Candida auris is an enigmatic yeast that provides substantial global risk in health care facilities and intensive care units. A unique phenotype exhibited by certain isolates of C. auris is their ability to form small clusters of cells known as aggregates, which have been to a limited extent described in the context of pathogenic traits. In this study, we screened several nonaggregative and aggregative C. auris isolates for biofilm formation, where we observed a level of heterogeneity among the different phenotypes. Next, we utilized an RNA sequencing approach to investigate the transcriptional responses during biofilm formation of a nonaggregative and aggregative isolate of the initial pool. Observations from these analyses indicate unique transcriptional profiles in the two isolates, with several genes identified relating to proteins involved in adhesion and invasion of the host in other fungal species. From these findings, we investigated for the first time the fungal recognition and inflammatory responses of a three-dimensional skin epithelial model to these isolates. In these models, a wound was induced to mimic a portal of entry for C. auris. We show that both phenotypes elicited minimal response in the model minus induction of the wound, yet in the wounded tissue, both phenotypes induced a greater response, with the aggregative isolate more proinflammatory. This capacity of aggregative C. auris biofilms to generate such responses in the wounded skin highlights how this opportunistic yeast is a high risk within the intensive care environment where susceptible patients have multiple indwelling lines. IMPORTANCE Candida auris has recently emerged as an important cause of concern within health care environments due to its ability to persist and tolerate commonly used antiseptics and disinfectants, particularly when attached to a surface (biofilms). This yeast is able to colonize and subsequently infect patients, particularly those that are critically ill or immunosuppressed, which may result in death. We have undertaken analysis on two different phenotypic types of this yeast, using molecular and immunological tools to determine whether either of these has a greater ability to cause serious infections. We describe that both isolates exhibit largely different transcriptional profiles during biofilm development. Finally, we show that the inability to form small aggregates (or clusters) of cells has an adverse effect on the organism’s immunostimulatory properties, suggesting that the nonaggregative phenotype may exhibit a certain level of immune evasion.

The gingival epithelium is a physical and immunological barrier to the microbiota of the oral cavity, which interact through soluble mediators with the immune cells that patrol the tissue at the gingival epithelium. We sought to develop a three-dimensional gingivae-biofilm interface model using a commercially available gingival epithelium to study the tissue inflammatory response to oral biofilms associated with “health”, “gingivitis” and “periodontitis”. These biofilms were developed by sequential addition of microorganisms to mimic the formation of supra- and sub-gingival plaque in vivo. Secondly, to mimic the interactions between gingival epithelium and immune cells in vivo, we integrated peripheral blood mononuclear cells and CD14+ monocytes into our three-dimensional model and were able to assess the inflammatory response in the immune cells cultured with and without gingival epithelium. We describe a differential inflammatory response in immune cells cultured with epithelial tissue, and more so following incubation with epithelium stimulated by “gingivitis-associated” biofilm. These results suggest that gingival epithelium-derived soluble mediators may control the inflammatory status of immune cells in vitro, and therefore targeting of the epithelial response may offer novel therapies. This multi-cellular interface model, both of microbial and host origin, offers a robust in vitro platform to investigate host-pathogens at the epithelial surface.

1Candida auris is an enigmatic yeast that provides substantial global risk in 2 healthcare facilities and intensive care units. A unique phenotype exhibited by 3 certain isolates of C. auris is their ability to form small clusters of cells known as 4 aggregates, which have been to a limited extent described in the context of 5 pathogenic traits. In this study, we screened several non-aggregative and 6 aggregative C. auris isolates for biofilm formation, where we observed a level of 7 heterogeneity amongst the different phenotypes. Next, we utilised an RNA-8 sequencing approach to investigate the transcriptional responses during biofilm 9 formation of a non-aggregative and aggregative isolate of the initial pool. 10 Observations from these analyses indicate unique transcriptional profiles in the two 11 isolates, with several genes identified relating to proteins involved in adhesion and 12 invasion of the host in other fungal species. From these findings we investigated for 13 the first time the fungal recognition and inflammatory responses of a three-14 dimensional skin epithelial model to these isolates. In these models, a wound was 15 induced to mimic a portal of entry for C. auris. We show both phenotypes elicited 16 minimal response in the model minus induction of the wound, yet in the wounded 17 tissue both phenotypes induced a greater response, with the aggregative isolate 18 more pro-inflammatory. This capacity of aggregative C. auris biofilms to generate 19 such responses in the wounded skin highlights how this opportunistic yeast is a high 20 risk within the intensive care environment where susceptible patients have multiple 21 indwelling lines.22 23

