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.

Occurrence of Lymphangiogenesis in Peripheral Nerve Autografts Contrasts Schwann Cell-Induced Apoptosis of Lymphatic Endothelial Cells In Vitro.

Peripheral nerve injuries pose a major clinical concern world-wide, and functional recovery after segmental peripheral nerve injury is often unsatisfactory, even in cases of autografting. Although it is well established that angiogenesis plays a pivotal role during nerve regeneration, the influence of lymphangiogenesis is strongly under-investigated. In this study, we analyzed the presence of lymphatic vasculature in healthy and regenerated murine peripheral nerves, revealing that nerve autografts contained increased numbers of lymphatic vessels after segmental damage. This led us to elucidate the interaction between lymphatic endothelial cells (LECs) and Schwann cells (SCs) in vitro. We show that SC and LEC secretomes did not influence the respective other cell types' migration and proliferation in 2D scratch assay experiments. Furthermore, we successfully created lymphatic microvascular structures in SC-embedded 3D fibrin hydrogels, in the presence of supporting cells; whereas SCs seemed to exert anti-lymphangiogenic effects when cultured with LECs alone. Here, we describe, for the first time, increased lymphangiogenesis after peripheral nerve injury and repair. Furthermore, our findings indicate a potential lymph-repellent property of SCs, thereby providing a possible explanation for the lack of lymphatic vessels in the healthy endoneurium. Our results highlight the importance of elucidating the molecular mechanisms of SC-LEC interaction.

Transcriptome Signatures Predict Phenotypic Variations of Candida auris.

Health care facilities are facing serious threats by the recently emerging human fungal pathogen Candida auris owing to its pronounced antifungal multidrug resistance and poor diagnostic tools. Distinct C. auris clades evolved seemingly simultaneously at independent geographical locations and display both genetic and phenotypic diversity. Although comparative genomics and phenotypic profiling studies are increasing, we still lack mechanistic knowledge about the C. auris species diversification and clinical heterogeneity. Since gene expression variability impacts phenotypic plasticity, we aimed to characterize transcriptomic signatures of C. auris patient isolates with distinct antifungal susceptibility profiles in this study. First, we employed an antifungal susceptibility screening of clinical C. auris isolates to identify divergent intra-clade responses to antifungal treatments. Interestingly, comparative transcriptional profiling reveals large gene expression differences between clade I isolates and one clade II strain, irrespective of their antifungal susceptibilities. However, comparisons at the clade levels demonstrate that minor changes in gene expression suffice to drive divergent drug responses. Finally, we functionally validate transcriptional signatures reflecting phenotypic divergence of clinical isolates. Thus, our results suggest that large-scale transcriptional profiling allows for predicting phenotypic diversities of patient isolates, which may help choosing suitable antifungal therapies of multidrug-resistant C. auris.

The Two-Component Response Regulator Ssk1 and the Mitogen-Activated Protein Kinase Hog1 Control Antifungal Drug Resistance and Cell Wall Architecture of Candida auris.

Candida auris is an emerging multidrug-resistant human fungal pathogen refractory to treatment by several classes of antifungal drugs. Unlike other Candida species, C. auris can adhere to human skin for prolonged periods of time, allowing for efficient skin-to-skin transmission in the hospital environments. However, molecular mechanisms underlying pronounced multidrug resistance and adhesion traits are poorly understood. Two-component signal transduction and mitogen-activated protein (MAP) kinase signaling are important regulators of adherence, antifungal drug resistance, and virulence. Here, we report that genetic removal of SSK1 encoding a response regulator and the mitogen-associated protein kinase HOG1 restores the susceptibility to both amphotericin B (AMB) and caspofungin (CAS) in C. auris clinical strains. The loss of SSK1 and HOG1 alters membrane lipid permeability, cell wall mannan content, and hyperresistance to cell wall-perturbing agents. Interestingly, our data reveal variable functions of SSK1 and HOG1 in different C. auris clinical isolates, suggesting a pronounced genetic plasticity affecting cell wall function, stress adaptation, and multidrug resistance. Taken together, our data suggest that targeting two-component signal transduction systems could be suitable for restoring C. auris susceptibility to antifungal drugs.IMPORTANCECandida auris is an emerging multidrug-resistant (MDR) fungal pathogen that presents a serious global threat to human health. The Centers for Disease Control and Prevention (CDC) have classified C. auris as an urgent threat to public health for the next decade due to its major clinical and economic impact and the lack of effective antifungal drugs and because of future projections concerning new C. auris infections. Importantly, the Global Antimicrobial Resistance Surveillance System (GLASS) has highlighted the need for more robust and efficacious global surveillance schemes enabling the identification and monitoring of antifungal resistance in Candida infections. Despite the clinical relevance of C. auris infections, our overall understanding of its pathophysiology and virulence, its response to human immune surveillance, and the molecular basis of multiple antifungal resistance remains in its infancy. Here, we show a marked phenotypic plasticity of C. auris clinical isolates. Further, we demonstrate critical roles of stress response mechanisms in regulating multidrug resistance and show that cell wall architecture and composition are key elements that determine antifungal drug susceptibilities. Our data promise new therapeutic options to treat drug-refractory C. auris infections.

Type I Interferons Ameliorate Zinc Intoxication of Candida glabrata by Macrophages and Promote Fungal Immune Evasion.

Host and fungal pathogens compete for metal ion acquisition during infectious processes, but molecular mechanisms remain largely unknown. Here, we show that type I interferons (IFNs-I) dysregulate zinc homeostasis in macrophages, which employ metallothionein-mediated zinc intoxication of pathogens as fungicidal response. However, Candida glabrata can escape immune surveillance by sequestering zinc into vacuoles. Interestingly, zinc-loading is inhibited by IFNs-I, because a Janus kinase 1 (JAK1)-dependent suppression of zinc homeostasis affects zinc distribution in macrophages as well as generation of reactive oxygen species (ROS). In addition, systemic fungal infections elicit IFN-I responses that suppress splenic zinc homeostasis, thereby altering macrophage zinc pools that otherwise exert fungicidal actions. Thus, IFN-I signaling inadvertently increases fungal fitness both in vitro and in vivo during fungal infections. Our data reveal an as yet unrecognized role for zinc intoxication in antifungal immunity and suggest that interfering with host zinc homeostasis may offer therapeutic options to treat invasive fungal infections.