Whole genome sequencing of newly emerged fungal pathogen Aspergillus lentulus and its Azole resistance gene prediction

Abstract Aspergillus lentulus is an important newly recorded species in the A. fumigatus complex and its resistance to azole drugs and the high mortality rate of infected individuals have emerged as problems. Comprehensive understanding of the A. lentulus is limited due to lack of genome-wide fine mapping data. The aim of this study was to investigate the A. lentulus signature at the molecular level, analyze the genome-wide profile of this strain and predict its possible genes that execute azole resistance. In this study, a whole genome sequencing of a clinically isolated A. lentulus strain (named A. lentulus PWCAL1) was studied by PacBio Sequel sequencing platform. Azole resistance genes were predicted based on whole-genome sequencing data analysis, gene function annotation, comparative genomics analysis, and blastp alignment using the Mycology Antifungal Resistance Database (MARDy) to comprehensively understanding the genome-wide features, pathogenicity, and resistance mechanisms of A. lentulus . The results of whole genome sequencing demonstrated that the total length of A. lentulus PWCAL1 genome was 31255105 bp, the GC content was 49.24%, and 6883 coding genes were predicted. A total of 4565, 1824 and 6405 genes were annotated in GO, KOG and KEGG databases, respectively. In the PHI and DFVF databases, 949 and 259 interacting virulence factors were identified, respectively, with the main virulence factor-Mutant virulence phenotype was enriched in reduced virulence. Comparative genomics analysis showed that there were 5456 consensus Core Genes in this strain and four closely related strains of A. fumigatus complex, which were mainly involved in Human Diseases, Metabolism, Organismal Systems, etc. Among the three aligned A. lentulus strains, the number of unique genes of this bacterium was the highest with number of 171, and these genes were mainly associated with Carbohydrate metabolism, Cell growth and death. Resistance gene prediction demonstrated that the A5653 gene of this bacterium had F46Y/N248T double point mutations on the CYP51A gene, but no tandem repeat TR mutations in the promoter region were detected. Further more, twelve genes belonging to the fungal multidrug resistance ABC transporters were identified based on the complete genome sequence and phylogenetic analysis of A. lentulus, which belonged to the ALDp subfamily, the PDR subfamily (AtrB, CDR1, and CDR2), and the MDR subfamily (MDR1), respectively, and there were four genes that are annotated to the MFS multidrug transporter. Further phylogenetic tree classification of the ABC transporter subfamilies predicted in the nine selected A. fumigatus complex strains showed that these putative ABC proteins were divided into two main clusters, which belonged to PDR (CDR1, CDR2, AtrB, and AtrF) and MDR subfamilies (MDR1, MDR2, and MDR3). The distribution of these ABC proteins varies among different species of the A. fumigatus complex. The main result obtained from this study for the whole genome of A. lentulus provide new insights into better understanding the biological characteristics, pathogenicity and resistance mechanisms of this bacterium. In this study, two resistance mechanisms which include CYP51A gene mutation and multidrug-resistant ABC transporter were predicted in a single isolate. Based on the predicted CYP51A-F46Y/N248T site mutation combination, we speculate that the CYP51A gene of A. lentulus may be partially responsible for azole resistance. Based on the predicted ABC transporter family genes, we hypothesize that resistance to multiple azoles in A. lentulus is mediated, at least in part, by these ABC transporters with resistance.