Interacting with hemoglobin: Paracoccidioides spp. recruits hsp30 on its cell surface for enhanced ability to use this iron source

Paracoccidioides spp. are thermally dimorphic fungi that cause paracoccidioidomycosis and can affect both immunocompetent and immunocompromised individuals. The infection can lead to moderate or severe illness and death. Paracoccidioides spp. undergo micronutrients deprivation within the host, including iron. To overcome such cellular stress, this genus of fungi responds in multiple ways, such as the utilization of hemoglobin. A glycosylphosphatidylinositol (GPI)-anchored fungal receptor, Rbt5, has the primary role of acquiring the essential nutrient iron from hemoglobin. Conversely, it is not clear if additional proteins participate in the process of using hemoglobin by the fungus. Therefore, in order to investigate changes in the proteomic level of P. lutzii cell wall, we deprived the fungus of iron and then treated those cells with hemoglobin. Deprived iron cells were used as control. Next, we performed cell wall fractionation and the obtained proteins were submitted to nanoUPLC-MSE. Protein expression levels of the cell wall F1 fraction of cells exposed to hemoglobin were compared with the protein expression of the cell wall F1 fraction of iron-deprived cells. Our results showed that P. lutzii exposure to hemoglobin increased the level of adhesins expression by the fungus, according to the proteomic data. We confirmed that the exposure of the fungus to hemoglobin increased its ability to adhere to macrophages by flow cytometry. In addition, we found that HSP30 of P. lutzii is a novel hemoglobin-binding protein and a possible heme oxygenase. In order to investigate the importance of HSP30 in the Paracoccidioides genus, we developed a Paracoccidioides brasiliensis knockdown strain of HSP30 via Agrobacterium tumefaciens-mediated transformation and demonstrated that silencing this gene decreases the ability of P. brasiliensis to use hemoglobin as a nutrient source. Additional studies are needed to establish HSP30 as a virulence factor, which can support the development of new therapeutic and/or diagnostic approaches.We thank CAPES: CNPq and FAPEG for providing fellowships to A.F.d.S, M.V.T., K.S.F.eS., J.S.d.C, C.A.P., L.C.B., J.D.P. and R.A.G., F.R. was supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER) (NORTE-01-0145-FEDER-000013). C.M.d.A.S. and M.P. belong to the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). This work was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Estado de Goiás (FAPEG), Instituto Nacional de Ciência e Tecnologia (INCT) de Estratégias de Interação Patógeno-Hospedeiro (grant number 201810267000022/INCT-FAPEG)

Heterologous expression and immunolocalization of HSP30 and peroxisomal catalase proteins in the wall of Paracoccidioides spp. interacting with macrophages

