| Summary: | In this study, we have specifically blocked a key step of sphingolipid (SL) biosynthesis in <i>Candida glabrata</i> by disruption of the orthologs of ScIpt1 and ScSkn1. Based on their close homology with <i>S. cerevisiae</i> counterparts, the proteins are predicted to catalyze the addition of a phosphorylinositol group onto mannosyl inositolphosphoryl ceramide (MIPC) to form mannosyl diinositolphosphoryl ceramide (M(IP)<sub>2</sub>C), which accounts for the majority of complex SL structures in <i>S. cerevisiae</i> membranes. High throughput lipidome analysis confirmed the accumulation of MIPC structures in <i>ΔCgipt1</i> and <i>ΔCgskn1</i> cells, albeit to lesser extent in the latter. Noticeably, <i>ΔCgipt1</i> cells showed an increased susceptibility to azoles; however, <i>ΔCgskn1</i> cells showed no significant changes in the drug susceptibility profiles. Interestingly, the azole susceptible phenotype of <i>ΔCgipt1</i> cells seems to be independent of the ergosterol content. <i>ΔCgipt1</i> cells displayed altered lipid homeostasis, increased membrane fluidity as well as high diffusion of radiolabeled fluconazole (<sup>3</sup>H-FLC), which could together influence the azole susceptibility of <i>C. glabrata</i>. Furthermore, in vivo experiments also confirmed compromised virulence of the <i>ΔCgipt1</i> strain. Contrarily, specific functions of CgSkn1 remain unclear.
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