Nicole Fewings

Fungal Inositol Pyrophosphate IP7 Is Crucial for Metabolic Adaptation to the Host Environment and Pathogenicity

ABSTRACT Inositol pyrophosphates (PP-IPs) comprising inositol, phosphate, and pyrophosphate (PP) are essential for multiple functions in eukaryotes. Their role in fungal pathogens has never been addressed. Cryptococcus neoformans is a model pathogenic fungus causing life-threatening meningoencephalitis. We investigate the cryptococcal kinases responsible for the production of PP-IPs (IP7/IP8) and the hierarchy of PP-IP importance in pathogenicity. Using gene deletion and inositol polyphosphate profiling, we identified Kcs1 as the major IP6 kinase (producing IP7) and Asp1 as an IP7 kinase (producing IP8). We show that Kcs1-derived IP7 is the most crucial PP-IP for cryptococcal drug susceptibility and the production of virulence determinants. In particular, Kcs1 kinase activity is essential for cryptococcal infection of mouse lungs, as reduced fungal burdens were observed in the absence of Kcs1 or when Kcs1 was catalytically inactive. Transcriptome and carbon source utilization analysis suggested that compromised growth of the KCS1 deletion strain (Δkcs1 mutant) in the low-glucose environment of the host lung is due to its inability to utilize alternative carbon sources. Despite this metabolic defect, the Δkcs1 mutant established persistent, low-level asymptomatic pulmonary infection but failed to elicit a strong immune response in vivo and in vitro and was not readily phagocytosed by primary or immortalized monocytes. Reduced recognition of the Δkcs1 cells by monocytes correlated with reduced exposure of mannoproteins on the Δkcs1 mutant cell surface. We conclude that IP7 is essential for fungal metabolic adaptation to the host environment, immune recognition, and pathogenicity. IMPORTANCE Cryptococcus neoformans is responsible for 1 million cases of AIDS-associated meningitis and ~600,000 deaths annually. Understanding cellular pathways responsible for pathogenicity might have an impact on new drug development. We characterized the inositol polyphosphate kinases Kcs1 and Asp1, which are predicted to catalyze the production of inositol pyrophosphates containing one or two diphosphate moieties (PP-IPs). Using gene deletion analysis and inositol polyphosphate profiling, we confirmed that Kcs1 and Asp1 are major IP6 and IP7 kinases, respectively. Kcs1-derived IP7, but not Asp1-derived IP8, is crucial for pathogenicity. Global expression profiling and carbon source utilization testing suggest that IP7-deficient cryptococci cannot adapt their metabolism to allow growth in the glucose-poor environment of the host lung, and consequently, fungal burdens are significantly reduced. Persistent asymptomatic Δkcs1 mutant infection correlated with decreased mannoprotein exposure on the Δkcs1 mutant surface and reduced phagocytosis. We conclude that IP7 is crucial for the metabolic adaptation of C. neoformans to the host environment and for pathogenicity. Cryptococcus neoformans is responsible for 1 million cases of AIDS-associated meningitis and ~600,000 deaths annually. Understanding cellular pathways responsible for pathogenicity might have an impact on new drug development. We characterized the inositol polyphosphate kinases Kcs1 and Asp1, which are predicted to catalyze the production of inositol pyrophosphates containing one or two diphosphate moieties (PP-IPs). Using gene deletion analysis and inositol polyphosphate profiling, we confirmed that Kcs1 and Asp1 are major IP6 and IP7 kinases, respectively. Kcs1-derived IP7, but not Asp1-derived IP8, is crucial for pathogenicity. Global expression profiling and carbon source utilization testing suggest that IP7-deficient cryptococci cannot adapt their metabolism to allow growth in the glucose-poor environment of the host lung, and consequently, fungal burdens are significantly reduced. Persistent asymptomatic Δkcs1 mutant infection correlated with decreased mannoprotein exposure on the Δkcs1 mutant surface and reduced phagocytosis. We conclude that IP7 is crucial for the metabolic adaptation of C. neoformans to the host environment and for pathogenicity.

