GXMR-CAR containing distinct GXM-specific single-chain variable fragment (scFv) mediated the cell activation against Cryptococcus spp. And had difference in the strength of tonic signaling.

Cryptococcus spp. has a polysaccharide capsule composed of glucuronoxylomannan-GXM, a major virulence factor that can prevent the recognition of fungi by immune cells. Chimeric Antigen Receptor (CAR) redirects T cells to target Cryptococcus spp. as previously demonstrated by a CAR specific to GXM, GXMR-CAR. The current study evaluated the strength of the signal transduction triggered by GXMR-CAR, composed of a distinct antigen-binding domain sourced from a single-chain variable fragment (scFv). GXM-specific scFv derived from mAbs 2H1 and 18B7, 2H1-GXMR-CAR and 18B7-GXMR-CAR, respectively, were designed to express CD8 molecule as hinge/transmembrane, and the costimulatory molecule CD137 (4-1BB) coupled to CD3ζ. The 2H1-GXMR-CAR or 18B7-GXMR-CAR Jurkat cells recognized soluble GXM from C. gattii and C. neoformans, and the levels of IL-2 released by the modified cells did not differ between the GXMR-CAR constructs after exposure to Cryptococcus spp. 18B7-GXMR-CAR triggered tonic signaling was more pronounced in modified Jurkat cells, and a protein kinase inhibitor of the Src family (dasatinib) significantly reduced GXMR-CAR tonic signaling and inhibited cell activation against ligands. 18B7 scFv showed a structural modification of the variable heavy (VH) chain that clarified the difference in the strength of tonic signaling and the level of cell activation between 2H1-GXMR-CAR and 18B7-GXMR-CAR. GXMR-CAR constructs induced T-cell activation against clinical isolates of Cryptococcus spp. and serum from patients with cryptococcosis induced high levels of IL-2, mainly in cells modified with 18B7-GXMR-CAR. Thus, 18B7-GXMR-CAR and 2H1-GXMR-CAR mediated T cell activation against Cryptococcus spp. and 18B7 and 2H1 scFv influenced the strength of tonic signaling.

2H1-GXMR-CAR and 18B7-GXMR-CAR are efficiently expressed on the cell surface;2H1-GXMR-CAR and 18B7-GXMR-CAR redirected T cells toward the ligands;18B7-GXMR-CAR provided highest levels of tonic signaling;Binding pocket of 18B7 scFv favored the tonic signaling triggered by GXMR-CAR;Binding pocket of 18B7 scFv favored the tonic signaling triggered by GXMR-CAR.

Deacetylation by sirtuins is important for Aspergillus fumigatus pathogenesis and virulence.

Protein acetylation is a crucial post-translational modification that controls gene expression and a variety of biological processes. Sirtuins, a prominent class of NAD + -dependent lysine deacetylases, serve as key regulators of protein acetylation and gene expression in eukaryotes. In this study, six single knockout strains of fungal pathogen Aspergillus fumigatus were constructed, in addition to a strain lacking all predicted sirtuins (SIRTKO). Phenotypic assays suggest that sirtuins are involved in cell wall integrity, secondary metabolite production, thermotolerance, and virulence. AfsirE deletion resulted in attenuation of virulence, as demonstrated in murine and Galleria infection models. The absence of AfSirE leads to altered acetylation status of proteins, including histones and non-histones, resulting in significant changes in the expression of genes associated with secondary metabolism, cell wall biosynthesis, and virulence factors. These findings encourage testing sirtuin inhibitors as potential therapeutic strategies to combat A. fumigatus infections or in combination therapy with available antifungals.

Aspergillus fumigatus Extracellular Vesicles Display Increased Galleria mellonella Survival but Partial Pro-Inflammatory Response by Macrophages.

