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Aim: Melanin has been linked to pathogenesis in several fungi. They often produce melanin-like pigments in the presence of L-dihydroxyphenylalanine (L-DOPA), but this is poorly studied in Candida glabrata. Methods & materials: C. glabrata was grown in minimal medium with or without L-DOPA supplementation and submitted to a chemical treatment with denaturant and hot acid. Results: C. glabrata turned black when grown in the presence of L-DOPA, whereas cells grown without L-DOPA supplementation remained white. Biophysical properties demonstrated that the pigment was melanin. Melanized C. glabrata cells were effectively protected from azoles and amphotericin B, incubation at 42°C and macrophage killing. Conclusion: In the presence of L-DOPA, C. glabrata produces melanin, increases antifungal resistance and enhances host survival.

Candida parapsilosis is a leading cause of invasive mycoses and the major cause of nosocomial fungaemia amongst low and very low birth weight neonates. However, the molecular and physiological characteristics of this fungus remain understudied. To advance our knowledge about the pathobiology of this pathogen, we sought to develop and validate an effective method for chemical transformation of C. parapsilosis. Chemical transformation is the primary procedure for introducing foreign DNA into Candida yeast as it requires no special equipment, although its performance efficacy drops rapidly when the size of the transforming DNA increases. To define optimal conditions for chemical transformation in C. parapsilosis, we selected a leucine auxotroph laboratory strain. We identified optimal cell density for transformation, incubation times, inclusion of specific enhancing chemicals, and size and amounts of DNA fragments that resulted in maximized transformation efficiency. We determined that the inclusion of dimethyl sulfoxide was beneficial, but dithiothreitol pretreatment reduced colony recovery. As a result, the modified protocol led to a 20–55-fold increase in transformation efficiency, depending on the size of the transforming fragment. We validated the modified methodology with prototrophic isolates and demonstrated that the new approach resulted in the recovery of significantly more transformants in 5 of 6 isolates. Additionally, we identified a medium in which transformation competent yeast cells could safely be maintained at −80°C for up to 6 weeks that reduces laboratory work and shortens the overall procedure. These modifications will significantly aid further investigations into the genetic basis for virulence in C. parapsilosis.

With rising skin cancer rates and interest in preventing photoaging, adjuvants for sunscreens are in high demand. The potential of curcumin has been posited due to its anti-inflammatory, antioxidant and wound healing properties. In prior studies, curcumin decreased UV-induced inflammation, apoptotic changes in human keratinocytes and dermal fibroblasts, and the expression of matrix metalloproteinases. However, curcumin's utility has been hindered by poor aqueous solubility and rapid degradation in vivo. To overcome these limitations, we synthesized curcumin nanoparticles (curc-np), which offer sustained topical delivery and enhanced bioavailability. Curc-np and controls were applied to the skin of BALB/c mice prior to UVB irradiation. Twenty-four hours later, mice pre-treated with curc-np showed less erythema, induration and scale compared to controls. Histopathology showed fewer sunburn cells, and TUNEL assay indicated decreased apoptosis in curc-np treated mice. Immunohistochemistry illustrated less p53 expression in skin pre-treated with curc-np. Furthermore, cytokine analysis revealed significantly less IL-6 and significantly greater anti-inflammatory IL-10 in skin of curc-np-treated mice as compared to controls. Taken together, our results reinforce curcumin's established anti-inflammatory effects in the skin and highlight its potential as a photoprotective adjuvant when delivered through nanoparticles. Further investigation alongside sunscreens against UV-induced damage is warranted.

Graphical abstract, Candida auris has emerged as a serious worldwide threat by causing opportunistic infections that are frequently resistant to one or more conventional antifungal medications resulting in high mortality rates. Against this backdrop, health warnings around the world have focused efforts on understanding C. auris fungal biology and effective prevention and treatment approaches to combat this fungus. To date, there is little information about the differentially expressed genes when this fungus is treated with conventional antifungals, and caspofungin is a standard echinocandin deployed in the therapy against C. auris. In this work, we treated two distinct strains of C. auris for 24 h with caspofungin, and the cellular responses were evaluated at the morphological, translational and transcriptional levels. We first observed that the echinocandin caused morphological alterations, aggregation of yeast cells, and modifications in the cell wall composition of C. auris. Transcriptomic analysis revealed an upregulation of genes related to the synthesis of the cell wall, ribosome, and cell cycle after exposure to caspofungin. Supporting these findings, the integrated proteomic analysis showed that caspofungin-treated cells were enriched in ribosome-related proteins and cell wall, especially mannoproteins. Altogether, these results provide further insights into the biology of C. auris and expands our understanding regarding the antifungal activity of caspofungin and reveal cellular targets, as the mannose metabolism, that can be further explored for the development of novel antifungals.

As there are more than 6 million human deaths due to mycoses each year, there is an urgent need to develop fungal vaccines. Moreover, given the similarities among pathogenic fungi, it may be possible to create a multi-fungi vaccine. In this study, we combined immunoproteomic and immunopeptidomic methods, for which we have adapted a technique based on co-immunoprecipitation (Co-IP) that made it possible to map Histoplasma capsulatum epitopes for the first time in a natural context using murine dendritic cells (DCs) and macrophages (Mφ). Although polysaccharide epitopes exist, this research focused on mapping protein epitopes as these are more immunogenic. We used different algorithms to screen proteins and peptides identified by two-dimensional electrophoresis (2-D) and Co-IP. Seventeen proteins were revealed by 2-D gels, and 45 and 24 peptides from distinct proteins were presented by DCs and Mφ, respectively. We then determined which epitopes were restricted to MHC-I and II from humans and mice and showed high promiscuity, but lacked identity with human proteins. The 4 most promising peptides were synthesized, and the peptides with and without incorporation into glucan particles induced CD4+ and CD8+ T cell proliferation and produced a Th1 and Th17 response marked by the secretion of high levels of IFN-γ, IL-17 and IL-2. These epitopes were from heat shock protein 60, enolase, and the ATP-dependent molecular chaperone HSC82, and they each have a high degree of identity with proteins expressed by other medically important pathogenic fungi. Thus, the epitopes described in this study have the potential for use in the development of vaccines that could result in cross-protection among fungal species.

Melanins are one of the great natural pigments produced by a wide variety of fungal species that promote fitness and cell survival in diverse hostile environments, including during mammalian infection. In this study, we sought to demonstrate the production of melanin in the conidia and hyphae of saprophytic fungi, including dematiaceous and hyaline fungi. We showed that a melanin-specific monoclonal antibody (MAb) avidly labeled the cell walls of hyphae and conidia, consistent with the presence of melanin in these structures, in 14 diverse fungal species. The conidia of saprophytic fungi were treated with proteolytic enzymes, denaturant, and concentrated hot acid to yield dark particles, which were shown to be stable free radicals, consistent with their identification as melanins. Samples obtained from patients with fungal keratitis due to Fusarium falciforme, Aspergillus fumigatus, Aspergillus flavus, Curvularia lunata, Exserohilum rostratum, or Fonsecaea pedrosoi were found to be intensely labeled by the melanin-specific MAb at the fungal hyphal cell walls. These results support the hypothesis that melanin is a common component that promotes survival under harsh conditions and facilitates fungal virulence. Increased understanding of the processes of melanization and the development of methods to interfere with pigment formation may lead to novel approaches to combat these complex pathogens that are associated with high rates of morbidity and mortality.

