Welcome to IMC 2018 International Mycological Congress
Enabling tools for the study of neutrophil-fungi interactions
- D. Irimia
During infections with Aspergillus fumigatus or Candida albicans in healthy individuals, neutrophils accumulate fast and in large numbers. The interactions between neutrophils and fungi are key for protecting healthy tissues, by sealing off sites of infection and neutralizing the pathogens. Better understanding of these interactions may provide new capabilities for protection against infections in patients at risk. However, currently, the investigations of the interactions between neutrophil-fungi interactions can only be studied in vitro and in animal models which are limited by lack of temporal and spatial control over interactions. In this presentation, we will discuss new approaches for studying neutrophil-fungi interaction at single-cell resolution over time. These approaches are enabled by microfluidic tools, which create precisely controlled, repeatable conditions for the interactions between neutrophil and growing fungi. In one example, we studied the interactions between human neutrophils and Aspergillus and observed an evasive fungal behavior triggered by interaction with neutrophils. Interacting hyphae performed de novo tip formation to generate new hyphal branches, allowing the fungi to avoid the interaction point and continue invasive growth. The consequence of branch induction upon interaction outcome depends on the number and activity of neutrophils available: In the presence of sufficient neutrophils branching makes hyphae more vulnerable to destruction, while in the presence of limited neutrophils the interaction increases the number of hyphal tips, potentially making the infection more aggressive. In another example, we found that human neutrophils swarmed vigorously against Candida and significantly delayed the growth of C. albicans hyphae for up to 16 hours. Disruption of swarming mediators compromised the ability of neutrophils to swarm and limited the ability to contain C. albicans. Neutrophil extracellular traps were formed during neutrophil swarming against both Candida and Aspergillus. However, the disruption of NETs only compromised the protection against Candida and not against Aspergillus. The novel capabilities enabled by microfluidic devices have implications for our understanding of infections in neutrophil-deficient patients and open new avenues for treatments targeting opportunistic fungi.
Septins impact hyphal and nuclear morphology
- M. Momany
AbstractSeptins serve as scaffolds and diffusion barriers, organizing cellular morphology. In several filamentous fungal pathogens loss of septins results in the emergence of extra protrusions from the hypha and decreased virulence. In A. nidulans deletion of septins aspA, aspB, or aspC causes extra protrusions to emerge from hyphae. In contrast deletion of aspD does not have a major impact of hyphal morphology; instead nuclei take on a “stringy” appearance and colony sectoring increases. A close examination of nuclei from aspD deletion strains shows that nuclear organization is disturbed, including improper positioning of the nucleolus.
Elucidating three pathways that contribute to directional growth regulation in Candida albicans hyphae
- A. Brand
- M. Almeida
- A. Lopez
- T. Bedekovic
Introduction: The production of hyphae is strongly linked to pathogenesis during superficial mucosal infections and the life-threatening disseminated disease, invasive candidiasis. Hyphae constitute the ‘Special Weapons And Tactics’ capability deployed by C. albicans during mucosal and endothelial cell layer invasion. Hyphae are equipped with adhesins and secreted effectors but these are only effective if hyphal guidance mechanisms are operational to direct penetrative growth into host tissue.
Purpose: Our aim is to elucidate the signalling pathways involved in hyphal guidance. We have identified three distinct hyphal growth phenotypes – kinked, chaotic and straight - in which the ability to respond normally to external cues is attenuated or lost. Each of the three phenotypes is produced by a specific grouping of mutant strains. The functional links within some groupings are emerging and we are undertaking proteomics screens to extend our understanding of each pathway. Our overall aim is to find out how these pathways integrate to regulate the directional growth of hyphae.
Materials and Methods: We have generated null mutant strains for proteins representative of the three phenotypes and GFP-tagged them to establish their cellular localisation. We use live-cell imaging and microfabricated topographies to test for aberrant hyphal growth responses in mutant strains. The tagged strains are used as pull-down baits to identify interacting proteins.
Results: Hyphae with kinked or chaotic growth trajectories are generated by deletion of the fungal Paxillin homolog (Pxl1) or the small Ras-like GTPase, Rsr1, respectively. Proteomics suggests Pxl1 interacts with the Rho1 GTPase and β-glucan synthase, which are involved in cell-wall biosynthesis, while Rsr1 interacts with proteins involved in membrane organisation.
Conclusions: An emerging theme in this study is the involvement of tip-localised GTPases, which cycle through active and inactive states. This mechanism for binding and releasing effector proteins may allow constitutively polarised cells to adjust the site of growth in response to external cues.
