Vaccine Strategies for Cryptococcus neoformans.

Cryptococcus neoformans infections are a major worldwide concern as current treatment strategies are becoming less effective in alleviating the infection. The most extreme and fatal cases are those of immunocompromised individuals. Clinical treatments for cryptococcosis are limited to a few classes of approved drugs, and due to a rise in drug resistance, these drugs are becoming less effective. Therefore, it is essential to develop innovative ways to control this infection. Vaccinations have emerged as a safe, viable, and cost-effective solution to treat a number of diseases over the years. Currently, there are no clinically available vaccines to treat cryptococcal infections, but a number of studies have shown promising results in animal models. Here, we present step-by-step experimental protocols using live-attenuated or heat-killed C. neoformans cells as a vaccination strategy in a preventive or in a therapeutic murine model of cryptococcosis.

Mass Spectrometry Analysis Reveals Lipids Induced by Oxidative Stress in Candida albicans Extracellular Vesicles.

Candida albicans is a commensal fungus in healthy humans that causes infection in immunocompromised individuals through the secretion of several virulence factors. The successful establishment of infection is owing to elaborate strategies to cope with defensive molecules secreted by the host, including responses toward oxidative stress. Extracellular vesicle (EV) release is considered an alternative to the biomolecule secretory mechanism that favors fungal interactions with the host cells. During candidiasis establishment, the host environment becomes oxidative, and it impacts EV release and cargo. To simulate the host oxidative environment, we added menadione (an oxidative stress inducer) to the culture medium, and we explored C. albicans EV metabolites by metabolomics analysis. This study characterized lipidic molecules transported to an extracellular milieu by C. albicans after menadione exposure. Through Liquid Chromatography coupled with Mass Spectrometry (LC-MS) analyses, we identified biomolecules transported by EVs and supernatant. The identified molecules are related to several biological processes, such as glycerophospholipid and sphingolipid pathways, which may act at different levels by tuning compound production in accordance with cell requirements that favor a myriad of adaptive responses. Taken together, our results provide new insights into the role of EVs in fungal biology and host-pathogen interactions.

Targeting Sterylglucosidase A to Treat Aspergillus fumigatus Infections.

Invasive fungal infections are a leading cause of death in immunocompromised patients. Current therapies have several limitations, and innovative antifungal agents are critically needed. Previously, we identified the fungus-specific enzyme sterylglucosidase as essential for pathogenesis and virulence of Cryptococcus neoformans and Aspergillus fumigatus (Af) in murine models of mycoses. Here, we developed Af sterylglucosidase A (SglA) as a therapeutic target. We identified two selective inhibitors of SglA with distinct chemical scaffolds that bind in the active site of SglA. Both inhibitors induce sterylglucoside accumulation and delay filamentation in Af and increase survival in a murine model of pulmonary aspergillosis. Structure-activity relationship (SAR) studies identified a more potent derivative that enhances both in vitro phenotypes and in vivo survival. These findings support sterylglucosidase inhibition as a promising antifungal approach with broad-spectrum potential. IMPORTANCE Invasive fungal infections are a leading cause of death in immunocompromised patients. Aspergillus fumigatus is a fungus ubiquitously found in the environment that, upon inhalation, causes both acute and chronic illnesses in at-risk individuals. A. fumigatus is recognized as one of the critical fungal pathogens for which a substantive treatment breakthrough is urgently needed. Here, we studied a fungus-specific enzyme, sterylglucosidase A (SglA), as a therapeutic target. We identified selective inhibitors of SglA that induce accumulation of sterylglucosides and delay filamentation in A. fumigatus and increase survival in a murine model of pulmonary aspergillosis. We determined the structure of SglA, predicted the binding poses of these inhibitors through docking analysis, and identified a more efficacious derivative with a limited SAR study. These results open several exciting avenues for the research and development of a new class of antifungal agents targeting sterylglucosidases.

Chitin Biosynthesis in Aspergillus Species.

