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.

Copper overload in Paracoccidioides lutzii results in the accumulation of ergosterol and melanin.

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.

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, Gaussia 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.