Mucor germinans, a novel dimorphic species resembling Paracoccidioides in a clinical sample: questions on ecological strategy.

Dimorphism is known among the etiologic agents of endemic mycoses as well as in filamentous Mucorales. Under appropriate thermal conditions, mononuclear yeast forms alternate with multi-nucleate hyphae. Here, we describe a dimorphic mucoralean fungus obtained from the sputum of a patient with Burkitt lymphoma and ongoing graft-versus-host reactions. The fungus is described as Mucor germinans sp. nov. Laboratory studies were performed to simulate temperature-dependent dimorphism, with two environmental strains Mucor circinelloides and Mucor kunryangriensis as controls. Both strains could be induced to form multinucleate arthrospores and subsequent yeast-like cells in vitro. Multilateral yeast cells emerge in all three Mucor species at elevated temperatures. This morphological transformation appears to occur at body temperature since the yeast-like cells were observed in the lungs of our immunocompromised patient. The microscopic appearance of the yeast-like cells in the clinical samples is easily confused with that of Paracoccidioides. The ecological role of yeast forms in Mucorales is discussed.IMPORTANCEMucormycosis is a devastating disease with high morbidity and mortality in susceptible patients. Accurate diagnosis is required for timely clinical management since antifungal susceptibility differs between species. Irregular hyphal elements are usually taken as the hallmark of mucormycosis, but here, we show that some species may also produce yeast-like cells, potentially being mistaken for Candida or Paracoccidioides. We demonstrate that the dimorphic transition is common in Mucor species and can be driven by many factors. The multi-nucleate yeast-like cells provide an effective parameter to distinguish mucoralean infections from similar yeast-like species in clinical samples.

Celecoxib exhibits antifungal effect against Paracoccidioides brasiliensis both directly and indirectly by activating neutrophil responses.

BACKGROUND: Celecoxib, an anti-inflammatory drug, combined therapies using antimicrobials and immune modulator drugs are being studied. OBJECTIVE: To assess whether Celecoxib has direct in vitro antifungal effect against the Paracoccidioides brasiliensis, the causative agent of Paracoccidioidomycosis-(PCM) and also if it improves the in vivo activity of neutrophils-(PMN) in an experimental murine subcutaneous-(air pouch) model of the disease. METHODS: The antifungal activity of Celecoxib(6 mg/mL) on P. brasiliensis-(Pb18) was evaluated using the microdilution technique. Splenocytes co-cultured with Pb18 and treated with Celecoxib(6 mg/mL) were co-cultured for 24, 48 and 72-hours. Swiss mice were inoculated with Pb18 and treated with Celecoxib(6 mg/kg) in the subcutaneous air pouch. Neutrophils were collected from the air pouch. Mitochondrial activity, reactive oxygen production, catalase, peroxidase, cytokines and chemokines, nitrogen species, total protein, microbicidal activity of PMNs and viable Pb18 cells numbers were analyzed. RESULTS: Celecoxib had no cytotoxic effect on splenocytes co-cultured with Pb18, but had a marked direct antifungal effect, inhibiting fungal growth both in vitro and in vivo. Celecoxib interaction with immune system cells in the air pouch, it leads to activation of PMNs, as confirmed by several parameters (mitochondrial activity, reactive oxygen species, peroxidase, KC and IL-6 increase, killing constant and phagocytosis). Celecoxib was able to reduce IL-4, IL-10 and IL-12 cytokine production. The number of recovered viable Pb18 decreased dramatically. CONCLUSIONS: This is the first report of the direct antifungal activity of Celecoxib against P. brasiliensis. The use of Celecoxib opens a new possibility for future treatment of PCM.

Talaromyces marneffei, Coccidioides species, and Paracoccidioides species-a systematic review to inform the World Health Organization priority list of fungal pathogens.

