Computational analysis of the genetic and environmental contributions to disease-related human phenotypes: From predicting adverse lipid response of HIV patients receiving antiretroviral therapy to genome-wide association studies of cardiovascular and lipid related disorders

Genetic variants influence the risk to develop certain diseases or give rise to differences in drug response. Recent progresses in cost-effective, high-throughput genome-wide techniques, such as microarrays measuring Single Nucleotide Polymorphisms (SNPs), have facilitated genotyping of large clinical and population cohorts. Combining the massive genotypic data with measurements of phenotypic traits allows for the determination of genetic differences that explain, at least in part, the phenotypic variations within a population. So far, models combining the most significant variants can only explain a small fraction of the variance, indicating the limitations of current models. In particular, researchers have only begun to address the possibility of interactions between genotypes and the environment. Elucidating the contributions of such interactions is a difficult task because of the large number of genetic as well as possible environmental factors.In this thesis, I worked on several projects within this context. My first and main project was the identification of possible SNP-environment interactions, where the phenotypes were serum lipid levels of patients from the Swiss HIV Cohort Study (SHCS) treated with antiretroviral therapy. Here the genotypes consisted of a limited set of SNPs in candidate genes relevant for lipid transport and metabolism. The environmental variables were the specific combinations of drugs given to each patient over the treatment period. My work explored bioinformatic and statistical approaches to relate patients' lipid responses to these SNPs, drugs and, importantly, their interactions. The goal of this project was to improve our understanding and to explore the possibility of predicting dyslipidemia, a well-known adverse drug reaction of antiretroviral therapy. Specifically, I quantified how much of the variance in lipid profiles could be explained by the host genetic variants, the administered drugs and SNP-drug interactions and assessed the predictive power of these features on lipid responses. Using cross-validation stratified by patients, we could not validate our hypothesis that models that select a subset of SNP-drug interactions in a principled way have better predictive power than the control models using "random" subsets. Nevertheless, all models tested containing SNP and/or drug terms, exhibited significant predictive power (as compared to a random predictor) and explained a sizable proportion of variance, in the patient stratified cross-validation context. Importantly, the model containing stepwise selected SNP terms showed higher capacity to predict triglyceride levels than a model containing randomly selected SNPs. Dyslipidemia is a complex trait for which many factors remain to be discovered, thus missing from the data, and possibly explaining the limitations of our analysis. In particular, the interactions of drugs with SNPs selected from the set of candidate genes likely have small effect sizes which we were unable to detect in a sample of the present size (<800 patients).In the second part of my thesis, I performed genome-wide association studies within the Cohorte Lausannoise (CoLaus). I have been involved in several international projects to identify SNPs that are associated with various traits, such as serum calcium, body mass index, two-hour glucose levels, as well as metabolic syndrome and its components. These phenotypes are all related to major human health issues, such as cardiovascular disease. I applied statistical methods to detect new variants associated with these phenotypes, contributing to the identification of new genetic loci that may lead to new insights into the genetic basis of these traits. This kind of research will lead to a better understanding of the mechanisms underlying these pathologies, a better evaluation of disease risk, the identification of new therapeutic leads and may ultimately lead to the realization of "personalized" medicine.

A stroma-related gene signature predicts resistance to neoadjuvant chemotherapy in breast cancer.

To better understand the relationship between tumor-host interactions and the efficacy of chemotherapy, we have developed an analytical approach to quantify several biological processes observed in gene expression data sets. We tested the approach on tumor biopsies from individuals with estrogen receptor-negative breast cancer treated with chemotherapy. We report that increased stromal gene expression predicts resistance to preoperative chemotherapy with 5-fluorouracil, epirubicin and cyclophosphamide (FEC) in subjects in the EORTC 10994/BIG 00-01 trial. The predictive value of the stromal signature was successfully validated in two independent cohorts of subjects who received chemotherapy but not in an untreated control group, indicating that the signature is predictive rather than prognostic. The genes in the signature are expressed in reactive stroma, according to reanalysis of data from microdissected breast tumor samples. These findings identify a previously undescribed resistance mechanism to FEC treatment and suggest that antistromal agents may offer new ways to overcome resistance to chemotherapy.