Fungal extracellular vesicles (EVs) mediate intra- and interspecies communication and are critical in host-fungus interaction, modulating inflammation and immune responses. In this study, we evaluated the in vitro pro- and anti-inflammatory properties of Aspergillus fumigatus EVs over innate leukocytes. A. fumigatus EVs induced a partial proinflammatory response by macrophages, characterized by increased tumor necrosis factor-alpha production, and increased gene expression of induced nitric oxide synthase and adhesion molecules. EVs induce neither NETosis in human neutrophils nor cytokine secretion by peripheral mononuclear cells. However, prior inoculation of A. fumigatus EVs in Galleria mellonella larvae resulted in increased survival after the fungal challenge. Taken together, these findings show that A. fumigatus EVs play a role in protection against fungal infection, although they induce a partial pro-inflammatory response.

Antifungal effect of Arnica montana and Hamamelis virginiana against Candida species

Objetivo ­ Avaliar a atividade antifúngica dos extratos glicólicos de Arnica montana e Hamamelis virginiana contra cepas de Candida spp. A candidíase é uma infecção fúngica comum, portanto, a pesquisa de novos agentes antifúngicos tem sido um alvo interessante. Várias plantas apresentaram atividades biológicas e, portanto, podem ser fontes promissoras de produtos naturais com atividades an-tifúngicas. Métodos ­ As atividades antifúngicas dos extratos glicólicos foram avaliadas por meio da determinação da concentração inibitória mínima (CIM) de acordo com o protocolo M27-S3 do Clinical and Laboratory Standards Institute (2008). Resultados ­ O ex-trato glicólico de A. montana apresentou a atividade antifúngica mais forte contra C. tropicalis, com concentração inibitória mínima (CIM) de 10% v/v e concentração fungicida mínima (MFC) de 80% v/v, seguido por C. krusei e C. glabrata, com valores de MIC e MFC de 20% v/v. Além disso, avaliamos a toxicidade dos dois extratos glicólicos no modelo Galleria mellonella usando as curvas de sobre-vivência de larvas tratadas com os extratos. Nossos resultados demonstraram que os extratos glicólicos de A. montana e H. virginiana não exibiram toxicidade contra larvas de G. mellonella e demonstraram atividade antifúngica contra espécies de Candida spp. Con-clusão ­ Assim, ambos os extratos são candidatos promissores para o desenvolvimento de novos agentes antifúngicos.

Objective ­ To evaluate the antifungal activity of Arnica montana and Hamamelis virginiana glycolic extracts against Candida strains. Methods ­ The antifungal activities of glycolic extracts were investigated by determination of the minimum inhibitory concentration (MIC) according to protocol M27-S3 of Clinical and Laboratory Standards Institute (2008). Results ­ A. montana glycolic extract showed the strongest antifungal activity against C. tropicalis, with a minimum inhibitory concentration (MIC) of 10% v/v and a minimum fungicidal concentration (MFC) of 80% v/v, then C. krusei and C. glabrata, with MIC and MFC values of 20% v/v. H. virginiana glycolic extract ex-hibited stronger activity against C. albicans and C. tropicalis, with MIC and MFC values of 10% v/v, than against C. glabrata, C. krusei, and C. parapsilosis, with MIC and MFC values of 20% v/v. Moreover, we evaluated the toxicity of the two glycolic extracts in the Galleria mellonella model using the survival curves of larvae treated with the two extracts. Our results demonstrated that the glycolic extracts of A. montana and H. virginiana exhibited no toxicity against G. mellonella larvae and demonstrated antifungal activity against Candida spe-cies. Conclusion ­ Thus, both extracts are promising candidates for the development of novel antifungal agents.

Addressing Microbial Resistance Worldwide: Challenges over Controlling Life-Threatening Fungal Infections.

Fungal infections are a serious global concern because of their ability to spread and colonize host tissues in immunocompromised individuals. Such infections have been frequently reported worldwide and are currently gaining clinical research relevance owing to their resistant character, representing a bottleneck in treating affected people. Resistant fungi are an emergent public health threat. The upsurge of such pathogens has led to new research toward unraveling the destructive potential evoked by these species. Some fungi-grouped into Candida, Aspergillus, and Cryptococcus-are causative agents of severe and systemic infections. They are associated with high mortality rates and have recently been described as sources of coinfection in COVID-hospitalized patients. Despite the efforts to elucidate the challenges of colonization, dissemination, and infection severity, the immunopathogenesis of fungal diseases remains a pivotal characteristic in fungal burden elimination. The struggle between the host immune system and the physiological strategies of the fungi to maintain cellular viability is complex. In this brief review, we highlight the relevance of drug resistance phenotypes in fungi of clinical significance, taking into consideration their physiopathology and how the scientific community could orchestrate their efforts to avoid fungal infection dissemination and deaths.