The fungal cell wall (FCW) is a dynamic structure responsible for the maintenance of cellular homeostasis, and is essential for modulating the interaction of the fungus with its environment. It is composed of proteins, lipids, pigments and polysaccharides, including chitin. Chitin synthesis is catalyzed by chitin synthases (CS), and up to eight CS-encoding genes can be found in Aspergillus species. This review discusses in detail the chitin synthesis and regulation in Aspergillus species, and how manipulation of chitin synthesis pathways can modulate fungal growth, enzyme production, virulence and susceptibility to antifungal agents. More specifically, the metabolic steps involved in chitin biosynthesis are described with an emphasis on how the initiation of chitin biosynthesis remains unknown. A description of the classification, localization and transport of CS was also made. Chitin biosynthesis is shown to underlie a complex regulatory network, with extensive cross-talks existing between the different signaling pathways. Furthermore, pathways and recently identified regulators of chitin biosynthesis during the caspofungin paradoxical effect (CPE) are described. The effect of a chitin on the mammalian immune system is also discussed. Lastly, interference with chitin biosynthesis may also be beneficial for biotechnological applications. Even after more than 30 years of research, chitin biosynthesis remains a topic of current interest in mycology.

Vaccination with Live or Heat-Killed Aspergillus fumigatus ΔsglA Conidia Fully Protects Immunocompromised Mice from Invasive Aspergillosis.

Aspergillus fumigatus causes invasive aspergillosis (IA) in immunocompromised patients, resulting in high mortality rates. Currently, no vaccine formulations to promote immune protection in at-risk individuals have been developed. In this work, we deleted the sterylglucosidase-encoding gene, sglA, in Aspergillus fumigatus and investigated its role in fungal virulence and host vaccine protection. The ΔsglA mutant accumulated sterylglucosides (SGs), newly studied immunomodulatory glycolipids, and exhibited reduced hyphal growth and altered compositions of cell wall polysaccharides. Interestingly, the ΔsglA mutant was avirulent in two murine models of IA and was fully eliminated from the lungs. Both corticosteroid-induced immunosuppressed and cyclophosphamide-induced leukopenic mice vaccinated with live or heat-killed ΔsglA conidia were fully protected against a lethal wild-type A. fumigatus challenge. These results highlight the potential of SG-accumulating strains as safe and promising vaccine formulations against invasive fungal infections. IMPORTANCE Infections by Aspergillus fumigatus occur by the inhalation of environmental fungal spores called conidia. We found that live mutant conidia accumulating glycolipids named sterylglucosides are not able to cause disease when injected into the lung. Interestingly, these animals are now protected against a secondary challenge with live wild-type conidia. Remarkably, protection against a secondary challenge persists even with vaccination with heat-killed mutant conidia. These results will significantly advance the field of the research and development of a safe fungal vaccine for protection against the environmental fungus A. fumigatus.

HIV-1 Gag and Vpr impair the inflammasome activation and contribute to the establishment of chronic infection in human primary macrophages.

The successful establishment of HIV-1 infection is related to inflammasome blocking or inactivation, which can result in the viral evasion of the immune responses and formation of reservoirs in several tissues. In this sense, we aimed to evaluate the viral and cellular mechanisms activated during HIV-1 infection in human primary macrophages that allow an effective viral replication in these cells. We found that resting HIV-1-infected macrophages, but not those activated in classical or alternative patterns, released IL-1ß and other pro-inflammatory cytokines, and showed increased CXCL10 expression, without changes in the NLRP3, AIM2 or RIG-I inflammasome pathways. Also, similar levels of Casp-1, phosphorylated NF-κB (p65) and NLRP3 proteins were found in uninfected and HIV-1-infected macrophages. Likewise, no alterations were detected in ASC specks released in the culture supernatant after HIV-1 infection, suggesting that macrophages remain viable after infection. Using in silico prediction studies, we found that the HIV-1 proteins Gag and Vpr interact with several host proteins. Comparable levels of trans-LTB4 were found in the supernatants of uninfected and HIV-1-infected macrophages, whereas ROS production was impaired in infected cells, which was not reversed after the PMA stimulus. Immunofluorescence analysis showed structural alterations in the mitochondrial architecture and an increase of BIM in the cytoplasm of infected cells. Our data suggest that HIV-1 proteins Gag and Vpr, through interacting with cellular proteins in the early steps of infection, preclude the inflammasome activation and the development of effective immune responses, thus allowing the establishment of the infection.

The Expression of Tax and HBZ Genes in Serum-Derived Extracellular Vesicles From HTLV-1 Carriers Correlates to Proviral Load and Inflammatory Markers.