The World Health Organization, in response to the growing burden of fungal disease, established a process to develop a fungal pathogen priority list. This systematic review aimed to evaluate the epidemiology and impact of infections caused by Talaromyces marneffei, Coccidioides species, and Paracoccidioides species. PubMed and Web of Sciences databases were searched to identify studies published between 1 January 2011 and 23 February 2021 reporting on mortality, complications and sequelae, antifungal susceptibility, preventability, annual incidence, and trends. Overall, 25, 17, and 6 articles were included for T. marneffei, Coccidioides spp. and Paracoccidioides spp., respectively. Mortality rates were high in those with invasive talaromycosis and paracoccidioidomycosis (up to 21% and 22.7%, respectively). Hospitalization was frequent in those with coccidioidomycosis (up to 84%), and while the duration was short (mean/median 3-7 days), readmission was common (38%). Reduced susceptibility to fluconazole and echinocandins was observed for T. marneffei and Coccidioides spp., whereas >88% of T. marneffei isolates had minimum inhibitory concentration values ≤0.015 µg/ml for itraconazole, posaconazole, and voriconazole. Risk factors for mortality in those with talaromycosis included low CD4 counts (odds ratio 2.90 when CD4 count <200 cells/µl compared with 24.26 when CD4 count <50 cells/µl). Outbreaks of coccidioidomycosis and paracoccidioidomycosis were associated with construction work (relative risk 4.4-210.6 and 5.7-times increase, respectively). In the United States of America, cases of coccidioidomycosis increased between 2014 and 2017 (from 8232 to 14 364/year). National and global surveillance as well as more detailed studies to better define sequelae, risk factors, outcomes, global distribution, and trends are required.

Carbon nanoparticle-based lateral flow assay for the detection of specific double-tagged DNA amplicons of Paracoccidioides spp.

To overcome the limitations of current methods for diagnosing paracoccidioidomycosis (PCM), it is critical to develop novel diagnostic strategies that can be implemented in low-resource settings and dramatically improve turnaround times. This study focused on the development of a portable molecular test to screen for Paracoccidioides spp. The proposed approach integrated double-tagging polymerase chain reaction (PCR) and a paper-based lateral flow assay (LFA) for readout, using carbon nanoparticles as a signal generation system. Primers tagged with biotin and digoxigenin were employed to conduct the double-tagging PCR, which can be conveniently carried out on portable thermocyclers. This method can generate billions of tagged DNA copies from a single target molecule, which can be rapidly detected by the LFA platform, providing results within minutes. Avidin-modified carbon nanoparticles served as a signal generation system, enabling detection in the immunochromatographic assay. The LFA demonstrated the capability to detect double-tagged amplicons as low as 0.21 ng or 0.10 ng, depending on whether the results were assessed visually or with a smartphone equipped with an image processor. These findings suggest that the proposed approach holds great promise as a point-of-care diagnostic tool for the early and accurate detection of PCM in low-resource settings. The diagnostic test is rapid and inexpensive, requires minimal handling and can be easily introduced into the general practitioner's armoury for ambulatory screening of infection. This innovative approach has the potential to make a substantial contribution to PCM diagnosis, ultimately reducing morbidity and mortality associated with this disease.

Extracellular vesicles from virulent P. brasiliensis induce TLR4 and dectin-1 expression in innate cells and promote enhanced Th1/Th17 response.

Extracellular vesicles (EVs) are membrane-enclosed nanoparticles that transport several biomolecules and are involved in important mechanisms and functions related to the pathophysiology of fungal diseases. EVs from Paracoccidioides brasiliensis, the main causative agent of Paracoccidioidomycosis (PCM), modulate the immune response of macrophages. In this study, we assessed the EVs proteome from a virulent P. brasiliensis isolated from granulomatous lesions and compared their immunomodulatory ability with EVs isolated from the fungus before the animal passage (control EVs) when challenging macrophages and dendritic cells (DCs). Proteome showed that virulent EVs have a higher abundance of virulence factors such as GP43, protein 14-3-3, GAPDH, as well as virulence factors never described in PCM, such as aspartyl aminopeptidase and a SidJ analogue compared with control EVs. Virulent extracellular vesicles induced higher expression of TLR4 and Dectin-1 than control EVs in macrophages and dendritic cells (DCs). In opposition, a lower TLR2 expression was induced by virulent EVs. Additionally, virulent EVs induced lower expression of CD80, CD86 and TNF-α, but promoted a higher expression of IL-6 and IL-10, suggesting that EVs isolated from virulent P. brasiliensis-yeast promote a milder DCs and macrophage maturation. Herein, we showed that EVs from virulent fungi stimulated a higher frequency of Th1/Tc1, Th17, and Treg cells, which gives new insights into fungal extracellular vesicles. Taken together, our results suggest that P. brasiliensis utilizes its EVs as virulence bags that manipulate the immune system in its favour, creating a milder immune response and helping with fungal evasion from the immune system.

