| Aspergillus is widely distributed in nature, which comprised of a diversity of species. In medical field, along with widespread used broad-spectrum antibiotic, deep-going carried antineoplaston, continuous application of organ transplantation and other surgical intervention, invasive aspergillosis infection on the rise. The treatment of aspergillosis infection progress slow, compared to a wide variety of other antimicrobial agents.Itraconazole and voriconazole became the first line azole introduced for the treatment of aspergillosis. While many azole-resistant strains of A. fumigatus have been isolated and characterized. The resistance mechanisms of these isolate, include the activation of drug efflux pumps, an alteration of the drug target, or alterations in stress response pathways.Previous work has demonstrated that calcium based signaling system may change the free Ca2+concentration in the cytoplasm in response to environment stimuli and physiological signals by calcium channels or calcium transporters, Ca2+-binding proteins and other downstream proteins. Calcium ions almost involve in all physiological activity, including growth, development, proliferation, secretion, transportation and stress-response reaction. Based on previous researches, we identified that Ca2+signaling pathway was involved in Aspergillus’ drug stress-response reactions.We used calcium chelators combined with inactivated mutations in calcium signaling pathway to test the susceptibility of Aspergillus nidulans and Aspergillus fumigatus to azole. Just as our expected, the addition of calcium chelator (2mM EGTA)with ITZ(0.1μg/ml)or VRC(0.12μg/ml) made the strain more sensitive than azole only. By using commercially available E-test strips (AB Biodisk), we determined that EGTA can enhance ITZ activity by two fold. Calcineurin (CnaA) and its target transcription factor CrzA mutant strains demonstrated increased sensitive to0.1μg/ml ITZ and0.12μg/ml VRC relative to the parent strains. While Ca2+-channels mutant strains, ΔmidA, ΔcchA were a little tolerance to azole than wild type in minimal medium. To further completely understand the regulation mechanism. We employed the Rh123-based assay, used a fluorescent substrate Rh123for the pumps to verify the functional activity of efflux pumps in the presence of calcium chelators EGTA, which can reduce the intracellular calcium. The addition of EGTA making an increase in Rh123accumulation was observed in Aspergillus nidulans. Then we measured the content level of ergosterol located in the cell membrane by HPLC. The total cellular ergosterol content of Δcna mycelia decreased to88.6%as compared to wild type. Reciprocally, in ΔmidA/Δ cchA double deletion strain, the total cellular ergosterol content increased by7%. And then, we inferred that the results achieved in Aspergillus nidulans can be also applied to Aspergillus fumigatus. Aspergillus fumigatus showed increased sensitivity when treated by0.7μg/ml ITZ and5mM EGTA than by ITZ only. To evaluate the benefits of combination therapy, we used a stringent model involving injection of a large fungal burden that results in rapid killing of larvae. The result showed that combination therapy can delay the rate of death in larvae.From the data upon, in Aspergillus free intracellular calcium concentration affected the activity of efflux pumps, Ca2+-channels and their interaction proteins, accompanied with its transcription factors can alter the drug target which is the primary mechanism of drug resistance. In the model of Galleria mellonella, the drug combination played a better role in the treatment of aspergillosis than ITZ only. |
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