Made available in DSpace on 2022-04-29T08:31:22Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-07-15 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Filamentous fungi such as Trichophyton rubrum and T. mentagrophytes, the main causative agents of onychomycosis, have been recognized as biofilm-forming microorganisms. Nitric oxide-releasing nanoparticles (NO-np) are currently in development for the management of superficial and deep bacterial and fungal infections, with documented activity against biofilms. In this context, this work aimed to evaluate, for the first time, the in vitro anti-T. rubrum biofilm potential of NO-np using standard ATCC MYA-4438 and clinical BR1A strains and compare it to commonly used antifungal drugs including fluconazole, terbinafine and efinaconazole. The biofilms formed by the standard strain produced more biomass than those from the clinical strain. NO-np, fluconazole, terbinafine, and efinaconazole inhibited the in vitro growth of planktonic T. rubrum cells. Similarly, NO-np reduced the metabolic activities of clinical strain BR1A preformed biofilms at the highest concentration tested (SMIC50 = 40 mg/mL). Scanning electron and confocal microscopy revealed that NO-np and efinaconazole severely damaged established biofilms for both strains, resulting in collapse of hyphal cell walls and reduced the density, extracellular matrix and thickness of the biofilms. These findings suggest that biofilms should be considered when developing and testing new drugs for the treatment of dermatophytosis. Development of a biofilm phenotype by these fungi may explain the resistance of dermatophytes to some antifungals and why prolonged treatment is usually required for onychomycosis. Department of Medicine Division of Infectious Diseases Albert Einstein College of Medicine Deparment of Clinical Analysis School of Pharmaceutical Sciences Sao Paulo State University (UNESP) Department of Oral Biology College of Dentistry University of Florida Department of Para-Clinic School of Veterinary Universidade Eduardo Mondlane (UEM) Department of Physiology and Biophysics Albert Einstein College of Medicine Department of Dermatology George Washington School of Medicine and Health Sciences Department of Medicine Division of Dermatology Albert Einstein College of Medicine Department of Microbiology and Immunology Albert Einstein College of Medicine Deparment of Clinical Analysis School of Pharmaceutical Sciences Sao Paulo State University (UNESP)

Publisher Copyright: Copyright © 2021 Horváth et al. Oral epithelial cells monitor microbiome composition and initiate immune response upon dysbiosis, as in the case of Candida imbalances. Candida species, such as C. albicans and C. parapsilosis, are the most prevalent yeasts in the oral cavity. Comparison of healthy oral epithelial cell responses revealed that while C. albicans infection robustly activated inflammation cascades, C. parapsilosis primarily activated various inflammation-independent pathways. In posttranscriptional regulatory processes, several miRNAs were altered by both species. For C. parapsilosis, the dose of yeast cells directly correlated with changes in transcriptomic responses with higher fungal burdens inducing significantly different and broader changes. MicroRNAs (miRNAs) associated with carbohydrate metabolism-, hypoxia-, and vascular development-related responses dominated with C. parapsilosis infection, whereas C. albicans altered miRNAs linked to inflammatory responses. Subsequent analyses of hypoxia-inducible factor 1a (HIF1-a) and hepatic stellate cell (HSC) activation pathways predicted target genes through which miRNA-dependent regulation of yeast-specific functions may occur, which also supported the observed species-specific responses. Our findings suggest that C. parapsilosis is recognized as a commensal at low doses by the oral epithelium; however, increased fungal burden activates different pathways, some of which overlap with the inflammatory processes robustly induced by C. albicans. IMPORTANCE A relatively new topic within the field of immunology involves the role of miRNAs in innate as well as adaptive immune response regulation. In recent years, posttranscriptional regulation of host-pathogenic fungal interactions through miRNAs was also suggested. Our study reveals that the distinct nature of human oral epithelial cell responses toward C. parapsilosis and C. albicans is possibly due to species-specific fine-tuning of host miRNA regulatory processes. The findings of this study also shed new light on the nature of early host cell transcriptional responses to the presence of C. parapsilosis and highlight the species’ potential inflammation-independent host activation processes. These findings contribute to our better understanding of how miRNA deregulation at the oral immunological barrier, in non-canonical immune cells, may discriminate between fungal species, particularly Candida species with high or low pathogenic potential.

Paracoccidioidomycosis (PCM) is an endemic mycosis in Latin America caused by the thermodimorphic fungi of the genus Paracoccidioides spp. Paracoccidioides lutzii (PL) is one of the 5 species that constitute the Paracoccidioides genus. PL expresses low amounts of glycoprotein (Gp) 43 (PLGp43) and PLGp43 displays few epitopes in common with the P. brasiliensis (PB) immunodominant antigen PBGp43, which is commonly used for serological diagnosis of PCM. This difference in structure between the glycoproteins markedly reduces the efficiency of serological diagnosis in patients infected with PL. We previously demonstrated that peptide 10 (P10) from the PBGp43 induces protective immune responses in in vitro and in vivo models of PB PCM. Since, P10 has proven to be a promising therapeutic to combat PB, we sought to identify peptides in PL that could similarly be applied for the treatment of PCM. PL yeast cell proteins were isolated from PL: dendritic cell co-cultures and subjected to immunoproteomics. This approach identified 18 PL peptides that demonstrated in silico predictions for immunogenicity. Eight of the most promising peptides were synthesized and applied to lymphocytes obtained from peptide-immunized or PL-infected mice as well as to in vitro cultures with peptides or dendritic cells pulsed the peptides. The peptides LBR5, LBR6 and LBR8 efficiently promoted CD4+ and CD8+ T cell proliferation and dendritic cells pulsed with LBR1, LBR3, LBR7 or LBR8 stimulated CD4+ T cell proliferation. We observed increases of IFN-γ in the supernatants from primed T cells for the conditions with peptides without or with dendritic cells, although IL-2 levels only increased in response to LBR8. These novel immunogenic peptides derived from PL will be employed to develop new peptide vaccine approaches and the proteins from which they are derived can be used to develop new diagnostic assays for PL and possibly other Paracoccidioides spp. These findings identify and characterize new peptides with a promising therapeutic profile for future against this important neglected systemic mycosis.

Talaromyces marneffei is a thermally dimorphic fungus that causes opportunistic systemic mycoses in patients with AIDS or other immunodeficiency syndromes. The purpose of this study was to develop an immunochromatographic strip test (ICT) based on a solid phase sandwich format immunoassay for the detection of T. marneffei antigens in clinical urine specimens. The T. marneffei yeast phase specific monoclonal antibody 4D1 (MAb4D1) conjugated with colloidal gold nanoparticle was used as a specific signal reporter. Galanthus nivalis Agglutinin (GNA) was adsorbed onto nitrocellulose membrane to serve as the test line. Similarly, a control line was created above the test line by immobilization of rabbit anti-mouse IgG. The immobilized GNA served as capturing molecule and as non-immune mediated anti-terminal mannose of T. marneffei antigenic mannoprotein. The MAb4D1–GNA based ICT showed specific binding activity with yeast phase antigen of T. marneffei, and it did not react with other common pathogenic fungal antigens. The limit of detection of this ICT for T. marneffei antigen spiked in normal urine was approximately 0.6 μg/ml. The diagnostic performance of the ICT was validated using 341 urine samples from patents with culture- confirmed T. marneffei infection and from a control group of healthy individuals and patients with other infections in an endemic area. The ICT exhibited 89.47% sensitivity, 100% specificity, and 97.65% accuracy. Our results demonstrate that the urine-based GNA–MAb4D1 based ICT produces a visual result within 30 minutes and that the test is highly specific for the diagnosis of T. marneffei infection. The findings validate the deployment of the ICT for clinical use., Author summary Talaromycosis (Penicilliosis marneffei) is a neglected disease that causes an opportunistic systemic mycoses in AIDS and other immune-deficient patients living in Southeast Asia, China and the Indian subcontinent. Although laboratory culture remains a gold standard diagnostic method, it lacks sensitivity and is time-consuming, which results in delayed patient’s treatment and needed care. In this study, we develop an immunochromatographic strip test (ICT) by utilizing a yeast phase specific monoclonal antibody 4D1 and Galanthus nivalis agglutinin for detection of T. marneffei antigens in clinical urine specimens. Our data showed that the assay exhibited high sensitivity (89.47%) and specificity (100%), with its result available within 30 minutes. In addition, this diagnostic assay is inexpensive, reproducible, and simple to perform. Therefore, the T. marneffei ICT should be considered for clinical application in the context of rapid and affordable point-of-care diagnostic test to reduce the burden of talaromycosis mortality in patients in low resource countries.

