ABSTRACT
Pathogenic microbes are exposed to a number of potential DNA-damaging stimuli during interaction with the host immune system. Microbial survival in this situation depends on a fine balance between the maintenance of DNA integrity and the adaptability provided by mutations. In this study, we investigated the association of the DNA repair response with the virulence of Cryptococcus neoformans, a basidiomycete that causes life-threatening meningoencephalitis in immunocompromised individuals. We focused on the characterization of C. neoformansAPN1 and APN2 putative genes, aiming to evaluate a possible role of the predicted Apurinic/apyrimidinic (AP) endonucleases 1 and 2 of the base excision repair (BER) pathway on C. neoformans response to stress conditions and virulence. Our results demonstrated the involvement of the putative AP-endonucleases Apn1 and Apn2 in the cellular response to DNA damage induced by alkylation and by UV radiation, in melanin production, in tolerance to drugs and in virulence of C. neoformans in vivo. We also pointed out the potential use of DNA repair inhibitor methoxy-amine in combination with conventional antifungal drugs, for the development of new therapeutic approaches against this human fungal pathogen. This work provides new information about the DNA damage response of the highly important pathogenic fungus C. neoformans.
ABSTRACT
Cryptococcus is a globally distributed fungal pathogen that primarily afflicts immunocompromised individuals. The therapeutic options are limited and include mostly amphotericin B or fluconazole, alone or in combination. The extensive usage of antifungals allowed the selection of resistant pathogens posing threats to global public health. Histone deacetylase genes are involved in Cryptococcus virulence, and in pathogenicity and resistance to azoles in Candida albicans. Aiming to assess whether histone deacetylase genes are involved in antifungal response and in synergistic drug interactions, we evaluated the activity of amphotericin B, fluconazole, sulfamethoxazole, sodium butyrate or trichostatin A (histone deacetylase inhibitors), and hydralazine or 5- aza-2'-deoxycytidine (DNA methyl-transferase inhibitors) against different Cryptococcus neoformans strains, C. neoformans histone deacetylase null mutants and Cryptococcus gattii NIH198. The drugs were employed alone or in different combinations. Fungal growth after photodynamic therapy mediated by an aluminium phthalocyanine chloride nanoemulsion, alone or in combination with the aforementioned drugs, was assessed for the C. neoformans HDAC null mutant strains. Our results showed that fluconazole was synergistic with sodium butyrate or with trichostatin A for the hda1Δ/hos2Δ double mutant strain. Sulfamethoxazole was synergistic with sodium butyrate or with hydralazine also for hda1Δ/hos2Δ. These results clearly indicate a link between HDAC impairment and drug sensitivity. Photodynamic therapy efficacy on controlling the growth of the HDAC mutant strains was increased by amphotericin B, fluconazole, sodium butyrate or hydralazine. This is the first study in Cryptococcus highlighting the combined effects of antifungal drugs, histone deacetylase or DNA methyltransferase inhibitors and photodynamic therapy in vitro.
Subject(s)
Antifungal Agents/metabolism , Bacterial Proteins/genetics , Cryptococcosis/drug therapy , Cryptococcus neoformans/enzymology , Epigenesis, Genetic/drug effects , Histone Deacetylases/genetics , Indoles/metabolism , Organometallic Compounds/metabolism , Photochemotherapy/methods , Amphotericin B/chemistry , Butyric Acid/chemistry , Drug Synergism , Emulsions/chemistry , Fluconazole/chemistry , Gene Expression Regulation, Bacterial/drug effects , Humans , Hydroxamic Acids/chemistry , Indoles/pharmacology , Nanoparticles/chemistry , Organometallic Compounds/pharmacology , Sulfamethoxazole/chemistryABSTRACT
Cryptococcosis is a disseminated infection caused mainly by C. neoformans and C. gattii. Limitations for the treatment involve the selection of isolates resistant to conventional antifungal drugs, prolonged treatment time and drugs side effects. This study evaluated the combined effect of histone deacetylase inhibitors (HDACi) and photodynamic therapy (PDT) on the growth of C. neoformans and C. gattii in vitro. Results showed that PDT inhibited yeasts proliferation and enhanced the HDACi-mediated cell viability impairment in Cryptococcus spp.