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.

IFNγ regulates NAD+ metabolism to promote the respiratory burst in human monocytes

Katelyn J. McCann, Stephen M. Christensen, Devon H. Colby, Peter J. McGuire, Ian A. Myles, Christa S. Zerbe, Clifton L. Dalgard, Gauthaman Sukumar, Warren J. Leonard, Beth A. McCormick, Steven M. Holland

Interferon γ (IFNγ) is an essential and pleiotropic activator of human monocytes, but little is known about the changes in cellular metabolism required for IFNγ-induced activation. We sought to elucidate the mechanisms by which IFNγ reprograms monocyte metabolism to support its immunologic activities. We found that IFNγ increased oxygen consumption rates (OCR) in monocytes, indicative of reactive oxygen species generation by both mitochondria and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Transcriptional profiling revealed that this oxidative phenotype was driven by IFNγ-induced reprogramming of NAD+ metabolism, which is dependent on nicotinamide phosphoribosyltransferase (NAMPT)-mediated NAD+ salvage to generate NADH and NADPH for oxidation by mitochondrial complex I and NADPH oxidase, respectively. Consistent with this pathway, monocytes from patients with gain-of-function mutations in STAT1 demonstrated higher-than-normal OCR, whereas chemical or genetic disruption of mitochondrial complex I (rotenone treatment or Leigh syndrome patient monocytes) or NADPH oxidase (diphenyleneiodonium treatment or chronic granulomatous disease [CGD] patient monocytes) reduced OCR. Interestingly, inhibition of NAMPT in healthy monocytes completely abrogated the IFNγ-induced oxygen consumption, comparable to levels observed in CGD monocytes. These data identify an IFNγ-induced, NAMPT-dependent, NAD+ salvage pathway that is critical for IFNγ activation of human monocytes.

Safety evaluation of Fy Protein™ (Nutritional Fungi Protein), a macroingredient for human consumption

Brian Furey, Kathleen Slingerland, Mark R. Bauter, Celeste Dunn, Richard E. Goodman, Sophia Koo

Abstract

Fy Protein™ (Nutritional Fungi Protein) is a macro-ingredient produced from the fermentation of the fungal microorganism Fusarium strain flavolapis, isolated from springs in Yellowstone National Park. Fy Protein contains all of the essential amino acids plus fiber, fat, carbohydrates, vitamins, and minerals and is developed as an alternative to animal-based protein foods such as meat and dairy.

Fy Protein's nutritional, digestibility, genotoxicity, allergenicity, toxicity, secondary metabolites, and pathogenicity were evaluated. Fy Protein did not show mutagenic or genotoxic potential in in vitro tests. In an allergenicity review, Fy Protein was found to be of low allergenic potential. In a 90-day sub chronic dietary study in rats, administration of Fy Protein did not produce any significant toxicologic manifestations, and the No Observed Adverse Effect Level (NOAEL) was the highest-level fed of 150,000 ppm (15% in the diet). Regulated secondary metabolites from fungi (termed mycotoxins) were non-detectable and below regulated levels using quantitative analytical techniques. A literature review was completed to identify the potential human pathogenicity of Fusarium sp., showing that Fusarium rarely infects humans, with infections seldom developing even in immunocompromised individuals.

The results of these studies confirm that Fy Protein from fermented F. str. flavolapis has low toxicological, genotoxic, pathogenic, and allergenic potential under the conditions tested and anticipated use.

New genomic resources and comparative analyses reveal differences in floral gene expression in selfing and outcrossing Collinsia sister species

Lauren J. Frazee, Joanna Rifkin 2 Dinusha C. Maheepala, Alannie-Grace Grant, Stephen Wright , Susan Kalisz, Amy Litt, and Rachel Spigler

