1. Understanding Fungal Pathogenesis with High-throughput Sequencing

Vincent Bruno

The invention of massively parallel DNA sequencing and the subsequent development of RNA-seq, ChIP-seq and microbiome sequencing have radically changed the landscape of molecular and systems biology by allowing researchers to address biological problems with a sensitivity and scope that was not previously possible. This chapter discusses how each of these high-throughput sequencing (HTS)-based approaches is being used to address biological questions within the field of human fungal pathogenesis. As the cost of sequencing becomes more affordable, the amount of sequence depth coverage that will be attainable will increase significantly allowing researchers to apply these genomic techniques to more in vivo models and clinical samples for which a great deal of coverage is required. Integrated approaches, which combine multiple HTS-based experiments, will provide necessary insight into the complex interactions between the host, microbiota and fungal pathogens.

2. Comparative Genomics and Evolutionary Analyses of Human Fungal Pathogens

David A. Fitzpatrick

Recent advances in next generation sequencing techniques have dramatically increased the availability of genomic data. Due to their relatively small genome size and importance as human and crop pathogens, over one hundred fungal genomes have been completely sequenced and published to date. This number is expected to increase dramatically over the coming years with individual institutions sequencing their own fungi of interest and the initiation of an ambitious project to sequence 1000 neglected fungal genomes (https://1000.fungalgenomes.org/). Currently a significant proportion of the available genomes are human pathogens as well as closely related non-pathogenic species. This type of data allows us to perform comparative genomic analyses between pathogenic and non-pathogenic species in an effort to uncover molecular mechanisms related to disease. The availability of full genome sequences also allows us to investigate many evolutionary processes in fungi such as horizontal gene transfer, gene fusions/fissions and intron gains and losses. The following chapter will discuss a selection of key comparative genomic analyses that have been performed on a number of the most common human pathogens and also illustrate how complete fungal genomes have helped us understand some of the mechanisms that have shaped fungal evolution.

3. Data-driven Systems Biology of Fungal Infections

Fabian Horn, Vito Valiante, Reinhard Guthke and Axel A. Brakhage

Recent technical advancements in biotechnology unlocked a new fascinating period of research. High-throughput data can be obtained within biomolecular experiments with reasonable efforts and costs. This enables the investigation of the host-fungal interactions at a system-wide level. The bottleneck in the experiment workflow has shifted from obtaining the data to its proper analysis. The analysis of high-throughput data can be described by the operational protocol of data-driven systems biology. The protocol includes experimental design, data pre-processing, feature selection, computational modeling, and biological evaluation. Throughout this procedure, data need to be analyzed within the corresponding biological context. The goal of the analysis pipeline is to obtain model variables and parameters in order to make new hypotheses for further experiments. The intertwined nature of the host-pathogen interaction is also reflected in the complex data analysis where first experiences are currently gathered and new methods developed. Pressing challenges are the integration of different levels of omics-data, i.e. modeling should consider multiple molecular levels and the integration of different technologies at each level. Advances in systems biology are expected to help translate in vivo knowledge about fungal infections into clinical applications.

4. Comparative Pathogenesis: Transcriptomic Analyses of Host Cell-Fungus Interactions

Elaine Bignell

The contribution of host activities to positive, and negative, outcomes of fungal disease is increasingly recognised. Technological advances are permitting scientists to unravel gene-by-gene the complexities of the host-pathogen interaction, an objective which will remain a crucial component of infectious disease research for the foreseeable future, and will likely yield a new generation of immunotherapeutic antifungal entities. The fungal host-pathogen interaction includes a remarkable array of behaviours from pathogen-mediated killing of host cells, to intracellular occupancy and lytic expulsion, behaviours which drive the outcomes of disease by governing pathogen replication, immune evasion, survival, and dissemination. This chapter will explore the fruits of transcriptomic analyses of fungal host-pathogen interactions.

5. Animal Models of Human Fungal Infection

Donna M. MacCallum

Fungal infection is a major cause of morbidity and mortality, with most infections caused by dermatophytes, Candida species, Aspergillus species, Cryptococcus neoformans and Pneumocystis jirovecii. Whilst dermatophytes can be considered primary pathogens, the majority of fungal infections are caused by opportunistic fungal species. Candida infections tend to originate from commensal sources, but Aspergillus, Cryptococcus and Pneumocystis infections are acquired from the environment. These infections are found in severely ill patients or in immunosuppressed individuals, particularly those with HIV/AIDS. Mortality associated with these infections remains high, partially due to difficulties in diagnosis, but also due to limited therapeutic choices. In order to facilitate development of these badly needed diagnostics and antifungal agents, a greater understanding of fungal pathogenesis is required. Animal models provide opportunities to investigate fungal disease initiation and progression and to evaluate novel antifungal agents. Animal models of dermatophyte, Candida, Aspergillus, Cryptococcus and Pneumocystis infections are described and their contribution to our current understanding of these important fungal infections discussed.

