Ubiquitin Ligase TRIM62 Regulates CARD9-Mediated Anti-fungal Immunity and Intestinal Inflammation.

CARD9 is a central component of anti-fungal innate immune signaling via C-type lectin receptors, and several immune-related disorders are associated with CARD9 alterations. Here, we used a rare CARD9 variant that confers protection against inflammatory bowel disease as an entry point to investigating CARD9 regulation. We showed that the protective variant of CARD9, which is C-terminally truncated, acted in a dominant-negative manner for CARD9-mediated cytokine production, indicating an important role for the C terminus in CARD9 signaling. We identified TRIM62 as a CARD9 binding partner and showed that TRIM62 facilitated K27-linked poly-ubiquitination of CARD9. We identified K125 as the ubiquitinated residue on CARD9 and demonstrated that this ubiquitination was essential for CARD9 activity. Furthermore, we showed that similar to Card9-deficient mice, Trim62-deficient mice had increased susceptibility to fungal infection. In this study, we utilized a rare protective allele to uncover a TRIM62-mediated mechanism for regulation of CARD9 activation.

Human inborn errors of immunity underlying superficial or invasive candidiasis.

Candida species, including C. albicans in particular, can cause superficial or invasive disease, often in patients with known acquired immunodeficiencies or iatrogenic conditions. The molecular and cellular basis of these infections in patients with such risk factors remained largely elusive, until the study of inborn errors of immunity clarified the basis of the corresponding inherited and "idiopathic" infections. Superficial candidiasis, also known as chronic mucocutaneous candidiasis (CMC), can be caused by inborn errors of IL-17 immunity. Invasive candidiasis can be caused by inborn errors of CARD9 immunity. In this chapter, we review both groups of inborn errors of immunity, and discuss the contribution of these studies to the deciphering of the critical mechanisms of anti-Candida immunity in patients with other conditions.

Novel bi-allelic splice mutations in CARD9 causing adult-onset Candida endophthalmitis.

CARD9 deficiency (CANDF2; OMIM# 212050) is an autosomal-recessive monogenic inborn error of immunity conferring susceptibility to invasive fungal diseases, including the very distinct syndrome of spontaneous central nervous system candidiasis, in which opportunistic yeast of the genus Candida infect the central nervous system (either brain parenchyma and/or meninges) in the absence of trauma, chemotherapy or underlying systemic disease. We present a patient with spontaneous endophthalmitis of the right eye due to Candida albicans; further investigations revealed concomitant cerebral abscess. She had a history of left endophthalmitis due to the dematiaceous mould, Aureobasidium pullulans, 15 years earlier. Targeted sequencing of the CARD9 gene revealed 2 novel variants (c.184G>A and c.288C>T). Analysis in silico predicted each variant altered splicing, which was confirmed by sequencing of cDNA from proband and carrier offsprings: c.184G>A results in a 4-base pair frameshift deletion with loss of allelic expression; c.288C>T results in an in-frame 36-bp pair deletion with detectable protein. CARD9 deficiency can present with a phenotype of spontaneous candidal endophthalmitis. We report 2 novel mutations in CARD9, both affecting splicing, expanding the range of morbid variants causing CARD9 deficiency, emphasising the importance of both genomic and cDNA sequencing for this condition.

Impaired RASGRF1/ERK-mediated GM-CSF response characterizes CARD9 deficiency in French-Canadians.

BACKGROUND: Caspase recruitment domain-containing protein 9 (CARD9) deficiency is an autosomal recessive primary immunodeficiency conferring human susceptibility to invasive fungal disease, including spontaneous central nervous system candidiasis (sCNSc). However, clinical characterization of sCNSc is variable, hindering its recognition. Furthermore, an in-depth understanding of the bases for this susceptibility has remained elusive. OBJECTIVES: We sought to comprehensively characterize sCNSc and to dissect the mechanisms by which a hypomorphic CARD9 mutation causes susceptibility to Candida species. METHODS: We describe the clinical and radiologic findings of sCNSc caused by CARD9 deficiency in a French-Canadian cohort. We performed genetic, cellular, and molecular analyses to further decipher its pathophysiology. RESULTS: In our French-Canadian series (n = 4) sCNSc had onset in adulthood (median, 38 years) and was often misinterpreted radiologically as brain malignancies; 1 patient had additional novel features (eg, endophthalmitis and osteomyelitis). CARD9 deficiency resulted from a hypomorphic p.Y91H mutation and allelic imbalance established in this population through founder effects. We demonstrate a consistent cellular phenotype of impaired GM-CSF responses. The ability of CARD9 to complex with B-cell CLL/lymphoma 10 (BCL10) and mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is intact in our series, arguing against its involvement in susceptibility to fungi. Instead, we show that the p.Y91H mutation impairs the ability of CARD9 to complex with Ras protein-specific guanine nucleotide-releasing factor 1 (RASGRF1), leading to impaired activation of nuclear factor κB and extracellular signal-regulated kinase (ERK) in monocytes and subsequent GM-CSF responses. Successful treatment of a second patient with adjunctive GM-CSF bolsters the clinical relevance of these findings. CONCLUSIONS: Hypomorphic CARD9 deficiency caused by p.Y91H results in adult-onset disease with variable penetrance and expressivity. Our findings establish the CARD9/RASGRF1/ERK/GM-CSF axis as critical to the pathophysiology of sCNSc.

