Hyphal organisms are a commonly used model system for studies of polarised growth. While growing hyphal tips offer a good example of polarised growth, little detail of the process of polarisation can be determined from them. Hyphal branching offers a good example of the development of polarity, however to date it has been largely impractical to study hyphal branching, due to the irregular timing and location along the hypha of natural branch formation. Chemical induction of branches circumnavigates this problem, using a localised concentration of nutrients adjacent to the growing hypha to stimulate controlled branching. Using previous studies of hyphal branching combined with the current understanding of hyphal tip growth, a model of the branching process was established (Jackson et al. 2001). Reception of a branching cue leads to the formation of a radial F-actin array at the new branch site. This, by means of either delivery of cell wall softening enzymes or direct mechanical pressure, leads in turn to the emergence of a visible bump in the hyphal wall. This bump enlarges and then progresses into the branch proper. The bump stage of the branching process is perhaps the least understood, with existing studies giving detail of pre- and post-bump events. The research described in this thesis suggests that bump emergence is a two stage process; an early bump stage, where localised cell wall softening leads to turgor pressure in the cell pushing out the bump, and a late bump, where F-actin is arranged into the developing branch. The addition of an F-actin inhibitor to the induction solution confirmed that the early bump stage is relatively independent of the F-actin cytoskeleton, however this experiment was unable to test F-actin's role in full branch development.

The aims of this work were to investigate the signal transduction pathways controlling the yeast-hyphal morphological transition of Candida albicans, and to gain a clearer understanding of the importance of hyphal formation of the virulence of this organism. The work can be divided into two areas. Firstly, the recently discovered species Candida dubliniensis, the only species in addition to C. albicans capable of forming true non-constricted hyphae, was examined in comparison to C. albicans to compare their virulence capability in vitro and in vivo. The two species were compared with respect to hypha formation, adherence, possession of SAP genes and virulence in the mouse model of systemic candidosis. C. dubliniensis possessed at least a homologue to each of the nine known C. albicans SAP genes, adhered to human cells to a greater degree on exposure in glucose, formed hyphae slightly less efficiently than C. albicans and was less virulent in mice. C. dubliniensis has been isolated particularly from the mouths of HIV positive and AIDS patients. The results of the virulence assessment could be interpreted as reflecting its epidemiological occurrence, - increased adherence on exposure to glucose may be a response to dietary sugar and the reduced virulence would explain in part its association with immunocompromised hosts. Secondly, the role of phosphoinositide signalling in control of the yeast-hyphal transition was investigated by the cloning and characterisation of two putative phosphatidylinositol 4-kinase genes from C. albicans, named CaPIK1 and CaPIK2. Both genes were cloned through their homology to the S. cerevisiae PIK1 gene. Disruption of the CaPIK1 gene in C. albicans indicated that it had no obvious role in the control of hypha formation.

Hyphal organisms are a commonly used model system for studies of polarised growth. While growing hyphal tips offer a good example of polarised growth, little detail of the process of polarisation can be determined from them. Hyphal branching offers a good example of the development of polarity, however to date it has been largely impractical to study hyphal branching, due to the irregular timing and location along the hypha of natural branch formation. Chemical induction of branches circumnavigates this problem, using a localised concentration of nutrients adjacent to the growing hypha to stimulate controlled branching. Using previous studies of hyphal branching combined with the current understanding of hyphal tip growth, a model of the branching process was established (Jackson et al. 2001). Reception of a branching cue leads to the formation of a radial F-actin array at the new branch site. This, by means of either delivery of cell wall softening enzymes or direct mechanical pressure, leads in turn to the emergence of a visible bump in the hyphal wall. This bump enlarges and then progresses into the branch proper. The bump stage of the branching process is perhaps the least understood, with existing studies giving detail of pre- and post-bump events. The research described in this thesis suggests that bump emergence is a two stage process; an early bump stage, where localised cell wall softening leads to turgor pressure in the cell pushing out the bump, and a late bump, where F-actin is arranged into the developing branch. The addition of an F-actin inhibitor to the induction solution confirmed that the early bump stage is relatively independent of the F-actin cytoskeleton, however this experiment was unable to test F-actin's role in full branch development.

