Fungal and host protein persulfidation are functionally correlated and modulate both virulence and antifungal response.

Aspergillus fumigatus is a human fungal pathogen that can cause devastating pulmonary infections, termed "aspergilloses," in individuals suffering immune imbalances or underlying lung conditions. As rapid adaptation to stress is crucial for the outcome of the host-pathogen interplay, here we investigated the role of the versatile posttranslational modification (PTM) persulfidation for both fungal virulence and antifungal host defense. We show that an A. fumigatus mutant with low persulfidation levels is more susceptible to host-mediated killing and displays reduced virulence in murine models of infection. Additionally, we found that a single nucleotide polymorphism (SNP) in the human gene encoding cystathionine γ-lyase (CTH) causes a reduction in cellular persulfidation and correlates with a predisposition of hematopoietic stem cell transplant recipients to invasive pulmonary aspergillosis (IPA), as correct levels of persulfidation are required for optimal antifungal activity of recipients' lung resident host cells. Importantly, the levels of host persulfidation determine the levels of fungal persulfidation, ultimately reflecting a host-pathogen functional correlation and highlighting a potential new therapeutic target for the treatment of aspergillosis.

Engineering Nanogels for Drug Delivery to Pathogenic Fungi Aspergillus fumigatus by Tuning Polymer Amphiphilicity.

Invasive aspergillosis is a serious threat to immunodeficient and critically ill patients caused mainly by the fungus Aspergillus fumigatus. Here, poly(glycidol)-based nanogels (NGs) are proposed as delivery vehicles for antifungal agents for sustained drug release. NGs are formed by simple self-assembly of random copolymers, followed by oxidative cross-linking of thiol functionalities. We investigate the impact of copolymer amphiphilicity on NG interaction with mature fungal hyphae in order to select the optimal drug delivery system for model antifungal drug amphotericin B. The results show that drug-loaded NGs decrease minimal inhibitory concentration (MIC) for around four times and slow down the fungal biofilm synthesis at concentrations lower than MIC. Our results suggest that amphiphilicity of nanoparticle's polymer matrix is an important factor in understanding the action of nanocarriers toward fungal cells and should be considered in the development of nanoparticle-based antifungal therapy.

Mating-type factor-specific regulation of the fumagillin/pseurotin secondary metabolite supercluster in Aspergillus fumigatus.

In the human pathogenic mold Aspergillus fumigatus, sexual identity is determined by the mating-type idiomorphs MAT1-1 and MAT1-2 residing at the MAT locus. Upon crossing of compatible partners, a heterothallic mating is executed to eventually form cleistothecia that contain recombinant ascospores. Given that the MAT1 gene products are DNA binding master regulators that govern this complex developmental process, we monitored the MAT1-driven transcriptomes of A. fumigatus by conditional overexpression of either MAT1 gene followed by RNA-seq analyses. Numerous genes related to the process of mating were found to be under transcriptional control, such as pheromone production and recognition. Substantial differences between the MAT1-1- and MAT1-2-driven transcriptomes could be detected by functional categorization of differentially expressed genes. Moreover, a significant and distinct impact on expression of genetic clusters of secondary metabolism became apparent, which could be verified on the product level. Unexpectedly, specific cross-regulation of the fumagillin/pseurotin supercluster was evident, thereby uncoupling its co-regulatory characteristic. These insights imply a tight interconnection of sexual development accompanied by ascosporogenesis with secondary metabolite production of a pathogenic fungus and impose evolutionary constraints that link these two fundamental aspects of the fungal lifestyle.

The novel Aspergillus fumigatus MAT1-2-4 mating-type gene is required for mating and cleistothecia formation.

Sexual propagation accompanied by recombination and the formation of spore-containing fruiting bodies is a cornerstone of fungal genetics and biology. In the human pathogen Aspergillus fumigatus sexual identity has previously been shown to be determined by MAT1-1-1 or MAT1-2-1 genes which act as transcriptional regulators and are present within idiomorphs found at the MAT locus. We here report the identification and first characterization of a further novel gene, termed MAT1-2-4, that is present in the MAT1-2 idiomorph of A. fumigatus. A mating-type swapping strategy was used to achieve an unbiased deletion of the MAT1-2-4 gene with no impact on MAT1-2-1 gene expression. Phenotypical characterization of the resulting strain revealed an inability to mate with the compatible MAT1-1 progenitor, demonstrating that the MAT1-2-4 gene product is a genuine mating-type factor required for correct sexual development. A GPI-anchored protein of unknown function was identified as interaction partner. However, no functional role in the mating process or ascosporogenesis could be demonstrated by deletion analysis for this latter protein, although a role in heterokaryon formation is suggested. Bioinformatic analysis also demonstrated the presence of MAT1-2-4 homologues in some, but not all, other Aspergillus species and the evolutionary origins and implications of the MAT1-2-4 gene are discussed.

Beyond the Charge: Interplay of Nanogels' Functional Group and Zeta-Potential for Antifungal Drug Delivery to Human Pathogenic Fungus Aspergillus Fumigatus.