Submitted by Luciana Ferreira ([email protected]) on 2019-02-11T10:19:53Z No. of bitstreams: 2 Dissertação - Christie Ataides Pereira - 2018.pdf: 2029195 bytes, checksum: b9fbd3f81e3957f4e060b51e3f5c3392 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5)Approved for entry into archive by Luciana Ferreira ([email protected]) on 2019-02-11T10:24:46Z (GMT) No. of bitstreams: 2 Dissertação - Christie Ataides Pereira - 2018.pdf: 2029195 bytes, checksum: b9fbd3f81e3957f4e060b51e3f5c3392 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5)Made available in DSpace on 2019-02-11T10:24:46Z (GMT). No. of bitstreams: 2 Dissertação - Christie Ataides Pereira - 2018.pdf: 2029195 bytes, checksum: b9fbd3f81e3957f4e060b51e3f5c3392 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-03-09Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPESThe genus Paracoccidioides comprises thermodymorphic ascomycete’s fungi, causative agents of Paracoccidioidomycosis (PCM). PCM is an endemic granulomatous systemic mycosis in Latin America. The pathogen ability to interact and adhere to host surface structures is critical to the colonization, invasion, growth, and hematogenous spread of the fungus to tissues. Fungi use a variety of surface molecules to bind to the components of the host's extracellular matrix and defense cells, such as macrophages, so they can survive in these environments. A total of 94 cell wall proteins of Paracoccidioides spp. interacting with macrophages were identified through mass spectrometry studies. In this sense it becomes important the production of those possible adhesins via heterologous expression and localization of these proteins, aiming to perform adhesion studies. Therefore, HSP30 and peroxisomal catalase proteins of Paracoccidioides brasiliensis were expressed in a bacterial heterologous system, Escherichia coli. Open reading frames (ORFs) of the genes encoding HSP30 and peroxisomal catalase were cloned into pGEX-4T3 expression vector and the respective clones were used in the transformation of E. coli pLySs cells. The recombinant proteins were used in the production of polyclonal antibodies in mice. Anti-HSP30 and anti-CatP polyclonal antibodies were used in immunofluorescence assays, to obtain confirmation of the cellular location of HSP30 and CatP proteins. The obtained data allowed the localization of those proteins in the cell wall of the fungi cells, corroborating with the proteomic analyzes. The aim of this work is to perform additional macrophage interaction experiments to evaluate the potential of both proteins as adhesins.O gênero Paracoccidioides compreende fungos ascomicetos termodimórficos, que causam a doença Paracoccidioidomicose (PCM). A PCM é uma micose sistêmica granulomatosa e endêmica na América Latina. A capacidade do patógeno de interagir e aderir à matriz extracelular do hospedeiro é fundamental para a colonização, invasão, crescimento e disseminação hematogênica do fungo para tecidos. Os fungos utilizam uma variedade de moléculas de superfície para se aderirem à componentes da matriz extracelular do hospedeiro e células de defesa, como macrófagos, podendo assim sobreviver nesses ambientes. Foi identificado por meio de estudos de espectrometria de massa, um total de 94 proteínas da parede celular de Paracoccidioides spp. interagindo com macrófagos. Nesse sentido torna-se importante a produção dessas possíveis adesinas via expressão heteróloga e localização dessas proteínas, visando estudos posteriores de adesão. Neste estudo, foram expressas as proteínas HSP30 e catalase peroxissomal de Paracoccidioides brasiliensis em sistema heterólogo bacteriano, Escherichia coli. Os quadros abertos de leitura (ORFs) dos genes codificadores de HSP30 e catalase peroxissomal foram clonados em vetor de expressão pGEX-4T3 e os respectivos clones foram utilizados na transformação de células de E. coli pLySs. As proteínas recombinantes resultantes foram utilizadas na produção de anticorpos policlonais,em camundongos. Os anticorpos policlonais anti-HSP30 e anti-CatP foram utilizados em ensaios de imunofluorescência para confirmação da localização celular das proteínas HSP30 e CatP. Com os resultados pode-se observar que as proteínas estão localizadas na parede celular do fungo corroborando assim com os dados de análises proteômicas previamente publicados. A perspectiva deste trabalho é realizar experimentos adicionais de interação com macrófagos para avaliar o potencial de ambas as proteínas como adesinas

Bioorthogonal Protein Conjugation: Application to the Development of a Highly Sensitive Bioluminescent Immunoassay for the Detection of Interferon‑γ

Bioorthogonal conjugation eliminates the shortcomings of classical conjugation methods. The conjugation of antibodies to reporter proteins, such as bioluminescent protein, can be controlled with orthogonal conjugation methods. Here we report a bioluminescent immunoassay for the sensitive detection of interferon-γ (IFN-γ) that utilizes orthogonal conjugation of bioluminescent protein, <i>Gaussia</i> luciferase to anti-IFN-γ antibody. The IFN-γ is produced by the immune system and the detection of the IFN-γ is pivotal for the detection of persistent viral and bacterial infections. A bioorthogonal conjugation approach is used to conjugate an anti-IFN-γ antibody with a GLuc mutant containing the N-terminal tyrosine using formylbenzene diazonium hexafluorophosphate reagent (FBDP) in hydrophilic mild pH environment yielding high conjugation efficiency (60%). This reagent is shown to be specific for tyrosine (Tyr) residues. Therefore, conjugation through Tyr was orthogonal and not detrimental to the bioluminescence activity of GLuc. The immunoassay described in this paper is a sandwich type assay and involves a capture and a detection antibody. The assay was validated for its robustness, precision, accuracy, limit of detection, and recovery