The aim of this study was to develop a novel lateral flow immunochromatoghaphic strip test (ICT) for detecting cryptococcal polysaccharide capsular antigens using only a single specific monoclonal antibody, mAb 18B7. The mAb 18B7 is a well characterized antibody that specifically binds repeating epitopes displayed on the cryptococcal polysaccharide glucuronoxylomannan (GXM). We validated the immunoreactivities of mAb 18B7 against capsular antigens of different cryptococcal serotypes. The mAb 18B7 ICT was constructed as a sandwich ICT strip and the antibody serving in the mobile phase (colloidal gold conjugated mAb 18B7) to bind one of the GXM epitopes while the stationary phase antibody (immobilized mAb18B7 on test line) binding to other remaining unoccupied epitopes to generate a positive visual readout. The lower limit of detection of capsular antigens for each of the Cryptococcus serotypes tested was 0.63 ng/mL. No cross-reaction was found against a panel of antigens isolated from cultures of other pathogenic fungal, except the crude antigen of Trichosporon sp. with the lower limit of detection of 500 ng/mL (~800 times higher than that for cryptococcal GXM). The performance of the mAb 18B7 ICT strip was studied using cerebrospinal fluid (CSF) and serum and compared to commercial diagnostic kits (latex agglutination CALAS and CrAg IMMY). The sensitivity, specificity and accuracy of the mAb18B7 ICT with CSF from patients with confirmed cryptococcal meningitis were 92.86%, 100% and 96.23%, respectively. No false positives were observed with samples from non-cryptococcosis patients. With serum samples, the mAb 18B7 ICT gave a sensitivity, specificity and accuracy of 96.15%, 97.78% and 96.91%, respectively. Our results show that the mAb 18B7 based ICT was reliable, reproducible, and cost-effective as a point-of-care immunodiagnostic test for cryptococcosis. The mAb 18B7 ICT may be particularly useful in countries where commercial kits are not available or affordable.

Heat shock proteins (Hsps) are highly conserved molecules that are constitutively expressed and upregulated in response to physiological stress conditions. These immunogenic chaperones can have essential functions in fungi, particularly in dimorphic pathogens. Histoplasma capsulatum and Paracoccidioides species are dimorphic fungi that are the causative agents of histoplasmosis and paracoccidioidomycosis, respectively, which are systemic mycoses with significant rates of morbidity and mortality. Current treatment consists of long-term antifungal agents, and there is an urgent need for new therapeutic approaches with higher efficacy, lower toxicity, better biodistribution and improved selectivity. We engineered an immunoglobulin G1 (IgG1) isotype chimeric mouse-human monoclonal antibody, titled ch-MAb 4E12, from the parental IgG2a MAb 4E12, a monoclonal antibody to H. capsulatum Hsp60 that is protective in experimental histoplasmosis and paracoccidioidomycosis models elicited by H. capsulatum var. capsulatum and Paracoccidioides lutzii, respectively. The ch-MAb 4E12 increased phagolysosomal fusion and enhanced the yeasts uptake by PMA differentiated human THP1 macrophage cells in vitro. At low concentrations, the chimeric antibody significantly reduced the pulmonary and splenic fungal burden compared to an irrelevant antibody or no treatment. These results are the first to show that a chimeric mouse-human antibody can modify infection caused by dimorphic fungi.

Oral squamous cell carcinoma (OSCC) is a serious health issue worldwide. OSCC is highly associated with oral candidiasis, although it is unclear whether the fungus promotes the genesis and progression of OSCC or cancer facilitates the growth of the fungus. Therefore, we investigated whether Candida could directly influence OSCC development and progression. Our in vitro results suggest that the presence of live C. albicans, but not C. parapsilosis, enhances the progression of OSCC by stimulating the production of matrix metalloproteinases, oncometabolites, pro-tumor signaling routes, and overexpression of prognostic marker genes associated with metastatic events. We also found that oral candidiasis triggered by C. albicans enhanced the progression of OSCC in vivo through the induction of inflammation and overexpression of metastatic genes and markers of epithelial-mesenchymal transition. Taken together, these results suggest that C. albicans actively participates in the complex process of OSCC progression.

The prevalence of fungal infections has increased in immunocompromised patients, leading to millions of deaths annually. Arachidonic acid (AA) metabolites, such as eicosanoids, play important roles in regulating innate and adaptative immune function, particularly since they can function as virulence factors enhancing fungal colonization and are produced by mammalian and lower eukaryotes, such as yeasts and other fungi (Candida albicans, Histoplasma capsulatum and Cryptococcus neoformans). C. albicans produces prostaglandins (PG), Leukotrienes (LT) and Resolvins (Rvs), whereas the first two have been well documented in Cryptococcus sp. and H. capsulatum. In this review, we cover the eicosanoids produced by the host and fungi during fungal infections. These fungal-derived PGs have immunomodulatory functions analogous to their mammalian counterparts. Prostaglandin E2 (PGE2) protects C. albicans and C. parapsilosis cells from the phagocytic and killing activity of macrophages. H. capsulatum PGs augment the fungal burden and host mortality rates in histoplasmosis. However, PGD2 potentiates the effects and production of LTB4, which is a very potent neutrophil chemoattractant that enhances host responses. Altogether, these data suggest that eicosanoids, mainly PGE2, may serve as a new potential target to combat diverse fungal infections.

Oral epithelial cells monitor microbiome composition and initiate immune response upon dysbiosis, as in case of Candida imbalances. Comparison of healthy oral epithelial cell responses revealed that the inability of C. parapsilosis to induce a robust antifungal response was due to activation of various inflammation-independent pathways, while C. albicans robustly activated inflammation cascades. Regarding posttranscriptional regulation, several miRNAs were altered by both species. For C. parapsilosis, the applied dose directly correlated with changes in transcriptomic responses. Carbohydrate metabolism, hypoxia- and cardiovascular development-related responses dominate after C. parapsilosis stimulus, whereas C. albicans altered inflammatory responses. Subsequent analyses of HIF1-α and HSC-activation pathways predicted target genes through which miRNA-dependent regulation of yeast-specific functions may occur, supporting the observed responses. Thus, C. parapsilosis is recognized as a commensal at low doses by the oral epithelium; however, increased fungal burden activates different pathways, some of which overlap with inflammatory processes induced by C. albicans.Impact statementAltered miRNA regulation discriminates between C. albicans and C. parapsilosis in human oral epithelial cells

The cell wall is a ubiquitous structure in the fungal kingdom, with some features varying depending on the species. Additional external structures can be present, such as the capsule of Cryptococcus neoformans (Cn), its major virulence factor, mainly composed of glucuronoxylomannan (GXM), with anti-phagocytic and anti-inflammatory properties. The literature shows that other cryptococcal species and even more evolutionarily distant species, such as the Trichosporon asahii, T. mucoides, and Paracoccidioides brasiliensis can produce GXM-like polysaccharides displaying serological reactivity to GXM-specific monoclonal antibodies (mAbs), and these complex polysaccharides have similar composition and anti-phagocytic properties to cryptococcal GXM. Previously, we demonstrated that the fungus Histoplasma capsulatum (Hc) incorporates, surface/secreted GXM of Cn and the surface accumulation of the polysaccharide enhances Hc virulence in vitro and in vivo. In this work, we characterized the ability of Hc to produce cellular-attached (C-gly-Hc) and secreted (E-gly) glycans with reactivity to GXM mAbs. These C-gly-Hc are readily incorporated on the surface of acapsular Cn cap59; however, in contrast to Cn GXM, C-gly-Hc had no xylose and glucuronic acid in its composition. Mapping of recognized Cn GXM synthesis/export proteins confirmed the presence of orthologs in the Hc database. Evaluation of C-gly and E-gly of Hc from strains of distinct monophyletic clades showed serological reactivity to GXM mAbs, despite slight differences in their molecular dimensions. These C-gly-Hc and E-gly-Hc also reacted with sera of cryptococcosis patients. In turn, sera from histoplasmosis patients recognized Cn glycans, suggesting immunogenicity and the presence of cross-reacting antibodies. Additionally, C-gly-Hc and E-gly-Hc coated Cn cap59 were more resistant to phagocytosis and macrophage killing. C-gly-Hc and E-gly-Hc coated Cn cap59 were also able to kill larvae of Galleria mellonella. These GXM-like Hc glycans, as well as those produced by other pathogenic fungi, may also be important during host-pathogen interactions, and factors associated with their regulation are potentially important targets for the management of histoplasmosis.

