Nystatin-like Pseudonocardia polyene B1, a novel disaccharide-containing antifungal heptaene antibiotic

Abstract

Polyene macrolides such as nystatin A1 and amphotericin B belong to a large family of very valuable antifungal polyketide compounds typically produced by soil actinomycetes. Recently, nystatin-like Pseudonocardia polyene (NPP) A1 has been identified as a unique disaccharide-containing tetraene antifungal macrolide produced by Pseudonocardia autotrophica. Despite its significantly increased water solubility and decreased hemolytic activity, its antifungal activity remains limited compared with that of nystatin A1. In this study, we developed NPP B1, a novel NPP A1 derivative harboring a heptaene core structure, by introducing two amino acid substitutions in the putative NADPH-binding motif of the enoyl reductase domain in module 5 of the NPP A1 polyketide synthase NppC. The low level NPP B1 production yield was successfully improved by eliminating the native plasmid encoding a polyketide biosynthetic gene cluster present in P. autotrophica. In vitro and in vivo antifungal activity and toxicity studies indicated that NPP B1 exhibited comparable antifungal activity against Candida albicans and was less toxic than the most potent heptaene antifungal, amphotericin B. Moreover, NPP B1 showed improved pharmacokinetic parameters compared to those of amphotericin B, suggesting that NPP B1 could be a promising candidate for development into a pharmacokinetically improved and less-toxic polyene antifungal antibiotic.

Figure 1

Polyene macrolides investigated in this study. Nystatin A1, a typical tetraene polyene macrolide produced by Streptomyces noursei; amphotericin B, the most potent polyene macrolide produced by Streptomyces nodosus; nystatin-like Pseudonocardia polyene (NPP) A1, produced by Pseudonocardia autotrophica wild-type; NPP B1, produced by enoyl reductase (ER) domain in module 5 (ER5) inactivation mutant of P. autotrophica.

Figure 2

Development of nystatin-like Pseudonocardia polyene (NPP) B1 overproducing strains in Pseudonocardia autotrophica. (A) Inactivation scheme of enoyl reductase (ER) domain in module 5 (ER5) of nppC gene; KS, ketosynthase; AT, acyltransferase; DH, dehydratase; ER, enoyl reductase; KR, ketoreductase; ACP, acyl carrier protein. (B) Deletion scheme of P. autotrophica originated plasmid. (C) Comparison of NPP B1 production yields with newly constructed NPP B1 overproducing strains.

Figure 3

In vivo efficacy of polyene macrolides. The survival rates of mice infected with Candida albicans after treatment with low and high concentrations (left and right, respectively) of polyene macrolides.