Influence of Galectin-3 on the Innate Immune Response during Experimental Cryptococcosis.

Cryptococcus neoformans, the causative agent of cryptococcosis, is the primary fungal pathogen that affects the immunocompromised individuals. Galectin-3 (Gal-3) is an animal lectin involved in both innate and adaptive immune responses. The present study aimed to evaluate the influence of Gal-3 on the C. neoformans infection. We performed histopathological and gene profile analysis of the innate antifungal immunity markers in the lungs, spleen, and brain of the wild-type (WT) and Gal-3 knockout (KO) mice during cryptococcosis. These findings suggest that Gal-3 absence does not cause significant histopathological alterations in the analyzed tissues. The expression profile of the genes related to innate antifungal immunity showed that the presence of cryptococcosis in the WT and Gal-3 KO animals, compared to their respective controls, promoted the upregulation of the pattern recognition receptor (PRR) responsive to mannose/chitin (mrc1) and a gene involved in inflammation (ccr5), as well as the downregulation of the genes related to signal transduction (card9, fos, ikbkb, jun) and PRRs (cd209a, colec12, nptx1). The absence of Gal-3, in fungal infection, a positively modulated gene involved in phagocytosis (sftpd) and negatively genes involved in signal transduction (syk and myd88), proinflammatory cytokines il-1ß and il-12b and cd209a receptor. Therefore, our results suggest that Gal-3 may play an essential role in the development of antifungal immune responses against cryptococcosis.

Altered expression of genes related to innate antifungal immunity in the absence of galectin-3.

Galectin-3 (Gal-3) is the most studied member of the animal galectin family, which comprises ß-galactoside-binding lectins and participates in several cellular events. Its expression in cells involved in innate and adaptive immunity is related to anti- and proinflammatory functions, signaling an important role in inflammatory, infectious, and tumorigenesis processes. Mice deficient in Gal-3 exhibit important phenotypes, but it is unclear whether these phenotypes reflect an impairment of the functions of this protein. Gal-3 plays an important role in modulating the immune response to different pathogenic microorganisms. However, the role of Gal-3 in immunity to infection is still poorly understood. Therefore, we investigated the effects of Gal-3 deletion on the expression of genes involved in the innate immune response in the lungs, spleens, and brains of Gal-3 KO mice. Gene profiling expression analysis suggested that Gal-3 deletion resulted in differentially modulated expression of the genes encoding beta-glucan, mannose and chitin-responsive pattern recognition receptors, signal transduction, inflammation, and phagocytosis. Our data thus suggest the importance of Gal-3 expression in the host innate immune system.

Antifungal Agents in Agriculture: Friends and Foes of Public Health.

Fungal diseases have been underestimated worldwide but constitute a substantial threat to several plant and animal species as well as to public health. The increase in the global population has entailed an increase in the demand for agriculture in recent decades. Accordingly, there has been worldwide pressure to find means to improve the quality and productivity of agricultural crops. Antifungal agents have been widely used as an alternative for managing fungal diseases affecting several crops. However, the unregulated use of antifungals can jeopardize public health. Application of fungicides in agriculture should be under strict regulation to ensure the toxicological safety of commercialized foods. This review discusses the use of antifungals in agriculture worldwide, the need to develop new antifungals, and improvement of regulations regarding antifungal use.

Galectin-3 Inhibits Paracoccidioides brasiliensis Growth and Impacts Paracoccidioidomycosis through Multiple Mechanisms.