Human T-lymphotropic virus 1 (HTLV-1) is the etiologic agent of adult cell leukemia/lymphoma (ATL) and HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). One of the major questions in HTLV-1 studies is related to the understanding of causes that lead to different clinical manifestations. However, it is well known that the viral genes tax and HTLV-1 basic leucine zipper factor (HBZ) are related to viral infectivity and the development of neurological and hematological diseases. Currently, there is evidence that HTLV-1 infected cells can release small extracellular vesicles (sEVs) involved in the mechanisms of viral particles spreading. Therefore, we evaluated the expression levels of tax and HBZ viral transcripts in serum-derived sEVs from HTLV-1 carriers, as well as the role of these vesicles in the modulation of the immune response. Three HAM/TSP carriers presented detectable levels of tax and HBZ transcripts in sEVs and were positively correlated to the proviral load (PVL) in peripheral blood mononuclear cells (PBMCs). The viral transcripts were only detectable in individuals with a PVL higher than 6,000/105 PBMCs. Additionally, it was observed that HBZ presented a 2-12-folds increase over tax expression units. Gene expression and secretory protein analysis indicated that PBMCs from blood donors and HTLV-1 carriers exposed to increasing doses of tax+ HBZ+ sEVs showed a dose-dependent increase in interferon (IFN)-γ and interleukin (IL)-8 transcripts and proteins. Interestingly, the increase in IL-8 levels was close to those seen in HTLV-1-infected PBMCs with high PVL. Taken together, these findings indicate that the expression of viral transcripts in serum-derived sEVs of HTLV-1 carriers is related to the PVL presented by the infected individual. Additionally, tax+ HBZ+ sEVs can induce the production of inflammatory cytokines in patients with low PVL, which may be related to the development of symptoms in HTLV-1 infection.

Gasdermin D inhibition prevents multiple organ dysfunction during sepsis by blocking NET formation.

Multiple organ dysfunction is the most severe outcome of sepsis progression and is highly correlated with a worse prognosis. Excessive neutrophil extracellular traps (NETs) are critical players in the development of organ failure during sepsis. Therefore, interventions targeting NET release would likely effectively prevent NET-based organ injury associated with this disease. Herein, we demonstrate that the pore-forming protein gasdermin D (GSDMD) is active in neutrophils from septic humans and mice and plays a crucial role in NET release. Inhibition of GSDMD with disulfiram or genic deletion abrogated NET formation, reducing multiple organ dysfunction and sepsis lethality. Mechanistically, we demonstrate that during sepsis, activation of the caspase-11/GSDMD pathway controls NET release by neutrophils during sepsis. In summary, our findings uncover a novel therapeutic use for disulfiram and suggest that GSDMD is a therapeutic target to improve sepsis treatment.

The influence of dehydroepiandrosterone on effector functions of neutrophils

Dehydroepiandrosterone (DHEA) is a steroid hormone secreted by the adrenal glands, gonads and brain. It is a precursor to sex hormones and also is known to have immune modulatory activity. However, little is known about the relationship between DHEA and neutrophils and thus our study evaluates the influence of DHEA in the effector functions of neutrophils. Human neutrophils were treated in vitro with DHEA and further infected with Salmonella enterica serovar Typhimurium. The treatment of neutrophils with 0.01 µM of DHEA increased the phagocytosis of Salmonella independent of TLR4 as the treatment did not modulate the TLR4 expression. Additionally, DHEA caused a decrease in ROS (reactive oxygen species) production and did not influence the formation of the neutrophil extracellular trap (NET). Steroid treated neutrophils, infected or stimulated with LPS (lipopolysaccharide), showed reduced production of IL-8, compared to untreated cells. Also, the protein levels of p-NFκB were decreased in neutrophils treated with DHEA, and this reduction could explain the reduced levels of IL-8. These results led us to conclude that the steroid hormone DHEA has important modulatory functions in neutrophils

Extracellular Vesicles from Aspergillus flavus Induce M1 Polarization In Vitro.