Paracoccidioidomycosis (PCM), caused by the Paracoccidioides species, is a systemic disease endemic in several Latin American countries, mainly in Brazil, Colombia, Argentina, and Venezuela. Current treatment approaches are challenging as they require prolonged durations of antifungal drugs that have potential toxicities, and despite antifungals, relapses are common. Hence, new therapeutic approaches, such as vaccines, are being investigated. The therapeutic vaccine consisting of peptide P10 associated with lipid cationic DODAB (P10+DODAB) is effective in murine models of PCM. However, the specific immune mechanisms required for the protective response has not been fully elucidated. The present work aims at evaluating the participation of neutrophils in the immune response induced by P10+DODAB. We found that the vaccine reduced both the influx of pulmonary neutrophils and the fungal load in comparison to infected animals that did not receive this treatment. The parenchymal architecture of the lungs of P10+DODAB-treated animals was largely preserved with only a few granulomas present, and tissue cytokine analysis showed a Th1 cytokine profile with augmented levels of IL-12, IFN-γ and TNF-α, and low levels of IL-4. When neutrophils were depleted 24 h prior to each treatment, the effectiveness of the P10+DODAB vaccine was completely lost as the fungal burdens remained high and histological examination showed a marked inflammation and fungal dissemination with a dysregulated cytokine response. In conclusion, these findings indicate that neutrophils are vital to ensure the triggering of an effective immune response to P10+DODAB.

Fungal infections of the heart occur significantly less frequently than bacterial infections, but they carry an excessively high mortality rate. Fungal endocarditis has an estimate mortality rate of ~50% while bacterial causes result in an ~18% in-hospital mortality (Murdoch et al., 2009). This article covers the cardiac involvement, particularly endocarditis and myocarditis, of common invasive fungi pathogens.

The peptide P10 is a vaccine candidate for Paracoccidioidomycosis, a systemic mycosis caused by fungal species of the genus Paracoccidioides spp. We have previously shown that peptide P10 vaccination, in the presence of several different adjuvants, induced a protective cellular immune response mediated by CD4+ Th1 lymphocytes that was associated with the increased production of IFN-&gamma, in mice challenged with a virulent isolate of Paracoccidoides brasiliensis. Cationic liposomes formulated with dioctadecyldimethylammonium and trehalose dibehenate (DDA/TDB, termed also CAF01&ndash, cationic adjuvant formulation) have been developed for safe administration in humans and CAF01 liposomes are utilized as an adjuvant for modulating a robust Th1/Th17 cellular response. We evaluated the efficacy of the adsorption of peptide P10 to CAF01 cationic liposomes and used the generated liposomes to vaccinate C57Bl/6 mice infected with P. brasiliensis. Our results showed that P10 was efficiently adsorbed onto CAF01 liposomes. The vaccination of infected mice with cationic liposomes formulated with DDA/TDB 250/50 &micro, g/mL and 20 &micro, g of P10 induced an effective cellular immune response with increased levels of Th17 cytokines, which correlated with significant decreases in the fungal burdens in lungs and protective granulomatous tissue responses. Hence, cationic liposomes of DDA/TDB 250/50 &micro, g/mL with 20 &micro, g of P10 are a promising therapeutic for safely and effectively improving the treatment of paracoccidioidomycosis.

Talaromyces marneffei is a dimorphic fungus that has emerged as an opportunistic pathogen particularly in individuals with HIV/AIDS. Since its dimorphism has been associated with its virulence, the transition from mold to yeast-like cells might be important for fungal pathogenesis, including its survival inside of phagocytic host cells. We investigated the expression of yeast antigen of T.marneffei using a yeast-specific monoclonal antibody (MAb) 4D1 during phase transition. We found that MAb 4D1 recognizes and binds to antigenic epitopes on the surface of yeast cells. Antibody to antigenic determinant binding was associated with time of exposure, mold to yeast conversion, and mammalian temperature. We also demonstrated that MAb 4D1 binds to and recognizes conidia to yeast cells’ transition inside of a human monocyte-like THP-1 cells line. Our studies are important because we demonstrated that MAb 4D1 can be used as a tool to study T.marneffei virulence, furthering the understanding of the therapeutic potential of passive immunity in this fungal pathogenesis.

Cryptococcus neoformans is an encapsulated yeast that causes disease mainly in immunosuppressed hosts. It is considered a facultative intracellular pathogen because of its capacity to survive and replicate inside phagocytes, especially macrophages. This ability is heavily dependent on various virulence factors, particularly the glucuronoxylomannan (GXM) component of the polysaccharide capsule. Inflammasome activation in phagocytes is usually protective against fungal infections, including cryptococcosis. Nevertheless, recognition of C. neoformans by inflammasome receptors requires specific changes in morphology or the opsonization of the yeast, impairing proper inflammasome function. In this context, we analyzed the impact of molecules secreted by C. neoformans B3501 strain and its acapsular mutant Δcap67 in inflammasome activation in an in vitro model. Our results showed that conditioned media derived from B3501 was capable of inhibiting inflammasome-dependent events (i.e., IL-1β secretion and LDH release via pyroptosis) more strongly than conditioned media from Δcap67, regardless of GXM presence. We also demonstrated that macrophages treated with conditioned media were less responsive against infection with the virulent strain H99, exhibiting lower rates of phagocytosis, increased fungal burdens, and enhanced vomocytosis. Moreover, we showed that the aromatic metabolite DL-Indole-3-lactic acid (ILA) and DL-p-Hydroxyphenyllactic acid (HPLA) were present in B3501’s conditioned media and that ILA alone or with HPLA is involved in the regulation of inflammasome activation by C. neoformans. These results were confirmed by in vivo experiments, where exposure to conditioned media led to higher fungal burdens in Acanthamoeba castellanii culture as well as in higher fungal loads in the lungs of infected mice. Overall, the results presented show that conditioned media from a wild-type strain can inhibit a vital recognition pathway and subsequent fungicidal functions of macrophages, contributing to fungal survival in vitro and in vivo and suggesting that secretion of aromatic metabolites, such as ILA, during cryptococcal infections fundamentally impacts pathogenesis.

As the early epicenter of the COVID-19 pandemic, New York City's medical schools experienced dramatic disruptions in every aspect of medical education. Remote learning was created, seemingly overnight, clerkships were disrupted, licensing examinations were cancelled, teaching faculty were redeployed, student volunteers rallied, and everyone was required to shelter at home. Seismic changes were required to adapt the authors' educational programs to a constantly evolving, unpredictable, and ever-worsening public health crisis. Entirely new communication strategies were adopted and thousands of decisions had to be made, often with little time to carefully reflect on the consequences of those decisions. What allowed each school to navigate these treacherous waters was a set of guiding principles that were used to ground each conversation, and inform every decision. While the language varied somewhat between schools, the core principles were universal and framed a way forward at a time when information, data, precedent, and best practices did not exist. The authors share these guiding principles in the hope that colleagues at other medical schools will find them to be a useful framework as we all continue to cope with the impact of COVID-19 on the future of medical education.

Cryptococcus neoformans is a human pathogenic fungus that mainly afflicts immunocompromised patients. One of its virulence strategies is the production of extracellular vesicles (EVs), containing cargo with immunomodulatory properties. We evaluated EV&rsquo, s characteristics produced by capsular and acapsular strains of C. neoformans (B3501 and &Delta, Cap67, respectively) growing in nutritionally poor or rich media and co-cultures with bone marrow-derived macrophages or dendritic cells from C57BL/6 mice. EVs produced under a poor nutritional condition displayed a larger hydrodynamic size, contained more virulence compounds, and induced a more robust inflammatory pattern than those produced in a rich nutritional medium, independently of strain. We treated infected mice with EVs produced in the rich medium, and the EVs inhibited more genes related to the inflammasome than untreated infected mice. These findings suggest that the EVs participate in the pathogenic processes that result in the dissemination of C. neoformans. Thus, these results highlight the versatility of EVs&rsquo, properties during infection by C. neoformans in different tissues and support ongoing efforts to harness EVs to prevent and treat cryptococcosis.