Paracoccidioides spp.: the structural characterization of extracellular matrix, expression of glucan synthesis and associated genes and adhesins during biofilm formation.

The genus Paracoccidioides includes Paracoccidioides lutzii and the Paracoccidioides brasiliensis complex, which comprises four phylogenetic species. A key feature distinguishing planktonic growth from biofilm is the presence of a 3D extracellular matrix (ECM). Therefore, in this study, we analyzed biofilm formation in different species of Paracoccidioides yeast phase, characterized the structural elements of the matrix of P. brasiliensis (Pb18), P. lutzii (Pl01 and 8334) and P. restrepiensis (339 and 192) and evaluated the expression of glucan genes, according to the stage of biofilm evolution for P. brasiliensis. The strains were cultivated in planktonic and biofilm form for 24-144 h. The fungi biomass and metabolic activity were determined by crystal violet and tetrazolium salt reduction (XTT) tests and colony-forming unit (CFU) by plating. The biofilm structure was designed using scanning electron microscopy and confocal laser scanning microscopy techniques. The extracellular matrix of P. brasiliensis and P. lutzii biofilms was extracted by sonication, and polysaccharides, proteins, and extracellular DNA (eDNA) were quantified. The RNA was extracted with the Trizol® reagent and quantified; then, the cDNA was synthesized to analyze the enolase expression, 14-3-3, FKS1, AGS1, GEL3, and KRE6 genes by real-time PCR. All strains of Paracoccidioides studied form a biofilm with more significant metabolic activity and biomass values in 144 h. The extracellular matrix of P. brasiliensis and P. lutzii had a higher content of polysaccharides in their composition, followed by proteins and eDNA in smaller quantities. The P. brasiliensis biofilm kinetics of formation showed greater expression of genes related to glucan's synthesis and its delivery to the external environment in addition adhesins during the biofilm's adhesion, initiation, and maturation. The GEL3 and enolase genes increased in expression within 24 h and during the biofilm maturation period, there was an increase in 14-3-3, AGS1, and FKS1. Furthermore, at 144 h, there was a decrease in KRE6 expression and an increase in GEL3. This study highlights the potential for biofilm formation for three species of Paracoccidioides and the main components of the extracellular matrix that can contribute to a better understanding of biofilm organization.

Clinical Characteristics and Outcomes of Endemic Mycoses After Solid Organ Transplantation: A Comprehensive Review.

Background: Geographically endemic fungi can cause significant disease among solid organ transplant (SOT) recipients. We provide an update on the epidemiology, clinical presentation, and outcomes of 5 endemic mycoses in SOT recipients. Methods: Multiple databases were reviewed from inception through May 2023 using key words for endemic fungi (eg, coccidioidomycosis or Coccidioides, histoplasmosis or Histoplasma, etc). We included adult SOT recipients and publications in English or with English translation. Results: Among 16 cohort studies that reported on blastomycosis (n = 3), coccidioidomycosis (n = 5), histoplasmosis (n = 4), and various endemic mycoses (n = 4), the incidence rates varied, as follows: coccidioidomycosis, 1.2%-5.8%; blastomycosis, 0.14%-0.99%; and histoplasmosis, 0.4%-1.1%. There were 204 reports describing 268 unique cases of endemic mycoses, including 172 histoplasmosis, 31 blastomycosis, 34 coccidioidomycosis, 6 paracoccidioidomycosis, and 25 talaromycosis cases. The majority of patients were male (176 of 261 [67.4%]). Transplanted allografts were mostly kidney (192 of 268 [71.6%]), followed by liver (n = 39 [14.6%]), heart (n = 18 [6.7%]), lung (n = 13 [4.9%]), and combined kidney-liver and kidney-pancreas (n = 6 [2.7%]). In all 5 endemic mycoses, most patients presented with fever (162 of 232 [69.8%]) and disseminated disease (179 of 268 [66.8%]). Cytopenias were frequently reported for histoplasmosis (71 of 91 [78.0%]), coccidioidomycosis (8 of 11 [72.7%]) and talaromycosis (7 of 8 [87.5%]). Graft loss was reported in 12 of 136 patients (8.8%). Death from all-causes was reported in 71 of 267 (26.6%); half of the deaths (n = 34 [50%]) were related to the underlying mycoses. Conclusions: Endemic mycoses commonly present with fever, cytopenias and disseminated disease in SOT recipients. There is a relatively high all-cause mortality rate, including many deaths that were attributed to endemic mycoses.

