Environmental Mapping of Paracoccidioides spp. in Brazil Reveals New Clues into Genetic Diversity,Biogeography and Wild Host Association

BackgroundParacoccidioides brasiliensis and Paracoccidioides lutzii are the etiological agents of Paracoccidioidomycosis (PCM), and are easily isolated from human patients. However, due to human migration and a long latency period, clinical isolates do not reflect the spatial distribution of these pathogens. Molecular detection of P. brasiliensis and P. lutzii from soil, as well as their isolation from wild animals such as armadillos, are important for monitoring their environmental and geographical distribution. This study aimed to detect and, for the first time, evaluate the genetic diversity of P. brasiliensis and P. lutzii for Paracoccidioidomycosis in endemic and non-endemic areas of the environment, by using Nested PCR and in situ hybridization techniques.Conclusions/SignificanceData may reflect the actual occurrence of Paracoccidioides species in their saprobic habitat, despite their absence/non-detection in seven armadillos evaluated in regions with high prevalence of PCM infection by P. lutzii. These results may indicate a possible ecological difference between P. brasiliensis and P. lutzii concerning their wild hosts.     

Paracoccidioides brasiliensis Interferes on Dendritic Cells Maturation by Inhibiting PGE2 Production

Paracoccidioidomycosis (PCM) is a systemic mycosis, endemic in most Latin American countries, especially in Brazil, whose etiologic agent is the thermodimorphic fungus of the genus Paracoccidioides, comprising cryptic species of Paracoccidioides brasiliensis, S1, PS2, PS3 and Paracoccidioides lutzii. The mechanisms involved in the initial interaction of the fungus with cells of the innate immune response, as dendritic cells (DCs), deserve to be studied. Prostaglandins (PGs) are eicosanoids that play an important role in modulating functions of immune cells including DCs. Here we found that human immature DCs derived from the differentiation of monocytes cultured with GM-CSF and IL-4 release substantial concentrations of PGE₂, which, however, were significantly inhibited after challenge with P. brasiliensis. In vitro blocking of pattern recognition receptors (PRRs) by monoclonal antibodies showed the involvement of mannose receptor (MR) in PGE₂ inhibition by the fungus. In addition, phenotyping assays showed that after challenge with the fungus, DCs do not change their phenotype of immature cells to mature ones, as well as do not produce IL-12 p70 or adequate concentrations of TNF-α. Assays using exogenous PGE₂ confirmed an association between PGE₂ inhibition and failure of cells to phenotypically mature in response to P. brasiliensis. We conclude that a P. brasiliensis evasion mechanism exists associated to a dysregulation on DC maturation. These findings may provide novel information for the understanding of the complex interplay between the host and this fungus.     

Characterization of PbPga1, an Antigenic GPI-Protein in the Pathogenic Fungus Paracoccidioides brasiliensis

Paracoccidioides brasiliensis is the etiologic agent of paracoccidioidomycosis (PCM), one of the most prevalent mycosis in Latin America. P. brasiliensis cell wall components interact with host cells and influence the pathogenesis of PCM. Cell wall components, such as glycosylphosphatidylinositol (GPI)-proteins play a critical role in cell adhesion and host tissue invasion. Although the importance of GPI-proteins in the pathogenesis of other medically important fungi is recognized, little is known about their function in P. brasiliensis cells and PCM pathogenesis. We cloned the PbPga1 gene that codifies for a predicted GPI-anchored glycoprotein from the dimorphic pathogenic fungus P. brasiliensis. PbPga1 is conserved in Eurotiomycetes fungi and encodes for a protein with potential glycosylation sites in a serine/threonine-rich region, a signal peptide and a putative glycosylphosphatidylinositol attachment signal sequence. Specific chicken anti-rPbPga1 antibody localized PbPga1 on the yeast cell surface at the septum between the mother cell and the bud with stronger staining of the bud. The exposure of murine peritoneal macrophages to rPbPga1 induces TNF-α release and nitric oxide (NO) production by macrophages. Furthermore, the presence of O-glycosylation sites was demonstrated by β-elimination under ammonium hydroxide treatment of rPbPga1. Finally, sera from PCM patients recognized rPbPga1 by Western blotting indicating the presence of specific antibodies against rPbPga1. In conclusion, our findings suggest that the PbPga1gene codifies for a cell surface glycoprotein, probably attached to a GPI-anchor, which may play a role in P. brasiliensis cell wall morphogenesis and infection. The induction of inflammatory mediators released by rPbPga1 and the reactivity of PCM patient sera toward rPbPga1 imply that the protein favors the innate mechanisms of defense and induces humoral immunity during P. brasiliensis infection.     

