Proline catabolism is a key factor facilitating Candida albicans pathogenicity.

Candida albicans, the primary etiology of human mycoses, is well-adapted to catabolize proline to obtain energy to initiate morphological switching (yeast to hyphal) and for growth. We report that put1-/- and put2-/- strains, carrying defective Proline UTilization genes, display remarkable proline sensitivity with put2-/- mutants being hypersensitive due to the accumulation of the toxic intermediate pyrroline-5-carboxylate (P5C), which inhibits mitochondrial respiration. The put1-/- and put2-/- mutations attenuate virulence in Drosophila and murine candidemia models and decrease survival in human neutrophils and whole blood. Using intravital 2-photon microscopy and label-free non-linear imaging, we visualized the initial stages of C. albicans cells infecting a kidney in real-time, directly deep in the tissue of a living mouse, and observed morphological switching of wildtype but not of put2-/- cells. Multiple members of the Candida species complex, including C. auris, are capable of using proline as a sole energy source. Our results indicate that a tailored proline metabolic network tuned to the mammalian host environment is a key feature of opportunistic fungal pathogens.

Crosstalk between the calcineurin and cell wall integrity pathways prevents chitin overexpression in Candida albicans.

Echinocandins such as caspofungin are frontline antifungal drugs that compromise ß-1,3 glucan synthesis in the cell wall. Recent reports have shown that fungal cells can resist killing by caspofungin by upregulation of chitin synthesis, thereby sustaining cell wall integrity (CWI). When echinocandins are removed, the chitin content of cells quickly returns to basal levels, suggesting that there is a fitness cost associated with having elevated levels of chitin in the cell wall. We show here that simultaneous activation of the calcineurin and CWI pathways generates a subpopulation of Candida albicans yeast cells that have supra-normal chitin levels interspersed throughout the inner and outer cell wall, and that these cells are non-viable, perhaps due to loss of wall elasticity required for cell expansion and growth. Mutations in the Ca2+-calcineurin pathway prevented the formation of these non-viable supra-high chitin cells by negatively regulating chitin synthesis driven by the CWI pathway. The Ca2+-calcineurin pathway may therefore act as an attenuator that prevents the overproduction of chitin by coordinating both chitin upregulation and negative regulation of the CWI signaling pathway. This article has an associated First Person interview with the first author of the paper.

Comparative transcriptomics coupled to developmental grading via transgenic zebrafish reporter strains identifies conserved features in neutrophil maturation.

Neutrophils are evolutionarily conserved innate immune cells playing pivotal roles in host defense. Zebrafish models have contributed substantially to our understanding of neutrophil functions but similarities to human neutrophil maturation have not been systematically characterized, which limits their applicability to studying human disease. Here we show, by generating and analysing transgenic zebrafish strains representing distinct neutrophil differentiation stages, a high-resolution transcriptional profile of neutrophil maturation. We link gene expression at each stage to characteristic transcription factors, including C/ebp-ß, which is important for late neutrophil maturation. Cross-species comparison of zebrafish, mouse, and human samples confirms high molecular similarity of immature stages and discriminates zebrafish-specific from pan-species gene signatures. Applying the pan-species neutrophil maturation signature to RNA-sequencing data from human neuroblastoma patients reveals association between metastatic tumor cell infiltration in the bone marrow and an overall increase in mature neutrophils. Our detailed neutrophil maturation atlas thus provides a valuable resource for studying neutrophil function at different stages across species in health and disease.

Decontamination of High-Efficiency Mask Filters From Respiratory Pathogens Including SARS-CoV-2 by Non-thermal Plasma.

The current pandemic resulted in a rapidly increasing demand for personal protective equipment (PPE) initially leading to severe shortages of these items. Hence, during an unexpected and fast virus spread, the possibility of reusing highly efficient protective equipment could provide a viable solution for keeping both healthcare professionals and the general public equipped and protected. This requires an efficient decontamination technique that preserves functionality of the sensitive materials used for PPE production. Non-thermal plasma (NTP) is a decontamination technique with documented efficiency against select bacterial and fungal pathogens combined with low damage to exposed materials. We have investigated NTP for decontamination of high-efficiency P3 R filters from viral respiratory pathogens in comparison to other commonly used techniques. We show that NTP treatment completely inactivates SARS-CoV-2 and three other common human respiratory viruses including Influenza A, Rhinovirus and Adenovirus, revealing an efficiency comparable to 90°C dry heat or UVC light. Unlike some of the tested techniques (e.g., autoclaving), NTP neither influenced the filtering efficiency nor the microstructure of the filter. We demonstrate that NTP is a powerful and economic technology for efficient decontamination of protective filters and other sensitive materials from different respiratory pathogens.

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.

Intestinal Shedding of SARS-CoV-2 in Children: No Evidence for Infectious Potential.

The clinical courses of COVID-19 in children are often mild and may remain undiagnosed, but prolonged intestinal virus shedding has been documented, thus potentially enabling fecal-oral transmission. However, the infectious potential of SARS-CoV-2 viruses excreted with feces has remained unclear. Here, we investigated 247 stool specimens from 213 pediatric patients to assess the prevalence of intestinal SARS-CoV-2 shedding in hospitalized children without or with COVID-19 and determined the infectious capacity of stool-borne viruses. Upon RT-qPCR screening, the infectivity of virus-positive samples was tested in cell culture using the Vero-E6 permissive cell line. SARS-CoV-2 RNA was detected by RT-qPCR in 32 (13%) stool specimens, but the analysis of virus-positive samples in cell culture revealed no cytopathic effects attributable to SARS-CoV-2-related cell damage. Our findings do not support the notion of potential fecal-oral SARS-CoV-2 spreading, thus questioning the role of hygienic measures designed to prevent this mode of viral transmission.

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.

Pathogenetic Impact of Bacterial-Fungal Interactions.

Polymicrobial infections are of paramount importance because of the potential severity of clinical manifestations, often associated with increased resistance to antimicrobial treatment. The intricate interplay with the host and the immune system, and the impact on microbiome imbalance, are of importance in this context. The equilibrium of microbiota in the human host is critical for preventing potential dysbiosis and the ensuing development of disease. Bacteria and fungi can communicate via signaling molecules, and produce metabolites and toxins capable of modulating the immune response or altering the efficacy of treatment. Most of the bacterial-fungal interactions described to date focus on the human fungal pathogen Candida albicans and different bacteria. In this review, we discuss more than twenty different bacterial-fungal interactions involving several clinically important human pathogens. The interactions, which can be synergistic or antagonistic, both in vitro and in vivo, are addressed with a focus on the quorum-sensing molecules produced, the response of the immune system, and the impact on clinical outcome.

Immunological Identification of Fungal Species.

Immunodetection is described in this chapter as a technique for producing specific antibodies for antigen detection of the major human fungal pathogens. In the case of Candida spp., heat-killed cells are used to immunize mice over a couple of weeks and then splenocytes are isolated and further fused with myelomas to easily propagate the antibodies produced in the mice. The resulting antibodies follow a purification process where antibody levels and concentrations are determined. Fungal cells are also lysed to obtain whole cell extracts as a prior step for identification of antigens using immunoprecipitation. Finally, this method permits the production of specific antibodies against fungi and the identification of the respective antigens in an in vivo model.