Inflammatory Protein Profiles in Plasma of Candidaemia Patients and the Contribution of Host Genetics to Their Variability.

Circulatory inflammatory proteins play a significant role in anti-Candida host immune defence. However, little is known about the genetic variation that contributes to the variability of inflammatory responses in response to C. albicans. To systematically characterize inflammatory responses in Candida infection, we profiled 91 circulatory inflammatory proteins in peripheral blood mononuclear cells (PBMCs) stimulated with C. albicans yeast isolated from 378 individuals of European origin from the 500 Functional Genomics (500FG) cohort of the Human Functional Genomics Project (HFGP) and Lifelines Deep cohort. To identify the genetic factors that determine variation in inflammatory protein responses, we correlated genome-wide single nucleotide polymorphism (SNP) genotypes with protein abundance (protein quantitative trait loci, pQTLs) produced by the Candida-stimulated PBMCs. Furthermore, we investigated whether differences in survival of candidaemia patients can be explained by modulating levels of inflammatory proteins. We identified five genome-wide significant pQTLs that modulate IL-8, MCP-2, MMP-1, and CCL3 in response to C. albicans. In addition, our genetic analysis suggested that GADD45G from rs10114707 locus that reached genome-wide significance could be a potential core gene that regulates a cytokine network upon Candida infection. Last but not least, we observed that a trans-pQTL marked from SNP rs7651677 at chromosome 3 that influences urokinase plasminogen activator (uPA) is strongly associated with patient survival (Psurvival = 3.52 x 10-5, OR 3). Overall, our genetic analysis showed that genetic variation determines the abundance of circulatory proteins in response to Candida infection.

Integrating GWAS with bulk and single-cell RNA-sequencing reveals a role for LY86 in the anti-Candida host response.

Candida bloodstream infection, i.e. candidemia, is the most frequently encountered life-threatening fungal infection worldwide, with mortality rates up to almost 50%. In the majority of candidemia cases, Candida albicans is responsible. Worryingly, a global increase in the number of patients who are susceptible to infection (e.g. immunocompromised patients), has led to a rise in the incidence of candidemia in the last few decades. Therefore, a better understanding of the anti-Candida host response is essential to overcome this poor prognosis and to lower disease incidence. Here, we integrated genome-wide association studies with bulk and single-cell transcriptomic analyses of immune cells stimulated with Candida albicans to further our understanding of the anti-Candida host response. We show that differential expression analysis upon Candida stimulation in single-cell expression data can reveal the important cell types involved in the host response against Candida. This confirmed the known major role of monocytes, but more interestingly, also uncovered an important role for NK cells. Moreover, combining the power of bulk RNA-seq with the high resolution of single-cell RNA-seq data led to the identification of 27 Candida-response QTLs and revealed the cell types potentially involved herein. Integration of these response QTLs with a GWAS on candidemia susceptibility uncovered a potential new role for LY86 in candidemia susceptibility. Finally, experimental follow-up confirmed that LY86 knockdown results in reduced monocyte migration towards the chemokine MCP-1, thereby implying that this reduced migration may underlie the increased susceptibility to candidemia. Altogether, our integrative systems genetics approach identifies previously unknown mechanisms underlying the immune response to Candida infection.

A Genome-Wide Functional Genomics Approach Identifies Susceptibility Pathways to Fungal Bloodstream Infection in Humans.

BACKGROUND: Candidemia, one of the most common causes of fungal bloodstream infection, leads to mortality rates up to 40% in affected patients. Understanding genetic mechanisms for differential susceptibility to candidemia may aid in designing host-directed therapies. METHODS: We performed the first genome-wide association study on candidemia, and we integrated these data with variants that affect cytokines in different cellular systems stimulated with Candida albicans. RESULTS: We observed strong association between candidemia and a variant, rs8028958, that significantly affects the expression levels of PLA2G4B in blood. We found that up to 35% of the susceptibility loci affect in vitro cytokine production in response to Candida. Furthermore, potential causal genes located within these loci are enriched for lipid and arachidonic acid metabolism. Using an independent cohort, we also showed that the numbers of risk alleles at these loci are negatively correlated with reactive oxygen species and interleukin-6 levels in response to Candida. Finally, there was a significant correlation between susceptibility and allelic scores based on 16 independent candidemia-associated single-nucleotide polymorphisms that affect monocyte-derived cytokines, but not with T cell-derived cytokines. CONCLUSIONS: Our results prioritize the disturbed lipid homeostasis and oxidative stress as potential mechanisms that affect monocyte-derived cytokines to influence susceptibility to candidemia.