Aspergillus flavus, a ubiquitous and saprophytic fungus, is the second most common cause of aspergillosis worldwide. Several mechanisms contribute to the establishment of the fungal infection. Extracellular vesicles (EVs) have been described as "virulence factor delivery bags" in several fungal species, demonstrating a crucial role during the infection. In this study, we evaluated production of A. flavus EVs and their immunomodulatory functions. We verified that A. flavus EVs induce macrophages to produce inflammatory mediators, such as nitric oxide, tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and IL-1ß. Furthermore, the A. flavus EVs enhance phagocytosis and killing by macrophages and induce M1 macrophage polarization in vitro In addition, a prior inoculation of A. flavus EVs in Galleria mellonella larvae resulted in a protective effect against the fungal infection. Our findings suggest that A. flavus EVs are biologically active and affect the interaction between A. flavus and host immune cells, priming the innate immune system to eliminate the fungal infection. Collectively, our results suggest that A. flavus EVs play a crucial role in aspergillosis.IMPORTANCE Immunocompromised patients are susceptible to several fungal infections. The genus Aspergillus can cause increased morbidity and mortality. Developing new therapies is essential to understand the fungal biology mechanisms. Fungal EVs carry important virulence factors, thus playing pivotal roles in fungal pathophysiology. No study to date has reported EV production by Aspergillus flavus, a fungus considered to be the second most common cause of aspergillosis and relevant food contaminator found worldwide. In this study, we produced A. flavus EVs and evaluated the in vitro immunomodulatory effects of EVs on bone marrow-derived macrophages (BMDMs) and in vivo effects in a Galleria mellonella model.

Functional Characterization of Clinical Isolates of the Opportunistic Fungal Pathogen Aspergillus nidulans.

Aspergillus nidulans is an opportunistic fungal pathogen in patients with immunodeficiency, and virulence of A. nidulans isolates has mainly been studied in the context of chronic granulomatous disease (CGD), with characterization of clinical isolates obtained from non-CGD patients remaining elusive. This study therefore carried out a detailed biological characterization of two A. nidulans clinical isolates (CIs), obtained from a patient with breast carcinoma and pneumonia and from a patient with cystic fibrosis that underwent lung transplantation, and compared them to the reference, nonclinical FGSC A4 strain. Both CIs presented increased growth in comparison to that of the reference strain in the presence of physiologically relevant carbon sources. Metabolomic analyses showed that the three strains are metabolically very different from each other in these carbon sources. Furthermore, the CIs were highly susceptible to cell wall-perturbing agents but not to other physiologically relevant stresses. Genome analyses identified several frameshift variants in genes encoding cell wall integrity (CWI) signaling components. Significant differences in CWI signaling were confirmed by Western blotting among the three strains. In vivo virulence studies using several different models revealed that strain MO80069 had significantly higher virulence in hosts with impaired neutrophil function than the other strains. In summary, this study presents detailed biological characterization of two A. nidulanssensu stricto clinical isolates. Just as in Aspergillus fumigatus, strain heterogeneity exists in A. nidulans clinical strains that can define virulence traits. Further studies are required to fully characterize A. nidulans strain-specific virulence traits and pathogenicity.IMPORTANCE Immunocompromised patients are susceptible to infections with opportunistic filamentous fungi from the genus Aspergillus Although A. fumigatus is the main etiological agent of Aspergillus species-related infections, other species, such as A. nidulans, are prevalent in a condition-specific manner. A. nidulans is a predominant infective agent in patients suffering from chronic granulomatous disease (CGD). A. nidulans isolates have mainly been studied in the context of CGD although infection with A. nidulans also occurs in non-CGD patients. This study carried out a detailed biological characterization of two non-CGD A. nidulans clinical isolates and compared the results to those with a reference strain. Phenotypic, metabolomic, and genomic analyses highlight fundamental differences in carbon source utilization, stress responses, and maintenance of cell wall integrity among the strains. One clinical strain had increased virulence in models with impaired neutrophil function. Just as in A. fumigatus, strain heterogeneity exists in A. nidulans clinical strains that can define virulence traits.

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.

SET protein accumulation prevents cell death in head and neck squamous cell carcinoma through regulation of redox state and autophagy.

