RESUMO
Inteins are genetic mobile elements that are inserted within protein-coding genes, which are usually housekeeping genes. They are transcribed and translated along with the host gene, then catalyze their own splicing out of the host protein, which assumes its functional conformation thereafter. As Prp8 inteins are found in several important fungal pathogens and are absent in mammals, they are considered potential therapeutic targets since inhibiting their splicing would selectively block the maturation of fungal proteins. We developed a target-based drug screening system to evaluate the splicing of Prp8 intein from the yeast pathogen Cryptococcus neoformans (CnePrp8i) using Saccharomyces cerevisiae Ura3 as a non-native host protein. In our heterologous system, intein splicing preserved the full functionality of Ura3. To validate the system for drug screening, we examined cisplatin, which has been described as an intein splicing inhibitor. By using our system, new potential protein splicing inhibitors may be identified and used, in the future, as a new class of drugs for mycosis treatment. Our system also greatly facilitates the visualization of CnePrp8i splicing dynamics in vivo.
RESUMO
BACKGROUND: Histoplasmosis is a neglected disease that affects mainly immunocompromised patients, presenting a progressive dissemination pattern and a high mortality rate, mainly due to delayed diagnosis, caused by slow fungal growth in culture. Therefore, a fast, suitable and cost-effective assay is required for the diagnosis of histoplasmosis in resource-limited laboratories. This study aimed to develop and evaluate two new molecular approaches for a more cost-effective diagnosis of histoplasmosis. METHODOLOGY: Seeking a fast, suitable, sensitive, specific and low-cost molecular detection technique, we developed a new Loop-mediated Isothermal Amplification (LAMP) assay and nested PCR, both targeting the Internal Transcribed Spacer (ITS) multicopy region of Histoplasma capsulatum. The sensitivity was evaluated using 26 bone marrow and 1 whole blood specimens from patients suspected to have histoplasmosis and 5 whole blood samples from healthy subjects. All specimens were evaluated in culture, as a reference standard test, and Hcp100 nPCR, as a molecular reference test. A heparin-containing whole blood sample from a heathy subject was spiked with H. capsulatum cells and directly assayed with no previous DNA extraction. RESULTS: Both assays were able to detect down to 1 fg/µL of H. capsulatum DNA, and ITS LAMP results could also be revealed to the naked-eye by adding SYBR green to the reaction tube. In addition, both assays were able to detect all clades of Histoplasma capsulatum cryptic species complex. No cross-reaction with other fungal pathogens was presented. In comparison with Hcp100 nPCR, both assays reached 83% sensitivity and 92% specificity. Furthermore, ITS LAMP assay showed no need for DNA extraction, since it could be directly applied to crude whole blood specimens, with a limit of detection of 10 yeasts/µL. CONCLUSION: ITS LAMP and nPCR assays have the potential to be used in conjunction with culture for early diagnosis of progressive disseminated histoplasmosis, allowing earlier, appropriate treatment of the patient. The possibility of applying ITS LAMP, as a direct assay, with no DNA extraction and purification steps, makes it suitable for resource-limited laboratories. However, more studies are necessary to validate ITS LAMP and nPCR as direct assay in other types of clinical specimens.