Neutrophils in Oral Paracoccidioidomycosis and the Involvement of Nrf2

Neutrophils have been implicated in granuloma formation in several infectious diseases, in addition to their main phagocytic and pathogen destruction role. It has been demonstrated that Nrf2 regulates antioxidant protection in neutrophils, attenuating inflammation without compromising the hosts bacterial defense. In this study, we analyzed the presence of neutrophils in Paracoccidioides brasiliensis mycosis (PCM), as well as the immunoexpression of Nrf2. Thirty-nine cases of oral PCM were classified according to quantity of fungi and to the presence of loose or well-organized granulomas and microabscesses. An Nrf2 antibody was used for immunohistochemical analysis. The results showed that neutrophils are present in microabscesses and loose granulomas, but were absent in structured granulomas. A greater quantity of fungi was shown in cases with only loose granulomas when compared to loose and well organized granulomas. Nrf2 was observed in the nuclei of neutrophils of loose granulomas and abscesses, with its expression in loose granulomas maintained despite the additional presence of well organized granulomas in the same specimen. This study suggests that neutrophils participate in P. brasiliensis granuloma formation and that Nrf2 has a possible role in neutrophil survival, via modulation of the inflammatory response.     

Low-level Laser Therapy to the Mouse Femur Enhances the Fungicidal Response of Neutrophils against Paracoccidioides brasiliensis

Neutrophils (PMN) play a central role in host defense against the neglected fungal infection paracoccidioidomycosis (PCM), which is caused by the dimorphic fungus Paracoccidioides brasiliensis (Pb). PCM is of major importance, especially in Latin America, and its treatment relies on the use of antifungal drugs. However, the course of treatment is lengthy, leading to side effects and even development of fungal resistance. The goal of the study was to use low-level laser therapy (LLLT) to stimulate PMN to fight Pb in vivo. Swiss mice with subcutaneous air pouches were inoculated with a virulent strain of Pb or fungal cell wall components (Zymosan), and then received LLLT (780 nm; 50 mW; 12.5 J/cm2; 30 seconds per point, giving a total energy of 0.5 J per point) on alternate days at two points on each hind leg. The aim was to reach the bone marrow in the femur with light. Non-irradiated animals were used as controls. The number and viability of the PMN that migrated to the inoculation site was assessed, as well as their ability to synthesize proteins, produce reactive oxygen species (ROS) and their fungicidal activity. The highly pure PMN populations obtained after 10 days of infection were also subsequently cultured in the presence of Pb for trials of protein production, evaluation of mitochondrial activity, ROS production and quantification of viable fungi growth. PMN from mice that received LLLT were more active metabolically, had higher fungicidal activity against Pb in vivo and also in vitro. The kinetics of neutrophil protein production also correlated with a more activated state. LLLT may be a safe and non-invasive approach to deal with PCM infection.     

Hepatic Disease with Portal Hypertension and Acute Juvenile Paracoccidioidomycosis: A Report of Two Cases and Literature Review

Paracoccidioidomycosis (PCM) is a neglected systemic mycosis endemic to Latin America caused by dimorphic fungi of the genus Paracoccidioides. The acute juvenile PCM is a severe type of presentation that usually affects young vulnerable patients and rarely progresses to portal hypertension. Here, two cases of liver disease and portal hypertension as complications of acute juvenile PCM are reported. Diagnosis of PCM was performed by isolation of the fungus and molecular identification of the strains provided through partial sequencing of two protein encoding genes, arf and gp43. Genotypic analysis revealed that Paracoccidioides brasiliensis S1 was the phylogenic species involved in both cases. Patients presented a good clinical response to amphotericin B and sulfamethoxazole–trimethoprim. These results highlight the importance of the interdisciplinary approach in patients with severe forms of PCM to avoid and treat complications, and the necessity of further investigations focusing on host-pathogen interaction in order to explain the broad clinical spectrum in PCM as well as the severity and poor outcome in some clinical cases.     