Analysis of the genetic diversity of Candida isolates obtained from diabetic patients and kidney transplant recipients.

Yeasts of the genus Candida have high genetic variability and are the most common opportunistic pathogenic fungi in humans. In this study, we evaluated the genetic diversity among 120 isolates of Candida spp. obtained from diabetic patients, kidney transplant recipients and patients without any immune deficiencies from Paraná state, Brazil. The analysis was performed using the ITS1-5.8S-ITS2 region and a partial sequence of 28S rDNA. In the phylogenetic analysis, we observed a consistent separation of the species C. albicans, C. dubliniensis, C. glabrata, C. tropicalis, C. parapsilosis, C. metapsilosis and C. orthopsilosis, however with low intraspecific variability. In the analysis of the C. albicans species, two clades were formed. Clade A included the largest number of isolates (91.2%) and the majority of isolates from GenBank (71.4%). The phylogenetic analysis showed low intraspecific genetic diversity, and the genetic polymorphisms between C. albicans isolates were similar to genetic divergence found in other studies performed with isolates from Brazil. This low genetic diversity of isolates can be explained by the geographic proximity of the patients evaluated. It was observed that yeast colonisation was highest in renal transplant recipients and diabetic patients and that C. albicans was the species most frequently isolated.

Inherited CARD9 deficiency in otherwise healthy children and adults with Candida species-induced meningoencephalitis, colitis, or both.

BACKGROUND: Invasive infections of the central nervous system (CNS) or digestive tract caused by commensal fungi of the genus Candida are rare and life-threatening. The known risk factors include acquired and inherited immunodeficiencies, with patients often displaying a history of multiple infections. Cases of meningoencephalitis, colitis, or both caused by Candida species remain unexplained. OBJECTIVE: We studied 5 previously healthy children and adults with unexplained invasive disease of the CNS, digestive tract, or both caused by Candida species. The patients were aged 39, 7, 17, 37, and 26 years at the time of infection and were unrelated, but each was born to consanguineous parents of Turkish (2 patients), Iranian, Moroccan, or Pakistani origin. Meningoencephalitis was reported in 3 patients, meningoencephalitis associated with colitis was reported in a fourth patient, and the fifth patient had colitis only. METHODS: Inherited caspase recruitment domain family, member 9 (CARD9) deficiency was recently reported in otherwise healthy patients with other forms of severe disease caused by Candida, Trichophyton, Phialophora, and Exophiala species, including meningoencephalitis but not colitis caused by Candida and Exophiala species. Therefore we sequenced CARD9 in the 5 patients. RESULTS: All patients were found to be homozygous for rare and deleterious mutant CARD9 alleles: R70W and Q289* for the 3 patients with Candida albicans-induced meningoencephalitis, R35Q for the patient with meningoencephalitis and colitis caused by Candida glabrata, and Q295* for the patient with Candida albicans-induced colitis. Regardless of their levels of mutant CARD9 protein, the patients' monocyte-derived dendritic cells responded poorly to CARD9-dependent fungal agonists (curdlan, heat-killed C albicans, Saccharomyces cerevisiae, and Exophiala dermatitidis). CONCLUSION: Invasive infections of the CNS or digestive tract caused by Candida species in previously healthy children and even adults might be caused by inherited CARD9 deficiency.

Clinical perspectives on echinocandin resistance among Candida species.

