RESUMO
OBJECTIVES: Historically, the Brazilian Central-West region has had high numbers of paracoccidioidomycosis (PCM) cases caused by the dimorphic fungus Paracoccidioides lutzii. METHODS: This epidemiological, observational, analytical, cross-sectional study was performed to investigate the clinical and laboratory data of 44 PCM patients with a culture-proven P. lutzii infection. All patients were referred to the Systemic Mycosis Center, Júlio Muller University Hospital, Cuiabá, Brazil, during January 2017 to March 2020. The neutrophil to lymphocyte ratio (NLR) was calculated and dichotomized by its median value to include in the identification of factors associated with severity. RESULTS: At admission, 13 (31.7%) patients showed the disseminated multifocal chronic form of PCM and 16 (36.4%) patients met the clinical severity criteria. Treatment prescribed on admission did not follow the recommendations of the Brazilian Guideline for the Clinical Management of Paracoccidioidomycosis in 26% of the severe PCM cases (prevalence ratio 0.26, 95% confidence interval 0.14-0.49; P < 0.0001). Patients with severe PCM had a higher NLR that was greater than the median (≥4.11). CONCLUSIONS: The NLR biomarker complements the criteria for PCM severity. Applying the low-cost NLR test can greatly increase the diagnostic sensitivity when screening patients for PCM and contribute to better control of the disease, management of complications, and therapeutic strategies.
Assuntos
Paracoccidioides/fisiologia , Paracoccidioidomicose/epidemiologia , Índice de Gravidade de Doença , Adulto , Estudos Transversais , Humanos , Masculino , Pessoa de Meia-Idade , Neutrófilos/citologia , Paracoccidioidomicose/diagnóstico , Paracoccidioidomicose/imunologia , PrevalênciaRESUMO
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.
Assuntos
Antígenos de Fungos/metabolismo , Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/imunologia , Proteínas Fúngicas/metabolismo , Imunoterapia/métodos , Macrófagos/imunologia , Paracoccidioides/fisiologia , Paracoccidioidomicose/imunologia , Animais , Antígenos de Fungos/genética , Proliferação de Células , Células Cultivadas , Resistência à Doença , Proteínas Fúngicas/genética , Humanos , Ativação Linfocitária , Ativação de Macrófagos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Paracoccidioidomicose/terapia , Peptídeos/genética , Peptídeos/metabolismoRESUMO
We aimed to investigate the effects of ethanol and its metabolites (ß-hydroxybutyrate and sodium acetate) in the effector functions of macrophages in response to Paracoccidioides brasiliensis yeast cells and to determine their influence in the development of the adaptive response. Purified peripheral blood monocytes were differentiated into macrophages and were treated with ethanol, ß-hydroxybutyrate, and sodium acetate, and stimulated with P. brasiliensis yeast cells and evaluated for their phenotypic characteristics, functional activity, and capability to induce T cells activation/differentiation. We found that the ethanol treatment diminished the expression of HLA-AB, HLA-DR, CD80, and CD86, modulating the expression of dectin-1, as well as Syk phosphorylation. The ethanol treatment increased the phagocytic activity, expression of CD206, and IL-10 production; however, reduced ROS production, fungicidal activity, caspase-1 cleavage, and IL-1ß and IL-6 production. Our data also showed that the presence of ethanol reduced the differentiation of Th1 and Th17 cells and increased the frequency of Th2 cells. Our results indicated that ethanol exposure could suppress effector function of macrophages, possibly leading to the polarization of M2 macrophages. The ethanol modulates the expression of costimulatory and antigen-presentation molecules and interferes with the NLRP3 inflammasome. Altogether, these alterations affect the development of the adaptive response, decreasing the frequency of IL-17, IL-22, and IFN- γ producing cells, and increasing the frequency of IL-4 producing cells. Therefore, exposure to ethanol can impair the capability of macrophages to exert their effector functions and activate the acquired response related to resistance to P. brasiliensis infection.
Assuntos
Etanol/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/microbiologia , Paracoccidioides/fisiologia , Paracoccidioidomicose/imunologia , Imunidade Adaptativa/efeitos dos fármacos , Antifúngicos/farmacologia , Complexo CD3/análise , Caspase 1/análise , Citocinas/análise , Citocinas/efeitos dos fármacos , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Inflamassomos/efeitos dos fármacos , Inflamassomos/metabolismo , Receptores de Lipopolissacarídeos/análise , Macrófagos/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/efeitos dos fármacos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Peróxidos/metabolismo , Fagocitose/efeitos dos fármacosRESUMO
Paracoccidioidomycosis (PCM) is the most prevalent systemic mycosis in Latin America and may be caused by the species Paracoccidioides brasiliensis. In the lungs, this fungus interacts with epithelial cells, activating host cell signalling pathways, resulting in the production of inflammatory mediators. This event may be initiated through the activation of Pattern-Recognition Receptors such as Toll-like Receptors (TLRs). By interacting with cell wall components, TLR2 is frequently related to fungal infections. In this work, we show that, after 24 h post-infection with P. brasiliensis, A549 lung epithelial cells presented higher TLR2 levels, which is important for IL-8 secretion. Besides, integrins may also participate in pathogen recognition by host cells. We verified that P. brasiliensis increased α3 integrin levels in A549 cells after 5 h of infection and promoted interaction between this receptor and TLR2. However, after 24 h, surprisingly, we verified a decrease of α3 integrin levels, which was dependent on direct contact between fungi and epithelial cells. Likewise, we observed that TLR2 is important to downmodulate α3 integrin levels after 24 h of infection. Thus, P. brasiliensis can modulate the host inflammatory response by exploiting host cell receptors and cell signalling pathways.