Saccharomyces cerevisiae Expressing Gp43 Protects Mice against Paracoccidioides brasiliensis Infection

The dimorphic fungus Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis (PCM). It is believed that approximately 10 million people are infected with the fungus and approximately 2% will eventually develop the disease. Unlike viral and bacterial diseases, fungal diseases are the ones against which there is no commercially available vaccine. Saccharomyces cerevisiae may be a suitable vehicle for immunization against fungal infections, as they require the stimulation of different arms of the immune response. Here we evaluated the efficacy of immunizing mice against PCM by using S. cerevisiae yeast expressing gp43. When challenged by inoculation of P. brasiliensis yeasts, immunized animals showed a protective profile in three different assays. Their lung parenchyma was significantly preserved, exhibiting fewer granulomas with fewer fungal cells than found in non-immunized mice. Fungal burden was reduced in the lung and spleen of immunized mice, and both organs contained higher levels of IL-12 and IFN-γ compared to those of non-vaccinated mice, a finding that suggests the occurrence of Th1 immunity. Taken together, our results indicate that the recombinant yeast vaccine represents a new strategy to confer protection against PCM.     

Serology of Paracoccidioidomycosis Due to Paracoccidioides lutzii

Paracoccidioides lutzii is a new agent of paracoccidioidomycosis (PCM) and has its epicenter localized to the Central-West region of Brazil. Serological diagnosis of PCM caused by P. lutzii has not been established. This study aimed to develop new antigenic preparations from P. lutzii and to apply them in serological techniques to improve the diagnosis of PCM due to P. lutzii. Paracoccidioides lutzii exoantigens, cell free antigen (CFA), and a TCA-precipitated antigen were evaluated in immunodiffusion (ID) tests using a total of 89 patient sera from the Central-West region of Brazil. Seventy-two sera were defined as reactive for P. brasiliensis using traditional antigens (AgPbB339 and gp43). Non-reactive sera for traditional antigens (n = 17) were tested with different P. lutzii preparations and P. lutzii CFA showed 100% reactivity. ELISA was found to be a very useful test to titer anti-P. lutzii antibodies using P. lutzii-CFA preparations. Sera from patients with PCM due to P. lutzii presented with higher antibody titers than PCM due to P. brasiliensis and heterologous sera. In western blot, sera from patients with PCM due to P. lutzii were able to recognize antigenic molecules from the P. lutzii-CFA antigen, but sera from patients with PCM due to P. brasiliensis could not recognize any P. lutzii molecules. Due to the facility of preparing P. lutzii CFA antigens we recommend its use in immunodiffusion tests for the diagnosis of PCM due to P. lutzii. ELISA and western blot can be used as complementary tests.     

A secreted serine protease of <Emphasis Type="Italic">Paracoccidioides brasiliensis</Emphasis> and its interactions with fungal proteins

Background  

Paracoccidioides brasiliensis is a thermodimorphic fungus, the causative agent of paracoccidioidomycosis (PCM). Serine proteases are widely distributed and this class of peptidase has been related to pathogenesis and nitrogen starvation in pathogenic fungi.     