6. Host Responses to Fungal Infection

David L. Moyes, Jonathan P. Richardson and Julian R. Naglik

The three major opportunistic fungal pathogens of humans are Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans. These pathogens primarily infect immunocompromised individuals either as a result of immunodeficiency (e.g. neutropenia, HIV infection) or intervention therapy (e.g. leukemia, organ transplant, cancer). This highlights the importance of host immune defences in controlling or preventing fungal infections. In health, these fungal pathogens initially interact with mucosal surfaces which may lead to colonisation and establishment of commensalism, but in the immunocompromised these fungi can cause serious superficial infections and may disseminate to cause life-threatening systemic infections. To combat these fungal infections, the host utilises vast communication network of cells, proteins and chemical signals distributed in blood and tissues, which constitute innate and adaptive immunity. In this chapter we will review how the host recognises these fungi, the events induced by fungal cells, and the host immune defences that ultimately resolve the infections during health. The overview will primarily target C. albicans, the most common fungal pathogen of humans.

7. A Molecular Update on the Pathogenesis of Candidiasis

Duncan Wilson and Bernhard Hube

In contrast to most of the human pathogenic fungi described in this book, pathogenic species of the genus Candida do not normally exist in ecological niches such as soil or compost. Instead, these fungi have evolved in close association with warm-blooded animals, such as humans. For most people, at most times, these yeasts exist as harmless members of the microbiome. However, some species are capable of causing devastating infections in their human hosts. In this chapter we summarise and discuss important recent developments that have been made in our understanding of the molecular basis of Candida pathogenicity, with an emphasis on the dominant pathogenic species, Candida albicans.

8. Virulence Characteristics of Aspergillus fumigatus

Rebecca A. Owens, Grainne O'Keeffe, Karen A. O'Hanlon, Lorna Gallagher and Sean Doyle

Aspergillus fumigatus is an opportunistic fungal pathogen which causes serious disease, broadly termed aspergillosis, in immunocompetent and immunocompromised individuals. Advances in the diagnosis of aspergillosis exploit detection of fungal nucleic acid via PCR and secreted galactomannan by ELISA. New diagnostic strategies are emerging based on detection of secreted low molecular mass metabolites. Drug treatments, which interfere with membrane biosynthesis and integrity, as well as cell wall function, are available to treat infection with A. fumigatus. No single characteristic appears to confer complete virulence on A. fumigatus, rather, it appears to depend on a combination of survival factors, in part derived from its function as a saprophyte, to facilitate host colonisation and fungal growth. These attributes include a robust cell wall, small hydrophobic conidia and biofilm architecture; resistance to oxidative stress, hypoxia and iron starvation; secondary metabolite biosynthesis and secretion, as well as thermotolerance and an ability to secrete degradative enzymes for nutrient uptake. Attenuation of the efficiency of these survival strategies offers possibilities for the development of new classes of antifungal drugs.

9. Cryptococcus

Rocío García-Rodas, Radames J.B. Cordero and Oscar Zaragoza

Cryptococcus neoformans is a cosmopolitan pathogen of major concern among HIV-infected patients and has a significant incidence in developing countries. Cryptococcus neoformans blastoconidia or desiccated spores are inhaled and can cause pneumonia and meningitis in immunodeficient individuals, being fatal if not treated. Its sibling species Cryptococcus gattii is more restricted to tropical areas and infects mainly immunocompetent individuals. Cryptococcus interactions with the human host have been widely studied, in particular its capacity to modulate the immune response, replicate within macrophages, disseminate from the lungs to the central nervous system, and/or remain in a latent state for years. Our current understanding of the virulence determinants that facilitate Cryptococcus to cause infection, particularly its polysaccharide capsule and melanin accumulation, as well as other traits that confer adaptation to the host will be reviewed in this chapter.

10. Dermatophytes as Saprophytes and Pathogens

Michel Monod, Bernard Mignon and Peter Staib

Dermatophytes infect the stratum corneum, nails and hair and are the most common agents of superficial mycoses in humans and animals. At present the genome of seven species has been sequenced. Between 22.5 and 24 Mb, the dermatophyte genomes are smaller in size than those of Coccidioides spp., Histoplasma spp. and Aspergillus spp. They are enriched for particular families of genes encoding secreted proteases, fungal specific kinases and proteins containing the LysM domain that is known to bind chitin. Different tools were recently developed to improve genetic analyses of dermatophytes, including efficient systems for targeted gene inactivation, gene silencing and broad transcriptional profiling techniques. Unexpectedly, gene expression profiles in the skin and hair of infected guinea pigs were found to be very different from those during in vitro growth using hard keratin as a substrate. Instead of the major in vitro expressed protease genes, others were found to only be activated in the skin of infected animals. In other words, the expression of putative virulence genes in dermatophytoses is more complex than previously assumed and likely depends on the site and type of infection. Further broad transcriptional profiling approaches during infections will give new insights into the pathogenic mechanisms of dermatophytes.

