fold. By comparing the lipid profiles of select isolates, we were able to observe gradual and statistically significant changes in several lipid classes, particularly in plasma membrane microdomain specific lipids such as mannosylinositolphosphorylceramides and ergosterol, and in a mitochondrial specific phosphoglyceride, phosphatidyl glycerol. Superimposed with these quantitative and qualitative changes in the lipid profiles, were simultaneous changes at the molecular lipid species levels which again coincided with the development of resistance to FLC. Reverse transcriptase-PCR of the key genes of the lipid metabolism validated lipidomic picture. Taken together, this study illustrates how the gradual corrective changes in Candida lipidome correspond to the development of FLC tolerance. Our study also shows a first instance of the mitochondrial membrane dysfunction and defective cell wall (CW) in clinical AR isolates of C. albicans, and provides evidence of a cross-talk between mitochondrial lipid homeostasis, CW integrity and azole tolerance

Proteomic Analysis of Rta2p-Dependent Raft-Association of Detergent-Resistant Membranes in Candida albicans

In Candida albicans, lipid rafts (also called detergent-resistant membranes, DRMs) are involved in many cellular processes and contain many important proteins. In our previous study, we demonstrated that Rta2p was required for calcineurin-mediated azole resistance and sphingoid long-chain base release in C. albicans. Here, we found that Rta2p was co-localized with raft-constituted ergosterol on the plasma membrane of C. albicans. Furthermore, this membrane expression pattern was totally disturbed by inhibitors of either ergosterol or sphingolipid synthesis. Biochemical fractionation of DRMs together with immunoblot uncovered that Rta2p, along with well-known DRM-associated proteins (Pma1p and Gas1p homologue), was associated with DRMs and their associations were blocked by inhibitors of either ergosterol or sphingolipid synthesis. Finally, we used the proteomic analysis together with immunoblot and identified that Rta2p was required for the association of 10 proteins with DRMs. These 5 proteins (Pma1p, Gas1p homologue, Erg11p, Pmt2p and Ali1p) have been reported to be DRM-associated and also that Erg11p is a well-known target of azoles in C. albicans. In conclusion, our results showed that Rta2p was predominantly localized in lipid rafts and was required for the association of certain membrane proteins with lipid rafts in C. albicans

Candida albicans AGE3, the Ortholog of the S. cerevisiae ARF-GAP-Encoding Gene GCS1, Is Required for Hyphal Growth and Drug Resistance

BACKGROUND: Hyphal growth and multidrug resistance of C. albicans are important features for virulence and antifungal therapy of this pathogenic fungus. METHODOLOGY/PRINCIPAL FINDINGS: Here we show by phenotypic complementation analysis that the C. albicans gene AGE3 is the functional ortholog of the yeast ARF-GAP-encoding gene GCS1. The finding that the gene is required for efficient endocytosis points to an important functional role of Age3p in endosomal compartments. Most C. albicans age3Delta mutant cells which grew as cell clusters under yeast growth conditions showed defects in filamentation under different hyphal growth conditions and were almost completely disabled for invasive filamentous growth. Under hyphal growth conditions only a fraction of age3Delta cells shows a wild-type-like polarization pattern of the actin cytoskeleton and lipid rafts. Moreover, age3Delta cells were highly susceptible to several unrelated toxic compounds including antifungal azole drugs. Irrespective of the AGE3 genotype, C-terminal fusions of GFP to the drug efflux pumps Cdr1p and Mdr1p were predominantly localized in the plasma membrane. Moreover, the plasma membranes of wild-type and age3Delta mutant cells contained similar amounts of Cdr1p, Cdr2p and Mdr1p. CONCLUSIONS/SIGNIFICANCE: The results indicate that the defect in sustaining filament elongation is probably caused by the failure of age3Delta cells to polarize the actin cytoskeleton and possibly of inefficient endocytosis. The high susceptibility of age3Delta cells to azoles is not caused by inefficient transport of efflux pumps to the cell membrane. A possible role of a vacuolar defect of age3Delta cells in drug susceptibility is proposed and discussed. In conclusion, our study shows that the ARF-GAP Age3p is required for hyphal growth which is an important virulence factor of C. albicans and essential for detoxification of azole drugs which are routinely used for antifungal therapy. Thus, it represents a promising antifungal drug target