Assuntos
Células Epiteliais/metabolismo , Integrina alfa3/metabolismo , Pulmão/metabolismo , Receptor 2 Toll-Like/metabolismo , Células A549 , Western Blotting , Células Epiteliais/microbiologia , Interações Hospedeiro-Patógeno , Humanos , Pulmão/microbiologia , Pulmão/patologia , Paracoccidioides/fisiologiaRESUMO
Fungi that belong to the genus Paracoccidioides are the etiologic agents of paracoccidioidomycosis, a human systemic mycosis, which occurs in Latin America. Epithelial cell is one of the first cells that interact with these fungi and responds by secreting inflammatory mediators such as cytokines. In the present study, we demonstrate that yeasts of different isolates of Paracoccidioides brasiliensis (Pb18 and Pb03) and Paracoccidioides lutzii (Pb01) distinctly promoted interleukin (IL)-8 secretion by the lung epithelial cell line A549. Depending on the isolate, this cytokine release may rely on the epithelial cell interaction with fungal secreted components or direct contact with the pathogen. In addition, adhesion of yeasts to the pulmonary epithelial cells was also different among Paracoccidioides isolates, and the highest percentage of A549 cells with adhered fungi was observed with P. lutzii. All Paracoccidioides isolates induced an expression increase of α3 and α5 integrins in A549 cells and, using small interfering RNA, we observed that the integrin silencing promoted a reduction of P. lutzii adhesion, which suggests the involvement of integrins in this event. Together, these results indicate that host epithelial cell response may depend on the isolate of Paracoccidioides.
Assuntos
Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/microbiologia , Interleucina-8/biossíntese , Paracoccidioides/fisiologia , Paracoccidioidomicose/metabolismo , Paracoccidioidomicose/microbiologia , Células A549 , Adesão Celular , Sobrevivência Celular , Células Cultivadas , Citocinas/metabolismo , Inativação Gênica , Humanos , Integrinas/genéticaRESUMO
Paracoccidioides spp. are thermodimorphic fungi that cause a neglected tropical disease (paracoccidioidomycosis) that is endemic to Latin America. These fungi inhabit the soil, where they live as saprophytes with no need for a mammalian host to complete their life cycle. Despite this, they developed sophisticated virulence attributes allowing them not only to survive in host tissues but also to cause disease. A hypothesis for selective pressures driving the emergence or maintenance of virulence of soil fungi is their interaction with soil predators such as amoebae and helminths. We evaluated the presence of environmental amoeboid predators in soil from armadillo burrows where Paracoccidioides had been previously detected and tested if the interaction of Paracoccidioides with amoebae selects for fungi with increased virulence. Nematodes, ciliates, and amoebae-all potential predators of fungi-grew in cultures from soil samples. Microscopical observation and ITS sequencing identified the amoebae as Acanthamoeba spp, Allovahlkampfia spelaea, and Vermamoeba vermiformis. These three amoebae efficiently ingested, killed and digested Paracoccidioides spp. yeast cells, as did laboratory adapted axenic Acanthamoeba castellanii. Sequential co-cultivation of Paracoccidioides with A. castellanii selected for phenotypical traits related to the survival of the fungus within a natural predator as well as in murine macrophages and in vivo (Galleria mellonella and mice). These changes in virulence were linked to the accumulation of cell wall alpha-glucans, polysaccharides that mask recognition of fungal molecular patterns by host pattern recognition receptors. Altogether, our results indicate that Paracoccidioides inhabits a complex environment with multiple amoeboid predators that can exert selective pressure to guide the evolution of virulence traits.