Tip growth is the primary form of growth in hyphal organisms and some plant cells. Tip growth in hyphae is highly dependent on F-actin, which acts to regulate and support growth. One of the models suggested for tip growth, the amebae model of tip growth, suggests that F-actin may also be the primary source of protrusive force for tip growth in some conditions, and that proteins with a similar function to animal integrins would be present an involved in tip growth (Heath and Steinberg 1999). In this thesis we examine the role of F-actin in the growth of the oomycete Phytophthora cinnamomi and the effects on growth of the F-actin disrupting compound Latrunculin B. We demonstrate that F-actin plays a critical role in the tip growth of Phytophthora cinnamomi with it's disruption causing rapid cessation in directional growth, followed by significant subapical swelling. Further more we examine Phytophthora cinnamomi for the presence of an B4 integrin like protein that has been previously reported in the oomycete Achlya bisexualis (Chitcholtan & Garrill 2005) and show that the B4 integrin like protein is not present in Phytophthora cinnamomi. These experiments help further our understanding of tip growth in Phytophthora cinnamomi an economically important plant pathogen.

Tip growth is the primary form of growth in hyphal organisms and some plant cells. Tip growth in hyphae is highly dependent on F-actin, which acts to regulate and support growth. One of the models suggested for tip growth, the amebae model of tip growth, suggests that F-actin may also be the primary source of protrusive force for tip growth in some conditions, and that proteins with a similar function to animal integrins would be present an involved in tip growth (Heath and Steinberg 1999). In this thesis we examine the role of F-actin in the growth of the oomycete Phytophthora cinnamomi and the effects on growth of the F-actin disrupting compound Latrunculin B. We demonstrate that F-actin plays a critical role in the tip growth of Phytophthora cinnamomi with it's disruption causing rapid cessation in directional growth, followed by significant subapical swelling. Further more we examine Phytophthora cinnamomi for the presence of an B4 integrin like protein that has been previously reported in the oomycete Achlya bisexualis (Chitcholtan & Garrill 2005) and show that the B4 integrin like protein is not present in Phytophthora cinnamomi. These experiments help further our understanding of tip growth in Phytophthora cinnamomi an economically important plant pathogen.

Arbuscular mycorrhizal fungi (AMF) are an ancient group of obligate symbionts, colonizing the roots of over 72% of land plants, increasing the uptake of nutrients from the soil, and providing many fitness benefits to their host plants. The multinucleate and coenocytic nature of AMF have interested researchers for decades, leading to many theories of the evolution, and genetic organization of these organisms. Recent findings propose that AMF carry two types of strains, identified based on putative MAT-loci, as either homokaryotic, carrying multiple and genetically similar nuclei, or dikaryotic with co-existing nuclei deriving from two parental strains. In other fungi, hyphal fusions, or anastomosis, between compatible strains results in nuclear transfer, creating heterokaryotic spore progeny. It has been hypothesized that dikaryotic AMF strains arose from the anastomosis between compatible homokaryons harbouring different nucleotypes. The goal of this research is to determine whether anastomosis events, known to occur in other fungi, like homokaryon-homokaryon, homokaryon-dikaryon (Buller phenomenon), and/or dikaryon-dikaryon nuclear exchanges also occur in AMF. To achieve this, the anastomosis frequencies between 15 crosses of homokaryotic and dikaryotic strains of the model AMF species Rhizophagus irregularis were examined using microscopy and droplet digital PCR (ddPCR) to determine if nuclear transfer between strains is possible. Overall, these experiments build on the existing evidence of compatible interactions between strains of R. irregularis.

