Transcriptomics and Phenotyping Define Genetic Signatures Associated with Echinocandin Resistance in Candida auris.

Candida auris emerged as a human fungal pathogen only during the past decade. Remarkably, C. auris displays high degrees of genomic diversity and phenotypic plasticity, with four major clades causing hospital outbreaks with high mortality and morbidity rates. C. auris can show clinical resistance to all classes of antifungal drugs, including echinocandins that are usually recommended as first-line therapies for invasive candidiasis. Here, we exploit transcriptomics coupled with phenotypic profiling to characterize a set of clinical C. auris isolates displaying pronounced echinocandin resistance (ECN-R). A hot spot mutation in the echinocandin FKS1 target gene is present in all resistant isolates. Moreover, ECN-R strains share a core signature set of 362 genes differentially expressed in ECN-R isolates. Among others, mitochondrial gene expression and genes affecting cell wall function appear to be the most prominent, with the latter correlating well with enhanced adhesive traits, increased cell wall mannan content, and altered sensitivity to cell wall stress of ECN-R isolates. Moreover, ECN-R phenotypic signatures were also linked to pathogen recognition and interaction with immune cells. Hence, transcriptomics paired with phenotyping is a suitable tool to predict resistance and fitness traits as well as treatment outcomes in pathogen populations with complex phenotypic diversity. IMPORTANCE The surge in antimicrobial drug resistance in some bacterial and fungal pathogens constitutes a significant challenge to health care facilities. The emerging human fungal pathogen Candida auris has been particularly concerning, as isolates can display pan-antifungal resistance traits against all drugs, including echinocandins. However, the mechanisms underlying this phenotypic diversity remain poorly understood. We identify transcriptomic signatures in C. auris isolates resistant to otherwise fungicidal echinocandins. We identify a set of differentially expressed genes shared by resistant strains compared to unrelated susceptible isolates. Moreover, phenotyping demonstrates that resistant strains show distinct behaviors, with implications for host-pathogen interactions. Hence, this work provides a solid basis to identify the mechanistic links between antifungal multidrug resistance and fitness costs that affect the interaction of C. auris with host immune defenses.

Quantification of zinc intoxication of Candida glabrata after phagocytosis by primary macrophages.

Zinc (Zn2+) is a trace element, playing pivotal roles during host-pathogen interactions. Macrophages can sequester Zn2+ and restrict bioavailability or increase phagolysosomal Zn2+ to kill pathogens. This method quantifies Zn2+-mediated clearance of the human fungal pathogen C. glabrata after phagocytosis by innate immune cells. Double staining with propidium iodide and a zinc-specific fluorescence dye allows for discrimination of live versus dead pathogens inside phagolysosomes. Moreover, elevated phagolysosomal Zn2+ decreases fungal viability as a function of intracellular Zn2+ concentrations in macrophages. For complete details on the use and execution of this protocol, please refer to Riedelberger et al. (2020).

Induction of aquaporin 4-reactive antibodies in Lewis rats immunized with aquaporin 4 mimotopes.

Most cases of neuromyelitis optica spectrum disorders (NMOSD) harbor pathogenic autoantibodies against the water channel aquaporin 4 (AQP4). Binding of these antibodies to AQP4 on astrocytes initiates damage to these cells, which culminates in the formation of large tissue destructive lesions in the central nervous system (CNS). Consequently, untreated patients may become permanently blind or paralyzed. Studies on the induction and breakage of tolerance to AQP4 could be of great benefit for NMOSD patients. So far, however, all attempts to create suitable animal models by active sensitization have failed. We addressed this challenge and identified peptides, which mimic the conformational AQP4 epitopes recognized by pathogenic antibodies of NMOSD patients. Here we show that these mimotopes can induce the production of AQP4-reactive antibodies in Lewis rats. Hence, our results provide a conceptual framework for the formation of such antibodies in NMOSD patients, and aid to improve immunization strategies for the creation of animal models suitable for tolerance studies in this devastating disease.

Type I Interferon Response Dysregulates Host Iron Homeostasis and Enhances Candida glabrata Infection.

Type I interferons (IFNs-I) fulfil multiple protective functions during pathogenic infections, but they can also cause detrimental effects and enhance immunopathology. Here, we report that IFNs-I promote the dysregulation of iron homeostasis in macrophages during systemic infections with the intracellular pathogen Candida glabrata, leading to fungal survival and persistence. By engaging JAK1, IFNs-I disturb the balance of the transcriptional activator NRF2 and repressor BACH1 to induce downregulation of the key iron exporter Fpn1 in macrophages. This leads to enhanced iron accumulation in the phagolysosome and failure to restrict fungal access to iron pools. As a result, C. glabrata acquires iron via the Sit1/Ftr1 iron transporter system, facilitating fungal intracellular replication and immune evasion. Thus, IFNs-I are central regulators of iron homeostasis, which can impact infection, and restricting iron bioavailability may offer therapeutic strategies to combat invasive fungal infections.

Circulating AQP4-specific auto-antibodies alone can induce neuromyelitis optica spectrum disorder in the rat.

It is well established that the binding of pathogenic aquaporin-4 (AQP4)-specific autoantibodies to astrocytes may initiate a cascade of events culminating in the destruction of these cells and in the formation of large tissue-destructive lesions typical for patients with neuromyelitis optica spectrum disorders (NMOSD). To date, not a single experimental study has shown that the systemic presence of the antibody alone can induce any damage to the central nervous system (CNS), while pathological studies on brains of NMOSD patients suggested that there might be ways for antibody entry and subsequent tissue damage. Here, we systemically applied a highly pathogenic, monoclonal antibody with high affinity to AQP4 over prolonged period of time to rats, and show that AQP4-abs can enter the CNS on their own, via circumventricular organs and meningeal or parenchymal blood vessels, that these antibodies initiate the formation of radically different lesions with AQP4 loss, depending on their mode and site of entry, and that lesion formation is much more efficient in the presence of encephalitogenic T-cell responses. We further demonstrate that the established tissue-destructive lesions trigger the formation of additional lesions by short and far reaching effects on blood vessels and their branches, and that AQP4-abs have profound effects on the AQP4 expression in peripheral tissues which counter-act possible titer loss by antibody absorption outside the CNS. Cumulatively, these data indicate that directly induced pathological changes caused by AQP4-abs inside and outside the CNS are efficient drivers of disease evolution in seropositive organisms.