Raphidiophrys Contractilis

Kobe University Repository : Thesis Mechanism of β-1,3-glucan mediated food uptake in the protozoon 学位論文題目 Raphidiophrys contractilis(原生生物Raphidiophrys contractilis におけ Title るβ-1, 3-グルカンが介在する捕食機構) 氏名 MOUSUMI BHADRA Author 専攻分野博士（理学） Degree 学位授与の日付 2017-09-25 Date of Degree 公開日 2018-09-25 Date of Publication 資源タイプ Thesis or Dissertation / 学位論文 Resource Type 報告番号甲第7000号 Report Number 権利 Rights JaLCDOI URL http://www.lib.kobe-u.ac.jp/handle_kernel/D1007000 ※当コンテンツは神戸大学の学術成果です。無断複製・不正使用等を禁じます。著作権法で認められている範囲内で、適切にご利用ください。 PDF issue: 2021-10-07 Doctoral Dissertation Mechanism of β-1, 3-glucan mediated food uptake in the protozoon Raphidiophrys contractilis 原生生物 Raphidiophrys contractilis における β-1, 3-グルカンが介在する捕食機構 July 2017 Graduate School of Science Kobe University Mousumi Bhadra Contents Acknowledgements ............................................................................................................ 2 Summary ........................................................................................................................... 3 Chapter 1: Introductory review............................................................................................ 7 Chapter 2: Proteins required for food capturing in Raphidiophrys contractilis..................... 11 2.1. Introduction ....................................................................................................... 11 2.2. Materials and methods ........................................................................................ 15 2.3. Results ............................................................................................................... 24 2.4. Discussion.......................................................................................................... 32 2.5. Tables and figures .............................................................................................. 38 Chapter 3: Concluding remarks and future perspectives ..................................................... 58 References ....................................................................................................................... 60 1 | P a g e Acknowledgements Firstly, I would like to express my sincere gratitude to my supervisor Prof. Dr. Toshinobu Suzaki for his continuous support in my PhD study and related research, and also for his patience, motivation, and immense knowledge. His guidance helped me in all the time of research and writing of this thesis. I could not have imagined having a better advisor and mentor for my PhD study. I would like to thank the rest of my thesis committee for their insightful comments and encouragement, and also for the difficult questions which incented me to widen my research from various perspectives. I am also honored to be able to thank Professor Madoka Kitagawa, Kobe University, and Professor Motonori Ando of Okayama University for their helpful support to my experiments. I thank my fellow labmates of Suzaki laboratory: Chisato Yoshimura, Osamu Yagiu, Jun Makimoto, Rina Matsumoto, Kyoko Nakata, Akane Chihara, Kento Nagao, and Munehiro Karasawa for stimulating discussions, for the time we were working together before deadlines, and for all the fun we have had in the last three years. In particular, I am grateful to Dr. Chihong Song, Dr. Lin Chen, and Dr. Mayumi Kobayashi for enlightening me the first glance of research. I'm actually at a loss for words to express my gratitude to my parents; my father, late Sushil Ranjan Bhadra who dreamed of my higher studies and my mother; Rita Bhadra who always dedicates her happiness for my raising. I am also thankful to my father and mother in- laws. Last but not the least, I would like to thank my beloved sisters, my brothers and all my family members, friends for supporting me spiritually throughout writing this thesis and my life in general. Special thanks to my husband Dr. Ranjan Kumar Mitra, for your caring love, understanding and all supports you have given me to complete this dissertation. 