High-resolution fingerprinting of Candida parapsilosis isolates suggests persistence and transmission of infections among neonatal intensive care unit patients in Kuwait.

Candida parapsilosis causes ~35% of all candidemia cases in neonates. High-resolution fingerprinting of C. parapsilosis isolates from neonatal intensive care unit (NICU) patients in Maternity Hospital (MH) was performed to identify epidemiologically related strains. Sixty-eight bloodstream/colonizing strains isolated from 59 NICU patients, two isolates from health care workers (HCWs) from MH and 18 bloodstream isolates from two other hospitals were used. Six microsatellite markers were employed, isolates were assigned a numerical microsatellite genotype (MSG), dendrogram was constructed and similarities between genotypes were visualized by minimum spanning tree. Fifty bloodstream isolates from MH yielded 37 MSGs with 20 isolates clustering in 7 MSGs. Duplicate isolates and colonizing strains yielded same/highly similar MSG as bloodstream isolates. Colonizing strains from two non-candidemia patients yielded unique MSGs while others belonged to a cluster. All isolates from HCWs and from two other hospitals belonged to unique MSGs. Cluster isolates came from patients in NICU-1 or from neonates in NICU-1 and other NICUs. Clonal complexes comprising closely related genotypes indicative of microevolution were also detected. Our data show that some C. parapsilosis strains have persisted in MH environment over several years and these endemic genotypes were transmitted to other patients in NICU-1 and/or other nearby NICUs.

An integrative genomics approach identifies novel pathways that influence candidaemia susceptibility.

Candidaemia is a bloodstream infection caused by Candida species that primarily affects specific groups of at-risk patients. Because only small candidaemia patient cohorts are available, classical genome wide association cannot be used to identify Candida susceptibility genes. Therefore, we have applied an integrative genomics approach to identify novel susceptibility genes and pathways for candidaemia. Candida-induced transcriptome changes in human primary leukocytes were assessed by RNA sequencing. Genetic susceptibility to candidaemia was assessed using the Illumina immunochip platform for genotyping of a cohort of 217 patients. We then integrated genetics data with gene-expression profiles, Candida-induced cytokine production capacity, and circulating concentrations of cytokines. Based on the intersection of transcriptome pathways and genomic data, we prioritized 31 candidate genes for candidaemia susceptibility. This group of genes was enriched with genes involved in inflammation, innate immunity, complement, and hemostasis. We then validated the role of MAP3K8 in cytokine regulation in response to Candida stimulation. Here, we present a new framework for the identification of susceptibility genes for infectious diseases that uses an unbiased, hypothesis-free, systems genetics approach. By applying this approach to candidaemia, we identified novel susceptibility genes and pathways for candidaemia, and future studies should assess their potential as therapeutic targets.

Vav Proteins Are Key Regulators of Card9 Signaling for Innate Antifungal Immunity.

Fungal infections are major causes of morbidity and mortality, especially in immunocompromised individuals. The innate immune system senses fungal pathogens through Syk-coupled C-type lectin receptors (CLRs), which signal through the conserved immune adaptor Card9. Although Card9 is essential for antifungal defense, the mechanisms that couple CLR-proximal events to Card9 control are not well defined. Here, we identify Vav proteins as key activators of the Card9 pathway. Vav1, Vav2, and Vav3 cooperate downstream of Dectin-1, Dectin-2, and Mincle to engage Card9 for NF-κB control and proinflammatory gene transcription. Although Vav family members show functional redundancy, Vav1/2/3-/- mice phenocopy Card9-/- animals with extreme susceptibility to fungi. In this context, Vav3 is the single most important Vav in mice, and a polymorphism in human VAV3 is associated with susceptibility to candidemia in patients. Our results reveal a molecular mechanism for CLR-mediated Card9 regulation that controls innate immunity to fungal infections.

Immunochip SNP array identifies novel genetic variants conferring susceptibility to candidaemia.

Candidaemia is the fourth most common cause of bloodstream infection, with a high mortality rate of up to 40%. Identification of host genetic factors that confer susceptibility to candidaemia may aid in designing adjunctive immunotherapeutic strategies. Here we hypothesize that variation in immune genes may predispose to candidaemia. We analyse 118,989 single-nucleotide polymorphisms (SNPs) across 186 loci known to be associated with immune-mediated diseases in the largest candidaemia cohort to date of 217 patients of European ancestry and a group of 11,920 controls. We validate the significant associations by comparison with a disease-matched control group. We observe significant association between candidaemia and SNPs in the CD58 (P = 1.97 × 10(-11); odds ratio (OR) = 4.68), LCE4A-C1orf68 (P = 1.98 × 10(-10); OR = 4.25) and TAGAP (P = 1.84 × 10(-8); OR = 2.96) loci. Individuals carrying two or more risk alleles have an increased risk for candidaemia of 19.4-fold compared with individuals carrying no risk allele. We identify three novel genetic risk factors for candidaemia, which we subsequently validate for their role in antifungal host defence.