Chitin Assembly in the Cell Wall of two Trichoderma species
- L. Kappel
- V. Bulone
- S. Gruber
The cell wall of (filamentous) fungi serves as armor against attacks by other organisms or hostile environments as well as a disguise in (phyto-)pathogenic and plant beneficial species to circumvent host defense mechanisms. Next to glucans, chitin is one of the major components of the cell wall that confers strength and rigidity but contributes also to the flexibility of the wall. The homopolymeric chitin chains, composed of β-1,4 linked N-acetylglucosamine units, constitute the innermost layer of the cell wall and are connected to the glucan matrix via β-1,3 and -1,4 links. Chitosan, the partially deacetylated form of chitin, is another important component of the fungal wall, which is present in minute to high amounts (up to 40% in Mucorales) depending on the fungal family, cellular component or growth stage. Trichoderma spp. are a cosmopolitan group of fungi, with a multitude of adaptions to a variety of environmental niches. Trichoderma reesei, for example, is a saprophytic fungus, which is used as potent cellulase producer in the industries. T. atroviride and T. virens are mycoparasites, with plant beneficial attributes, and thus, are important as biocontrol agents. Although Trichoderma spp. had been studied extensively over the past decades, little was known about their cell wall composition. Here we provide a first insight in the composition of the cell wall of T. reesei and T. atroviride. We show that de novo chitin and chitosan synthesis involves coordinated regulation of members of chitin synthesizing and chitin modifying enzyme families, rendering the fungi capable of fast adaption to a variety of environmental stresses and growth conditions. Chitin biosynthesis requires a set of chitin synthases that belong to the glycosyl transferase family (GT) 2. Although a central catalytic domain is shared by these isoenzymes the N- and C- terminal ends can vary considerably. Trichoderma, furthermore, contains a variety of putatively secreted and intracellular chitin modifying enzymes, which contribute to the cell wall plasticity. We provide new insights into the assembly of chitin and chitosan in Trichoderma spp. Eight chitin synthases and more than 15 additional enzymes - deacetylases, chitinolytic enzymes and accessory proteins, which are important for correct assembly and turnover of chitin and chitosan - are involved in chitin metabolism in Trichoderma spp. Defining the mechanisms of fungal chitin and chitosan synthesis facilitates a guided approach in cell wall reconstruction, which contributes to understanding the mycoparasitic capability of Trichoderma as biocontrol agents and their adaptability to changing environments.
Cell-cell contact during fusion triggers self/non-self recognition in a social microbe
- A. Gonçalves
- J. Heller
- E. Span
- M. Marletta
- N. Glass
Cell fusion is required for the development of the hyphal network made by Neurospora crassa. Fusion involves chemotropism, remodeling of the cell wall and merging of the plasma membranes. In the wild, a cell will encounter numerous prospective partners with variable degrees of genetic similarity and fusing with them can be beneficial or detrimental for fitness. Therefore, in an attempt to judge between ‘good’ and ‘bad’ consequences, cells have developed genetic barriers that are put in place during cell fusion events. We have showed that among a wild population of Neurospora isolates, a long-distance kind recognition system defined by the allelic variation at the doc locus functions at the level of germling communication; only cells that belong to the same doc haplotype group are able to communicate with each other. Interestingly, our recent data indicates that communication specificity is not enough to guarantee successful fusion due to a second recognition system functions at the cell wall dissolution stage. Germling pairs that harbor compatible doc genes but are dissimilar at a locus that we called ‘cell wall remodeling checkpoint’ or cwr, display an arrest phenotype following contact accompanied by the accumulation of cell wall material and are unable to proceed with fusion. The cwr locus, encompassing cwr-1, cwr-2 and cwr-3, showed high allelic diversity and trans-species polymorphism in populations of Neurospora, two features consistent with allorecognition mechanisms. Fusion assays revealed that cwr-2 is dispensable for non-self recognition, while a Δcwr-1Δcwr-3 mutant undergoes cell wall remodeling and fusion with both compatible and formerly incompatible partners. Our current investigations aim to establish the mode of action of CWR proteins during non-self discrimination. In summary, we show that microbes have developed sophisticated barriers to avoid unwanted confrontations. The novel self/non-self surveillance system that functions at the cell wall dissolution step demonstrates that in the fungal world talking the same dialect is not sufficient for effective fusion.
Investigating appressorium-mediated plant infection by the rice blast fungus Magnaporthe oryzae
- M. Osés-Ruiz
- C. Rodriguez Herrero
- L. Ryder
- W. Sakulkoo
- M. Martin-Urdiroz
- N. Talbot