Efflux in Fungi: La Pièce de Résistance

Pathogens must be able to overcome both host defenses and antimicrobial treatment in order to successfully infect and maintain colonization of the host. One way fungi accomplish this feat and overcome intercellular toxin accumulation is efflux pumps, in particular ATP-binding cassette transporters and transporters of the major facilitator superfamily. Members of these two superfamilies remove many toxic compounds by coupling transport with ATP hydrolysis or a proton gradient, respectively. Fungal genomes encode a plethora of members of these families of transporters compared to other organisms. In this review we discuss the role these two fungal superfamilies of transporters play in virulence and resistance to antifungal agents. These efflux transporters are responsible not only for export of compounds involved in pathogenesis such as secondary metabolites, but also export of host-derived antimicrobial compounds. In addition, we examine the current knowledge of these transporters in resistance of pathogens to clinically relevant antifungal agents

Unexpected effects of azole transporter inhibitors on antifungal susceptibility in Candida glabrata and other pathogenic Candida species

The pathogenic fungus Candida glabrata is often resistant to azole antifungal agents. Drug efflux through azole transporters, such as Cdr1 and Cdr2, is a key mechanism of azole resistance and these genes are under the control of the transcription factor Pdr1. Recently, the monoamine oxidase A (MAO-A) inhibitor clorgyline was shown to inhibit the azole efflux pumps, leading to increased azole susceptibility in C. glabrata. In the present study, we have evaluated the effects of clorgyline on susceptibility of C. glabrata to not only azoles, but also to micafungin and amphotericin B, using wild-type and several mutant strains. The addition of clorgyline to the culture media increased fluconazole susceptibility of a C. glabrata wild-type strain, whereas micafungin and amphotericin B susceptibilities were markedly decreased. These phenomena were also observed in other medically important Candida species, including Candida albicans, Candida parapsilosis, Candida tropicalis, and Candida krusei. Expression levels of CDR1, CDR2 and PDR1 mRNAs and an amount of Cdr1 protein in the C. glabrata wild-type strain were highly increased in response to the treatment with clorgyline. However, loss of Cdr1, Cdr2, Pdr1, and a putative clorgyline target (Fms1), which is an ortholog of human MAO-A, or overexpression of CDR1 did not affect the decreased susceptibility to micafungin and amphotericin B in the presence of clorgyline. The presence of other azole efflux pump inhibitors including milbemycin A4 oxime and carbonyl cyanide 3-chlorophenylhydrazone also decreased micafungin susceptibility in C. glabrata wild-type, Δcdr1, Δcdr2, and Δpdr1 strains. These findings suggest that azole efflux pump inhibitors increase azole susceptibility but concurrently induce decreased susceptibility to other classes of antifungals independent of azole transporter functions

Indexed donor cardiac output for improved size matching in heart transplantation: A United Network for Organ Sharing database analysisCentral MessagePerspective

Objective: Implantation of an appropriately sized donor heart is critical for optimal outcomes after heart transplantation. Although predicted heart mass has recently gained consideration, there remains a need for improved granularity in size matching, particularly among small donor hearts. We sought to determine if indexed donor cardiac output is a sensitive metric to assess the adequacy of a donor heart for a given recipient. Methods: A retrospective analysis was performed (2003-2021) in isolated orthotopic heart transplant recipients from the United Network for Organ Sharing database. Donor cardiac output was divided by recipient body surface area to compute cardiac index (donor cardiac index). Predicted heart mass ratio was computed as donor/recipient predicted heart mass. The primary outcome was mortality 1 year after transplant. Results: Among transplant recipients, median donor cardiac output was 7.3 (5.8-9.0) liters per minute and donor cardiac index was 3.7 (3.0-4.6) liters per minute/m2. Predicted heart mass ratio was 1.01 (0.91-1.13). After multivariable adjustment, higher donor cardiac index was associated with lower 1-year mortality risk (odds ratio, 0.92, P = .042). Recipients with predicted heart mass ratio less than 0.80 (n = 255) had a lower median donor cardiac index than those with a predicted heart mass ratio of 0.80 or greater (3.2 vs 3.7, P < .001). As predicted, heart mass ratio became smaller and the association between donor cardiac index and 1-year mortality became progressively stronger. Conclusions: Higher donor cardiac index was associated with a lower probability of 1-year mortality among patients undergoing heart transplantation and served to further quantify mortality risk among those with a small predicted heart mass ratio. Donor cardiac index appears to be an effective tool for size matching and may serve as an adjunctive strategy among small donor hearts with a low predicted heart mass ratio