Leukemias containing Mixed Lineage Leukemiagene rearrangement (MLL-r) account for 5-10% of human acute leukemia cases and are associated with a poor prognosis. The unmet clinical need and the lack of an effective targeted therapy emphasizes the need for novel approaches for these malignancies.Recent studies have discovered an essential role for the histone H3 lysine 79 (H3K79) methyltransferase DOT1L in the maintenance of MLL-r leukemias. Phase-I clinical trials (NCT01684150 and NCT02141828) have demonstrated the safety and clinical activity of the DOT1L-specific small molecule inhibitor EPZ5676 (Pinometostat, Epizyme Inc.). However, the variable response of the MLL-r patients in these clinical trials indicates the need to further understand the mechanism of action and resistance to DOT1L inhibitors. To address this issue, we designed a high-density CRISPR screen approach (Fig 1) to dissect the function of Dot1l coding regions in mouse MLL-AF9 leukemic cells, a well-established mouse model that mimics human MLL-r acute myeloid leukemia (AML). We constructed a library of 602 sgRNA that targets most of the "NGG" protospacer adjacent motifs (PAM) within Dot1l exons. The average targeting density is 7.7 bp per sgRNA (i.e. 2.5 a.a. per sgRNA). We delivered this sgRNA library into Cas9-expressing MLL-AF9 AML cells using lentiviral transduction, and then compared the frequencies of each integrated sgRNA sequence before vs. after 12 days of culture using high-throughput sequencing (NextSeq, Illumina Inc.). The results revealed a significant depletion of clusters of sgRNA targeting the known functional regions including the lysine methyltransferase (KMT) core and the AF9-binding domains of DOT1L. We mapped the CRISPR scan score to the previously solved three-dimensional structures of DOT1L and found the high-density CRISPR scan clearly distinguishes the substrate binding pocket from the less important regions within the KMT domain. Furthermore, we identified CRISPR hotspots that coincide with amino acid residues directly contacting the enzymatic substrate S-adenosylmethionine (SAM). Similarly, CRISPR scan identifies amino acid hot spots in DOT1L that establish a direct interaction with the cofactor AF9. These results implicate the utility of the high-density CRISPR scan to pinpoint the functional elements within a protein to a sub-domain resolution. To identify regions in DOT1L that mediate response of MLL-r leukemia to DOT1L-inhibitors, we conducted parallel CRISPR scans by culturing the Dot1l sgRNA library transduced MLL-AF9-Cas9+cells in either DMSO (vehicle) or the DOT1L inhibitor (1 uM EPZ5676) for 12 days. These paired screens revealed distinct patterns of the CRISPR scan scores between the two culture conditions. Surprisingly, a cluster of 31 sgRNA targeting the amino acid residues T560 - S661 of DOT1L was significantly enriched only in the presence of the DOT1L inhibitor.Expression of individual sgRNA targeting this region confirmed the EPZ5676-resistant phenotype observed in the CRISPR scans. Computational modeling of the structure of DOT1L suggests this newly identified region overlaps with two alpha-helixes motifs in a predicted coiled-coil domain. Finally, we examined eight clinically observed DOT1L missense mutations in this region (cBioPortal database; 54,510 patients) and foundthree DOT1L-mutant constructs (A591V, G594S and L626P) when expressed in the MLL-AF9 leukemia cells can confer resistance to EPZ5676. These findings suggest the utility of the high-density CRISPR scan for de novoidentification of drug-resistant mutant alleles in the human population, which maydirect future clinical decisions and benefit patients with specific genomic backgrounds. Disclosures No relevant conflicts of interest to declare.