The thermodimorphic pathogenic fungi Paracoccidioides brasiliensis and Paracoccidioides lutzii are the etiologic causes of paracoccidioidomycosis (PCM), the most prevalent systemic mycosis in Latin America. Galectin-3 (Gal-3), an animal ß-galactoside-binding protein, modulates important roles during microbial infections, such as triggering a Th2-polarized immune response in PCM. Herein, we demonstrate that Gal-3 also plays other important roles in P. brasiliensis infection. We verified that Gal-3 levels are upregulated in human and mice infections and established that Gal-3 inhibited P. brasiliensis growth by inhibiting budding. Furthermore, Gal-3 affected disruption and internalization of extracellular vesicles (EVs) from P. brasiliensis by macrophages. Our results suggest important protective roles for Gal-3 in P. brasiliensis infection, indicating that increased Gal-3 production during P. brasiliensis infection may affect fungal growth and EV stability, thus promoting beneficial effects that could influence the course of PCM. The finding that Gal-3 has effects against P. brasiliensis together with previously reported effects against Cryptococcus neoformans suggests that molecule has a general antifungal role in innate defenses against fungal pathogens.IMPORTANCE Paracoccidioidomycosis (PCM) is the most prevalent systemic mycosis in Latin America. Although the immune mechanisms to control PCM are still not fully understood, several events of the host innate and adaptive immunity are crucial to determine the progress of the infection. Mammalian ß-galactoside-binding protein galectin-3 (Gal-3) plays significant roles during microbial infections and has been studied for its immunomodulatory roles, but it can also have direct antimicrobial effects. We asked whether this protein plays a role in Paracoccidioides brasiliensis We report herein that Gal-3 indeed has direct effects on the fungal pathogen, inhibiting fungal growth and reducing extracellular vesicle stability. Our results suggest a direct role for Gal-3 in P. brasiliensis infection, with beneficial effects for the mammalian host.

Extracellular Vesicles From the Dermatophyte Trichophyton interdigitale Modulate Macrophage and Keratinocyte Functions.

The release of biomolecules critically affects all pathogens and their establishment of diseases. For the export of several biomolecules in diverse species, the use of extracellular vesicles (EVs) is considered to represent an alternative transport mechanism, but no study to date has investigated EVs from dermatophytes. Here, we describe biologically active EVs from the dermatophyte Trichophyton interdigitale, a causative agent of mycoses worldwide. EV preparations from T. interdigitale were examined using nanoparticle-tracking analysis, which revealed vesicular structures 20-380 nm in diameter. These vesicles induced the production of proinflammatory mediators by bone marrow-derived macrophages (BMDMs) and keratinocytes in a dose-dependent manner, and an addition of the EVs to BMDMs also stimulated the transcription of the M1-polarization marker iNOS (inducible nitric oxide synthase) and diminished the expression of the M2 markers arginase-1 and Ym-1. The observed M1 macrophages' polarization triggered by EVs was abolished in cells obtained from knockout Toll-like receptor-2 mice. Also, the EVs-induced productions of pro-inflammatory mediators were blocked too. Furthermore, the EVs from T. interdigitale enhanced the fungicidal activity of BMDMs. These results suggest that EVs from T. interdigitale can modulate the innate immune response of the host and influence the interaction between T. interdigitale and host immune cells. Our findings thus open new areas of investigation into the host-parasite relationship in dermatophytosis.

Galectin-3 impacts Cryptococcus neoformans infection through direct antifungal effects.

Cryptococcus neoformans is an encapsulated fungal pathogen that causes cryptococcosis, which is a major opportunistic infection in immunosuppressed individuals. Mammalian ß-galactoside-binding protein Galectin-3 (Gal-3) modulates the host innate and adaptive immunity, and plays significant roles during microbial infections including some fungal diseases. Here we show that this protein plays a role also in C. neoformans infection. We find augmented Gal-3 serum levels in human and experimental infections, as well as in spleen, lung, and brain tissues of infected mice. Gal-3-deficient mice are more susceptible to cryptococcosis than WT animals, as demonstrated by the higher fungal burden and lower animal survival. In vitro experiments show that Gal-3 inhibits fungal growth and exerts a direct lytic effect on C. neoformans extracellular vesicles (EVs). Our results indicate a direct role for Gal-3 in antifungal immunity whereby this molecule affects the outcome of C. neoformans infection by inhibiting fungal growth and reducing EV stability, which in turn could benefit the host.