Extracellular vesicles (EVs) are lipid bilayered compartments released by virtually all living cells, including fungi. Among the diverse molecules carried by fungal EVs, a number of immunogens, virulence factors and regulators have been characterized. Within EVs, these components could potentially impact disease outcomes by interacting with the host. From this perspective, we previously demonstrated that EVs from C. albicans could be taken up by and activate macrophages and dendritic cells to produce cytokines and express costimulatory molecules. Moreover, pre-treatment of Galleria mellonella larvae with fungal EVs protected the insects against a subsequent lethal infection with C. albicans yeasts. These data indicate that C. albicans EVs are multi-antigenic compartments that activate the innate immune system and could be exploited as vaccine formulations. Here we investigated whether immunization with C. albicans EVs induces a protective effect against murine candidiasis in immunosuppressed mice. Total and fungal antigen-specific serum IgG antibodies increased by 21 days after immunization, confirming the efficacy of the protocol. Vaccination decreased fungal burden in the liver, spleen and kidney of mice challenged with C. albicans. Splenic levels of cytokines indicated a lower inflammatory response in mice immunized with EVs when compared with EVs+Freund's adjuvant (ADJ). Higher levels of IL-12p70, TNFα and IFNγ were detected in mice vaccinated with EVs+ADJ, while IL-12p70, TGFβ, IL-4 and IL-10 were increased when no adjuvants were added. Full protection of lethally challenged mice was observed when EVs were administered, regardless the presence of adjuvant. Physical properties of the EVs were also investigated and EVs produced by C. albicans were relatively stable after storage at 4, -20 or -80 0 C, keeping their ability to activate dendritic cells and to protect G. mellonella against a lethal candidiasis. Our data suggest that fungal EVs could be a safe source of antigens to be exploited in vaccine formulations. This article is protected by copyright. All rights reserved.

Vaccination is one of the greatest public health achievements in the past century, protecting and improving the quality of life of the population worldwide. However, a safe and effective vaccine for therapeutic or prophylactic treatment of fungal infections is not yet available. The lack of a vaccine for fungi is a problem of increasing importance as the incidence of diverse species, including Paracoccidioides, Aspergillus, Candida, Sporothrix, and Coccidioides, has increased in recent decades and new drug-resistant pathogenic fungi are emerging. In fact, our antifungal armamentarium too frequently fails to effectively control or cure mycoses, leading to high rates of mortality and morbidity. With this in mind, many groups are working towards identifying effective and safe vaccines for fungal pathogens, with a particular focus of generating vaccines that will work in individuals with compromised immunity who bear the major burden of infections from these microbes. In this review, we detail advances in the development of vaccines for pathogenic fungi, and highlight new methodologies using immunoproteomic techniques and bioinformatic tools that have led to new vaccine formulations, like peptide-based vaccines.

Candida auris (C. auris) is an emerging pathogenic fungal species that is especially worrisome due to its high mortality rates and widespread antifungal resistance. Previous studies have demonstrated the efficacy of nitric oxide (NO) nanoparticles on Candida species, and, to our knowledge, this is the first study to investigate the antifungal effects of a NO-generating nanoparticle on C. auris. Six C. auris strains were incubated with a nanoparticle (NAC-SNO-np), which releases N-acetylcysteine S-nitrosothiol (NAC-SNO) and N-acetylcysteine (NAC), and generates NO, through colony forming unit (CFU) assays, and confocal laser scanning microscopy. NAC-SNO-np effectively eradicates planktonic and biofilm C. auris. Across all six strains, 10 mg/mL NAC-SNO-np significantly reduced the number of CFUs (p &lt, 0.05) and demonstrated a &gt, 70% decrease in biofilm viability (p &lt, 0.05). NAC-SNO-np effectively eradicates planktonic C. auris and significantly reduces C. auris biofilm formation. Hence, this novel NO-releasing nanoparticle shows promise as a future therapeutic.

Lipids play a fundamental role in fungal cell biology, being essential cell membrane components and major targets of antifungal drugs. A deeper knowledge of lipid metabolism is key for developing new drugs and a better understanding of fungal pathogenesis. Here we built a comprehensive map of theHistoplasma capsulatumlipid metabolic pathway by incorporating proteomic and lipidomic analyses. We performed genetic complementation and overexpression ofH. capsulatumgenes inSaccharomyces cerevisiaeto validate reactions identified in the map and to determine enzymes responsible for catalyzing orphan reactions. The map led to the identification of both the fatty acid desaturation and the sphingolipid biosynthesis pathways as targets for drug development. We found that the sphingolipid biosynthesis inhibitor myriocin, the fatty acid desaturase inhibitor thiocarlide and the fatty acid analog 10-thiastearic acid inhibitH. capsulatumgrowth in nanomolar to low micromolar concentrations. These compounds also reduced the intracellular infection in an alveolar macrophage cell line. Overall, this lipid metabolic map revealed pathways that can be targeted for drug development.

Human fungal diseases differ fundamentally from other infections in diverse ways. As eukaryotic pathogens, fungi share many similarities with their host cells, which impairs the development of antifungal compounds. Fungal tropism is highly variable, as pathogens infect a wide range of cell types. A single fungal pathogen can infect multiple tissues in the same patient (depending on the host’s immunological status) and can undergo morphogenic shifts during infection. Fungi are still underappreciated as major pathogens by both the public and public health officials. Diseases caused by protozoa, bacteria, and viruses have been recognized as important public health issues for centuries. For instance, syphilis, influenza, and Chagas disease have been documented for over 100 years [1], while invasive mycoses were only widely acknowledged as medically important pathogens in the 1980s [2].

Paracoccidioidomycosis is a highly prevalent systemic mycosis in Latin America, caused by fungi of the genus Paracoccidioides. Copper is essential for eukaryotes and bacteria. This micronutrient is used in many vital biochemical processes, although metal excess levels can be toxic for organisms. Pathways underlying copper overload are poorly understood in members of the Paracoccidioides complex. The responses of Paracoccidioides lutzii yeast cells to copper overload were here evaluated. The results showed that under copper overload, cells presented a dark brown pigment, identified as melanin. Proteomic analyses identified mainly the accumulation of proteins related to amino acids metabolism, ergosterol synthesis and melanin production, suggesting that P. lutzii responds to copper overload by changing aspects of its metabolism and also plasma membrane and cell wall remodeling. Proteomic data were confirmed by biochemical analysis.

Invasive fungal infections (IFI) cause devastating morbidity and mortality, especially in organ transplant patients, cancer patients and patients in intensive care units, with the number of IFIs more than tripling since 1979. Radioimmunotherapy (RIT) utilizes antigen-antibody interaction to deliver lethal doses of ionizing radiation to cells and has demonstrated efficacy in several types of cancer. Our laboratories were the first to demonstrate that microorganism-specific monoclonal antibodies are highly effective in treatment of experimental fungal, bacterial and viral infections. Later we proposed to utilize surface expressed antigens shared by major IFI-causing pathogens such as melanin, heat shock protein 60 (HSP60) and beta-glucans as targets for radiolabeled antibodies. Here we evaluated in vivo RIT targeting pan-antigens in IFIs. Blastomycosis dermatitidis was chosen as a model for this work as it causes serious infections in immunocompromised patients, immunocompetent individuals and in companion dogs. B. dermatitidis cells were treated with the 400-2 antibody to (1→3)-β-glucans which was radiolabeled with the beta particles emitter 177Lutetium (177Lu) and alpha particles emitter 213Bismuth (213Bi) and the efficacy of cell kill was determined by CFUs. To determine the antigen-specific localization of the 400-2 antibody in vivo, C57BL6 mice were infected intratracheally with 2×105 B. dermatitidis cells and given 111In-400-2 antibody 24 hr later. To evaluate the killing of B. dermatitidis cells with RIT, intratracheally infected mice were treated with 150 µCi 213Bi-400-2 and their lungs analyzed for CFUs 96 hrs post-infection. 213Bi-400-2 proved to be more effective in killing B. dermatitidis cells in vitro than 177Lu-400-2. Three times more 111In-400-2 accumulated in the lungs of infected mice, than in the non-infected ones. 213Bi-400-2 lowered the fungal burden in the lungs of infected mice more than 2 logs in comparison with non-treated infected controls. In conclusion, our results demonstrate the ability of an anti-(1-3)-beta-D-glucan antibody armed with an alpha-emitter 213Bi to selectively kill B. dermatitidis cells in vitro and in vivo. These results provide strong evidence of the effectiveness of RIT targeting pan-antigens on fungal pathogens.