The Effect of Geoclimatic Factors on the Distribution of Paracoccidioidomycosis in Mato Grosso do Sul, Brazil.

The incidence of paracoccidioidomycosis (PCM) varies in Latin America, and it is influenced by environmental factors. This study evaluated the distribution of PCM acute/subacute form (AF) cases and their correlation with geoclimatic factors in the Mato Grosso do Sul (MS) state. The study included 81 patients diagnosed with the PCM/AF at the University Hospital of the Federal University of Mato Grosso do Sul between January 1980 and February 2022. Geographic coordinates, health microregion of patient's residence, compensated average temperature, relative air humidity (RH), El Niño Southern Oscillation (ENSO), and average global temperature were analyzed. The highest incidence was observed in the Aquidauana (7/100,000 inhabitants), while Campo Grande, the state's capital, had the highest number (n = 34; 42.4%) and density (4.4 cases/km2) of cases. The number of cases increased during extended periods of the El Niño phenomenon. A positive correlation was found between higher RH and PCM/AF cases. Most PCM/AF cases were found in areas with loamy soils and RH ranging from 60.8 to 73.6%. In MS, the health microregions of PCM/AF patients are characterized by deforestation for agricultural and pasture use, coupled with loamy soils and specific climatic phenomena leading to higher soil humidity.

Unveiling the functional significance of the 14.3.3 protein: A key player in Paracoccidioides brasiliensis biofilm formation.

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by Paracoccidioides spp. The interaction mediated by the presence of adhesins on the fungal surface and receptors in the extracellular matrix of the host, as well as the biofilm formation, is essential in its pathogenesis. Adhesins such as gp43, enolase, GAPDH (glyceraldehyde-3-phosphate dehydrogenase), and 14-3-3 have been demonstrated in the Paracoccidioides brasiliensis (Pb18) strain and recognized as necessary in the fungus-host interaction. The Pb 18 strain silenced to 14-3-3 showed changes in morphology, virulence, and adhesion capacity. The study aimed to evaluate the role of adhesin 14-3-3 in P. brasiliensis biofilm formation and the differential expression of genes related to adhesins, comparing planktonic and biofilm forms. The presence of biofilm was also verified in sutures in vitro and in vivo. The silenced strain (Pb14-3-3 aRNA) was compared with the wild type Pb18, determining the differential metabolic activity between the strains by the XTT reduction assay; the biomass by violet crystal and the polysaccharides by safranin, even as morphological differences by microscopic techniques. Differential gene expression for adhesins was also analyzed, comparing the relative expression of these in planktonic and biofilm forms at different times. The results suggested that the silencing of 14-3-3 protein altered the ability to form biofilm and its metabolism. The quantity of biomass was similar in both strains; however, the formation of exopolymeric substances and polysaccharide material was lower in the silenced strain. Our results showed increased expression of enolase, GAPDH, and 14-3-3 genes in the first periods of biofilm formation in the Pb18 strain. In contrast, the silenced strain showed a lower expression of these genes, indicating that gene silencing can influence the expression of other genes and be involved in the biofilm formation of P. brasiliensis. In vitro and in vivo assays using sutures confirmed this yeast's ability to form biofilm and may be implicated in the pathogenesis of paracoccidioidomycosis.

Structure of glyceraldehyde-3-phosphate dehydrogenase from Paracoccidioides lutzii in complex with an aldonic sugar acid.

The pathogen Paracoccidioides lutzii (Pb01) is found in South America countries Colombia, Ecuador, Venezuela and Brazil, especially in the central, west, and north regions of the latter. It belongs to the Ajellomycetaceae family, Onygenales order, and is typically thermodimorphic, presenting yeast cells when it grows in animal tissues, but mycelia when in the environment, where it produces the infectious propagule. This fungus is one of the etiologic agents of Paracoccidioidomycosis (PCM), the most important endemic fungal infection in Latin America. Investigations on its genome have contributed to a better understanding about its metabolism and revealed the complexity of several metabolic glycolytic pathways. Glyceraldehyde-3-Phosphate Dehydrogenase from Paracoccidioides lutzii (PlGAPDH) is considered a moonlighting protein and participates in several biological processes of this pathogen. The enzyme was expressed and purified, as seen in SDS-PAGE gel, crystallized and had its three dimensional structure (3D) determined in complex with NAD+, a sulphate ion and d-galactonic acid, therefore, a type of 'GAA site'. It is the first GAPDH structure to show this chemical type in this site and how this protein can bind an acid derived from oxidation of a linear hexose.