Striatin family proteins contain a caveolin binding domain, a coiled-coil motif, a calmodulin binding domain, and a WD-repeat domain. Homologs of striatin protein have been However, our knowledge of the function and the molecular mechanism of fungal striatin homologs is limited. Based on the conserved sequences of functional domains, I hypothesized that the fungal striatin orthologs also act as scaffolding proteins that are functionally conserved among fungal species and involved in multiple types of development in the diverse kingdom Mycota. I used reverse genetic strategies to study the function of the Aspergillus nidulans striatin ortholog (strA) and the Colletotrichum graminicola striatin ortholog (str1). In assays of sexual development, the strA deletion strain (ΔstrA) produces fewer ascospores with smaller cleistothecia, while the str1 deletion strain (Δstr1) is defective in perithecia development. The ΔstrA phenotypes indicate that StrA is associated with ascosporogenesis in cleistothecia. Both ΔstrA and Δstr1 are reduced in radial growth and in conidia production. The Δstr1 strain is also altered in its spiral growth pattern and morphology of conidia and hyphopodia, but it produces appressoria similar to wild type. The pairing of nitrate non-utilizing mutants demonstrates that Str1 is required for hyphal fusion. In pathogenicity, Δstr1 is less virulent in maize anthracnose leaf blight and stalk rot. The phenotypes of Δstr1 are complemented by the Fusarium verticillioides striatin ortholog (fsr1), indicating that Fsr1 and Str1 are functionally conserved. Over-expression of StrA reveals its positive role in conidiation and the sexual production. StrA::eGFP localizes mainly to the endoplasmic reticulum. After comparing the results from these two species and other studied fungal species, I suggest that fungal striatins are involved in five types of development including hyphal growth, hyphal fusion, conidiation, sexual development, and virulence, and propose a model of fungal striatin protein interactions to account for these diverse phenotypes.

Incidences of fungal infections are on the rise in immunosuppressed people. Predominant causative agents for these mycoses are species of the genus Candida, including Candida albicans, Candida glabrata and Candida dublieniensis. Despite a wide range of emerging pathogens, C. albicans remains the leading cause. According to recent epidemiological studies, blood stream infections with C. albicans cause annually ~55% mortality in approximately 300,000 patients from intensive care units worldwide. Furthermore, the percentage of morbidity linked to oral, esophageal and vulvovaginal mycoses cause by C. albicans reach up to 90%. Reasons for these medical concerns are the lack of efficient diagnostics and antifungal therapy. Here, we therefore sought to find novel antifungal strategies inspired by innate immune cells, such as neutrophils. These phagocytes are able to block the fungal pathogenicity. Neutrophils are bloodstream leukocytes serving as the first line of defense against pathogenic microbes. It has been shown that neutrophils have a strong antifungal activity by impairing the conversion of the dimorphic C. albicans from yeast to hyphal form (Y-H). Consequently, we raised the question whether other immune cells, such as mast cells, with less phagocytic cabapilities may have similar activity to neutrophils. Mast cells are tissue-dwelling cells. Mucosal tissue is rich in mast cells and usually constitutes the entry ports for fungal pathogens into the human body. A contribution of mast cells in antifungal defense is, thus, very likely. We human explored mast cell functions upon encounter with fungal pathogens. Interestingly, human mast cells show a transient potential to impair fungal viability. To understand the mechanism behind this impairment we analyzed the human mast cell functions in more detail. We found that human mast cells challenged with C. albicans, immediately degranulate and secrete distinct cytokines and chemokines in an orchestrated manner. The chemokines secreted attract neutrophils. Mast cells moreover are able to internalize fungal cells and to ‘commit suicide’ by releasing extracellular DNA traps that ensnare the pathogen.   The effectiveness of future antifungals is depended on targeting the pathogen virulence with more efficiency. The dimorphism of C. albicans is proven to be essential its virulence. Blockage of this switching ability could render the pathogen avirulent. Consequently, we screened for compounds that mimic the neutrophils anti-dimorphic activity by screening small chemical molecule libraries that block Y-H transition. The screening of big chemical libraries requires a reliable, reproducible and rapid high-throughput screening assay (HTS). We developed an HTS assay based on automated microscopy and image analysis, thereby allowing to distinguish between yeast and filamentous forms. In order to find the ideal Y-H blocker, we also evaluated the cell viability via the count of ATP levels when challenged with the respective small chemical molecules.   Drug development is an elaborate and expensive process. We therefore applied our screening setup to identify antidimorphic/antifungal activity in compounds from two different chemical libraries including FDA-approved drugs. The study disclosed 7 off-patent antifungal drugs that have potent antimycotic activity, including 4 neoplastic agents, 2 antipsychotic drugs and 1 antianemic medication. In a nutshell, we aimed to mimic the anti-dimorphic/antifungal activity of neutrophils with small chemical molecules. Furthermore, we elucidated how immune cells contribute to antifungal defense to exploit these mechanisms for the development of novel antifungal therapies. Thus, this thesis provides novel tools for the discovery of more efficient compounds, identifies previously unknown antifungal aspect of off-patent FDA-approved drugs and highlights the interplay of mast cells with pathogenic fungi with the aim to define new screening strategies.

