CHARACTERIZING THE GENOME AND MECHANISMS OF ANTIFUNGAL RESISTANCE IN LOMENTOSPORA PROLIFICANS

Lomentospora prolificans is a filamentous fungus capable of causing fungemia in profoundly immunocompromised patients. Such infections are often fatal due in large part to the organism’s pan-resistance to all systemic antifungal. Of the three families of systemic antifungals, azoles, echinocandins, and polyenes, a mechanism of resistance has been established only for echinocandins. One difficulty impeding research into L. prolificans has been the lack of a published whole genome sequence. In 2017, a whole genome sequence was published, but did not achieve chromosome-level assembly. To address the absence of a chromosome-level genome sequence of L. prolificans, we sequenced an additional two isolates and assembled these, as well as the reads from the published genome sequence. Additionally, pulsed-field gel electrophoresis was performed to confirm chromosome numbers of the three sequenced strains. To pursue the mechanisms of drug resistance in L. prolificans, RNA sequencing was performed on cultures treated with itraconazole, voriconazole, or amphotericin B, and compared to untreated cultures. Also, the role of the cell wall in amphotericin B resistance was assessed by comparing the susceptibility of protoplasts and intact cells, then developing a drug activity assay to localize the cell wall component responsible for interfering with amphotericin B activity. Whole genome sequencing revealed that the L. prolificans genome is ~37 Mb with 11 chromosomes. One of three strains shows evidence of two different chromosomal translocations. BUSCO results reveal all three strains’ sequences include >95% of the single copy genes expected for its phylum. We believe that these genome sequences, one chromosoel-level and two near chromosome-level, will enable future molecular work in L. prolificans. RNA sequencing shows that itraconazole, voriconazole and amphotericin B alter expression of 16, 4 and 1876 genes, respectively. All of the genes affected by voriconazole, including azole target CYP51, are also regulated by itraconazole. Many potential mediators of amphotericin B resistance were identified, including RTA-like and beta lactamase-like proteins. Inquiry into the cell wall shows that L. prolificans protoplasts were much more susceptible to amphotericin B treatment than intact cells. A soluble cell wall component with NMR shifts consistent with a glycolipid was capable of eliminating amphotericin B activity