Immunopathogenesis of Asthma and Atopic Diseases – The specific Role of a selected Panel of Genes in human T helper Cell Differentiation

T helper cell (Th) functions are crucial for proper immune defence against various intra- and extracellular pathogens. According to the specific immune responses, Th cells can be classified into subtypes, Th1 and Th2 cells being the most frequently characterized classes. Th1 and Th2 cells interact with other immune cells by regulating their functions with specific cytokine production. IFN, IL-2 and TNF- are the cytokines predominantly produced by Th1 cells whereas Th2 cells produce Th2-type cytokines, such as IL-4, IL-5 and IL-13. Upon TCR activation and in the presence of polarizing cytokines, Th cells differentiate into effector subtypes from a common precursor cell. IFN and IL-12 are the predominant Th1 polarizing cytokines whereas IL-4 directs Th2 polarization. The cytokines mediate their effects through specific receptor signalling. The differentiation process is complex, involving various signalling molecules and routes, as well as functions of the specific transcription factors. The functions of the Th1/Th2 cells are tightly regulated; however, knowledge on human Th cell differentiation is, as yet, fairly poor. The susceptibility for many immune-mediated disorders often originates from disturbed Th cell responses. Thus, research is needed for defining the molecular mechanisms involved in the differentiation and balanced functions of the Th cells. Importantly, the new information obtained will be crucial for a better understanding of the pathogenesis of immune-mediated disorders, such as asthma or autoimmune diseases. In the first subproject of this thesis, the role of genetic polymorphisms in the human STAT6, GATA3 and STAT4 genes were investigated for asthma or atopy susceptibility in Finnish asthma families by association analysis. These genes code for key transcription factors regulating Th cell differentiation. The study resulted in the identification of a GATA3 haplotype that associated with asthma and related traits (high serum IgE level). In the second subproject, an optimized method for human primary T cell transfection and enrichment was established. The method can be utilized for functional studies for the selected genes of interest. The method was also utilized in the third subproject, which aimed at the identification of novel genes involved in early human Th cell polarization (0-48h) using genome-wide oligonucleotide arrays. As a result, numerous genes and ESTs with known or unknown functions were identified in the study. Using an shRNA knockdown approach, a panel of novel IL-4/STAT6 regulated genes were identified in the functional studies of the genes. Moreover, one of the genes, NDFIP2, with a previously uncharacterized role in the human Th differentiation, was observed to promote IFN production of the differentiated Th1 cells. Taken together, the results obtained have revealed potential new relevant candidate genes serving as a basis for further studies characterizing the detailed networks involved in the human Th cell differentiation as well as in the genetic susceptibility of Th-mediated immune disorders.

Comparative genomics of emerging pathogens in the Candida glabrata clade

BACKGROUND: Candida glabrata follows C. albicans as the second or third most prevalent cause of candidemia worldwide. These two pathogenic yeasts are distantly related, C. glabrata being part of the Nakaseomyces, a group more closely related to Saccharomyces cerevisiae. Although C. glabrata was thought to be the only pathogenic Nakaseomyces, two new pathogens have recently been described within this group: C. nivariensis and C. bracarensis. To gain insight into the genomic changes underlying the emergence of virulence, we sequenced the genomes of these two, and three other non-pathogenic Nakaseomyces, and compared them to other sequenced yeasts. RESULTS: Our results indicate that the two new pathogens are more closely related to the non-pathogenic N. delphensis than to C. glabrata. We uncover duplications and accelerated evolution that specifically affected genes in the lineage preceding the group containing N. delphensis and the three pathogens, which may provide clues to the higher propensity of this group to infect humans. Finally, the number of Epa-like adhesins is specifically enriched in the pathogens, particularly in C. glabrata. CONCLUSIONS: Remarkably, some features thought to be the result of adaptation of C. glabrata to a pathogenic lifestyle, are present throughout the Nakaseomyces, indicating these are rather ancient adaptations to other environments. Phylogeny suggests that human pathogenesis evolved several times, independently within the clade. The expansion of the EPA gene family in pathogens establishes an evolutionary link between adhesion and virulence phenotypes. Our analyses thus shed light onto the relationships between virulence and the recent genomic changes that occurred within the Nakaseomyces.