PURPOSE OF REVIEW: We review and offer our clinical perspectives on the emergence of echinocandin-resistant Candida. RECENT FINDINGS: Candida FKS gene mutations attenuate echinocandin activity, but overall mutation rates among clinical isolates remain low (Candida glabrata, ∼4%; other species, <1%). Rates are higher with prior echinocandin exposure, exceeding 50% among C. glabrata or Candida albicans isolates causing breakthrough invasive candidiasis. The median duration of prior echinocandin exposure among FKS mutant isolates is ∼100 days. The clinical usefulness of echinocandin susceptibility testing is limited by the low overall prevalence of resistance, and uncertainties surrounding testing methods and interpretation of minimum inhibitory concentrations (MICs). In single-center studies, caspofungin resistance (defined using institution-specific MIC breakpoints) was 32-53% sensitive and 75-95% specific for predicting treatment outcomes of C. glabrata invasive candidiasis; corresponding values for the presence of an FKS mutation were 35-41% and 90-98%. Results were similar using anidulafungin and micafungin MICs. Clinical data are scarce for non-C. glabrata species. SUMMARY: Echinocandins remain preferred agents against invasive Candida infections. Susceptibility testing and FKS genotypic testing do not have roles in routine clinical practice, but may be useful in newly-diagnosed patients who are echinocandin-experienced or those who have not responded to echinocandin treatment.

Invasive fungal infection and impaired neutrophil killing in human CARD9 deficiency.

Caspase recruitment domain-containing protein 9 (CARD9) is an adaptor molecule in the cytosol of myeloid cells, required for induction of T-helper cells producing interleukin-17 (Th17 cells) and important in antifungal immunity. In a patient suffering from Candida dubliniensis meningoencephalitis, mutations in the CARD9 gene were found to result in the loss of protein expression. Apart from the reduced numbers of CD4(+) Th17 lymphocytes, we identified a lack of monocyte-derived cytokines in response to Candida strains. Importantly, CARD9-deficient neutrophils showed a selective Candida albicans killing defect with abnormal ultrastructural phagolysosomes and outgrowth of hyphae. The neutrophil killing defect was independent of the generation of reactive oxygen species by the reduced NAD phosphate oxidase system. Taken together, this demonstrates that human CARD9 deficiency results in selective defect in the host defense against invasive fungal infection, caused by an impaired phagocyte killing.

Genetic diversity of medically important and emerging Candida species causing invasive infection.

BACKGROUND: Genetic variation in the ribosomal DNA (rDNA) internal transcribed spacer (ITS) region has been studied among fungi. However, the numbers of ITS sequence polymorphisms in the various Candida species and their associations with sources of invasive fungal infections remain poorly investigated. Here, we characterized the intraspecific and interspecific ITS diversity of Candida spp. strains collected from patients with bloodstream or oroesophageal candidiasis. METHODS: We selected cultures of representative medically important species of Candida as well as some rare and emerging pathogens. Identification was performed by micromorphology and by biochemical testing using an ID32C system, as well as by the sequencing of rDNA ITS. The presence of intraspecific ITS polymorphisms was characterized based on haplotype networks, and interspecific diversity was characterized based on Bayesian phylogenetic analysis. RESULTS: Among 300 Candida strains, we identified 76 C. albicans, 14 C. dubliniensis, 40 C. tropicalis, 47 C. glabrata, 34 C. parapsilosis (sensu stricto), 31 C. orthopsilosis, 3 C. metapsilosis, 21 Meyerozyma guilliermondii (C. guilliermondii), 12 Pichia kudriavzevii (C. krusei), 6 Clavispora lusitaniae (C. lusitaniae), 3 C. intermedia, 6 Wickerhamomyces anomalus (C. pelliculosa), and 2 C. haemulonii strains, and 1 C. duobushaemulonii, 1 Kluyveromyces marxianus (C. kefyr), 1 Meyerozyma caribbica (C. fermentati), 1 Pichia norvegensis (C. norvegensis), and 1 Lodderomyces elongisporus strain. Out of a total of seven isolates with inconsistent ID32C profiles, ITS sequencing identified one C. lusitaniae strain, three C. intermedia strains, two C. haemulonii strains and one C. duobushaemulonii strain. Analysis of ITS variability revealed a greater number of haplotypes among C. albicans, C. tropicalis, C. glabrata and C. lusitaniae, which are predominantly related to endogenous sources of acquisition. Bayesian analysis confirmed the major phylogenetic relationships among the isolates and the molecular identification of the different Candida spp. CONCLUSIONS: Molecular studies based on ITS sequencing are necessary to identify closely related and emerging species. Polymorphism analysis of the ITS rDNA region demonstrated its utility as a genetic marker for species identification and phylogenetic relationships as well as for drawing inferences concerning the natural history of hematogenous infections caused by medically important and emerging Candida species.

Association of cytokine gene polymorphisms with susceptibility to invasive candidiasis.

