Candida albicans Genes Modulating Echinocandin Susceptibility of Caspofungin-Adapted Mutants Are Constitutively Expressed in Clinical Isolates with Intermediate or Full Resistance to Echinocandins.

The opportunistic fungus Candida albicans is the leading cause of invasive candidiasis in immune-compromised individuals. Drugs from the echinocandin (ECN) class, including caspofungin, are used as a first line of therapy against invasive candidiasis. The only known mechanism of clinical resistance to ECNs is point mutations in the FKS1 gene, which encodes the drug target. However, many clinical isolates developed decreased ECN susceptibilities in the absence of resistance-associated FKS1 mutations. We have identified 15 C. albicans genes that contribute to decreased drug susceptibility. We explored the expression of these 15 genes in clinical isolates with different levels of ECN susceptibility. We found that these 15 genes are expressed in clinical isolates with or without FKS1 mutations, including those strains that are less susceptible to ECNs. In addition, FKS1 expression was increased in such less susceptible isolates compared to highly susceptible isolates. Similarities of gene expression patterns between isolates with decreased ECN susceptibilities in the absence of FKS1 mutations and clinically resistant isolates with mutations in FKS1 suggest that clinical isolates with decreased ECN susceptibilities may be a precursor to development of resistance.

Identification of 10 genes on Candida albicans chromosome 5 that control surface exposure of the immunogenic cell wall epitope ß-glucan and cell wall remodeling in caspofungin-adapted mutants.

IMPORTANCE: Candida infections are often fatal in immuno-compromised individuals, resulting in many thousands of deaths per year. Caspofungin has proven to be an excellent anti-Candida drug and is now the frontline treatment for infections. However, as expected, the number of resistant cases is increasing; therefore, new treatment modalities are needed. We are determining metabolic pathways leading to decreased drug susceptibility in order to identify mechanisms facilitating evolution of clinical resistance. This study expands the understanding of genes that modulate drug susceptibility and reveals new targets for the development of novel antifungal drugs.

At least 10 genes on chromosome 5 of Candida albicans are downregulated in concert to control cell wall and to confer adaptation to caspofungin.

Candida albicans is part of normal microbiota, however, can cause superficial and life threatening infection in immune-compromised individuals. Drugs from echinocandin (ECN) class that disrupt cell wall synthesis, are being used as a major treatment strategy against candidiasis. As the use of ECNs for the treatment of candidiasis is increasing, resistance against ECNs is also emerging. Previously, we reported involvement of 5 chromosome 2 (Ch2) genes in adaptation to ECN drugs. Here, we explored 22 candidate-genes on Ch5 that are consistently downregulated in independent mutants adapted to caspofungin (CAS), for their role in ECN adaptation. We also compared cell wall remodelling in CAS-adapted mutants and in 10 knockouts (KOs) from Ch5. Independent KO experiments as combined with broth microdilution assay, demonstrated that, as expected, 10 out of 22 Ch5 genes decrease ECN susceptibility by controlling the levels of three major components of the cell wall, glucan, mannan, and chitin. Some KOs decreased glucan or increased chitin or both. Similar cell wall remodelling, decreased glucan and increased chitin, was found in CAS-adapted mutants with no ploidy change. Some other KOs had no glucan change, but increased the level of either mannan or chitin. Our results identify the function of two uncharacterized genes, orf19.970 and orf19.4149.1, and expand the functions of DUS4, RPS25B, UAP1, URA7, RPO26, HAS1 , and CKS1 . The function of CHT2 , as negative regulator of ECN susceptibility, has been previously established. Importantly, half of the above genes are essential indicating that essential processes are involved in cell wall remodelling for adaptation to ECNs. Also important, orf19.970 and orf19.4149.1 have no human orthologues. Finally, our work shows that multiple mechanisms are used by C. albicans cells to remodel cell wall in order to adapt to CAS. This work continues to identify common pathways that are involved in drug adaptation, as well as new genes controlling ECN susceptibility and reveals new targets for development of novel antifungal drugs.

Multiple Genes of Candida albicans Influencing Echinocandin Susceptibility in Caspofungin-Adapted Mutants.