Assuntos
Amoeba/fisiologia , Interações Hospedeiro-Patógeno/fisiologia , Paracoccidioides/fisiologia , Microbiologia do Solo , Acanthamoeba castellanii/fisiologia , Amoeba/citologia , Amoeba/microbiologia , Animais , Tatus , Cilióforos , Técnicas de Cocultura , Modelos Animais de Doenças , Fungos , Macrófagos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Nematoides , Paracoccidioides/patogenicidade , Paracoccidioidomicose/microbiologia , Fagocitose , Solo , Virulência , Fatores de Virulência/fisiologiaAssuntos
Doenças Negligenciadas/microbiologia , Paracoccidioidomicose/microbiologia , Adulto , Antifúngicos/uso terapêutico , Ensaios Clínicos como Assunto , Humanos , Masculino , Pessoa de Meia-Idade , Doenças Negligenciadas/diagnóstico , Doenças Negligenciadas/tratamento farmacológico , Doenças Negligenciadas/epidemiologia , Paracoccidioides/classificação , Paracoccidioides/genética , Paracoccidioides/isolamento & purificação , Paracoccidioides/fisiologia , Paracoccidioidomicose/diagnóstico , Paracoccidioidomicose/tratamento farmacológico , Paracoccidioidomicose/epidemiologia , Pobreza , América do Sul/epidemiologia , Organização Mundial da SaúdeRESUMO
Paracoccidioides brasiliensis is a temperature-dependent dimorphic fungus that cause paracoccidioidomycosis (PCM), the major systemic mycosis in Latin America. The capacity to evade the innate immune response of the host is due to P. brasiliensis ability to respond and to survive the nitrosative stress caused by phagocytic cells. However, the regulation of signal transduction pathways associated to nitrosative stress response are poorly understood. Ras GTPase play an important role in the various cellular events in many fungi. Ras, in its activated form (Ras-GTP), interacts with effector proteins and can initiate a kinase cascade. In this report, we investigated the role of Ras GTPase in P. brasiliensis after in vitro stimulus with nitric oxide (NO). We observed that low concentrations of NO induced cell proliferation in P. brasiliensis, while high concentrations promoted decrease in fungal viability, and both events were reversed in the presence of a NO scavenger. We observed that high levels of NO induced Ras activation and its S-nitrosylation. Additionally, we showed that Ras modulated the expression of antioxidant genes in response to nitrosative stress. We find that the Hog1 MAP kinase contributed to nitrosative stress response in P. brasiliensis in a Ras-dependent manner. Taken together, our data demonstrate the relationship between Ras-GTPase and Hog1 MAPK pathway allowing for the P. brasiliensis adaptation to nitrosative stress.
Assuntos
Proteínas Fúngicas/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Estresse Nitrosativo/fisiologia , Paracoccidioides/fisiologia , Proteínas ras/fisiologia , Sequência de Aminoácidos , Animais , Morte Celular/fisiologia , Proliferação de Células/fisiologia , Expressão Gênica/fisiologia , Masculino , Camundongos Endogâmicos BALB C , Proteínas Quinases Ativadas por Mitógeno/química , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Óxido Nítrico/química , Óxido Nítrico/farmacologia , Processamento de Proteína Pós-TraducionalRESUMO
Paracoccidioides spp. is a thermally dimorphic fungus endemic to Latin America and the etiological agent of paracoccidioidomycosis (PCM), a granulomatous disease acquired through fungal propagule inhalation by its mammalian host. The infection is established after successful mycelia to yeast transition in the host pulmonary alveoli. The challenging environment inside the host exposes the fungus to the need of adaptation in order to circumvent nutritional, thermal, oxidative, immunological and other stresses that can directly affect their survival. Considering that autophagy is a response to abrupt environmental changes and is induced by stress conditions, this study hypothesizes that this process might be crucially involved in the adaptation of Paracoccidioides spp. to the host and, therefore, it is essential for the proper establishment of the disease. By labelling autophagous vesicles with monodansylcadaverine, autophagy was observed as an early event in cells during the normal mycelium to yeast transition, as well as in yeast cells of P. brasiliensis under glucose deprivation, and under either rapamycin or 3-methyladenine (3-MA). Findings in this study demonstrated that autophagy is triggered in P. brasiliensis during the thermal-induced mycelium to yeast transition and by glucose-limited conditions in yeasts, both of which modulated by rapamycin or 3-MA. Certainly, further genetic and in vivo analyses are needed in order to finally address the contribution of autophagy for adaptation. Yet, our data propose that autophagy possibly plays an important role in Paracoccidioides brasiliensis virulence and pathogenicity.
Assuntos
Autofagia/genética , Nutrientes/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Paracoccidioides/genética , Adenina/análogos & derivados , Adenina/farmacologia , Regulação Fúngica da Expressão Gênica , Micélio/genética , Micélio/crescimento & desenvolvimento , Nutrientes/genética , Estresse Oxidativo/genética , Paracoccidioides/patogenicidade , Paracoccidioides/fisiologia , Paracoccidioidomicose/genética , Paracoccidioidomicose/microbiologia , Saccharomyces cerevisiae/genética , Sirolimo/farmacologiaRESUMO
Paracoccidioides brasiliensis is the agent of paracoccidioidomycosis (PCM), a cause of disease in healthy and immunocompromised persons in Latin America. The infection begins after inhalation of the fungal propagules and their thermo-dimorphic shift to yeast form. The development of the disease depends on factors associated with the host immune response and the infectious agent's characteristics, especially virulence. The oxidative stress response is an important virulence attribute in several fungi. In this study, we assessed the enzymatic repertoire of responses to oxidative stress in the Pb18 isolate with different degrees of virulence. The virulence of attenuated Pb18 (aPb18) strain was recovered after several animal passages. Virulent strain (vPb18) showed an effective fungal oxidative stress response and several genes involved in response to oxidative stress were up-regulated in this isolate. These genes expressed the same profile when we recovered the phenotypic virulence in attenuated strain aPb18. Our study demonstrated that attenuated P. brasiliensis recovered their virulence after serial animal passages (vPb18), and this process positively modulated the fungus's antioxidant repertoire.