Protection induced in BALB/c mice by the high-molecular-mass (hMM) fraction of <Emphasis Type="Italic">Paracoccidioides brasiliensis</Emphasis>

Paracoccidioidomycosis (PCM) is a granulomatous disease caused by a dimorphic fungus, Paracoccidioides brasiliensis. The present study investigated the protective activity of the P. brasiliensis high-molecular-mass (hMM) fraction (~380 kDa) in experimental murine PCM. In the first step, lymphocyte proliferation and production of IFNγ (but not IL-4) were observed in “in vitro” spleen cells (from female BALB/c mice infected (i.v.) with P. brasiliensis) that were stimulated with hMM fractions. In the second step, female BALB/c mice were previously immunized (s.c.) with hMM fraction (25 μg/protein = F-25 and 50 μg/protein = F-50), and the colony-forming units (CFU) of the lung and spleen, the histopathological characteristics of the granulomatous lesions, and plasmatic gp43 soluble antigens and anti-hMM IgG levels were analyzed at 28 and 56 days after infection. The lung and liver CFU were lower in mice previously immunized with the hMM fraction (P < 0.05). The granulomatous lesions revealed a greater degree of compaction and organization, with no dissemination of the fungus to other organs. Lower soluble antigen levels (P < 0.05) and higher IgG anti-hMM fraction (P < 0.05) were observed in immunized groups. The results for CFU, histopathology and antigenemia suggest that the hMM fraction has a protective effect in experimental paracoccidioidomycosis in BALB/c mice.     

Macrophage Interaction with Paracoccidioides brasiliensis Yeast Cells Modulates Fungal Metabolism and Generates a Response to Oxidative Stress

Macrophages are key players during Paracoccidioides brasiliensis infection. However, the relative contribution of the fungal response to counteracting macrophage activity remains poorly understood. In this work, we evaluated the P. brasiliensis proteomic response to macrophage internalization. A total of 308 differentially expressed proteins were detected in P. brasiliensis during infection. The positively regulated proteins included those involved in alternative carbon metabolism, such as enzymes involved in gluconeogenesis, beta-oxidation of fatty acids and amino acids catabolism. The down-regulated proteins during P. brasiliensis internalization in macrophages included those related to glycolysis and protein synthesis. Proteins involved in the oxidative stress response in P. brasiliensis yeast cells were also up-regulated during macrophage infection, including superoxide dismutases (SOD), thioredoxins (THX) and cytochrome c peroxidase (CCP). Antisense knockdown mutants evaluated the importance of CCP during macrophage infection. The results suggested that CCP is involved in a complex system of protection against oxidative stress and that gene silencing of this component of the antioxidant system diminished the survival of P. brasiliensis in macrophages and in a murine model of infection.     

High interleukin-4 expression and interleukin-4 gene polymorphisms are associated with susceptibility to human paracoccidioidomycosis

Paracoccidioidomycosis (PCM) is caused by dimorphic fungi from theParacoccidioides brasiliensis complex. Previous studies have demonstrated that the severity of disease is associated with a T-helper 2 immune response characterised by high interleukin (IL)-4 production. In the present study we analysed two polymorphisms in the IL-4 gene (-590 C/T and intron-3 microsatellite) in 76 patients with PCM and 73 control subjects from an endemic area. The production of IL-4 by peripheral blood mononuclear cells after antigen or phytohaemagglutinin stimulation was determined by ELISA. A significant correlation was observed between the RP2/RP2 intron-3 genotype and infection with Paracoccidioides sp. (p = 0.011), whereas the RP1/RP1 genotype was correlated with resistance. No significant correlation was observed for the IL-4 promoter polymorphism. Furthermore, the low IL-4 expression observed in the control group compared with patients was associated with the RP1/RP1 genotype. These results suggest that IL-4polymorphisms might be associated with the ability of the host to control Paracoccidioides sp. infection. The relevance of this polymorphism is supported by the observation that patients with disease produce high levels of IL-4 following mitogen or antigen stimulation. The IL-4 gene is located in the cytokine cluster region of chromosome 5 where other polymorphisms have also been described.     