Frequencies of candidate genes and associations with carcass and meat traits in Nellore and crossbred cattle

Abstract: The objective of this work was to estimate allelic frequencies of the polymorphisms IGF2/MboII (G > T) of the insulin-like growth factor 2 (IGF2) gene, DQ499531.1:g.134A > T of the pro-melanin-concentrating hormone (PMCH) gene, and DQ667048.1:g.3290G > T of the RARrelated orphan receptor C (RORC) gene in beef cattle of different genetic groups, and to evaluate the associations between these polymorphisms and traits related to carcass composition and meat quality. Data on carcass and meat quality of 499 animals was used: of 313 Nellore (Bos indicus) and of 186 Nellore crossed with different taurine (Bos taurus) breeds. For the IGF2/MboII polymorphism, the frequencies found for the G allele were 0.231 and 0.631 for Nellore and crossed breeds, respectively. For the DQ499531.1:g.134A > T polymorphism, the allelic frequencies of A were 0.850 for Nellore and 0.905 for crossed breeds. For the DQ667048.1:g.3290G > T polymorphism, the allelic frequencies of G were 0.797 and 0.460 for Nellore and crossed breeds, respectively. The evaluated single nucleotide polymorphisms (SNPs) are not significantly associated with carcass and meat traits (rib eye area, back fat thickness, shear force, total lipids, and myofibrillar fragmentation index), suggesting little utility of the analyzed polymorphisms of the IGF2, PMHC, and RORC genes as selection markers in the studied cattle populations.

Pseudoachondroplasia and multiple epiphyseal dysplasia: a 7-year comprehensive analysis of the known disease genes identify novel and recurrent mutations and provides an accurate assessment of their relative contribution.

Pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED) are relatively common skeletal dysplasias resulting in short-limbed dwarfism, joint pain, and stiffness. PSACH and the largest proportion of autosomal dominant MED (AD-MED) results from mutations in cartilage oligomeric matrix protein (COMP); however, AD-MED is genetically heterogenous and can also result from mutations in matrilin-3 (MATN3) and type IX collagen (COL9A1, COL9A2, and COL9A3). In contrast, autosomal recessive MED (rMED) appears to result exclusively from mutations in sulphate transporter solute carrier family 26 (SLC26A2). The diagnosis of PSACH and MED can be difficult for the nonexpert due to various complications and similarities with other related diseases and often mutation analysis is requested to either confirm or exclude the diagnosis. Since 2003, the European Skeletal Dysplasia Network (ESDN) has used an on-line review system to efficiently diagnose cases referred to the network prior to mutation analysis. In this study, we present the molecular findings in 130 patients referred to ESDN, which includes the identification of novel and recurrent mutations in over 100 patients. Furthermore, this study provides the first indication of the relative contribution of each gene and confirms that they account for the majority of PSACH and MED.

Additive effects of HLA alleles and innate immune genes determine viral outcome in HCV infection.

