gms | German Medical Science

Systemic infections caused by mucormycetes are often lethal. Recommended first line is amphotericin B (AMB) and posaconazole (PSC) is suggested as salvage therapy. Azole resistance was found to be species-dependent for mucormycetes. Therefore, underlying molecular mechanisms of the phenotypical observed PSC were studied. To this aim, Sanger sequence analysis of the two lanosterol 14-α demethylase orthologous genes were performed followed by structural analyses of protein.

A strain collection of 131 Mucorales isolates was catalogued for a) species identification using direct sequencing analysis of the internal transcribed spacer region (ITS), and b) determination of PSC in vitro susceptibility testing by following the CLSI guidelines. Full gene sequences of two CYP51 orthologues are available online for Rhizopus arrhizus, and Mucor circinelloides (http://www.broadinstitute.org/). These were set as reference for amplification and sequencing analysis of the genes encoding for lanosterol 14-α demethylase, known as primary structural target of azoles. All nucleic acid sequences of both PSC-susceptible and -resistant strains were screened for SNPs and translated into the amino acid sequence. Found missense mutations were in silico modeled based on the molecular 3D structure of the Erg11 protein of Saccharomyces cerevisae (Erg11p6xHis).

Three species [M. circinelloides (McCYP51A and McCYP51B), R. arrhizus (RaCYP51A and RaCYP51B), and R. microsporus (RmCYP51A and RmCYP51B)] were identified to contain subpopulations with reduced in vitro susceptibilities against PSC. These subpopulations plus the wild-type populations were genetically analyzed. From the eight amino acid variations found for R. arrhizus, one in the orthologous protein of CYP51B was of particular interest (I57V) as it might indirectly affect PSC binding. Furthermore, out of the 12 amino acid changes found for RmCYP51B, the amino acid change RYH might give rise to significant PSC resistance.

We provided for the first time, the full gene sequences of two orthologous loci of lanosterol 14-α demethylase in three clinically relevant Mucorales species. The amino acid substitution RYH found in R. microsporus may confer reduced PSC susceptibility. However, experimental validation of this new amino acid change on its impact on PSC resistance is needed.