Paracoccidioidomycosis Induced by Immunosuppressive Drugs in a Patient with Rheumatoid Arthritis and Bone Sarcoma: Case Report and Review of the Literature

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by Paracoccidioides brasiliensis, which is endemic in many regions of Latin America. We describe the case of a 60-year-old man from a region endemic for PCM who presented with a long history of left hip pain. He had been treated over the past 3 years with immunosuppressive drugs (methotrexate, leflunomide, and adalimumab) for rheumatoid arthritis (RA). A hip radiograph showed lytic bone lesions, and a chest radiograph showed an expansive excavated lesion in the left lung, suggestive of a lung cancer with bone metastases. A left hip joint biopsy was inconclusive, but histological analysis of a surgical lung biopsy specimen was consistent with P. brasiliensis infection. Treatment with intravenous amphotericin B (50 mg/day) and hydrocortisone (25 mg/day) was initiated. However, increasing hip pain resulted in the amputation of the left lower limb, and the analysis of the surgical specimen revealed a diagnosis of bone sarcoma. Postoperatively, the patient developed sepsis and died approximately 1 month later. To our knowledge, this is the first report of PCM in a patient with RA who had been treated with immunosuppressive drugs, in particular TNF-α blocking agents. The atypical presentation (left hip pain alone) emphasizes the importance of considering PCM in the differential diagnosis of patients with pulmonary lesions and osteolytic lesions who live in a region endemic for PCM. This case report also demonstrates that health professionals in these regions must pay close attention to patients receiving immunosuppressive drugs because of the possibility of reactivating quiescent P. brasiliensis lesions.     

Enhanced expression of NLRP3 inflammasome components by monocytes of patients with pulmonary paracoccidioidomycosis is associated with smoking and intracellular hypoxemia

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by thermally dimorphic fungi of the genus Paracoccidioides that affects predominantly 30-60-year-old male rural workers. The main clinical forms of the disease are acute/subacute, chronic (CF); almost all CF patients develop pulmonary fibrosis, and they also exhibit emphysema due to smoke. An important cytokine in this context, IL-1β, different from the others, is produced by an intracellular multimolecular complex called inflammasome that is activated by pathogens and/or host signs of damage. Inflammasome has been recognized for its contribution to chronic inflammatory diseases, from that, we hypothesized that this activation could be involved in paracoccidioidomycosis, contributing to chronic inflammation. While inflammasome activation has been demonstrated in experimental models of Paracoccidioides brasiliensis infection, no information is available in patients, leading us to investigate the participation of NLRP3-inflammasome machinery in CF/PCM patients from a Brazilian endemic area. Our findings showed increased priming in mRNA levels of NLRP3 inflammasome genes by monocytes of PCM patients in vitro than healthy controls. Similar intracellular protein expression of NLRP3, CASP-1, ASC, and IL-1β were also observed in freshly isolated monocytes of PCM patients and smoker controls. Increased expression of NLRP3 and ASC was observed in monocytes from PCM patients under hypoxia in comparison with smoker controls. For the first time, we showed that primed monocytes of CF-PCM patients were associated with enhanced expression of components of NLRP3-inflammasome due to smoke. Also, hypoxemia boosted this machinery. These findings reinforce the systemic low-grade inflammation activation observed in PCM during and after treatment.     

Paracoccidioidomycosis due to Paracoccidioides brasiliensis S1 associated with acquired immunodeficiency syndrome: A case report

BackgroundParacoccidioidomycosis (PCM) is an endemic disease in Latin America. In immunocompetent hosts, PCM occurs in two main clinical forms: acute and chronic. However, in HIV-infected patients PCM may show up simultaneous manifestations of acute and chronic forms.Case reportWe present the case of a patient diagnosed with HIV who had disseminated skin lesions and generalized lymphadenopathy, as well as respiratory and central nervous system involvement. The PCM diagnosis was confirmed by direct KOH examination, double immunodiffusion and the isolation of the fungus in samples of an abscess in the subcostal region. The isolate was identified as Paracoccidioides brasiliensis S1 by species-specific PCR using primers for protein-coding gene GP43 (exon 2) followed by PCR-RFLP of the alpha-tubulin gene.ConclusionsThere are few data in literature reporting species-specific molecular identification of Paracoccidioides in HIV/PCM patients. Therefore, this case report may contribute to improve the knowledge about this severe disease, its causative cryptic species, and its consequences to patients.     