Molecular alterations in cell death pathways and imbalances in regulators of up- or downstream signaling pathways can lead to resistance to cell death, which is one of the hallmarks of cancer. These signaling modifications are strategies that tumor cells use to resist chemotherapy and that contribute to the high recurrence rate of head and neck squamous cell carcinoma (HNSCC). The SET oncoprotein is a PP2A inhibitor that accumulates in HNSCC and represents a promising therapeutic target. Here we report the role that SET protein plays in resistance to death of two HNSCC cell lines: Cal 27 and HN13. SET protein regulated intracellular redox balance by controlling cellular localization of APE 1 - an endonuclease that is part of the SET complex and regulates antioxidant gene transcription. SET protein knockdown (siSET) associated with tert-butyl hydroperoxide-induced oxidative stress sensitized Cal 27 and HN13 cells to apoptosis via the extrinsic and intrinsic pathways, respectively. SET protein upregulated autophagy in HNSCC cells in a PP2A-dependent manner and apparently regulated ULK1 expression. The fact that siSET lowered Bcl-2 phosphorylation levels indicated that SET protein interfered with an alternative pathway that modulated autophagy in HNSCC cells. Overall, SET protein regulated intracellular redox state and sustained autophagy in HNSCC cells, which may explain resistance to death of HNSCC cells. Altogether, the findings reported herein support SET protein as therapeutic target for HNSCC.

Epigenetic alterations are associated with monocyte immune dysfunctions in HIV-1 infection.

Monocytes are key cells in the immune dysregulation observed during human immunodeficiency virus (HIV) infection. The events that take place specifically in monocytes may contribute to the systemic immune dysfunction characterized by excessive immune activation in infected individuals, which directly correlates with pathogenesis and progression of the disease. Here, we investigated the immune dysfunction in monocytes from untreated and treated HIV + patients and associated these findings with epigenetic changes. Monocytes from HIV patients showed dysfunctional ability of phagocytosis and killing, and exhibited dysregulated cytokines and reactive oxygen species production after M. tuberculosis challenge in vitro. In addition, we showed that the expression of enzymes responsible for epigenetic changes was altered during HIV infection and was more prominent in patients that had high levels of soluble CD163 (sCD163), a newly identified plasmatic HIV progression biomarker. Among the enzymes, histone acetyltransferase 1 (HAT1) was the best epigenetic biomarker correlated with HIV - sCD163 high patients. In conclusion, we confirmed that HIV impairs effector functions of monocytes and these alterations are associated with epigenetic changes that once identified could be used as targets in therapies aiming the reduction of the systemic activation state found in HIV patients.

Identification of promising plasma immune biomarkers to differentiate active pulmonary tuberculosis.

Although much research has been done related to biomarker discovery for tuberculosis infection, a set of biomarkers that can discriminate between active and latent TB diseases remains elusive. In the current study we correlate clinical aspects of TB disease with changes in the immune response as determined by biomarkers detected in plasma. Our study measured 18 molecules in human plasma in 17 patients with active disease (APTB), 14 individuals with latent tuberculosis infection (LTBI) and 16 uninfected controls (CTRL). We found that active tuberculosis patients have increased plasma levels of IL-6, IP-10, TNF-α, sCD163 and sCD14. Statistical analysis of these biomarkers indicated that simultaneous measurement of sCD14 and IL-6 was able to diagnose active tuberculosis infection with 83% accuracy. We also demonstrated that TNF-α and sCD163 were correlated with tuberculosis severity. We showed that the simultaneous detection of both plasma sCD14 and IL-6 is a promising diagnostic approach to identify APTB, and further, measurement of TNF-α and sCD163 can identify the most severe cases of tuberculosis.

HIV infection: focus on the innate immune cells.

Innate immune cells play a critical role during the onset of HIV infection and remain active until the final events that characterize AIDS. The viral impact on innate immune cell response may be a result of direct infection or indirect modulation, and each cell type responds in a specific manner to HIV. During HIV infection, the immune system works in a dynamic way, where innate and adaptive cells contribute with each other stimulating their function and modulating phenotypes and consequently infection resolution. Understanding the alterations in the cell populations induced by the virus is pivotal and can help to combat HIV at the time of infection and above all, to prevent the establishment of viral reservoirs. In this review, we will describe the frequency and the subtypes of infected cells such as of monocytes, DCs, neutrophils, eosinophils, mast cells/basophils, NK cells, NKT cells and γδ T cells, and we discuss the possibility of cell-targeting strategies. Our aim is to consolidate the existing knowledge of the interaction between HIV and cells that constitute the innate immune response.