Integration of host, pathogen and microbiome -omics data for studying infectious diseases

In an ever-growing worldwide population, human infectious diseases are an increasingly serious problem for public health. In particular, more than a million deaths and millions of infectious disease cases per year caused by fungal pathogens have been reported globally in recent years. Hence, more investments must be put into fungal research to overcome the problem. The opportunistic pathogen Candida albicans and the airborne Aspergillus fumigatus are the two most prevalent fungal pathogens causing serious issues in medical care units. Despite the recent advances in fungal research, there is little knowledge about the role of fungal metabolism in developing the infection when coexisting within the human body with microbial community members in different organs. This dissertation applied computational tools, and implemented systems biology approaches to uncover key factors in the colonization of the pathogens, especially C. albicans and A. fumigatus, from a systems biology perspective and unseen by wet-lab experiments alone. Next to multi-omics data analysis, a major effort was put into genome-scale metabolic models (GEMs) generation and analysis as a promising approach to shed light on the role of metabolism in developing the infection. In brief, this thesis sheds light on key factors leading to the inhibition or promotion of fungal growth. This especially includes the first available GEM reconstruction of C. albicans to theoretically study the intricate interaction of the fungus with the human host and the microbial community members. Lastly, a platform of 252 A. fumigatus GEMs at the strain resolution was generated. It revealed the phenotypic diversity of A. fumigatus strains isolated from different hospitals and farms in Germany and explained the contribution of the fungus to the shaping of the metabolic landscape of the lung microbiome in a favorable manner for fungal growth

Molecular characterization of an adaptive response to alkylating agents in the opportunistic pathogen Aspergillus fumigatus.

An adaptive response to alkylating agents based upon the conformational change of a methylphosphotriester (MPT) DNA repair protein to a transcriptional activator has been demonstrated in a number of bacterial species, but this mechanism appears largely absent from eukaryotes. Here, we demonstrate that the human pathogen Aspergillus fumigatus elicits an adaptive response to sub-lethal doses of the mono-functional alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). We have identified genes that encode MPT and O(6)-alkylguanine DNA alkyltransferase (AGT) DNA repair proteins; deletions of either of these genes abolish the adaptive response and sensitize the organism to MNNG. In vitro DNA repair assays confirm the ability of MPT and AGT to repair methylphosphotriester and O(6)-methylguanine lesions respectively. In eukaryotes, the MPT protein is confined to a select group of fungal species, some of which are major mammalian and plant pathogens. The evolutionary origin of the adaptive response is bacterial and rooted within the Firmicutes phylum. Inter-kingdom horizontal gene transfer between Firmicutes and Ascomycete ancestors introduced the adaptive response into the Fungal kingdom. Our data constitute the first detailed characterization of the molecular mechanism of the adaptive response in a lower eukaryote and has applications for development of novel fungal therapeutics targeting this DNA repair system

Genome-Wide Fitness Test and Mechanism-of-Action Studies of Inhibitory Compounds in Candida albicans

Candida albicans is a prevalent fungal pathogen amongst the immunocompromised population, causing both superficial and life-threatening infections. Since C. albicans is diploid, classical transmission genetics can not be performed to study specific aspects of its biology and pathogenesis. Here, we exploit the diploid status of C. albicans by constructing a library of 2,868 heterozygous deletion mutants and screening this collection using 35 known or novel compounds to survey chemically induced haploinsufficiency in the pathogen. In this reverse genetic assay termed the fitness test, genes related to the mechanism of action of the probe compounds are clearly identified, supporting their functional roles and genetic interactions. In this report, chemical–genetic relationships are provided for multiple FDA-approved antifungal drugs (fluconazole, voriconazole, caspofungin, 5-fluorocytosine, and amphotericin B) as well as additional compounds targeting ergosterol, fatty acid and sphingolipid biosynthesis, microtubules, actin, secretion, rRNA processing, translation, glycosylation, and protein folding mechanisms. We also demonstrate how chemically induced haploinsufficiency profiles can be used to identify the mechanism of action of novel antifungal agents, thereby illustrating the potential utility of this approach to antifungal drug discovery

Comparative genomics and transcriptomics elucidate virulence mechanisms and host responses in infectious diseases

The main thematic area of the present thesis is the development and application of bioinformatics pipelines, namely whole-genome sequence (WGS) analysis and transcriptome profile analysis. These pipelines were applied to study the fungal pathogen Aspergillus fumigatus (Manuscripts I, III, and IV) and the early human immune mechanisms activated in response to different types of pathogens (bacteria, fungi, and co-infections) in sepsis patients (Manuscript II). The comparative genomic and transcriptomic analyses applied in my thesis have significantly improved our understanding of fungal pathogenicity as well as the pathogen-specific immune response mechanisms of the human host. Next to a number of novel insights, my work included in this thesis has generated a large number of new hypotheses based on big-data analysis, offering the scientific community the possibility to design exciting new research to confirm them in future experimental studies and bring us closer to actual precision medicine for infectious diseases

Analysis of putative quadruplex-forming sequences in fungal genomes: Novel antifungal targets?