MiR-204/miR-211 downregulation contributes to candidemia-induced kidney injuries via derepression of Hmx1 expression.

AIMS: This study was aimed to exploit the role of heme oxygenase Hmx1 and the potential miRNA mechanisms in the kidney injuries induced by urinary tract infection by Candida species/Candidemia. MAIN METHODS: We employed a mouse model of systemic Candidiasis by injection of the Candida albicans strain SC5314 into C57BL/6 mice. Kidney injuries were assessed by measuring serum cystatin C (CysC), serum ß2-microglobulin (ß2-MG) and blood urea nitrogen (BUN). Validation of miRNA target gene was conducted by luciferase reporter gene assay, Western blot analysis and real-time RT-PCR. KEY FINDINGS: We showed here that Candidemia caused significant downregulation of microRNAs miR-204 and miR-211. In sharp contrast, Hmx1 expression was remarkably upregulated, particularly at the protein level. Computational analysis predicted Hmx1 as a target gene for both miR-204 and miR-211 that share the same seed site sequence. We then experimentally validated the targeting relationship between miR-204/miR-211 and Hmx1, which explains the reciprocal changes of expression of miR-204/miR-211 and Hmx1 in Candidemia. Administration of miR-204/miR-211 mimics substantially downregulated Hmx1 and mitigated the severity of the kidney injuries induced by Candidemia, as reflected by improved renal glomerular filtration rate (GFR) determined by serum cystatin C (CysC), serum ß2-microglobulin (ß2-MG) and blood urea nitrogen (BUN). Knockdown of miR-204/miR-211 worsened while forced expression of miR-204/miR-211 ameliorated kidney injuries in mice with systemic Candidiasis. SIGNIFICANCE: Our findings indicate that miR-204/miR-211 downregulation accounts at least partially for the Hmx1 upregulation and the miR-204/miR-211-Hmx1 signaling axis may contribute to immune-suppression in the host thereby the Candidemia-induced kidney dysfunction.

Candida identification: a journey from conventional to molecular methods in medical mycology.

The incidence of Candida infections have increased substantially in recent years due to aggressive use of immunosuppressants among patients. Use of broad-spectrum antibiotics and intravascular catheters in the intensive care unit have also attributed with high risks of candidiasis among immunocompromised patients. Among Candida species, C. albicans accounts for the majority of superficial and systemic infections, usually associated with high morbidity and mortality often caused due to increase in antimicrobial resistance and restricted number of antifungal drugs. Therefore, early detection of candidemia and correct identification of Candida species are indispensable pre-requisites for appropriate therapeutic intervention. Since blood culture based methods lack sensitivity, and species-specific identification by conventional method is time-consuming and often leads to misdiagnosis within closely related species, hence, molecular methods may provide alternative for accurate and rapid identification of Candida species. Although, several molecular approaches have been developed for accurate identification of Candida species but the internal transcribed spacer 1 and 2 (ITS1 and ITS2) regions of the rRNA gene are being used extensively in a variety of formats. Of note, ITS sequencing and PCR-RFLP analysis of ITS region seems to be promising as a rapid, easy, and cost-effective method for identification of Candida species. Here, we review a number of existing techniques ranging from conventional to molecular approaches currently in use for the identification of Candida species. Further, advantages and limitations of these methods are also discussed with respect to their discriminatory power, reproducibility, and ease of performance.

Functional genomics identifies type I interferon pathway as central for host defense against Candida albicans.

Candida albicans is the most common human fungal pathogen causing mucosal and systemic infections. However, human antifungal immunity remains poorly defined. Here by integrating transcriptional analysis and functional genomics, we identified Candida-specific host defence mechanisms in humans. Candida induced significant expression of genes from the type I interferon pathway in human peripheral blood mononuclear cells. This unexpectedly prominent role of type I interferon pathway in anti-Candida host defence was supported by additional evidence. Polymorphisms in type I interferon genes modulated Candida-induced cytokine production and were correlated with susceptibility to systemic candidiasis. In in vitro experiments, type I interferons skewed Candida-induced inflammation from a Th17 response towards a Th1 response. Patients with chronic mucocutaneous candidiasis displayed defective expression of genes in the type I interferon pathway. These findings indicate that the type I interferon pathway is a main signature of Candida-induced inflammation and has a crucial role in anti-Candida host defence in humans.