Candida albicans is an opportunistic human fungal pathogen that causes invasive infections in immunocompromised individuals. Despite the high anticandidal activity among the echinocandins (ECNs), a first-line therapy, resistance remains an issue. Furthermore, many clinical isolates display decreased ECN susceptibility, a physiological state which is thought to lead to resistance. Determining the factors that can decrease susceptibility is of high importance. We searched for such factors genome-wide by comparing the transcriptional profiles of five mutants that acquired decreased caspofungin susceptibility in vitro in the absence of canonical FKS1 resistance mutations. The mutants were derived from two genetic backgrounds and arose due to independent mutational events, some with monosomic chromosome 5 (Ch5). We found that the mutants exhibit common transcriptional changes. In particular, all mutants upregulate five genes from Ch2 in concert. Knockout experiments show that all five genes positively influence caspofungin and anidulafungin susceptibility and play a role in regulating the cell wall mannan and glucan contents. The functions of three of these genes, orf19.1766, orf19.6867, and orf19.5833, were previously unknown, and our work expands the known functions of LEU42 and PR26. Importantly, orf19.1766 and LEU42 have no human orthologues. Our results provide important clues as to basic mechanisms of survival in the presence of ECNs while identifying new genes controlling ECN susceptibility and revealing new targets for the development of novel antifungal drugs.

Candida albicans Strains Adapted to Caspofungin Due to Aneuploidy Become Highly Tolerant under Continued Drug Pressure.

Candida albicans is a prevalent fungal pathogen of humans. Understanding the development of decreased susceptibility to ECN drugs of this microbe is of substantial interest, as it is viewed as an intermediate step allowing the formation of FKS1 resistance mutations. We used six previously characterized mutants that decreased caspofungin susceptibility either by acquiring aneuploidy of chromosome 5 (Ch5) or by aneuploidy-independent mechanisms. When we exposed these caspofungin-adapted mutants to caspofungin again, we obtained 60 evolved mutants with further decreases in caspofungin susceptibility, as determined with CLSI method. We show that the initial adaptation to caspofungin is coupled with the adaptation to other ECNs, such as micafungin and anidulafungin, in mutants with no ploidy change, but not in aneuploid mutants, which become more susceptible to micafungin and anidulafungin. Furthermore, we find that the initial mechanism of caspofungin adaptation determines the pattern of further adaptation as parentals with no ploidy change further adapt to all ECNs by relatively small decreases in susceptibility, whereas aneuploid parentals adapt to all ECNs, primarily by large decrease in susceptibilities. Our data suggest that either distinct or common mechanisms can govern adaptation to different ECNs.

The caspase-like Gpi8 subunit of Candida albicans GPI transamidase is a metal-dependent endopeptidase.

GPI anchored proteins (GPI-APs) act at the frontiers of cells, decoding environmental cues and determining host-pathogen interactions in several lower eukaryotes. They are also essential for viability in lower eukaryotes. The GPI biosynthetic pathway begins at the ER and follows a roughly linear pathway to generate the complete precursor (CP) glycolipid. The GPI transamidase (GPIT) transfers this glycolipid to the C-terminal end of newly translated proteins after removing their GPI attachment signal sequence (SS). The GPIT subunit that cleaves SS is Gpi8, a protein with a conserved Cys/His catalytic dyad typical of cysteine proteases. A CaGPI8 heterozygous mutant accumulates CPs and has reduced cell surface GPI-APs. Using a simple cell-free assay, we demonstrate that the heterozygous CaGPI8 strain has low endopeptidase activity as well. The revertant strain is restored in all these phenotypes. CaGpi8 is also shown to be a metalloenzyme, whose protease activity is sensitive to agents that modify Cys/His residues.

Saccharomyces cerevisiae Gpi2, an accessory subunit of the enzyme catalyzing the first step of glycosylphosphatidylinositol (GPI) anchor biosynthesis, selectively complements some of the functions of its homolog in Candida albicans.

GPI2 encodes for one of the six accessory subunits of the GPI-N-acetylglucosaminyltransferase (GPI-GnT) complex that catalyzes the first step of GPI biosynthesis in S. cerevisiae and C. albicans. It has been previously reported in S. cerevisiae that this subunit physically interacts with and negatively modulates Ras signaling. On the other hand, studies from our lab have shown that the homologous subunit in C. albicans is a positive modulator of Ras signaling. Are the functions of this subunit therefore strictly species dependent? We present here functional complementation studies on GPI2 from S. cerevisiae and C. albicans that were carried out to address this issue. Expression of CaGPI2 in a ScGPI2 conditional lethal mutant could not restore its growth defects. Likewise, ScGPI2 overexpression in a CaGPI2 heterozygous mutant could not restore its deficient GPI-GnT activity or reverse defects in its cell wall integrity and could only poorly restore filamentation. However, interestingly, ScGPI2 could restore lanosterol demethylase (CaERG11) levels and reverse azole resistance of the CaGPI2 heterozygote. It appeared to do this by regulating levels of another GPI-GnT subunit, CaGPI19, which we have previously shown to be involved in cross-talk with CaERG11. Thus, the effect of CaGPI2 on sterol biosynthesis in C. albicans is independent of its interaction with the GPI-GnT complex and Ras signaling pathways. In addition, the interaction of Gpi2 with other subunits of the GPI-GnT complex as well as with Ras signaling appears to have evolved differently in the two organisms.