| Candida albicans (C. albicans) caused a sharp rise in the incidence of fungus infection. Synergy of the two drugs has many advantages such as increase the antifungal efficacy, reduce toxicity, provide a faster cure, prevent the emergence of resistance and provide broader-spectrum antifungal activity as compared with monotherapy regimens. Amino acids as therapeutic agents in clinical is very active currently, such as lysine hydrochloride and sodium chloride injection for cerebral insufficiency, acetylcysteinefor the treatment of chronic bronchitis, histidine-u-methionine for the treatment of vitamin ulcer disease, and so on. Therefore, amino acids are most likely as a source of antifungal sensitizer, we choose lysine and cysteine for the study and made a further study for the enhanced antifungal activity of lysine or cysteine in combination with amphotericin B and their possible mechanism. The purpose of this study is to make a research of the effect and mechanism of amino acids in combination with antifungals.Research in this study, we examined the impact of exogenous amino acids on antifungal agents showed that cysteine itself could significantly inhibit the growth of C. albicans, a different amino acid in combination with a different antifungal agents have different effects on growth of C. albicans in vitro. Results showd:①amino acids interaction with caspofungin (CAS):cysteine, lysine, threonine, isoleucine, arginine, methionine, serine, leucine, alanine acid, glycine, phenylalanine, tyrosine, proline can significantly enhance the role of CAS against C. albicans, aspartic acid can antagonize CAS against C. albicans;②amino acids interaction with miconazole (MCZ):cysteinecan significantly enhance the role of MCZ against C. albicans, serine, methionine can antagonize MCZ against C. albicans;③amino acids interaction with amphotericin B (AmB):cysteine, lysine, threonine, isoleucine, arginine, methionine, leucine, histidine, proline can significantly enhance the role of AmB against C. albicans, histidine, tyrosine can antagonize AmB against C. albicans;④amino acid interaction with5-fluorouracil (5-Fu):cysteine, aspartic acid, glutamic acid can significantly enhanced the role of5-Fu against C. albicans, tryptophan, valine can antagonize5-Fu against C. albicans;⑤amino acids interaction with terbinafine (TER):cystein, threonine, arginine can significantly enhance the role of TER against C. albicans, methionine, aspartic acid, glutamic acid, phenylalanine, tryptophan can antagonize TER against C. albicans;⑥amino acids interaction with shikonin (SK):cysteine, lysine, threonine, isoleucine, arginine, methionine, phenylalanine, tryptophan, ornithine, glycine, serine, alanine can significantly enhance the role of SK against C. albicans, aspartic acid, leucine can antagonize the role of SK against C. albicans.First of all, our study determined the effect of20kinds of different amino acids to various types of antifungal by the susceptibility testing. It was found that most of the amino acids do not affect the growth of C. albicans, the inhibition effect of threonine to the growth of C. albicans is weak, cysteine can significantly inhibit the growth of C. albicans, a different amino acid in combination with a different antifungal have different effects to the growth of C. albicans.Second, we demonstrated that100μg/ml lysine could enhance the effect of AmB (0.25μg/ml) against C. albicans in vitro, although lysine itself did not exert a fungicidal effect. In addition,0.5μg/ml AmB combined with200μg/ml lysine could provide an enhanced action against C. parapsilosis (0401380,392,90018,22090,22019,0201309) and Cryptococcus neoformans (0201309) compared with the use of0.5μg/ml AmB alone.50μg/ml cysteine has a strong influence to the growth of C. albicans,50μg/ml lysine could enhance the effect of AmB (0.25μg/ml) against C. albicans in vitro, it also has a strong effect on the growth of C. krusei ACCT2159, C. glabrata ACCT28226, C. parapsilosis22090. Growth curve test, spot aasay, survival further confirmed that lysine or cysteine can enhance the effect of AmB against C. albicans in vitro. We also investigated by XTT reduction assay, the effects of0.5μg/ml AmB and800μg/ml lysine in combination showed a strong effect of the anti-biofilm activity.At the same time, bio film formation experiment were performed to investigate the inhibition effect of biofilm formation when lysine in combination with AmB. The result showed that800μg/ml lysine in combination with0.5μg/ml AmB dramatically inhibited the biofilm formation. In addition, the growth of hypha experiment proved the impact of lysine in combination with AmB to C. albicans biofilms may be due to its inhibition effect on hypha formation.Next, through the study on the ultra-structure after drugs treatment of C. albicans, the flow cytometry to determine the cell cycle to observe the effect of drugs on cell cycle, the POLARstar multifunctional microplate to detect intracellular reactive oxygen species (ROS) generation, mitochondrial membrane potential detection (JC-1) method for the determination of mitochondrial membrane potential, we investigated the mechanism of action of lysine enhances the effect of AmB against C. albicans. Research results showed that, firstly, lysine in combination with AmB treatment increased generation of endogenous reactive oxygen species (ROS), decreased mitochondrial membrane potential level. We also used Real-time PCR investigated redox-related genes (Sod2, TRR1, GLR1, Cap1, CaMCA1, GRP2), and found that most of the redox related gene expression changed. Secondly, compared with AmB alone group, lysine combination with AmB group during cell division septum formation is not obvious. Lysine in combination with AmB can cause cell cycle arrest at the G2/M phase.Finally, not as same as the effect in vitro, the results in vivo showed that cysteine had no therapeutic efficacy on C. albicans infection. Consistent with the the effect in vitro, the therapeutic efficacy of cysteine in combination AmB is superior to amphotericin B alone treatment on C. albicans infection.In summary, this research has found that a variety of amino acids has the enhanced effect on antifungals against C. albicans in vitro, cysteine has a strong antifungal activity and cysteine in combination with AmB has the enhanced effect on antifungals against C. albicans both in vitro and in vivo. Lysine can be better enhance the sensitivity of AmB against C. albicans in vitro, and its main mechanism of action includes preventing the formation of the membrane during cell division, cell cycle arrest in the G2/M phase, increased intracellular reactive oxygen species and reduced the mitochondrial membrane potential. Amino acids as a synergist, can be developed into a promising new antifungal agents and provide a new idea for the study of fungi. |
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