Toll-like receptor 1 polymorphisms increase susceptibility to candidemia.

BACKGROUND: Candidemia is a severe invasive fungal infection with high mortality. Recognition of Candida species is mediated through pattern recognition receptors such as Toll-like receptors (TLRs). This study assessed whether genetic variation in TLR signaling influences susceptibility to candidemia. METHODS: Thirteen mostly nonsynonymous single nucleotide polymorphisms (SNPs) in genes encoding TLRs and signaling adaptors MyD88 and Mal/TIRAP were genotyped in 338 patients (237 white, 93 African American, 8 other race) with candidemia and 351 noninfected controls (263 white, 88 African American). The SNPs significant in univariate analysis were further analyzed with multivariable logistic regression to determine association with clinical outcomes. Functional consequences of these polymorphisms were assessed via in vitro stimulation assays. RESULTS: Analyses of TLR SNPs revealed that 3 TLR1 SNPs (R80T, S248N, I602S) were significantly associated with candidemia susceptibility in whites. This association was not found in African Americans, likely due to lower power in this smaller study population. Furthermore, these TLR1 polymorphisms displayed impaired cytokine release by primary monocytes. No associations with susceptibility to candidemia were observed for SNPs in TLR2, TLR4, TLR6, TLR9, MyD88, or TIRAP. CONCLUSIONS: Nonsynonymous SNPs in TLR1 are associated with impaired TLR1 function, decreased cytokine responses, and predisposition to candidemia in whites.

The impact of caspase-12 on susceptibility to candidemia.

Candida is one of the leading causes of sepsis, and an effective host immune response to Candida critically depends on the cytokines IL-1ß and IL-18, which need caspase-1 cleavage to become bioactive. Caspase-12 has been suggested to inhibit caspase-1 activation and has been implicated as a susceptibility factor for bacterial sepsis. In populations of African descent, CASPASE-12 is either functional or non-functional. Here, we have assessed the frequencies of both CASPASE-12 alleles in an African-American Candida sepsis patients cohort compared to uninfected patients with similar predisposing factors. African-American Candida sepsis patients (n = 93) and non-infected African-American patients (n = 88) were genotyped for the CASPASE-12 genotype. Serum cytokine concentrations of IL-6, IL-8, and IFNγ were measured in the serum of infected patients. Statistical comparisons were performed in order to assess the effect of the CASPASE-12 genotype on susceptibility to candidemia and on serum cytokine concentrations. Our findings demonstrate that CASPASE-12 does not influence the susceptibility to Candida sepsis, nor has any effect on the serum cytokine concentrations in Candida sepsis patients during the course of infection. Although the functional CASPASE-12 allele has been suggested to increase susceptibility to bacterial sepsis, this could not be confirmed in our larger cohort of fungal sepsis patients.

Genetic variation in the dectin-1/CARD9 recognition pathway and susceptibility to candidemia.

BACKGROUND: Candidemia is an important cause of morbidity and mortality in critically ill patients or patients undergoing invasive treatments. Dectin-1 is the main ß-glucan receptor, and patients with a complete deficiency of either dectin-1 or its adaptor molecule CARD9 display persistent mucosal infections with Candida albicans. The role of genetic variation of DECTIN-1 and CARD9 genes on the susceptibility to candidemia is unknown. METHODS: We assessed whether genetic variation in the genes encoding dectin-1 and CARD9 influence the susceptibility to candidemia and/or the clinical course of the infection in a large cohort of American and Dutch candidemia patients (n = 331) and noninfected matched controls (n = 351). Furthermore, functional studies have been performed to assess the effect of the DECTIN-1 and CARD9 genetic variants on cytokine production in vitro and in vivo in the infected patients. RESULTS: No significant association between the single-nucleotide polymorphisms DECTIN-1 Y238X and CARD9 S12N and the prevalence of candidemia was found, despite the association of the DECTIN-1 238X allele with impaired in vitro and in vivo cytokine production. CONCLUSIONS: Whereas the dectin-1/CARD9 signaling pathway is nonredundant in mucosal immunity to C. albicans, a partial deficiency of ß-glucan recognition has a minor impact on susceptibility to candidemia.