The ubiquitous mold Aspergillus fumigatus (A. fumigatus) is one of the main fungal pathogens causing invasive infections in immunocompromised humans. Conventional antifungal agents exhibit limited efficacy and often cause severe side effects. Nanoparticle-based antifungal delivery provides a promising alternative, which can increase local drug concentration; while, mitigating toxicity, thereby enhancing treatment efficacy. Previous research underscores the potential of poly(glycidol)-based nanogels (NG) with negative surface charge as carriers for delivering antifungals to A. fumigatus hyphae. In this study, NG is tailored with 2-carboxyethyl acrylate (CEA) or with phosphoric acid 2-hydroxyethyl acrylate (PHA). It is discovered that quenching with PHA clearly improves the adhesion of NG to hyphal surface and the internalization of NG into the hyphae under protein-rich conditions, surpassing the outcomes of non-quenched and CEA-quenched NG. This enhancement cannot be solely attributed to an increase in negative surface charge but appears to be contingent on the functional group of the quencher. Further, it is demonstrated that itraconazole-loaded, PHA-functionalized nanogels (NGxPHA-ITZ) show lower MIC in vitro and superior therapeutic effect in vivo against A. fumigatus compared to pure itraconazole. This confirms NGxPHA as a promising antifungal delivery system.

Effects of Dietary Supplementation With Clostridium butyricum on Growth Performance, Apparent Digestibility, Blood Metabolites, Ruminal Fermentation and Bacterial Communities of Fattening Goats.

Clostridium butyricum (C. butyricum) is currently widely used to improve the body health and productive performance of monogastric animals. However, there have been few reports on the effects and specific mechanism of action of Clostridium butyricum in ruminants. This study aimed to investigate the effects of Clostridium butyricum supplementation on the growth performance and digestive microbiota of fattening goats. Twenty-four healthy male Albas goats (body weight = 22 ± 2.03 kg) were randomly divided into 3 treatment groups with eight goats in each group. The treatments were as follows: control group (CON) (basal diet, concentrate to forage ratio = 65:35); low-dose Clostridium butyricum (LCB) (basal diet plus 2.0 × 108 CFU/kg Clostridium butyricum); and high-dose Clostridium butyricum (HCB) (basal diet plus 1.0 × 109 CFU/kg Clostridium butyricum). The experiment lasted for 8 weeks after a 2-week adaptation period. Therefore, growth performance and rumen and rectum microbiota were evaluated in goats supplemented with Clostridium butyricum and its metabolites. The results showed that dietary supplementation with Clostridium butyricum significantly increased the pH (P < 0.05), but had no significant effect on growth performance (P > 0.05). Compared with the control group, dietary Clostridium butyricum supplementation significantly increased the relative abundance of Prevotella_1, Christensenellaceae AE_R-7_Group and Prevotellaceae AE_UCG-003 (P < 0.05), and significantly decreased Succiniclasticum and Muribaculaceae_unclassified (P < 0.05). The relative abundance of Clostridium in the rumen was <1.0%. Moreover, 16S rDNA analysis showed that the fecal Clostridium or Clostridium butyricum count was significantly decreased (P < 0.05), and the relative abundance of Alistipes and Akkermansia was increased (P < 0.10) in the low-dose group compared with the control group. Supplementing Clostridium butyricum in a high-concentrate diet did not significantly affect the performance of goats, while regulation of the gastrointestinal microbiota and related metabolites was associated with rumen fermentation.

Direct Visualization of Fungal Burden in Filamentous Fungus-Infected Silkworms.

Invasive fungal infections (IFIs) are difficult to diagnose and to treat and, despite several available antifungal drugs, cause high mortality rates. In the past decades, the incidence of IFIs has continuously increased. More recently, SARS-CoV-2-associated lethal IFIs have been reported worldwide in critically ill patients. Combating IFIs requires a more profound understanding of fungal pathogenicity to facilitate the development of novel antifungal strategies. Animal models are indispensable for studying fungal infections and to develop new antifungals. However, using mammalian animal models faces various hurdles including ethical issues and high costs, which makes large-scale infection experiments extremely challenging. To overcome these limitations, we optimized an invertebrate model and introduced a simple calcofluor white (CW) staining protocol to macroscopically and microscopically monitor disease progression in silkworms (Bombyx mori) infected with the human pathogenic filamentous fungi Aspergillus fumigatus and Lichtheimia corymbifera. This advanced silkworm A. fumigatus infection model could validate knockout mutants with either attenuated, strongly attenuated or unchanged virulence. Finally, CW staining allowed us to efficiently visualize antifungal treatment outcomes in infected silkworms. Conclusively, we here present a powerful animal model combined with a straightforward staining protocol to expedite large-scale in vivo research of fungal pathogenicity and to investigate novel antifungal candidates.

Innovative therapies for invasive fungal infections in preclinical and clinical development.

