A correlative study of the genomic underpinning of virulence traits and drug tolerance of Candida auris

Authors: Bo Yang, Benjamin Vaisvil, Daniel Schmitt, Joseph Collins, Eric Young, Vinayak Kapatral, Reeta Rao

Candida auris is an opportunistic fungal pathogen with high mortality rates which presents a clear threat to public health. The risk of C. auris infection is high because it can colonize the body, resist antifungal treatment, and evade the immune system. The genetic mechanisms for these traits are not well known. Identifying them could lead to new targets for new treatments. To this end, we present an analysis of the genetics and gene expression patterns of C. auris carbon metabolism, drug resistance, and macrophage interaction. We chose to study two C. auris isolates simultaneously, one drug sensitive (B11220 from Clade II) and one drug resistant (B11221 from Clade III). Comparing the genomes, we confirm the previously reported finding that B11220 was missing a 12.8 kb region on chromosome VI. This region contains a gene cluster encoding proteins related to alternative sugar utilization. We show that B11221, which has the gene cluster, readily assimilates and utilizes D-galactose and L-rhamnose as compared to B11220, which harbors the deletion. B11221 exhibits increased adherence and drug resistance compared to B11220 when grown in these sugars. Transcriptomic analysis of both isolates grown on glucose or galactose showed that the gene cluster was upregulated when grown on D-galactose. These findings reinforce growing evidence of a link between metabolism and drug tolerance. B11221 resists phagocytosis by macrophages and exhibits decreased β-1,3-glucan exposure, a key determinant that allows Candida to evade the host immune system, as compared to B11220. In a transcriptomic analysis of both isolates co-cultured with macrophages, we find upregulation of genes associated with transport and transcription factors in B11221. Our studies show a positive correlation between membrane composition and immune evasion, alternate sugar utilization, and drug tolerance in C. auris.

Transcriptomics elucidates metabolic regulation and functional promoters in the basidiomycete red yeast Xanthophyllomyces dendrorhous CBS 6938

Emma E. Tobin, Joseph H. Collins, Celeste B. Marsan, Gillian T. Nadeau, Kim Mori, Anna Lipzen, Stephen Mondo, Igor V. Grigoriev, Eric M. Young

Genomics has become the primary way to explore microbial diversity, because genetic tools are currently difficult to develop in non-model organisms. Here, we demonstrate that -omics can be leveraged to accelerate genetic tool development for the basidiomycete yeast Xanthophyllomyces dendrorhous CBS 6938, the sole biotechnologically relevant organism in the Tremellomycete family. First, we sequence the genome. Then, we perform transcriptomics under a variety of conditions, focusing on light and oxidative stress. This data not only reveals novel photobiology and metabolic regulation, it also allows derivation of constitutive and regulated gene expression parts. Our analysis of X. dendrorhous photobiology shows for the first time that a complex system of white-collar and cryptochrome homologs mediate response to ultraviolet light (UV). Our analysis of metabolic regulation shows that UV activates DNA repair, aromatic amino acid and carotenoid biosynthesis and represses central carbon metabolism and the fungal-like apoptotic pathway. Thus, X. dendrorhous shows a dynamic response toward biosynthetic pathways for light-absorbing compounds and survival and away from energy production. We then define a modular cloning system, including antibiotic selections, integration sites, and reporter genes, and use the transcriptomics to derive strong constitutive and regulated promoters. Notably, we discover a novel promoter from a hypothetical gene that has 9-fold activation upon UV exposure. Thus, -omics-to-parts workflows can simultaneously provide useful genomic data and advance genetic tools for non-model microbes, particularly those without a closely related model organism. This approach will be broadly useful in current efforts to engineer diverse microbes.

Differential Gene Expression of Ca2+ Channel-Mediated Signaling Pathways in DPSCs under Hypoxic Condition

JOHN T. TRAN
Dental pulp stem cell (DPSC) survival, differentiation into odontoblast and deposition of matrix for mineralization is essential for vital pulp therapy (VPT) and regenerative endodontic therapy (RET). Calcium homeostasis in DPSC plays a critical role for its survival, longevity, and differentiation processes. Cells use this external source of signal calcium ions by activating various entry channels with different properties. The objective of this research is to investigate key gene candidates involved with calcium homeostasis in human dental pulp tissue and DPSCs.

DPSCs isolated from human dental pulp were cultured in hypoxic chamber (3%) for 21 days at 37°C in differentiation media and controls were cultured under normal conditions. Later, DPSCs collected were extracted for mRNA isolation. The mRNA was sequenced using NextSeq Illunima and was analyzed using the ERGOTM transcription tool using statistical methods such as Limma (Linear Model) and DESeq2 (Negative Binomial).

There were a total of 24 biomarkers identified with a 6-fold higher over expressed in hypoxic conditions compared to normoxia. These include key genes such as CALM2, MYLK, FGR1, ASPH, PDGFRA, ATP2B4, ATP2B1, PDGFRB, CALM1, and PPP3CA. The genes and pathways were identified in KEGG pathways overlaying expression values.

The release of calcium ions from internal stores and the influx of calcium ions from the environment cause a dramatic and rapid increase in cytoplasmic calcium concentration, which has been exploited for signal transduction. In combination with power statistical analysis such as DESeq2 and Limma, key pathways can be identified that are responsible for calcium homeostasis and calcium channel signaling as key diagnostic markers for VPT and RET.

Investigation of Apoptotic Biomarkers in Dental Pulp Stem Cells under Hypoxic Condition using RNA-Seq

Philip William Cannizzaro

Immature teeth can have halted root development through pathology associated with caries, trauma, or dental anomalies such as dens evaginatus. Pulp tissue under stress induces ischemia and subsequent necrosis. Through regenerative endodontic procedures, proper procedural protocol often leads to revitalization following blood clot formation. The aim of this study is focused on understanding the roles of caspase proteins within the inflammasome reaction leading to pulp necrosis by evaluating expression profile levels through RNA sequencing technology. Human dental pulp tissue was extracted from healthy adult third molars extracted prophylactically and healthy adult premolars extracted for orthodontics collected with IRB exemption. Tissue samples purified for mRNA allows expression analysis evaluation. DESeq2 and Limma analysis promoted statistical analysis of expression profile to determine whether or not significant differences in transcription and likely subsequent translation between diseased and healthy tissue. The study identified 27 genes above six-fold higher expression under hypoxia conditions, these markers include the following: TUBA1B, ACTB, NFKB, CTSD, CTSB, MCL1, JUN, TUBA1C, and others.