BACKGROUND: Chronic HCV infection is a leading cause of liver-related morbidity globally. The innate and adaptive immune responses are thought to be important in determining viral outcomes. Polymorphisms associated with the IFNL3 (IL28B) gene are strongly associated with spontaneous clearance and treatment outcomes. OBJECTIVE: This study investigates the importance of HLA genes in the context of genetic variation associated with the innate immune genes IFNL3 and KIR2DS3. DESIGN: We assess the collective influence of HLA and innate immune genes on viral outcomes in an Irish cohort of women (n=319) who had been infected from a single source as well as a more heterogeneous cohort (Swiss Cohort, n=461). In the Irish cohort, a number of HLA alleles are associated with different outcomes, and the impact of IFNL3-linked polymorphisms is profound. RESULTS: Logistic regression was performed on data from the Irish cohort, and indicates that the HLA-A*03 (OR 0.36 (0.15 to 0.89), p=0.027) -B*27 (OR 0.12 (0.03 to 0.45), p=<0.001), -DRB1*01:01 (OR 0.2 (0.07 to 0.61), p=0.005), -DRB1*04:01 (OR 0.31 (0.12 to 0.85, p=0.02) and the CC IFNL3 rs12979860 genotypes (OR 0.1 (0.04 to 0.23), p<0.001) are significantly associated with viral clearance. Furthermore, DQB1*02:01 (OR 4.2 (2.04 to 8.66), p=0.008), KIR2DS3 (OR 4.36 (1.62 to 11.74), p=0.004) and the rs12979860 IFNL3 'T' allele are associated with chronic infection. This study finds no interactive effect between IFNL3 and these Class I and II alleles in relation to viral clearance. There is a clear additive effect, however. Data from the Swiss cohort also confirms independent and additive effects of HLA Class I, II and IFNL3 genes in their prediction of viral outcome. CONCLUSIONS: This data supports a critical role for the adaptive immune response in the control of HCV in concert with the innate immune response.

Chromosome 7 gain and DNA hypermethylation at the HOXA10 locus are associated with expression of a stem cell related HOX-signature in glioblastoma.

BACKGROUND: HOX genes are a family of developmental genes that are expressed neither in the developing forebrain nor in the normal brain. Aberrant expression of a HOX-gene dominated stem-cell signature in glioblastoma has been linked with increased resistance to chemo-radiotherapy and sustained proliferation of glioma initiating cells. Here we describe the epigenetic and genetic alterations and their interactions associated with the expression of this signature in glioblastoma. RESULTS: We observe prominent hypermethylation of the HOXA locus 7p15.2 in glioblastoma in contrast to non-tumoral brain. Hypermethylation is associated with a gain of chromosome 7, a hallmark of glioblastoma, and may compensate for tumor-driven enhanced gene dosage as a rescue mechanism by preventing undue gene expression. We identify the CpG island of the HOXA10 alternative promoter that appears to escape hypermethylation in the HOX-high glioblastoma. An additive effect of gene copy gain at 7p15.2 and DNA methylation at key regulatory CpGs in HOXA10 is significantly associated with HOX-signature expression. Additionally, we show concordance between methylation status and presence of active or inactive chromatin marks in glioblastoma-derived spheres that are HOX-high or HOX-low, respectively. CONCLUSIONS: Based on these findings, we propose co-evolution and interaction between gene copy gain, associated with a gain of chromosome 7, and additional epigenetic alterations as key mechanisms triggering a coordinated, but inappropriate, HOX transcriptional program in glioblastoma.

A peroxisomal glutathione transferase of Saccharomyces cerevisiae is functionally related to sulfur amino acid metabolism