The aim of this study was to investigate the role of cytokine genes in the susceptibility to Candida infection. A total of 275 consecutive patients diagnosed with Candida infection were selected between May 2010 and May 2011, along with 305 uninfected controls. Genotyping of the IL-1ß gene polymorphisms (IL1ß) rs1143634, IL1ßrs16944, IL8 rs4073, IL10 rs1800872, and IL10 rs1800896 was carried out using a 384-well plate format on the Sequenom MassARRAY platform. Patients with invasive Candida infections were more likely to have had an immunocompromised state, hematopoietic stem cell transplantation, solid organ transplant, solid tumor, chemotherapy within the past three months, neutropenia, surgery within the past 30 days, acute renal failure, liver failure, and/or median baseline serum creatinine. Conditional logistic regression analyses found that individuals with the rs1800896 GG genotype were associated with a higher risk of invasive Candida infections than those carrying the AA genotype (odds ratio = 0.61, 95% confidence interval = 0.37-0.94). From the results of this case-control study, we suggest that the cytokine IL-10 gene rs1800896 polymorphism might play a role in the etiology of invasive Candida infections.

CARD9 deficiency and spontaneous central nervous system candidiasis: complete clinical remission with GM-CSF therapy.

We demonstrate autosomal-recessive Caspase Recruitment Domain-containing protein 9 (CARD9) deficiency in a patient with relapsing C. albicans meningoencephalitis. We identified a novel, hypomorphic mutation with intact Th17 responses, but impaired GM-CSF responses. We report complete clinical remission with adjunctive GM-CSF therapy, suggesting that a CARD9/GM-CSF axis contributes to susceptibility to candidiasis.

Association of lectin pathway proteins with intra-abdominal Candida infection in high-risk surgical intensive-care unit patients. A prospective cohort study within the fungal infection network of Switzerland.

OBJECTIVES: Human studies on the role of mannose-binding lectin (MBL) in patients with invasive candidiasis have yielded conflicting results. We investigated the influence of MBL and other lectin pathway proteins on Candida colonization and intra-abdominal candidiasis (IAC) in a cohort of high-risk patients. METHODS: Prospective observational cohort study of 89 high-risk intensive-care unit (ICU) patients. Levels of lectin pathway proteins at study entry and six MBL2 single-nucleotide polymorphisms were analyzed by sandwich-type immunoassays and genotyping, respectively, and correlated with development of heavy Candida colonization (corrected colonization index (CCI) ≥0.4) and occurrence of IAC during a 4-week period. RESULTS: Within 4 weeks after inclusion a CCI ≥0.4 and IAC was observed in 47% and 38% of patients respectively. Neither serum levels of MBL, ficolin-1, -2, -3, MASP-2 or collectin liver 1 nor MBL2 genotypes were associated with a CCI ≥0.4. Similarly, none of the analyzed proteins was found to be associated with IAC with the exception of lower MBL levels (HR 0.74, p = 0.02) at study entry. However, there was no association of MBL deficiency (<0.5 µg/ml), MBL2 haplo- or genotypes with IAC. CONCLUSION: Lectin pathway protein levels and MBL2 genotype investigated in this study were not associated with heavy Candida colonization or IAC in a cohort of high-risk ICU patients.

Initiation of phospholipomannan ß-1,2 mannosylation involves Bmts with redundant activity, influences its cell wall location and regulates ß-glucans homeostasis but is dispensable for Candida albicans systemic infection.

Pathogenic and non-pathogenic fungi synthesize glycosphingolipids, which have a crucial role in growth and viability. Glycosphingolipids also contribute to fungal-associated pathogenesis. The opportunistic yeast pathogen Candida albicans synthesizes phospholipomannan (PLM), which is a glycosphingolipid of the mannosylinositol phosphorylceramide family. Through its lipid and glycan moieties, PLM contributes to the initial recognition of the yeast, causing immune system disorder and persistent fungal disease through activation of host signaling pathways. The lipid moiety of PLM activates the deregulation signaling pathway involved in yeast phagocytosis whereas its glycan moiety, composed of ß-1,2 mannosides (ß-Mans), participates to inflammatory processes through a mechanism involving Galectin-3. Biosynthesis of PLM ß-Mans involves two ß-1,2 mannosyltransferases (Bmts) that initiate (Bmt5) and elongate (Bmt6) the glycan chains. After generation of double bmtsΔ mutants, we show that Bmt5 has redundant activity with Bmt2, which can replace Bmt5 in bmt5Δ mutant. We also report that PLM is located in the inner layer of the yeast cell wall. PLM seems to be not essential for systemic infection of the yeast. However, defect of PLM ß-mannosylation increases resistance of C. albicans to inhibitors of ß-glucans and chitin synthesis, highlighting a role of PLM in cell wall homeostasis.