BACKGROUND Bacteria, particularly in the biofilm state, may be implicated in the pathogenesis of chronic rhinosinusitis (CRS) and enhance antibiotic resistance. Nitric oxide (NO) is a gaseous immunomodulator with antimicrobial activity and a short half-life, complicating achievement of therapeutic concentrations. We hypothesized that a novel microparticle-based delivery platform, which allows for adjustable release of NO, could exhibit potent antibacterial effects. METHODS Porous organosilica microparticles (SNO-MP) containing nitrosylated thiol groups were formulated. Dissociation of the nitrosothiol groups generates NO at body temperature. The susceptibility of bacterial isolates from CRS patients to SNO-MP was evaluated through a colony forming unit (CFU) assay. Serial dilutions of SNO-MP in triplicate were incubated with isolates in suspension for 6 hours followed by plating on tryptic soy agar and overnight incubation followed by CFU quantification. Statistical analysis was performed with SPSS using one-way analysis of variance with Bonferroni correction. RESULTS SNO-MP displayed antibacterial activity against gram-positive (methicillin-resistant and -sensitive Staphylococcus aureus) and gram-negative (Pseudomonas aeruginosa, Enterobacter aerogenes, and Proteus mirabilis) isolates. SNO-MP induced dose-dependent reductions in CFU across all strains. Compared with controls and blank nanoparticles, SNO-MP (10 mg/mL) induced a 99.99%-100% reduction in CFU across all isolates, equivalent to a 5-9 log kill (p < 0.005). There was no statistically significant difference in CFU concentration between controls and blank microparticles. CONCLUSION SNO-MP demonstrates potent bactericidal effect against antibiotic-resistant CRS bacterial strains.

Metastatic melanoma remains difficult to treat despite recent approvals of several new drugs. Recently we reported encouraging results of Phase I clinical trial of radiolabeled with 188Re murine monoclonal IgM 6D2 to melanin in patients with Stage III/IV melanoma. Subsequently we generated a novel murine IgG 8C3 to melanin. IgGs are more amenable to humanization and cGMP (current Good Manufacturing Practice) manufacturing than IgMs. We performed comparative structural analysis of melanin-binding IgM 6D2 and IgG 8C3. The therapeutic efficacy of 213Bi- and 188Re-labeled 8C3 and its comparison with anti-CTLA4 immunotherapy was performed in B16-F10 murine melanoma model. The primary structures of these antibodies revealed significant homology, with the CDRs containing a high percentage of positively charged amino acids. The 8C3 model has a negatively charged binding surface and significant number of aromatic residues in its H3 domain, suggesting that hydrophobic interactions contribute to the antibody-melanin interaction. Radiolabeled IgG 8C3 showed significant therapeutic efficacy in murine melanoma, safety towards healthy melanin-containing tissues and favorable comparison with the anti-CTLA4 antibody. We have demonstrated that antibody binding to melanin relies on both charge and hydrophobic interactions while the in vivo data supports further development of 8C3 IgG as radioimmunotherapy reagent for metastatic melanoma.

The effect of size and release kinetics of doxorubicin-nanoparticles on anti-tumor efficacy was evaluated in a panel of human cancer cell lines, including triple-negative breast cancer (TNBC) cells that frequently demonstrate resistance to doxorubicin. Different nano-formulations of sol-gel-based Doxorubicin containing nanoparticles were synthesized. Increased cell kill in chemorefractory triple-negative breast cancer cells was associated with the smallest size of nanoparticles and the slowest release of Dox. Modeling of dose-response parameters in Dox-sensitive versus Dox-resistant lines demonstrated increased EMax and area under the curve in Dox-resistant mesenchymal TNBC cells, implying potentially favorable activity in this molecular subtype of breast cancer. Mesenchymal TNBC cells demonstrated a high rate of fluorescent bead uptake suggestive of increased endocytosis, which may partially account for the enhanced efficacy of Dox-np in this subtype. Thus, manipulation of size and release kinetics of this nanoparticle platform is associated with enhanced dose-response metrics and tumor cell kill in therapeutically recalcitrant TNBC cell models. This platform is easily customizable and warrants further exploration.

Invasive pulmonary aspergillosis is a frequent complication in immunocompromised individuals, and it continues to be an important cause of mortality in patients undergoing hematopoietic stem cell transplantation. In addition to antifungal therapy used for mycoses, immune-modulatory molecules such as cytokines and chemokines can modify the host immune response and exhibit a promising form of antimicrobial therapeutics to combat invasive fungal diseases. Cytokine and chemokine profiles may also be applied as biomarkers during fungal infections and clinical research has demonstrated different activation patterns of cytokines in invasive mycoses such as aspergillosis. In this review, we summarize different aspects of cytokines that have been described to date and provide possible future directions in research on invasive pulmonary aspergillosis following hematopoietic stem cell transplantation. These findings suggest that cytokines and chemokines may serve as useful biomarkers to improve diagnosis and monitoring of infection.

Fusarium spp. are recognized as the second most frequently filamentous fungi causing opportunistic infections and particularly important due to the increasing number of immunocompromised patients. F. keratoplasticum (a member of F. solani species complex) is one of the Fusarium species commonly associated with human infection, and therefore, studies on the virulence of this fungus are needed. This study aimed to confirm the presence of melanin in F. keratoplasticum from a patient with systemic fusariosis. Immunofluorescence labeling with anti-melanin monoclonal antibody (MAb) was used to examine an expression of melanin in F. keratoplasticum in vitro and during infection. Electron spin resonance identified the particles extracted from F. keratoplasticum as stable free radical consistent with melanin. Lesional skin from the sites with fusariosis contained hyphal structures that could be labeled by melanin-binding MAb, while digestion of the tissue yielded dark particles that were reactive. These findings suggest that F. keratoplasticum hyphae and chlamydospores can produce melanin in vitro and that hyphae can synthesize pigment in vivo. Given the potential role of melanin in virulence of other fungi, this pigment in F. keratoplasticum may play a role in the pathogenesis of fusariosis.

The increasing incidence in systemic fungal infections in humans has increased focus for the development of fungal vaccines and use of monoclonal antibodies. Invasive mycoses are generally difficult to treat, as most occur in vulnerable individuals, with compromised innate and adaptive immune responses. Mortality rates in the setting of our current antifungal drugs remain excessively high. Moreover, systemic mycoses require prolonged durations of antifungal treatment and side effects frequently occur, particularly drug-induced liver and/or kidney injury. The use of monoclonal antibodies with or without concomitant administration of antifungal drugs emerges as a potentially efficient treatment modality to improve outcomes and reduce chemotherapy toxicities. In this review, we focus on the use of monoclonal antibodies with experimental evidence on the reduction of fungal burden and prolongation of survival in in vivo disease models. Presently, there are no licensed monoclonal antibodies for use in the treatment of systemic mycoses, although the potential of such a vaccine is very high as indicated by the substantial promising results from several experimental models.