The low EOMES/TBX21 molecular phenotype in multiple sclerosis reflects CD56+ cell dysregulation and is affected by immunomodulatory therapies

Multiple Sclerosis (MS) is an autoimmune disease treated by therapies targeting peripheral blood cells. We previously identified that expression of two MS-risk genes, the transcription factors EOMES and TBX21 (ET), was low in blood from MS and stable over time. Here we replicated the low ET expression in a new MS cohort (p<0.0007 for EOMES, p<0.028 for TBX21) and demonstrate longitudinal stability (p<10(-4)) and high heritability (h(2)=0.48 for EOMES) for this molecular phenotype. Genes whose expression correlated with ET, especially those controlling cell migration, further defined the phenotype. CD56+ cells and other subsets expressed lower levels of Eomes or T-bet protein and/or were under-represented in MS. EOMES and TBX21 risk SNP genotypes, and serum EBNA-1 titres were not correlated with ET expression, but HLA-DRB1*1501 genotype was. ET expression was normalised to healthy control levels with natalizumab, and was highly variable for glatiramer acetate, fingolimod, interferon-beta, dimethyl fumarate.

Data characterizing the ZMIZ1 molecular phenotype of multiple sclerosis

The data presented in this article are related to the research article entitled “The autoimmune risk gene ZMIZ1 is a vitamin D responsived marker of a molecular phenotype of multiple sclerosis” Fewings et al. (2017) [1]. Here we identify the set of genes correlated with ZMIZ1 in multiple cohorts, provide phenotypic details on those cohorts, and identify the genes negatively correlated with ZMIZ1 and the cells predominantly expressing those genes. We identify the metabolic pathways in which the molecular phenotype genes are over-represented. Finally, we present the flow cytometry gating strategy we have used to identify the immune cells from blood which are producing ZMIZ1 and RPS6.

Differences in common heritable blood immune cell populations may underlie MS susceptibility and progression

A promising new avenue of MS research that may lead to a better understanding of pathogenesis, progression and therapeutic response, and to development of new therapies, comes from the recent identification of defined immune cell populations that are highly heritable. Such stable populations have been identified in three recent papers using extensive flow cytometric panels to investigate twin and family cohorts. They showed that while most of the variation in immune cell populations between individuals was not heritable, some was. This heritability was sometimes very high, and the authors concluded that it likely contributes to variability in response among individuals for disease and drug response traits.

Genetic evidence that the latency III stage of Epstein-Barr Virus infection is a therapeutic target for Multiple Sclerosis

Genome wide association studies have identified >200 susceptibility loci accounting for much of the heritability of Multiple Sclerosis (MS). Epstein Barr virus (EBV), a memory B cell tropic virus, has been identified as necessary but not sufficient for development of MS, with evidence for disease causation. The molecular and immunological basis for this has not been established. LCL proliferation is driven by signalling through the EBV produced cell surface protein LMP1, a homologue of the MS risk gene CD40. We show that the CD40 ligand, CD40L, potentially through competitive signalling with LMP1, reduces LCL proliferation (p<0.001). The MS risk variants of the LMP1 signalling inhibitor, TRAF3, had altered expression in B cells and LCLs. Both CD40 and TRAF3 risk SNPs are in binding sites for the EBV transcription factor EBNA2. We have investigated transcriptomes of B cells and EBV infected B cells at Latency III (LCLs) and identified 47 MS risk genes with altered expression, associated with the risk genotype. Overall these MS risk SNPs were overrepresented in target loci of the EBV transcription factor EBNA2 (p<10−16), in genes dysregulated between B and LCLs (p<10−5), and as targets for EBV miRNAs (p<10−4). The risk gene ZC3HAV1 is the putative target for multiple EBV miRNAs. It amplifies the interferon response, and was shown to have reduced expression in LCLs for the risk allele. These data indicate targeting EBV EBNA2, miRNAs, and MS risk genes on the LMP1/LMP2 pathways, and the pathways themselves, may be of therapeutic benefit in MS.

Distribution of monocyte subsets and polarization in preeclampsia and intrauterine fetal growth restriction

Monocytes are likely to play a significant role in the pathogenesis of preeclampsia (PE) and intrauterine fetal growth restriction (IUGR), given their role in homeostasis and tissue repair. Our aim was to study the gestational changes in monocytes in normal pregnancy and to determine whether monocyte subsets and phenotype are altered in pregnancy complications, such as PE and IUGR.