Fungal infections cause >1 million deaths annually and the emergence of antifungal resistance has prompted the exploration for novel antifungal targets. Quadruplexes are four-stranded nucleic acid secondary structures, which can regulate processes such as transcription, translation, replication and recombination. They are also found in genes linked to virulence in microbes, and ligands that bind to quadruplexes can eliminate drug-resistant pathogens. Using a computational approach, we quantified putative quadruplex-forming sequences (PQS) in 1359 genomes across the fungal kingdom and explored their presence in genes related to virulence, drug resistance and biological processes associated with pathogenicity in Aspergillus fumigatus. Here we present the largest analysis of PQS in fungi and identify significant heterogeneity of these sequences throughout phyla, genera and species. PQS were genetically conserved in Aspergillus spp. and frequently pathogenic species appeared to contain fewer PQS than their lesser/non-pathogenic counterparts. GO-term analysis identified that PQS-containing genes were involved in processes linked with virulence such as zinc ion binding, the biosynthesis of secondary metabolites and regulation of transcription in A. fumigatus. Although the genome frequency of PQS was lower in A. fumigatus, PQS could be found enriched in genes involved in virulence, and genes upregulated during germination and hypoxia. Moreover, PQS were found in genes involved in drug resistance. Quadruplexes could have important roles within fungal biology and virulence, but their roles require further elucidation

The Ins and Outs and ABCs of Antifungal Drug Transport: Characterizing the Role of Membrane Transporters in Pathogenic Fungi

Title from PDF of title page, viewed March 29, 2023Dissertation advisor: Theodore C. WhiteVitaIncludes bibliographical referencesDissertation (Ph.D)--Department of Cell Biology and Biophysics, Department of Molecular Biology and Biochemistry. University of Missouri--Kansas City, 2016Pathogenic fungi cause serious disease and even death in humans, animals and plants. In medicine and agriculture alike, fungal infections are widespread and represent a significant threat to global public health. The number and array of fungal species, each exhibiting diverse mechanisms of pathogenesis, makes the challenge of fungal infection prevention and treatment formidable. The current repertoire of effective antifungal treatment strategies is very limited. As a result of increased use of antifungals to treat and prevent clinical fungal infections in humans, as well as widespread use of fungicides in agriculture, fungal strains that are resistant to each of the classes of antifungals have emerged. A significant rise in the number of fungal infections in recent years, combined with an increasing amount of drug resistant fungal strains is great cause for concern and places urgency on the development of new and more effective fungal infection treatment and prevention strategies. New fungal drug targets may be discovered with a better understanding of basic fungal biological processes. New or improved fungal infection treatment strategies may stem from a more complete knowledge of fungal response to drug treatment, worldwide trends of fungal pathogenesis and development of resistance, and even fungal evolutionary relationships. The goal of this research was to characterize the most basic fungal/drug interactions, which includes the balance of uptake, retention, and efflux of antifungal drugs in the fungal cell. We analyzed a variety of environmental and cellular factors that affect antifungal drug uptake and retention in two medically and agriculturally important pathogenic fungi, Aspergillus fumigatus and Magnaporthe oryzae. We then identified and characterized a number of A. fumigatus plasma membrane ABC transporters that may contribute to antifungal drug resistance due to their role in the efflux of antifungal drugs. To analyze antifungal drug uptake and retention, we developed an assay to directly measure accumulation of radioactively-labeled azoles in A. fumigatus and M. oryzae. Our analysis of drug uptake under a variety of cellular and environmental conditions demonstrated that these filamentous fungi import azoles by a facilitated diffusion mechanism. Contrasts between the M. oryzae and A. fumigatus data revealed interesting differences that suggest variations in expression, induction, or function of efflux transporters in the two organisms. To analyze antifungal efflux, we cloned and expressed a selection of putative ABC transporter genes from the A. fumigatus genome and heterologously expressed each gene in S. cerevisiae for direct characterization of drug efflux potential. Our efflux transporter analysis showed differences in substrate specificity, drug susceptibilities, energy-dependent efflux activity, and effect of efflux-inhibitor treatment between the different transporters. These data illustrate the complexity of predicting and counteracting fungal drug treatment response, but also highlight the possibilities for identifying new drug targets.Introduction to azole drug resistance mechanisms in pathogenic fungi -- Azole drug import into the pathogenic fungus Aspergillus Fumigatus -- Azole drug import into the fungal plant pathogen Magnaporthe Oryzae -- Functional and inducible expression of A. Fumigatus putative efflux transporters in S. Cerevisiae -- Conclusions and future direction

OGEE v2: an update of the online gene essentiality database with special focus on differentially essential genes in human cancer cell lines

OGEE is an Online GEne Essentiality database. To enhance our understanding of the essentiality of genes, in OGEE we collected experimentally tested essential and non-essential genes, as well as associated gene properties known to contribute to gene essentiality. We focus on large-scale experiments, and complement our data with text-mining results. We organized tested genes into data sets according to their sources, and tagged those with variable essentiality statuses across data sets as conditionally essential genes, intending to highlight the complex interplay between gene functions and environments/experimental perturbations. Developments since the last public release include increased numbers of species and gene essentiality data sets, inclusion of non-coding essential sequences and genes with intermediate essentiality statuses. In addition, we included 16 essentiality data sets from cancer cell lines, corresponding to 9 human cancers; with OGEE, users can easily explore the shared and differentially essential genes within and between cancer types. These genes, especially those derived from cell lines that are similar to tumor samples, could reveal the oncogenic drivers, paralogous gene expression pattern and chromosomal structure of the corresponding cancer types, and can be further screened to identify targets for cancer therapy and/or new drug development. OGEE is freely available at http://ogee.medgenius.info