Saccharomyces cerevisiae cells contain three omega-class glutathione transferases with glutaredoxin activity (Gto1, Gto2, and Gto3), in addition to two glutathione transferases (Gtt1 and Gtt2) not classifiable into standard classes. Gto1 is located at the peroxisomes, where it is targeted through a PTS1-type sequence, whereas Gto2 and Gto3 are in the cytosol. Among the GTO genes, GTO2 shows the strongest induction of expression by agents such as diamide, 1-chloro-2,4-dinitrobenzene, tert-butyl hydroperoxide or cadmium, in a manner that is dependent on transcriptional factors Yap1 and/or Msn2/4. Diamide and 1-chloro-2,4-dinitrobenzene (causing depletion of reduced glutathione) also induce expression of GTO1 over basal levels. Phenotypic analyses with single and multiple mutants in the S. cerevisiae glutathione transferase genes show that, in the absence of Gto1 and the two Gtt proteins, cells display increased sensitivity to cadmium. A gto1-null mutant also shows growth defects on oleic acid-based medium, which is indicative of abnormal peroxisomal functions, and altered expression of genes related to sulfur amino acid metabolism. As a consequence, growth of the gto1 mutant is delayed in growth medium without lysine, serine, or threonine, and the mutant cells have low levels of reduced glutathione. The role of Gto1 at the S. cerevisiae peroxisomes could be related to the redox regulation of the Str3 cystathionine -lyase protein. This protein is also located at the peroxisomes in S. cerevisiae, where it is involved in transulfuration of cysteine into homocysteine, and requires a conserved cysteine residue for its biological activity.

The UlaG protein familly defines novel structural and functional motifs grafted on an ancient RNase fold

Background: Bacterial populations are highly successful at colonizing new habitats and adapting to changing environmental conditions, partly due to their capacity to evolve novel virulence and metabolic pathways in response to stress conditions and to shuffle them by horizontal gene transfer (HGT). A common theme in the evolution of new functions consists of gene duplication followed by functional divergence. UlaG, a unique manganese-dependent metallo-b-lactamase (MBL) enzyme involved in L-ascorbate metabolism by commensal and symbiotic enterobacteria, provides a model for the study of the emergence of new catalytic activities from the modification of an ancient fold. Furthermore, UlaG is the founding member of the so-called UlaG-like (UlaGL) protein family, a recently established and poorly characterized family comprising divalent (and perhaps trivalent)metal-binding MBLs that catalyze transformations on phosphorylated sugars and nucleotides. Results: Here we combined protein structure-guided and sequence-only molecular phylogenetic analyses to dissect the molecular evolution of UlaG and to study its phylogenomic distribution, its relatedness with present-day UlaGL protein sequences and functional conservation. Phylogenetic analyses indicate that UlaGL sequences are present in Bacteria and Archaea, with bona fide orthologs found mainly in mammalian and plant-associated Gramnegative and Gram-positive bacteria. The incongruence between the UlaGL tree and known species trees indicates exchange by HGT and suggests that the UlaGL-encoding genes provided a growth advantage under changing conditions. Our search for more distantly related protein sequences aided by structural homology has uncovered that UlaGL sequences have a common evolutionary origin with present-day RNA processing and metabolizing MBL enzymes widespread in Bacteria, Archaea, and Eukarya. This observation suggests an ancient origin for the UlaGL family within the broader trunk of the MBL superfamily by duplication, neofunctionalization and fixation. Conclusions: Our results suggest that the forerunner of UlaG was present as an RNA metabolizing enzyme in the last common ancestor, and that the modern descendants of that ancestral gene have a wide phylogenetic distribution and functional roles. We propose that the UlaGL family evolved new metabolic roles among bacterial and possibly archeal phyla in the setting of a close association with metazoans, such as in the mammalian gastrointestinal tract or in animal and plant pathogens, as well as in environmental settings. Accordingly, the major evolutionary forces shaping the UlaGL family include vertical inheritance and lineage-specific duplication and acquisition of novel metabolic functions, followed by HGT and numerous lineage-specific gene loss events.

Examining the virulence of Candida albicans transcription factor mutants using Galleria mellonella and mouse infection models.