The latitude-dependent autoimmune disease risk genes ZMIZ1 and IRF8 regulate mononuclear phagocytic cell differentiation in response to vitamin D

Epidemiological, molecular and genetic studies have indicated that high serum vitamin D levels are associated with lower risk of several autoimmune diseases. The vitamin D receptor (VDR) binding sites in monocytes and dendritic cells (DCs) are more common in risk genes for diseases with latitude dependence than in risk genes for other diseases. The transcription factor genes Zinc finger MIZ domain-containing protein 1 (ZMIZ1) and interferon regulatory factor 8 (IRF8)-risk genes for many of these diseases-have VDR binding peaks co-incident with the risk single nucleotide polymorphisms (SNPs). We show these genes are responsive to vitamin D: ZMIZ1 expression increased and IRF8 expression decreased, and this response was affected by genotype in different cell subsets. The IL10/IL12 ratio in tolerogenic DCs increased with vitamin D. These data indicate that vitamin D regulation of ZMIZ1 and IRF8 in DCs and monocytes contribute to latitude-dependent autoimmune disease risk.

Characterising the Molecular Phenotypes of MS: heredity, gene expression modules, dysregulated immune cell subsets and response to therapy

Background: Current MS therapies target the systemic circulation. We previously identified that genes encoding transcription factors were over-represented amongst MS risk factors, and that expression of several of these were altered in MS blood, including EOMES, TBX21 and ZMIZ1. Expression was stable over time, so that it defines a molecular phenotype. Objectives: Here we tested if this finding could be replicated in independent cohorts, if expression was heritable, longitudinally stable, affected by therapy, and associated with genetic and environmental risk factors. We sought the immune cell subsets expressing these transcription factors, and tested if protein expression was also altered. Methods: Whole blood mRNA expression was determined in new cohorts of untreated MS (n = 23, Sydney; n = 47, Perth) and healthy controls (n = 23) and protein expression determined by flow cytometry in PBMCs of untreated MS and healthy controls (n = 28, 30 respectively). Effect of major therapies and correlation of gene expression with risk SNP genotypes, and anti-Epstein Barr Virus EBNA-1 titres was assessed, and heritability tested in a large twin cohort. Results: MS Molecular Phenotypes were replicated in new cohorts. Modules of genes, whose expression correlated with EOMES/TBX21 or ZMIZ1, further defined the phenotypes. CD56+ cells, inflammatory monocytes and plasmacytoid dendritic cells expressed lower levels of Molecular Phenotype transcription factors. EOMES and TBX21 risk SNP genotypes, and serum EBNA-1 titres were not correlated with gene expression, but HLA-DRB1*1501 genotype was. Therapies altered expression levels (e.g. TBX21 returned to control levels on natalizumab) with significant variability between individuals. Conclusions: EOMES/TBX21 and ZMIZ1 tag molecular phenotypes of MS that are affected by therapy. The phenotypes are due to under-representation and altered state of CD56+ and inflammatory monocytes/pDCs respectively. The dysregulated immune cells are a novel target for therapy and, together with gene expression, represent potential biomarkers of therapeutic response.Background: Population based genome wide association studies have identified 110 single nucleotide polymorphisms (SNPs) that are associated with an increase in MS risk. These SNPs are all common, and have odds ratios of between 1.1 and 1.4. Most are found in non-protein coding regions, and their functions are largely unknown. Objectives: Importantly, recent work has shown that some non-coding SNPs can function by changing immune gene expression levels as a quantitative trait, termed expression quantitative trait loci (eQTL). We conducted studies to evaluate the effects of MS risk SNPs on gene expression in four main immune cell types. Methods: We isolated monocytes, B-cells, CD4- and CD8- T-cells from untreated relapsing MS cases (n=79) and healthy controls (n=101). To test for cis-eQTL associations, we selected all genes within +/-500kb of an MS risk SNP (2500 pairs in total). The Illumina Immunochip was used to genotype for MS risk SNPs, and gene expression was measured for each cell type by microarray. Results: We have identified MS risk eQTL associations in each immune cell type, some of which are cell type specific. We also present preliminary data showing that some MS risk SNPs could exert differential effects on gene expression in cases compared to controls. Here we report likely disease state specific eQTLs for all cell types with the top associations being: RNF26/rs9736016, B cells; MACROD1/rs694739, CD8 cells; SLC25A41/rs1077667, CD4 cells; GPR18/rs4772201, monocyte cells. Conclusions: We have shown that MS risk SNPs contribute to immune heterogeneity. It is hoped that through an understanding of the functions of individual common risk variants, it may be possible to uncover the processes and cell types that are most important for conveying the genetic risk of MS.