Targeting CBLB as a potential therapeutic approach for disseminated candidiasis.

Disseminated candidiasis has become one of the leading causes of hospital-acquired blood stream infections with high mobility and mortality. However, the molecular basis of host defense against disseminated candidiasis remains elusive, and treatment options are limited. Here we report that the E3 ubiquitin ligase CBLB directs polyubiquitination of dectin-1 and dectin-2, two key pattern-recognition receptors for sensing Candida albicans, and their downstream kinase SYK, thus inhibiting dectin-1- and dectin-2-mediated innate immune responses. CBLB deficiency or inactivation protects mice from systemic infection with a lethal dose of C. albicans, and deficiency of dectin-1, dectin-2, or both in Cblb(-/-) mice abrogates this protection. Notably, silencing the Cblb gene in vivo protects mice from lethal systemic C. albicans infection. Our data reveal that CBLB is crucial for homeostatic control of innate immune responses mediated by dectin-1 and dectin-2. Our data also indicate that CBLB represents a potential therapeutic target for protection from disseminated candidiasis.

Host genetics of invasive Aspergillus and Candida infections.

Invasive candidiasis and aspergillosis are major complications in surgical and onco-hematological patients, and still associated with an important morbidity and mortality. A large number of studies highlighted the potential role of host genetic polymorphisms that may influence susceptibility to fungal pathogens, but many were limited by insufficient statistical power, problematic design, and/or lack of replication. However, some relevant polymorphisms are now emerging from well-conducted studies whose associations have been replicated and/or are supported by strong biological evidence. Such polymorphisms together with other biomarkers may play a role in the prediction, diagnosis, and management of severe fungal infections in high-risk patients in the coming years.

Mannose-binding lectin codon 54 gene polymorphism in relation to risk of nosocomial invasive fungal infection in preterm neonates in the neonatal intensive care unit.

OBJECTIVE: Preterm neonates are susceptible to infection due to a combination of sub-optimal immunity and increased exposure to invasive organisms. Invasive fungal infections are associated with significant morbidity and mortality among preterm infants cared for in the neonatal intensive care unit (NICU). Mannose-binding lectin (MBL) is a component of the innate immune system, which may be especially important in the neonatal setting. The objective of this study was to investigate the presence of any association between MBL gene polymorphism and nosocomial invasive fungal infection in preterm neonates. METHODS: Codon 54 (B allele) polymorphism in exon 1 of the MBL gene was investigated in 31 patients diagnosed as nosocomial invasive fungal infection and 30 control preterm neonates. RESULTS: AB genotype was determined in 26% and 30% of patient and control groups, respectively, and the difference was not statistically significant. AA genotype was determined in 74% of the patient group and in 67% of the control group, and the difference was not statistically significant. B allele frequency was not different significantly in the patient group (13%) compared to the control group (18%). CONCLUSIONS: In our study, no relationship was found between MBL codon 54 gene polymorphism and the risk of nosocomial invasive fungal infection in preterm neonates in NICU.

Analysis of the genetic diversity of Candida isolates obtained from diabetic patients and kidney transplant recipients

Yeasts of the genus Candida have high genetic variability and are the most common opportunistic pathogenic fungi in humans. In this study, we evaluated the genetic diversity among 120 isolates of Candida spp. obtained from diabetic patients, kidney transplant recipients and patients without any immune deficiencies from Paraná state, Brazil. The analysis was performed using the ITS1-5.8S-ITS2 region and a partial sequence of 28S rDNA. In the phylogenetic analysis, we observed a consistent separation of the species C. albicans, C. dubliniensis, C. glabrata, C. tropicalis, C. parapsilosis, C. metapsilosis and C. orthopsilosis, however with low intraspecific variability. In the analysis of the C. albicans species, two clades were formed. Clade A included the largest number of isolates (91.2%) and the majority of isolates from GenBank (71.4%). The phylogenetic analysis showed low intraspecific genetic diversity, and the genetic polymorphisms between C. albicans isolates were similar to genetic divergence found in other studies performed with isolates from Brazil. This low genetic diversity of isolates can be explained by the geographic proximity of the patients evaluated. It was observed that yeast colonisation was highest in renal transplant recipients and diabetic patients and that C. albicans was the species most frequently isolated.