The aim of the present study was to identify Candida albicans transcription factors (TFs) involved in virulence. Although mice are considered the gold-standard model to study fungal virulence, mini-host infection models have been increasingly used. Here, barcoded TF mutants were first screened in mice by pools of strains and fungal burdens (FBs) quantified in kidneys. Mutants of unannotated genes which generated a kidney FB significantly different from that of wild-type were selected and individually examined in Galleria mellonella. In addition, mutants that could not be detected in mice were also tested in G. mellonella. Only 25% of these mutants displayed matching phenotypes in both hosts, highlighting a significant discrepancy between the two models. To address the basis of this difference (pool or host effects), a set of 19 mutants tested in G. mellonella were also injected individually into mice. Matching FB phenotypes were observed in 50% of the cases, highlighting the bias due to host effects. In contrast, 33.4% concordance was observed between pool and single strain infections in mice, thereby highlighting the bias introduced by the "pool effect." After filtering the results obtained from the two infection models, mutants for MBF1 and ZCF6 were selected. Independent marker-free mutants were subsequently tested in both hosts to validate previous results. The MBF1 mutant showed impaired infection in both models, while the ZCF6 mutant was only significant in mice infections. The two mutants showed no obvious in vitro phenotypes compared with the wild-type, indicating that these genes might be specifically involved in in vivo adapt.

Genome sequence of the Drosophila melanogaster male-killing Spiroplasma strain MSRO endosymbiont.

Spiroplasmas are helical and motile members of a cell wall-less eubacterial group called Mollicutes. Although all spiroplasmas are associated with arthropods, they exhibit great diversity with respect to both their modes of transmission and their effects on their hosts; ranging from horizontally transmitted pathogens and commensals to endosymbionts that are transmitted transovarially (i.e., from mother to offspring). Here we provide the first genome sequence, along with proteomic validation, of an endosymbiotic inherited Spiroplasma bacterium, the Spiroplasma poulsonii MSRO strain harbored by Drosophila melanogaster. Comparison of the genome content of S. poulsonii with that of horizontally transmitted spiroplasmas indicates that S. poulsonii has lost many metabolic pathways and transporters, demonstrating a high level of interdependence with its insect host. Consistent with genome analysis, experimental studies showed that S. poulsonii metabolizes glucose but not trehalose. Notably, trehalose is more abundant than glucose in Drosophila hemolymph, and the inability to metabolize trehalose may prevent S. poulsonii from overproliferating. Our study identifies putative virulence genes, notably, those for a chitinase, the H2O2-producing glycerol-3-phosphate oxidase, and enzymes involved in the synthesis of the eukaryote-toxic lipid cardiolipin. S. poulsonii also expresses on the cell membrane one functional adhesion-related protein and two divergent spiralin proteins that have been implicated in insect cell invasion in other spiroplasmas. These lipoproteins may be involved in the colonization of the Drosophila germ line, ensuring S. poulsonii vertical transmission. The S. poulsonii genome is a valuable resource to explore the mechanisms of male killing and symbiont-mediated protection, two cardinal features of many facultative endosymbionts. IMPORTANCE: Most insect species, including important disease vectors and crop pests, harbor vertically transmitted endosymbiotic bacteria. These endosymbionts play key roles in their hosts' fitness, including protecting them against natural enemies and manipulating their reproduction in ways that increase the frequency of symbiont infection. Little is known about the molecular mechanisms that underlie these processes. Here, we provide the first genome draft of a vertically transmitted male-killing Spiroplasma bacterium, the S. poulsonii MSRO strain harbored by D. melanogaster. Analysis of the S. poulsonii genome was complemented by proteomics and ex vivo metabolic experiments. Our results indicate that S. poulsonii has reduced metabolic capabilities and expresses divergent membrane lipoproteins and potential virulence factors that likely participate in Spiroplasma-host interactions. This work fills a gap in our knowledge of insect endosymbionts and provides tools with which to decipher the interaction between Spiroplasma bacteria and their well-characterized host D. melanogaster, which is emerging as a model of endosymbiosis.

New genes for focal epilepsies with speech and language disorders.