We engaged medical students with antimicrobial stewardship (AS) and resistance (AMR) through patient stories and a panel on AMR advocacy with experts from the Centers for Disease Control and Prevention and the Infectious Diseases Society of America. Students were surveyed on their perceptions about AS and AMR (response rate=139/166, 84%).

Histoplasma capsulatum is a dimorphic fungus that most frequently causes pneumonia, but can also disseminate and proliferate in diverse tissues. Histoplasma capsulatum has a complex secretion system that mediates the release of macromolecule-degrading enzymes and virulence factors. The formation and release of extracellular vesicles (EVs) are an important mechanism for non-conventional secretion in both ascomycetes and basidiomycetes. Histoplasma capsulatum EVs contain diverse proteins associated with virulence and are immunologically active. Despite the growing knowledge of EVs from H. capsulatum and other pathogenic fungi, the extent that changes in the environment impact the sorting of organic molecules in EVs has not been investigated. In this study, we cultivated H. capsulatum with distinct culture media to investigate the potential plasticity in EV loading in response to differences in nutrition. Our findings reveal that nutrition plays an important role in EV loading and formation, which may translate into differences in biological activities of these fungi in various fluids and tissues.

BACKGROUND Sporotrichosis is a subcutaneous mycosis caused by dimorphic pathogenic fungi belonging to the Sporothrix genus. Pathogenic Sporothrix species typically produce melanin, which is known to be a virulence factor. OBJECTIVES The aim of this study was to perform phenotypic, genotypic, and virulence analyses of two distinct Sporothrix brasiliensis strains isolated from the same lesion on a patient from Rio de Janeiro. METHODS AND FINDINGS Genotypic analyses by partial sequencing of the calmodulin, β-tubulin, and chitin synthase genes, as well as polymerase chain reaction (PCR)-fingerprinting by T3B, M13, and GACA, showed that the isolates were very similar but not identical. Both isolates had similar phenotypic characteristics and effectively produced melanin in their yeast forms, accounting for their ability of causing disease in a murine sporotrichosis model. Remarkably, isolate B was albino in its environmental form but caused more severe disease than the pigmented A isolate. CONCLUSIONS These findings indicate that the patient was infected by two genetically and biologically distinct S. brasiliensis that vary in their production of melanin in their environmental forms. The results underscore the importance of characterizing phenotypically different isolates found in the same clinical specimen or patient.

Paracoccidioidomycosis (PCM) is an endemic mycosis in Latin American caused by the thermodimorphic fungi of the genus Paracoccidioides spp. Notably, a Th1 immune response is required to control PCM. In this context, dendritic cells (DCs) seem to be essential players in capture, processing and presentation of Paracoccidioides antigens to naïve T cells and their further activation. We have previously demonstrated that differentiated DCs from bone marrow cells, pulsed with the immunoprotective peptide 10 (P10), effectively control experimental PCM immunocompetent and immunosuppressed mice. However, this procedure may not be infeasible or it is limited for the therapy of human patients. Therefore, we have sought a less invasive but equally effective approach that would better mimics the autologous transplant of DC in a human patient. Here, we isolated and generated monocyte differentiated dendritic cells (MoDCs) from infected mice, pulsed them with P-10, and used them in the therapy of PCM in syngeneic mice. Similar to the results using BMDCs, the P10-pulsed MoDCs stimulated the proliferation of CD4+ T lymphocytes, induced a mixed production of Th1/Th2 cytokines and decreased the fungal burden in murine lungs in the setting of PCM. The process of differentiating MoDCs derived from an infected host, and subsequently used for therapy of PCM is much simpler than that for obtaining BMDCs, and represents a more reasonable approach to treat patients infected with Paracoccidioides. The results presented suggest that P10-primed MoDC may be a promising strategy to combat complicated PCM as well as to significantly shorten the lengthy requirements for treatment of patients with this fungal disease.

BACKGROUND Bacterial biofilms are implicated in the pathogenesis of chronic rhinosinusitis. Nitric oxide (NO) is a key immune effector with potent antimicrobial effects, but a short half-life limits achievement of therapeutic concentrations. We hypothesized that manuka honey (MH) could induce sustained reduction of nitrite to NO causing biofilm disruption and that this effect would be enhanced with the addition of a NO-releasing microparticle. METHODS Porous organosilica microparticles containing nitrosylated thiol groups were formulated (SNO-MP). MH was combined with serial dilutions of nitrite. NO release was evaluated using a NO analyzer. The susceptibility of 2 strains of Pseudomonas aeruginosa biofilms to these NO-releasing platforms was evaluated using confocal microscopy. Cell viability and biofilm volume were quantified. Statistical analysis was performed using the Mann-Whitney U test with SPSS software. RESULTS MH with nitrite generated a linear increase in NO formation. SNO-MP induced a bolus release of NO within 5 minutes, followed by a sustained plateau phase. MH with nitrite combined with SNO-MP enhanced NO release during the plateau phase. MH with nitrite reduced biofilm live cells and volume by 88.5% to 96.9% and 95.1% to 95.6%, respectively, vs control (p < 0.0001). SNO-MP reduced live cells and volume by 61.0% to 98.5% and 74.7% to 85.7%, respectively, vs control (p < 0.0001). MH with nitrite combined with SNO-MP nearly eradicated biofilm, with a 98.3% to 99.8% (log 1.8-2.6) reduction in viability and a 91.4% to 97.7% decrease in volume (p < 0.0001 vs control). CONCLUSION A novel platform that generates NO using MH and nitrite produces a potent anti-biofilm effect, which can be further enhanced with the addition of SNO-MP.

Extracellular vesicles (EVs) play important roles in cellular communication and pathogenesis. The RNA molecules in EVs have been implicated in a variety of processes. EV-associated RNA classes have recently been described in pathogenic fungi; however, only a few reports of studies describing the RNAs in fungal EVs are available. Improved knowledge of EV-associated RNA will contribute to the understanding of their role during infection. In this study, we described the RNA content in EVs produced by two isolates of Histoplasma capsulatum. Our results add this important pathogen to the current short list of fungal species with the ability to use EVs for the extracellular release of RNA., Eukaryotic cells, including fungi, release extracellular vesicles (EVs). These lipid bilayered compartments play essential roles in cellular communication and pathogenesis. EV composition is complex and includes proteins, glycans, pigments, and RNA. RNAs with putative roles in pathogenesis have been described in EVs produced by fungi. Here we describe the RNA content in EVs produced by the G186AR and G217B strains of Histoplasma capsulatum, an important human-pathogenic fungal pathogen. A total of 124 mRNAs were identified in both strains. In this set of RNA classes, 93 transcripts were enriched in EVs from the G217B strain, whereas 31 were enriched in EVs produced by the G186AR strain. This result suggests that there are important strain-specific properties in the mRNA composition of fungal EVs. We also identified short fragments (25 to 40 nucleotides in length) that were strain specific, with a greater number identified in EVs produced by the G217B strain. Remarkably, the most highly enriched processes were stress responses and translation. Half of these fragments aligned to the reverse strand of the transcript, suggesting the occurrence of microRNA (miRNA)-like molecules in fungal EVs. We also compared the transcriptome profiles of H. capsulatum with the RNA composition of EVs, and no correlation was observed. Taking the results together, our study provided information about the RNA molecules present in H. capsulatum EVs and about the differences in composition between the strains. In addition, we found no correlation between the most highly expressed transcripts in the cell and their presence in the EVs, reinforcing the idea that the RNAs were directed to the EVs by a regulated mechanism. IMPORTANCE Extracellular vesicles (EVs) play important roles in cellular communication and pathogenesis. The RNA molecules in EVs have been implicated in a variety of processes. EV-associated RNA classes have recently been described in pathogenic fungi; however, only a few reports of studies describing the RNAs in fungal EVs are available. Improved knowledge of EV-associated RNA will contribute to the understanding of their role during infection. In this study, we described the RNA content in EVs produced by two isolates of Histoplasma capsulatum. Our results add this important pathogen to the current short list of fungal species with the ability to use EVs for the extracellular release of RNA.

