RESUMEN

The phenotypic plasticity of Cryptococcus neoformans is widely studied and demonstrated in vitro, but its influence on pathogenicity remains unclear. In this study, we investigated the dynamics of cryptococcal cell and transcriptional remodeling during pulmonary infection in a murine model. We showed that in Cryptococcus neoformans, cell size reduction (cell body ≤ 3 µm) is important for initial adaptation during infection. This change was associated with reproductive fitness and tissue invasion. Subsequently, the fungus develops mechanisms aimed at resistance to the host's immune response, which is determinant for virulence. We investigated the transcriptional changes involved in this cellular remodeling and found an upregulation of transcripts related to ribosome biogenesis at the beginning (6 h) of infection and a later (10 days) upregulation of transcripts involved in the inositol pathway, energy production, and the proteasome. Consistent with a role for the proteasome, we found that its inhibition delayed cell remodeling during infection with the H99 strain. Altogether, these results further our understanding of the infection biology of C. neoformans and provide perspectives to support therapeutic and diagnostic targets for cryptococcosis.

Asunto(s)

Criptococosis , Cryptococcus neoformans , Ratones , Animales , Cryptococcus neoformans/genética , Cryptococcus neoformans/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Modelos Animales de Enfermedad , Criptococosis/microbiología , Virulencia

RESUMEN

Cryptococcosis is a fungal disease of global significance for which new effective treatments are needed. The conjugation of the synthetic antimicrobial peptide fragment UBI 31-38 to a coumarin derivative showed to be an effective approach for the design of a novel anticryptococcal agent. In addition to antifungal activity, the conjugate exhibited intense fluorescence, which could be valuable for mechanistic investigations of this molecule. In this work, we studied the photophysical properties of the conjugate and confocal scanning laser microscopy was used to inspect the distribution of the peptide-coumarin conjugate in Cryptococcus cell. The synergism of this compound with amphotericin B or fluconazole against C. gattii and C. neoformans strains was also investigated. The results indicated that the fluorescent conjugate alone as well as its combination with amphotericin B are promising tools against cryptococcosis.

Asunto(s)

Anfotericina B/farmacología , Antifúngicos/farmacología , Cumarinas/farmacología , Criptococosis/tratamiento farmacológico , Cryptococcus/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Anfotericina B/química , Antifúngicos/síntesis química , Antifúngicos/química , Cumarinas/química , Cryptococcus/citología , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Humanos , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/química , Procesos Fotoquímicos , Relación Estructura-Actividad

RESUMEN

Influenza A virus (IAV) infects millions of people annually and predisposes to secondary bacterial infections. Inhalation of fungi within the Cryptococcus complex causes pulmonary disease with secondary meningo-encephalitis. Underlying pulmonary disease is a strong risk factor for development of C. gattii cryptococcosis though the effect of concurrent infection with IAV has not been studied. We developed an in vivo model of Influenza A H1N1 and C. gattii co-infection. Co-infection resulted in a major increase in morbidity and mortality, with severe lung damage and a high brain fungal burden when mice were infected in the acute phase of influenza multiplication. Furthermore, IAV alters the host response to C. gattii, leading to recruitment of significantly more neutrophils and macrophages into the lungs. Moreover, IAV induced the production of type 1 interferons (IFN-α4/ß) and the levels of IFN-γ were significantly reduced, which can be associated with impairment of the immune response to Cryptococcus during co-infection. Phagocytosis, killing of cryptococci and production of reactive oxygen species (ROS) by IAV-infected macrophages were reduced, independent of previous IFN-γ stimulation, leading to increased proliferation of the fungus within macrophages. In conclusion, IAV infection is a predisposing factor for severe disease and adverse outcomes in mice co-infected with C. gattii.

Asunto(s)

Causalidad , Coinfección , Criptococosis/complicaciones , Cryptococcus gattii/patogenicidad , Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Infecciones por Orthomyxoviridae/complicaciones , Acetilglucosaminidasa/metabolismo , Animales , Conducta Animal , Encéfalo/microbiología , Encéfalo/patología , Proliferación Celular , Quimiocinas/metabolismo , Coinfección/inmunología , Coinfección/microbiología , Coinfección/mortalidad , Coinfección/virología , Criptococosis/inmunología , Cryptococcus gattii/inmunología , Cryptococcus neoformans/inmunología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Perros , Femenino , Humanos , Subtipo H1N1 del Virus de la Influenza A/inmunología , Interferón gamma/metabolismo , Pulmón/enzimología , Pulmón/patología , Pulmón/virología , Macrófagos/metabolismo , Macrófagos/virología , Células de Riñón Canino Madin Darby , Ratones , Ratones Endogámicos C57BL , Neutrófilos , Óxido Nítrico/metabolismo , Infecciones por Orthomyxoviridae/inmunología , Peroxidasa/metabolismo , Ácido Peroxinitroso/metabolismo , Fagocitosis , Especies Reactivas de Oxígeno/metabolismo , Tasa de Supervivencia

RESUMEN

Antimicrobial peptides (AMPs) are currently being investigated as potential sources of novel therapeutics against an increasing number of microorganisms resistant to conventional antibiotics. The conjugation of an AMP to other bioactive compounds is an interesting approach for the development of new derivatives with increased antimicrobial efficiency and broader spectra of action. In this work, the synthesis of a new peptide-coumarin conjugate via copper(I)-catalyzed azide-alkyne cycloaddition is described. The conjugate was assayed for in vitro cytotoxicity and displayed antifungal activity against Cryptococcus gattii and Cryptococcus neoformans. Additionally, the conjugate exhibited increased antifungal efficacy when compared with the individual peptide, coumarin, or triazole moieties. Treatment of C. gattii with the peptide-coumarin conjugate enhanced the production of reactive oxygen species, suggesting that the oxidative burst plays an important role in the mechanism of action of the conjugate.