Contribution to the study of inflammasome and programmed cell death in paracoccidioidomycosis oral lesions.

BACKGROUND: Paracoccidioidomycosis is the most prevalent systemic mycosis in Latin America, with a high incidence in Brazil, Colombia and Venezuela, and constitutes a serious public health problem, a frequent cause of morbidity and disability for work. Some mechanisms of cell death are described as important tools in infectious processes. When apoptosis is blocked, RIPK (Receptor-interacting protein kinase) 3 dependent, a caspase-independent form of cell death, can limit the replication and spread of pathogens. Some molecules that mediate necroptosis include RIPK3 and have been extensively studied due to their signalling mechanism and pathological function. RIPK3 activates NLRP1 and NLRP3-mediated inflammasome formation. Caspase-1 has an important role in processing the cytokines ILß and IL18 to their active form. Such molecules are part of the inflammasome characterization, whose caspase-1-dependent activation promotes the death of pyroptotic cells and the secretion of proinflammatory cytokines. Knowledge about the mechanisms of pathogen-mediated cell death can be useful for understanding of the pathogenesis of infections and inflammatory conditions. OBJECTIVE: The objective of this work was to identify the mechanisms of programmed cell death and inflammasome components in human oral mucosal lesions of paracoccidioidomycosis through immunohistochemical methods and identification of RIPK-3, IL1ß, IL18, NLRP-1 and caspase-1. Thirty specimens were included, and a histopathological analysis of the lesions was performed using haematoxylin-eosin staining. RESULTS: Our results on in situ expression of inflammasome elements and programmed cell death showed increased expression of IL-1ß, NLRP-1, caspase-1 and RIPK-3. We suggest that inflammasome complex participate in the immunopathogenesis in paracoccidioidomycosis oral lesions in an interplay with RIPK3.

Evaluation of commercial immunodiffusion reagents for detecting serum anti-Paracoccidioides antibodies

ABSTRACT Background: Accurate diagnosis of paracoccidioidomycosis is crucial for improving patient outcomes. Paracoccidioides antibody detection by double immunodiffusion (DID) is a convenient diagnostic tool, but testing performance can vary based on certain factors. Methods: We assessed DID performance using a commercially prepared Paracoccidioides reagents (IMMY, USA), involving 40 serum specimens, including 20 from patients with proven paracoccidioidomycosis and 20 from patients without the disease. The DID test demonstrated a sensitivity of 90% (95% CI=68%-99%) and a specificity of 100% (95% CI=83%-100%). Conclusions: Our findings suggest that DID using commercial reagents may provide a feasible tool with satisfactory testing performance for anti-Paracoccidioides antibody detection.

Proteomic analysis reveals changes in the proteome of human THP-1 macrophages infected with Paracoccidioides brasiliensis.

Paracoccidioides spp. is the etiologic agent of Paracoccidioidomycosis (PCM), a systemic disease with wide distribution in Latin America. Macrophages are very important cells during the response to infection by P. brasiliensis. In this study, we performed a proteomic analysis to evaluate the consequences of P. brasiliensis yeast cells on the human THP-1 macrophage proteome. We have identified 443 and 2247 upregulated or downregulated proteins, respectively, in macrophages co-cultured with yeast cells of P. brasiliensis in comparison to control macrophages unexposed to the fungus. Proteomic analysis revealed that interaction with P. brasiliensis caused metabolic changes in macrophages that drastically affected energy production pathways. In addition, these macrophages presented regulated many factors related to epigenetic modifications and gene transcription as well as a decrease of many proteins associated to the immune system activity. This is the first human macrophage proteome derived from interactions with P. brasiliensis, which contributes to elucidating the changes that occur during the host response to this fungus. Furthermore, it highlights proteins that may be targets for the development of new therapeutic approaches to PCM.

Paracoccidoidomicosis: Reporte de 4 casos

La paracoccidioidomicosis (PCM) es una infección causada por hongos patógenos humanos del género Paracoccidioides. Es una micosis sistémica que puede afectar cualquier órgano. Se describen con este reporte cuatro casos de paracoccidioidomicosis con diferentes presentaciones clínicas, tiempo de evolución, con afectación mucocutánea, pulmonar, glándulas suprarrenales, sistema nervioso entre otros, principalmente en adultos varones de diferentes edades y profesiones, tanto inmunosuprimidos como inmunocompetentes, teniendo en común el contacto con el suelo. Se demuestra de esta manera la importancia de considerar esta patología ante una sospecha clínica de micosis sistémica para así realizar una confirmación temprana y tratamiento oportuno ya que presenta buena respuesta terapéutica antimicótica y mejoría clínica.