11. Pathogenesis Mechanisms of Histoplasma capsulatum

Chad A. Rappleye

The dimorphic fungal pathogen Histoplasma capsulatum causes respiratory and systemic disease in both immunocompromised and immunocompetent individuals. In mammalian hosts, Histoplasma grows as pathogenic yeasts which survive and replicate within phagocytes. Genetic studies have Identified a signaling kinase and 3 transcription factors factors that regulate the thermally-induced yeast-phase differentiation process. The mechanisms that facilitate Histoplasma pathogenesis (summarized in Figure 11.1) centre on three primary tasks facing intracellular pathogens: (1) concealment of molecular signatures to avoid detection by phagocytes and activation of immune defenses, (2) detoxification of antimicrobial defence molecules produced by phagocytes, and (3) acquisition of essential nutrients for growth and replication within the nutrient-limited phagosome. Demonstrated virulence factors accomplishing these required aspects of pathogenesis include synthesis of α-glucan which masks immunostimulatory yeast cell wall β-glucans, production and secretion of antioxidant molecules (extracellular superoxide dismutase and catalase) to defend specifically against phagocyte-derived reactive oxygen, and secretion of multiple factors for iron acquisition (siderophores and iron reductases). In addition, Histoplasma yeasts secrete several novel factors with undefined functions but which are likely linked to pathogenesis by virtue of their specific expression only by pathogenic-phase cells and their extracellular localization. Together, these mechanisms enable Histoplasma yeasts to successfully establish infections in spite of fully functional immune defences of the host.

12. Blastomyces dermatitidis and Blastomycosis

Gregory M. Gauthier

The etiologic agent of blastomycosis, Blastomyces dermatitidis, belongs to a group of ascomycete fungi that exhibit thermal dimorphism. In the soil (22-25°C), these fungi grow as filamentous mold that produce infectious conidia. Following the disruption of soil by activities such as construction, aerosolized conidia and mold fragments are inhaled into the lungs of a host (37°C) and convert into pathogenic yeast. In the yeast form, B. dermatitidis is capable of evading immune defenses by altering the composition of the fungal cell wall and impairing the host's cytokine response. Approximately 25-40% of patients infected with B. dermatitidis develop disseminated disease, which frequently involves the skin or bone. Diagnosis of blastomycosis requires a high degree of clinical suspicion coupled with the use of culture and non-culture diagnostics. Treatment requires the use of polyene or azole antifungals.

13. New Insights into Pathogenesis of Pneumocystis Pneumonia

Jakrapun Pupaiboo and Andrew H. Limper

Pneumocystis pneumonia (PCP) remains a major cause of morbidity and mortality in HIV-infected patients. In addition, the prevalence of PCP is increasing in non-HIV patients with suppressed immune status, following the use of potent immunosuppressive therapies. Our understanding of Pneumocystis is based on immunosuppressed animal models. Investigations of the Pneumocystis life cycle, pathogenesis, and host immune response provide attractive targets for the development of novel anti-Pneumocystis agents. Sulfamethoxazole-trimethoprim remains the first line regimen for treatment and prophylaxis of PCP. Many aspects of Pneumocystis biology remain unanswered or controversial, such as respiratory colonization, mode of transmission, clinical significance of mutations to currently available anti-Pneumocystis agents, and prophylaxis and therapy in non-HIV immunocompromised patients. There is a critical need to continue investigating this unique medically important pathogenic fungus in order to allow us to develop more effective strategies to prevent and treat Pneumocystis infection.

14. Paracoccidioides Mechanisms of Pathogenesis and Virulence

Juliana Alves Parente, Clayton Luiz Borges, Maristela Pereira, Alexandre Melo Bailão, Rosely Maria Zancopé Oliveira and Célia Maria de Almeida Soares

Members of the genus Paracoccidioides are the causative agents of one of the most prevalent systemic mycoses in Latin America, namely, paracoccidioidomycosis (PCM). Studies have reported different degrees of pathogenicity and virulence among the members of this genus and provided evidence of variation in host susceptibility to infection. The identification and validation of virulence factors has been hindered by difficulties in the genetic manipulation of this pathogen, although studies have demonstrated that Paracoccidioides pathogenicity is facilitated through a number of virulence attributes, including fungal dimorphism, the composition of the cell surface and adherence to host tissues, secretion of molecules comprising the virulence arsenal of the fungus, competition with the host for essential nutrients, such as iron, and the pathogen's consequent adaptation to the host environment. This chapter provides information on the current knowledge of the virulence of the genus Paracoccidioides.