The evolutionary transition from outcross- to self-fertilization is one of the most common in angiosperms and is often associated with a parallel shift in floral morphological and developmental traits, such as reduced flower size and pollen to ovule ratios, known as the “selfing syndrome.” How these convergent phenotypes arise, the extent to which they are shaped by selection, and the nature of their underlying genetic basis are unsettled questions in evolutionary biology. The genus Collinsia (Plantaginaceae) includes seven independent transitions from outcrossing or mixed mating to high selfing rates accompanied by selfing syndrome traits. Accordingly, Collinsia represents an ideal system for investigating this parallelism, but requires genomic resource development. We present a high quality de novo genome assembly for the highly selfing species Collinsia rattanii. To begin addressing the basis of selfing syndrome developmental shifts, we evaluate and contrast patterns of gene expression from floral transcriptomes across three stages of bud development for C. rattanii and its outcrossing sister species Collinsia linearis. Relative to C. linearis, total gene expression is less variable among individuals and bud stages in C. rattanii. In addition, there is a common pattern among differentially expressed genes: lower expression levels that are more constant across bud development in C. rattanii relative to C. linearis. Transcriptional regulation of enzymes involved in pollen formation specifically in early bud development may influence floral traits that distinguish selfing and outcrossing Collinsia species through pleiotropic functions. Future work will include additional Collinsia outcrossing-selfing species pairs to identify genomic signatures of parallel evolution. Keywords: Collinsia; RNA-seq; selfing syndrome; pollen; floral development; differential gene expression; DESeq2; dichogamy; evolutionary genomics; Hi-C scaffolding; parallel evolution

DAF-16 and SMK-1 Contribute to Innate Immunity During Adulthood in Caenorhabditis elegans

Daniel R. McHugh, Elena Koumis, Paul Jacob, Jennifer Goldfarb, Michelle Schlaubitz-Garcia, Safae Bennani, Paul Regan, Prem Patel, and Matthew J. Youngman

Aging is accompanied by a progressive decline in immune function termed “immunosenescence”. Deficient surveillance coupled with the impaired function of immune cells compromises host defense in older animals. The dynamic activity of regulatory modules that control immunity appears to underlie agedependent modifications to the immune system. In the roundworm Caenorhabditis elegans levels of PMK-1 p38 MAP kinase diminish over time, reducing the expression of immune effectors that clear bacterial pathogens. Along with the PMK-1 pathway, innate immunity in C. elegans is regulated by the insulin signaling pathway. Here we asked whether DAF-16, a Forkhead box (FOXO) transcription factor whose activity is inhibited by insulin signaling, plays a role in host defense later in life. While in younger C. elegans DAF-16 is inactive unless stimulated by environmental insults, we found that even in the absence of acute stress the transcriptional activity of DAF-16 increases in an age-dependent manner. Beginning in the reproductive phase of adulthood, DAF-16 upregulates a subset of its transcriptional targets, including genes required to kill ingested microbes. Accordingly, DAF-16 has little to no role in larval immunity, but functions specifically during adulthood to confer resistance to bacterial pathogens. We found that DAF-16-mediated immunity in adults requires SMK-1, a regulatory subunit of the PP4 protein phosphatase complex. Our data suggest that as the function of one branch of the innate immune system of C. elegans (PMK-1) declines over time, DAF-16- mediated immunity ramps up to become the predominant means of protecting adults from infection, thus reconfiguring immunity later in life.

Transcriptional Profiling the 150 kb Linear Megaplasmid of Borrelia turicatae Suggests a Role in Vector Colonization and Initiating Mammalian Infection

Hannah K. Wilder, Sandra J. Raffel, Alan G. Barbour, Stephen F. Porcella, Daniel E. Sturdevant, Benjamin Vaisvil, Vinayak Kapatral, Daniel P. Schmitt, Tom G. Schwan, Job E. Lopez

Adaptation is key for survival as vector-borne pathogens transmit between the arthropod and vertebrate, and temperature change is an environmental signal inducing alterations in gene expression of tick-borne spirochetes. While plasmids are often associated with adaptation, complex genomes of relapsing fever spirochetes have hindered progress in understanding the mechanisms of vector colonization and transmission. We utilized recent advances in genome sequencing to generate the most complete version of the Borrelia turicatae 150 kb linear megaplasmid (lp150). Additionally, a transcriptional analysis of open reading frames (ORFs) in lp150 was conducted and identified regions that were up-regulated during in vitro cultivation at tick-like growth temperatures (22°C), relative to bacteria grown at 35°C and infected murine blood. Evaluation of the 3’ end of lp150 identified a cluster of ORFs that code for putative surface lipoproteins. With a microbe’s surface proteome serving important roles in pathogenesis, we confirmed the ORFs expression in vitro and in the tick compared to spirochetes infecting murine blood. Transcriptional evaluation of lp150 indicates the plasmid likely has essential roles in vector colonization and/or initiating mammalian infection. These results also provide a much needed transcriptional framework to delineate the molecular mechanisms utilized by relapsing fever spirochetes during their enzootic cycle.

Published: February 4, 2016 DOI: 10.1371/journal.pone.0147707