Histoplasmosis is an infection caused by the dimorphic fungus Histoplasma capsulatum. Histoplasmosis is typically self-limited and presents asymptomatically in most people. Nevertheless, histoplasmosis can cause severe pulmonary disease and death. Histoplasmosis is increasingly found worldwide; however, it is best documented in the endemic region of the Mississippi river valley system in the Eastern part of the United States (US). Epidemiological studies from the US detailing the morbidity, mortality, and cost associated with histoplasmosis underscore the need to develop a vaccine. PURPOSE OF REVIEW: This review will detail some of the major developments in potential vaccines against histoplasmosis, with particular emphasis on those that could be used to immunize immunocompromised hosts. Additionally, this review will highlight some non-traditional vaccine-like ideas for the prevention of diverse mycoses. RECENT FINDINGS: Historically, immunization strategies against histoplasmosis have largely focused on identifying immunogenic proteins that confer protection in animal models. More recently, novel active, therapeutic, and immunomodulatory strategies have been explored as potential alternatives for those with various immune-deficiencies. SUMMARY: The studies summarized in this review demonstrate that more research is needed to clarify the immunobiology, clinical role and efficacy of each candidate vaccine in the ever-expanding potential armamentarium against histoplasmosis.

Eukaryotic cells, including fungi, release extracellular vesicles (EVs). These lipid bilayered compartments play essential roles in cellular communication and pathogenesis. EV composition is complex and includes proteins, glycans, pigments, and RNA. RNA classes with putative roles in pathogenesis have been described in EVs produced by fungi. Here we describe the RNA content in EVs produced by the G186AR and G217B strains ofHistoplasma capsulatum, an important human fungal pathogen. A total of 124 mRNA were identified in both strains. In this set of RNA classes, 93 transcripts were enriched in EVs from the G217B strain, while 31 enriched in EVs produced by the G186AR strain. This result suggests that there are important strain-specific properties in the mRNA composition of fungal EVs. We also identified short fragments (25-40 long) that were strain-specific, with a greater number of them identified in EVs produced by the G217B strain. Remarkably, the most enriched processes were stress responses and translation. Half of these fragments aligned to the reverse strand of the transcript, suggesting the occurrence of miRNA-like molecules in fungal EVs. We also compared the transcriptome profiles ofH. capsulatumwith the RNA composition of EVs and no correlation was observed. Altogether, our study provided information about the RNA molecules present inH. capsulatumEVs, and the differences in composition between the G186AR and G217B strains. In addition, we showed that the correlation between the most expressed transcripts in the cell and their presence in the EVs, reinforcing the idea that the RNAs were directed to the EVs by a regulated mechanism.ImportanceExtracellular vesicles (EVs) play important roles in cellular communication and pathogenesis. The RNA molecules in EVs have been implicated in a variety of processes. In pathogenic fungi, EV-associated RNA classes have recently been described; however, only a few studies describing the RNA in fungal EVs are available. An improved knowledge on EV-associated RNA will contribute to the understanding of their role during infection. In this study, we described the RNA content in EVs produced by two isolates ofHistoplasma capsulatum. Our results add this important pathogen to the current short list of fungal species with the ability to use EVs for the extracellular release of RNA.

Cryptococcus neoformansis an encapsulated yeast that causes disease mainly in immunosuppressed hosts. It is considered a facultative intracellular pathogen because of its capacity to survive and replicate inside phagocytes, especially macrophages. This capacity is heavily dependent on various virulence factors, particularly the glucuronoxylomannan (GXM) component of the polysaccharide capsule, that render the non- or poorly-activated macrophage ineffective against phagocytosed yeast. Strategies utilized by macrophages to prevent this scenario include pyroptosis (a rapid highly inflammatory cell death) and vomocytosis (the expulsion of the pathogen from the intracellular environment without lysis). Inflammasome activation in phagocytes is usually protective against fungal infections, including cryptococcosis. Nevertheless, recognition ofC. neoformansby inflammasome receptors requires specific changes in morphology or the opsonization of the yeast, impairing a proper inflammasome function. In this context, we analyzed the impact of molecules secreted byC. neoformansB3501 strain and its acapsular mutantΔcap67in an inflammasome activationin vitromodel. Our results showed that conditioned media derived from B3501 was capable of inhibiting inflammasome dependent events (i. e. IL-1β secretion and LDH release via pyroptosis) more strongly than conditioned media fromΔcap67, regardless of GXM presence. We also demonstrated that macrophages treated with conditioned media were less responsive against infection with the virulent strain H99, exhibiting lower rates of phagocytosis, increased fungal burdens and enhanced vomocytosis. Moreover, we showed that the aromatic metabolite DL-Indole-3-lactic acid (ILA) was present in B3501’s conditioned media and that this fungal metabolite is involved in the regulation of inflammasome activation byC. neoformans. Overall, the results presented show that conditioned media from a wild-type strain can inhibit an important recognition pathway and subsequent fungicidal functions of macrophages, contributing to fungal survivalin vitroand suggesting that this serves as an important role for secreted molecules during cryptococcal infections.Author’s SummaryCryptococcus neoformansis the agent of cryptococcal meningitis, a disease that can be life-threatening in immunocompromised hosts such as those infected with HIV. The infection thrives in hosts that poorly activate their immune system, mainly because of the yeast’s ability to survive inside macrophages and migrate towards the central nervous system. Emerging data indicate that cryptococci modulate the host immune response, but the underlying mechanisms remain largely uncharacterized. Here we show that secreted molecules from a wild-type strain ofC. neoformansimpair inflammatory responses driven by inflammasome activation, which in turn impact the macrophage antifungal activity. We further show that this inhibition does not involve GXM, the main constituent of the fungal capsule, but rather is partially dependent on DL-Indole-3-lactic acid (ILA), a metabolite not previously implicated in fungal virulence.

Fungal keratitis (FK) is a serious ocular infection that can result in various degrees of vision loss, including blindness. The aim of the study was to identify and retrospectively review all FK cases diagnosed between August 2012 and December 2020 at a tertiary care hospital in northern Thailand with a specific focus on epidemiologic features, including season, patient sex and age, the spectrum of pathogens, and presence of certain putative virulence factors. Of 1237 patients with corneal ulcers, 294 (23.8%) were confirmed by direct microscopic examination and/or fungal culture. For the positive cases, direct examinations of Calcofluor white (CW) stains and KOH mounts were found in 97.3% (286/294) and 76.5% (225/294), respectively (p &lt, 0.05). Of the cases diagnosed by microscopy and culture, fungi were isolated in 152 (51.7%), with Fusarium spp. being the most frequently identified (n = 69, 45.5%) followed by dematiaceous fungi (n = 45, 29.6%) and Aspergillus spp. (n = 18, 11.8%). The incidence of FK was higher in the rainy season of July to October. The mean age was 54.4 ± 14.4 (SD) years, with a range of 9–88 years. Males (75.8%) were affected significantly more than females (24.2%) (p &lt, 0.05). Of 294 patients, 132 (44.9%) were middle-aged adults (41–60 years) and 107 (36.4%) were older than 60 years. Trauma to the eye by soil or vegetative matter were the most common preceding factors (188/294, 64.0%). We assessed two virulence factors. First, 142 of the 152 culture-positive FK cases were due to molds, indicating that hyphal morphogenesis is extremely important in disease. We also demonstrated that fungal melanization occurs in the molds during the course of FK by applying a melanin-specific monoclonal antibody (MAb) that labeled fungal elements in corneal samples of patients, and melanin particles derived from the hyphae were also recovered after treatment of the samples with proteolytic enzymes, denaturant and hot concentrated acid. In summary, we demonstrate that northern Thailand has a high rate of FK that is influenced by season and males engaged in outside activities are at highest risk for disease. Moulds are significantly more commonly responsible for FK, in part due to their capacity to form hyphae and melanins. Future studies will examine models of fungal corneal interactions and assess additional factors of virulence, such as secreted enzymes, to more deeply decipher the pathogenesis of FK.