The dimorphic fungus Ustilago maydis is a pathogen of maize and it was used for decades to understand the molecular basis of plant pathogenicity aspects. Recently, much effort went into understanding the cell biology that underlies the virulence of U. maydis. It was shown previously that early endosomes (EEs) move bidirectionally within fungal hyphal cells. Although it was shown that the motility of EEs facilitates growth of the infectious hypha and mutants defective for kinesin-3 (Kin3), the major EE transporter, exhibit impaired polarized growth, the importance of EEs and their motility in plant colonization is not known. The first part of this thesis is focused on the role of EE motility during plant infection. In collaboration with Natalie Steinberg, who performed the plant infection assays, I used a synthetic molecular anchor, K1rPX, to block the motility of EEs at early and late stages during the host plant infection and I found that EE motility is essential during the first two days of pathogenic development, when infectious hyphae exhibit most prominent elongation, whereas blockage of EE motility after 3 days post infection does not inhibit plant colonization. Moreover, I documented that the blockage of EE motility during early stages of the infection causes high plant defence response, which means that the pathogen becomes recognized by the host plant defence system. These results indicate that EE motility is crucial during initial stages of the plant host infection and enables colonization by U. maydis and additionally suggests involvement of EEs in some defence response machinery. The second part of the thesis addresses the relationship between Kin3, the major motor for EE motility, and the microtubule (MT) array. I demonstrate here that Kin3 uses all MT tracks available in the cell, which is in contrast to published results in other systems. In the third part I focused on the interaction between Kin3 and the EEs. I found that the pleckstrin homology (PH) domain localized at the distal part of the Kin3 tail is of minor importance for EE association. This conclusion is supported by in vivo experiments, showing that truncated Kin3PH, which lacks the PH domain, was still able to bind to the organelles. By systematic truncation of parts of the Kin3 tail I found two adjacent regions, a DUF3694 domain and a "linker" region, that are important for binding of Kin3 to EEs. By using a synthetic anchor composed of Kin1 rigor domain and selected Kin3 domains I proved that both domains anchor the EEs to MTs and inhibit EE motility. I also showed that the PH domain is not able to block EE motility. In collaboration with Dr. Nicholas Harmer, who performed structural modelling of selected PH domains, I demonstrated that the PH domain is likely to interact with the motor domain of Kin3. This result was confirmed by using a yeast-two hybrid approach and a protein affinity assay. This indicates a globular organization of the Kin3 motor, which was confirmed by a split-YFP assay in living cells. Deletion of the PH domain and most probably lack of intramolecular interaction between the tail and motor domain reduces Kin3 motility parameters like velocity, frequency and run length indicating that the interaction of the PH domain with the motor domain has a role in the control of Kin3 motility.

京都大学 / 0048 / 新制・課程博士 / 博士(地球環境学) / 甲第22022号 / 地環博第186号 / 新制||地||96(附属図書館) / 京都大学大学院地球環境学舎地球環境学専攻 / (主査)准教授 岡田 直紀, 教授 舟川 晋也, 准教授 真常 仁志 / 学位規則第4条第1項該当 / Doctor of Global Environmental Studies / Kyoto University / DGAM