Paracoccidioidomycosis (PCM) is an infection caused by human pathogenic fungi of the genus Paracoccidioides. This report describes: 4 cases of paracoccidioidomycosis diagnosed with different clinical presentations, different times of evolution, with mucocutaneous, pulmonary, adrenal gland, and nervous system involvement, among others, mainly in male adults of different ages and professions, both immunosuppressed and immunocompetent, having in common contact with the ground. In this way, PCM is a systemic mycosis that can affect any organ and therefore the importance of considering this pathology when a diagnostic suspicion of systemic mycosis is presented in order to make an early diagnosis and timely treatment since it presents a good therapeutic response, antifungal and clinical improvement.

Dectin-2 is critical for phagocyte function and resistance to Paracoccidioides brasiliensis in mice.

Germline-encoded pattern recognition receptors, particularly C-type lectin receptors (CLRs), are essential for phagocytes to sense invading fungal cells. Among CLRs, Dectin-2 (encoded by Clec4n) plays a critical role in the antifungal immune response as it recognizes high-mannose polysaccharides on the fungal cell wall, triggering phagocyte functional activities and ultimately determining adaptive responses. Here, we assessed the role of Dectin-2 on the course of primary Paracoccidioides brasiliensis systemic infection in mice with Dectin-2-targeted deletion. Paracoccidioides brasiliensis constitutes the principal etiologic agent of paracoccidioidomycosis, the most prominent invasive mycosis in Latin American countries. The deficiency of Dectin-2 resulted in shortened survival rates, high lung fungal burden, and increased lung pathology in mice infected with P. brasiliensis. Consistently, dendritic cells (DCs) from mice lacking Dectin-2 infected ex vivo with P. brasiliensis showed impaired secretion of several proinflammatory and regulatory cytokines, including TNF-α, IL-1ß, IL-6, and IL-10. Additionally, when cocultured with splenic lymphocytes, DCs were less efficient in promoting a type 1 cytokine pattern secretion (i.e., IFN-γ). In macrophages, Dectin-2-mediated signaling was required to ensure phagocytosis and fungicidal activity associated with nitric oxide production. Overall, Dectin-2-mediated signaling is critical to promote host protection against P. brasiliensis infection, and its exploitation might lead to the development of new vaccines and immunotherapeutic approaches.

We report a critical role of the innate immune receptor Dectin-2 during Paracoccidioides brasiliensis infection. Fungal sensing by Dectin-2 improved the survival of mice and lowered fungal burden. Further, Dectin-2 was required for cytokine production, phagocytosis, and fungal killing by phagocytes.

The Response of Paracoccidioides lutzii to the Interaction with Human Neutrophils.

The fungal pathogen Paracoccidioides lutzii causes systemic mycosis Paracoccidioidomycosis (PCM), which presents a broad distribution in Latin America. Upon infection, the fungus undergoes a morphological transition to yeast cells and provokes an inflammatory granulomatous reaction with a high number of neutrophils in the lungs. In this work, we employed proteomic analysis to investigate the in vitro response of the fungus to the interaction with human neutrophils. Proteomic profiling of P. lutzii yeast cells harvested at 2 and 4 h post interaction with human polymorphonuclear cells allowed the identification of 505 proteins differentially accumulated. The data indicated that P. lutzii yeast cells underwent a shift in metabolism from glycolysis to Beta oxidation, increasing enzymes of the glyoxylate cycle and upregulating enzymes related to the detoxification of oxidative and heat shock stress. To our knowledge, this is the first study employing proteomic analysis in the investigation of the response of a member of the Paracoccidioides genus to the interaction with neutrophils.

Proteomics of Paracoccidioides lutzii: Overview of Changes Triggered by Nitrogen Catabolite Repression.