The last 2 years have seen exciting advances in the genetics of Landau-Kleffner syndrome and related disorders, encompassed within the epilepsy-aphasia spectrum (EAS). The striking finding of mutations in the N-methyl-D-aspartate (NMDA) receptor subunit gene GRIN2A as the first monogenic cause in up to 20 % of patients with EAS suggests that excitatory glutamate receptors play a key role in these disorders. Patients with GRIN2A mutations have a recognizable speech and language phenotype that may assist with diagnosis. Other molecules involved in RNA binding and cell adhesion have been implicated in EAS; copy number variations are also found. The emerging picture highlights the overlap between the genetic determinants of EAS with speech and language disorders, intellectual disability, autism spectrum disorders and more complex developmental phenotypes.

Candida albicans Inhibits Pseudomonas aeruginosa Virulence through Suppression of Pyochelin and Pyoverdine Biosynthesis.

Bacterial-fungal interactions have important physiologic and medical ramifications, but the mechanisms of these interactions are poorly understood. The gut is host to trillions of microorganisms, and bacterial-fungal interactions are likely to be important. Using a neutropenic mouse model of microbial gastrointestinal colonization and dissemination, we show that the fungus Candida albicans inhibits the virulence of the bacterium Pseudomonas aeruginosa by inhibiting P. aeruginosa pyochelin and pyoverdine gene expression, which plays a critical role in iron acquisition and virulence. Accordingly, deletion of both P. aeruginosa pyochelin and pyoverdine genes attenuates P. aeruginosa virulence. Heat-killed C. albicans has no effect on P. aeruginosa, whereas C. albicans secreted proteins directly suppress P. aeruginosa pyoverdine and pyochelin expression and inhibit P. aeruginosa virulence in mice. Interestingly, suppression or deletion of pyochelin and pyoverdine genes has no effect on P. aeruginosa's ability to colonize the GI tract but does decrease P. aeruginosa's cytotoxic effect on cultured colonocytes. Finally, oral iron supplementation restores P. aeruginosa virulence in P. aeruginosa and C. albicans colonized mice. Together, our findings provide insight into how a bacterial-fungal interaction can modulate bacterial virulence in the intestine. Previously described bacterial-fungal antagonistic interactions have focused on growth inhibition or colonization inhibition/modulation, yet here we describe a novel observation of fungal-inhibition of bacterial effectors critical for virulence but not important for colonization. These findings validate the use of a mammalian model system to explore the complexities of polymicrobial, polykingdom infections in order to identify new therapeutic targets for preventing microbial disease.

The Aspergillus fumigatus septins play pleiotropic roles in septation, conidiation, and cell wall stress, but are dispensable for virulence.

Septins are a conserved family of GTPases that regulate important cellular processes such as cell wall integrity, and septation in fungi. The requirement of septins for virulence has been demonstrated in the human pathogenic yeasts Candida albicans and Cryptococcus neoformans, as well as the plant pathogen Magnaporthe oryzae. Aspergillus spp. contains five genes encoding for septins (aspA-E). While the importance of septins AspA, AspB, AspC, and AspE for growth and conidiation has been elucidated in the filamentous fungal model Aspergillus nidulans, nothing is known on the role of septins in growth and virulence in the human pathogen Aspergillus fumigatus. Here we deleted all five A. fumigatus septins, and generated certain double and triple septin deletion strains. Phenotypic analyses revealed that while all the septins are dispensable in normal growth conditions, AspA, AspB, AspC and AspE are required for regular septation. Furthermore, deletion of only the core septin genes significantly reduced conidiation. Concomitant with the absence of an electron-dense outer conidial wall, the ΔaspB strain was also sensitive to anti-cell wall agents. Infection with the ΔaspB strain in a Galleria mellonella model of invasive aspergillosis showed hypervirulence, but no virulence difference was noted when compared to the wild-type strain in a murine model of invasive aspergillosis. Although the deletion of aspB resulted in increased release of TNF-α from the macrophages, no significant inflammation differences in lung histology was noted between the ΔaspB strain and the wild-type strain. Taken together, these results point to the importance of septins in A. fumigatus growth, but not virulence in a murine model.