INTRODUCTION: The incidence of life-threatening invasive fungal infections (IFIs) has increased significantly in recent years. Current therapeutic options for IFIs are limited. Only four major classes of antifungal agents are available to clinicians, namely polyenes, azoles, echinocandins, and flucytosine. These antifungals have particular drawbacks, including toxicity, drug-drug interactions, and increasing antifungal resistance. Consequently, there is an urgent need for new antifungals to combat IFIs. AREAS COVERED: This review illuminates new classes of synthetic antifungal drugs under preclinical and clinical investigations that have novel mechanisms of action; it also examines innovative strategies for the in vivo delivery of antifungal drugs. EXPERT OPINION: It is imperative to expand the pipeline of antifungals to tackle emerging fungal resistance against conventional antimycotic drugs, toxicity, and drug-drug interactions. This unmet medical need should not be underappreciated.

Three-Dimensional Light Sheet Fluorescence Microscopy of Lungs To Dissect Local Host Immune-Aspergillus fumigatus Interactions.

Aspergillus fumigatus is an opportunistic fungal pathogen that can cause life-threatening invasive lung infections in immunodeficient patients. The cellular and molecular processes of infection during onset, establishment, and progression of A. fumigatus infections are highly complex and depend on both fungal attributes and the immune status of the host. Therefore, preclinical animal models are of paramount importance to investigate and gain better insight into the infection process. Yet, despite their extensive use, commonly employed murine models of invasive pulmonary aspergillosis are not well understood due to analytical limitations. Here, we present quantitative light sheet fluorescence microscopy (LSFM) to describe fungal growth and the local immune response in whole lungs at cellular resolution within its anatomical context. We analyzed three very common murine models of pulmonary aspergillosis based on immunosuppression with corticosteroids, chemotherapy-induced leukopenia, or myeloablative irradiation. LSFM uncovered distinct architectures of fungal growth and degrees of tissue invasion in each model. Furthermore, LSFM revealed the spatial distribution, interaction, and activation of two key immune cell populations in antifungal defense: alveolar macrophages and polymorphonuclear neutrophils. Interestingly, the patterns of fungal growth correlated with the detected effects of the immunosuppressive regimens on the local immune cell populations. Moreover, LSFM demonstrates that the commonly used intranasal route of spore administration did not result in complete intra-alveolar deposition, as about 80% of fungal growth occurred outside the alveolar space. Hence, characterization by LSFM is more rigorous than by previously used methods employing murine models of invasive pulmonary aspergillosis and pinpoints their strengths and limitations.IMPORTANCE The use of animal models of infection is essential to advance our understanding of the complex host-pathogen interactions that take place during Aspergillus fumigatus lung infections. As in the case of humans, mice need to suffer an immune imbalance in order to become susceptible to invasive pulmonary aspergillosis (IPA), the most serious infection caused by A. fumigatus There are several immunosuppressive regimens that are routinely used to investigate fungal growth and/or immune responses in murine models of invasive pulmonary aspergillosis. However, the precise consequences of the use of each immunosuppressive model for the local immune populations and for fungal growth are not completely understood. Here, to pin down the scenarios involving commonly used IPA models, we employed light sheet fluorescence microscopy (LSFM) to analyze whole lungs at cellular resolution. Our results will be valuable to optimize and refine animal models to maximize their use in future research.

When green and red mycology meet: Impressions from an interdisciplinary forum on virulence mechanisms of phyto- and human-pathogenic fungi.

Fungal infections pose a constant threat to plants and humans, but detailed knowledge about pathogenesis, immunity, or virulence is rather scarce. Due to the fact that a certain overlap in the armoury of infection exists between plant- and human-pathogenic fungi, an interdisciplinary forum was held in October 2016 at the Institute for Clinical Microbiology, Immunology and Hygiene in Erlangen under the organisational umbrella from two special interest groups of German microbial societies. Scientific exchange and intense discussion of this timely topic was fostered by bringing together renowned experts in their respective fields to present their thoughts and recent findings in the course of a plenary lecture and six themed sessions, accompanied by oral and poster contributions of young researchers. By targeting the topic of fungal virulence mechanisms from various angles and in the context of plant and human hosts, some common grounds and exciting perspectives could be deduced during this vibrant scientific event.

Effect of TERT on the growth of fibrosarcoma via caspase-3, survivin and PKB.

The present study explored the effect of telomerase reverse transcriptase (TERT) on the growth and apoptosis of fibrosarcoma, and investigated the potential molecular signalling pathways underlying its effect. A plasmid was constructed in order to overexpress TERT and siRNA was used to knockdown TERT. The effect of TERT on fibrosarcoma cells in vitro was studied by performing reverse transcription-quantitative PCR and western blotting to determine the expression of p53, survivin, caspase-3, caspase-7 and PKB. Knockdown of TERT suppressed cell growth, decreased fibrosarcoma volume, decreased survivin and PKB expression, and increased caspase-3 expression. The results of the present study suggest that TERT regulates the growth of fibrosarcoma in vitro and in vivo, and that this is associated with the expression of caspase-3 and survivin, in addition to the PKB signalling pathway.