Members of the Paracoccidioides complex are the causative agents of Paracoccidioidomycosis (PCM), a human systemic mycosis endemic in Latin America. Upon initial contact with the host, the pathogen needs to uptake micronutrients. Nitrogen is an essential source for biosynthetic pathways. Adaptation to nutritional stress is a key feature of fungi in host tissues. Fungi utilize nitrogen sources through Nitrogen Catabolite Repression (NCR). NCR ensures the scavenging, uptake and catabolism of alternative nitrogen sources, when preferential ones, such as glutamine or ammonium, are unavailable. The NanoUPLC-MSE proteomic approach was used to investigate the NCR response of Paracoccidioides lutzii after growth on proline or glutamine as a nitrogen source. A total of 338 differentially expressed proteins were identified. P. lutzii demonstrated that gluconeogenesis, ß-oxidation, glyoxylate cycle, adhesin-like proteins, stress response and cell wall remodeling were triggered in NCR-proline conditions. In addition, within macrophages, yeast cells trained under NCR-proline conditions showed an increased ability to survive. In general, this study allows a comprehensive understanding of the NCR response employed by the fungus to overcome nutritional starvation, which in the human host is represented by nutritional immunity. In turn, the pathogen requires rapid adaptation to the changing microenvironment induced by macrophages to achieve successful infection.

Human Lobomycosis Caused by Paracoccidioides (Lacazia) loboi, Panama, 2022.

We report a patient from Panama who had lobomycosis caused by Paracoccidioides (Lacazia) loboi. We used combined clinical-epidemiologic and phylogenetic data, including a new gene sequence dataset on this fungus in Panama, for analysis. Findings contribute useful insights to limited knowledge of this fungal infection in the Mesoamerican Biologic Corridor.

Polypeptides Targeting Paracoccidioides brasiliensis Drk1.

Considering the toxicity of conventional therapeutic approaches and the importance of precise mechanistic targets, it is important to explore signaling pathways implicated in fungal pathobiology. Moreover, treatment of paracoccidioidomycosis, a systemic mycosis caused by a dimorphic fungus, requires prolonged therapeutic regimens. Among the numerous factors underpinning the establishment of Paracoccidioides spp. infection, the capacity to transition from the mycelial to the yeast form is of pivotal importance. The Drk1 protein of Paracoccidioides brasiliensis likely plays a decisive role in this morphological shift and subsequent virulence. We identified peptides with affinity for the PbDrk1 protein using the phage-display method and assessed the effects of these peptides on P. brasiliensis. The peptides were found to inhibit the phase transition of P. brasiliensis. Furthermore, a substantial proportion of these peptides prevented adhesion to pneumocytes. Although these peptides may not possess inherent antifungal properties, they can augment the effects of certain antifungal agents. Notably, the cell wall architecture of P. brasiliensis appears to be modulated by peptide intervention, resulting in a reduced abundance of glycosylated proteins and lipids. These peptides were also evaluated for their efficacy in a Galleria mellonella model and shown to contribute to enhanced larval survival rates. The role of PbDrk1, which is notably absent in mammals, should be further investigated to improve the understanding of its functional role in P. brasiliensis, which may be helpful for designing novel therapeutic modalities.

Identification and immunogenic potential of glycosylphosphatidylinositol-anchored proteins in Paracoccidioides brasiliensis.

In fungal pathogens the cell wall plays an important role in host-pathogen interactions because its molecular components (e.g., polysaccharides and proteins) may trigger immune responses during infection. GPI-anchored proteins represent the main protein class in the fungal cell wall where they can perform several functions, such as cell wall remodeling and adhesion to host tissues. Genomic analysis has identified the complement of GPI-anchored proteins in many fungal pathogens, but the function has remained unknown for most of them. Here, we conducted an RNA expression analysis of GPI-anchored proteins of Paracoccidioides brasiliensis which causes paracoccidioidomycosis (PCM), an important human systemic mycosis endemic in Latin America. The expression of the GPI-anchored proteins was analyzed by quantitative PCR in both the mycelium and yeast forms. qPCR analysis revealed that the transcript levels of 22 of them were increased in hyphae and 10 in yeasts, respectively, while 14 did not show any significant difference in either form. Furthermore, we cloned 46 open reading frames and purified their corresponding GPI-anchored proteins in the budding yeast. Immunoblot and ELISA analysis of four purified GPI-anchored proteins revealed immune reactivity of these proteins against sera obtained from PCM patients. The information obtained in this study provides valuable information about the expression of many GPI-anchored proteins of unknown function. In addition, based on our immune analysis, some GPI-anchored proteins are expressed during infection and therefore, they might serve as good candidates for the development of new diagnostic methods.