An update on the occurrence of Paracoccidioides species in the Midwest region,Brazil: Molecular epidemiology,clinical aspects and serological profile of patients from Mato Grosso do Sul State

BackgroundParacoccidioidomycosis (PCM) is a systemic and endemic fungal infection in Latin American, mainly in Brazil. The majority of PCM cases occur in large areas in Brazil, comprising the South, Southeast and Midwest regions, with the latter demonstrating a higher incidence of the species Paracoccidioides lutzii.Methodology and main findingsThis study presents clinical, molecular and serological data of thirteen new PCM cases during 2016 to 2019 from the state of Mato Grosso do Sul, located in the Midwest region, Brazil. From these thirteen cases, sixteen clinical isolates were obtained and their genomic DNAs were subjected to genotyping by tub1 -PCR-RFLP. Results showed Paracoccidioides brasiliensis sensu stricto (S1) (11/16; 68.8%), Paracoccidioides restrepiensis (PS3) (4/16; 25.0%) and P. lutzii (1/16; 6.2%) as Paracoccidiodes species. Therefore, in order to understand whether the type of phylogenetic species that are circulating in the state influence the reactivity profile of serological tests, we performed double agar gel immunodiffusion (DID), using exoantigens from genotyped strains found in this series of PCM cases. Overall, our DID tests have been false negative in about 30% of confirmed PCM cases. All patients were male, most with current or previous rural activity, with ages ranging from 17 to 59 years, with 11 patients (84.6%) over 40 years of age. No clinical or epidemiological differences were found between Paracoccidioides species. However, it is important to note that the only case of P. lutzii died as an outcome.ConclusionsThis study suggests P. brasiliensis sensu stricto (S1) as the predominant species, showing its wide geographic distribution in Brazil. Furthermore, our findings revealed, for the first time, the occurrence of P. restrepiensis (PS3) in the state of Mato Grosso do Sul, Brazil. Despite our setbacks, it would be interesting to provide the complete sequencing of these clinical isolates to complement the molecular information presented.     

Peptides from Paracoccidioides brasiliensis GP43 inhibit macrophage functions and inflammatory response

Paracoccidioidomycosis (PCM) is a systemic granulomatous disease caused by Paracoccidioides brasiliensis (Pb), a thermal dimorphic fungus. Its major antigen is a 43-kDa glycoprotein. Gp43 embodies different functions: it participates in evasion mechanisms during the installation of primary infection, stimulates granuloma-like formation in vitro and presents T-cell epitopes that induce protective response against the fungus. Here, we investigated epitopes from gp43 inhibitory of both, macrophage functions and inflammatory reaction. Different gp43 peptides, spanning the entire sequence of the molecule, were added to cultures of bone marrow-derived macrophages. After challenge with zymosan or Pb cells, phagocytic indexes were measured. Peptides expressed on the molecule surface were determined by graphic analysis using the Protean module; DNAstar Inc. Two peptides which decreased phagocytic index and were expressed at the surface of the molecule, P4 and P23, were selected for further studies. It was shown that both inhibited the release of NO by zymosan stimulated macrophages while enhanced release of H₂O₂. The release of TNF-α in culture supernatants from in vitro phagocytic tests showed different response depending of P4 concentration (data not shown). In vivo assays with Mycobacterium bovis – bacillus Calmette–Guérin (BCG) or Pb cells demonstrated that these peptides presented non-specific and specific anti-inflammatory properties.     

Cdc42p controls yeast-cell shape and virulence of Paracoccidioides brasiliensis

Paracoccidioides brasiliensis is characterized by a multiple budding phenotype and a polymorphic cell growth, leading to the formation of cells with extreme variations in shape and size. Since Cdc42 is a pivotal molecule in establishing and maintaining polarized growth for diverse cell types, as well as during pathogenesis of certain fungi, we evaluated its role during cell growth and virulence of the yeast-form of P. brasiliensis. We used antisense technology to knock-down PbCDC42’s expression in P. brasiliensis yeast cells, promoting a decrease in cell size and more homogenous cell growth, altering the typical polymorphism of wild-type cells. Reduced expression levels also lead to increased phagocytosis and decreased virulence in a mouse model of infection. We provide genetic evidences underlying Pbcdc42p as an important protein during host–pathogen interaction and the relevance of the polymorphic nature and cell size in the pathogenesis of P. brasiliensis.