Mitigating the Risk: Transfusion or Reoperation for Bleeding After Cardiac Surgery

Background: Several studies have established morbidity associated with bleeding after cardiac surgery. Although reoperation has been implicated as the marker for this morbidity, there remains limited understanding regarding relative morbidities of reoperation and substantial transfusion. Methods: The Society of Thoracic Surgeons (STS) Maryland Adult Cardiac Surgery Database (July 2011-September 2018) was reviewed (N = 23,240). Substantial transfusion was defined as requiring greater than the reoperation group median red blood cells (5 units) and non-red blood cells (4 units). Patients were stratified into 4 subgroups: group 1, no reoperation without substantial transfusion (n = 22,365); group 2, reoperation without substantial transfusion (n = 351); group 3, no reoperation with substantial transfusion (n = 350); and group 4, reoperation with substantial transfusion (n = 167). Operative morbidity and mortality were compared. Results: Reoperation patients were older with a higher STS predicted risk of mortality (1.8% vs 1.2%, P \u3c .001). Multivariable analysis demonstrated that group 4 increased the odds of renal failure (odds ratio [OR] 7.36, P \u3c .001), stroke (OR 3.24, P = .002), and operative mortality (OR 8.68, P \u3c .001) compared with group 1. Both group 2 and group 3 increased the odds of mortality and renal failure compared with group 1. However, group 3 had greater risk for renal failure (OR 3.48, P \u3c .001) and mortality (OR 2.91, P \u3c .001) than group 2. Conclusions: Although reoperation for bleeding is associated with morbidity after cardiac surgery, substantial transfusion without reoperation appears to increase morbidity compared with a limited-transfusion reoperative approach. Better timing for reoperation and guided transfusion approaches may mitigate morbidity compared with substantial transfusion alone

Racial Disparity in Cardiac Surgery Risk and Outcome: Report From a Statewide Quality Initiative

Background: Racial disparities persist in health care. Our study objective was to evaluate racial disparity in cardiac surgery in Maryland. Methods: A statewide database was used to identify patients. Demographics, comorbidities, and predicted risk of death were compared between races. Crude mortality and incidence of complications were compared between groups, as were risk-adjusted odds for mortality and major morbidity or mortality. Results: The study included 23,094 patients. Most patients were white (75.8%), followed by African American (16.3%), Asian (3.8%), and other races (4.1%). African Americans had a higher preoperative risk for mortality based on The Society of Thoracic Surgeons predictive models compared with white patients (3.0% vs 2.3%, P \u3c .001). African Americans also had higher prevalence of diabetes mellitus, hypertension, peripheral vascular disease, and cerebral vascular disease than white patients. After adjustment for preoperative risk, there was no difference in 30-day mortality between African Americans (odds ratio [OR], 1.26; 95% confidence interval [CI], 0.99-1.59), Asians (OR, 1.22; 95% CI, 0.75-1.97), and other races (OR, 1.18; 95% CI, 0.74-1.89) compared with whites. African Americans had lower risk-adjusted odds of major morbidity or mortality compared with whites (OR, 0.83; 95% CI, 0.75-0.93). Conclusions: African American cardiac surgical patients have the highest preoperative risk in Maryland. Patients appeared to receive excellent cardiac surgical care, regardless of race, as risk-adjusted mortality did not differ between groups, and African American patients had lower risk-adjusted odds of major morbidity or mortality than white patients. Future interventions in Maryland should be aimed at reducing preoperative risk disparity in cardiac surgical patients