2 | P a g e Summary For heterotrophic protists, self and nonself discrimination is necessary for correct targeting of the predatory protozoa to prey organisms. The overall objective of this research was to find out proteins that take part in the food capturing system of the heliozoon Raphidiophrys contractilis. In this research, β-1,3-glucan binding proteins were found to be involved in the food capturing system of R. contractilis. In chapter 1, after briefly mentioned about prey/predator interactions in heterotrophic protozoon organisms, I summarized the importance of β-1,3-glucan in self/nonself discrimination in unicellular eukaryotes. β-1,3-glucan, in general, plays important roles in self/nonself discrimination in eukaryotes. In both innate and acquired immunity, β-1,3-glucan plays roles in activating components of the immune systems. For example, β-1,3-glucan works in the innate immune system by binding to the surface receptors on macrophages and activating their availability to identify and destroy foreign organisms. β-1,3-glucan-binding proteins are employed in the immune systems that recognize or attack other organisms that has β-1,3-glucan molecules on the surface. These proteins take part in discrimination between self and nonself, which is also indispensable in heterotrophic unicellular organisms for correct targeting of the predator to prey organisms. Heliozoons are unicellular protists which obtain nutrition by predatorial feeding, using special granulated organelles called extrusomes. R. contractilis is an example of heliozoans that possess extrusomes in axopodia, or long and slender cytoplasmic projections radiating from the spherical cell body. Contents of the extrusomes are expelled outside when the heliozoons make contact with prey, by which prey organisms stop swimming and firmly entrapped by the predator. In chapter 2, I demonstrated that two β-1,3-glucan binding proteins are involved in the food capturing system of R. contractilis for detecting and capturing prey cells. R. contractilis were cultured monoxenically with Chlorogonium capillatum as food. The ability of β-1,3- 3 | P a g e glucan recognition by R. contractilis was shown by microscopic observation. β-1,3-glucan molecules were found to be recognized by R. contractilis as food in both living conditions (living yeast cells) and as naturally-derived substances (curdlan and zymosan). These materials were finally engulfed into phagosomes for intracellular digestion. Experiments were carried out to find out specific proteins that bind to β-1,3-glucan by pull-down purification with curdlan (insoluble β-1,3-glucan) gel. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed to detect glucan-binding proteins. A protein band of an apparent molecular mass of 100 kDa appeared as a sole band that showed β-1,3-glucan binding. Mass spectrometry was carried out with a reference nucleotide database (contig database) constructed by a transcriptome analysis. Several protein fragments were successfully detected from the 100 kDa protein band, and BLAST search suggested that all of the sequences were parts of the major vault protein (MVP). Then, these contigs were carefully aligned with one of the top-hit MVP protein sequence. Some contigs were found to be overlapped with others, strongly suggesting that multiple MVP proteins are included in the 100 kDa protein band. After alignment, the sequence gaps were filled by PCR amplification using primers targeting parts of the neighboring contigs. The full-length sequences of the transcripts were determined by 3'- and 5'-RACE methods. Finally, two protein sequences were determined (named as RAC (meaning R. contractilis) MVP-1 and RAC MVP-2), both belonging to the MVP protein family with consensus multiple “MVP repeats” and a “MVP shoulder” domain. The full-length cDNA of RAC MVP-1 had 2,669 base pairs (bp) with an open reading frame of 2,493 bp. The full-length cDNA of RAC MVP- 2 had 2,629 bp, and 2,535 bp for the open reading frame. MVP is a main component of the vault, a ubiquitous and a very large cytoplasmic ribonucleoprotein particle with yet undetermined function. It was also suggested that the RAC MVP-1 and RAC MVP-2 are neither transmembrane nor secreted proteins. The MVPs in R. contractilis might form 4 | P a g e polymers, because “MVP shoulder” domains of both RAC MVP-1 and MVP-2 contain “oligomer interface regions”, as in MVPs of other organisms. These proteins were further verified as MVP with antibodies against anti-human MVP. According to a neighbor joining phylogenetic tree, both RAC MVP-1 and RAC MVP-2 were positioned next to each other (bootstrap value 100), and were included in the family of MVP, closely related to Opisthokonta and Amoebozoa supergroups. The specific association of RAC MVPs to β-1,3-glucan was demonstrated by competitive inhibition using laminarin (soluble β-1,3-glucan). This result can be explained if association of MVP and curdlan is competitively inhibited by the presence of another glucan species, and is considered as an additional evidence that RAC MVPs act as glucan-binding proteins. RAC MVPs were found to be secreted from R. contractilis cells during feeding. In this experiment, R. contractilis was mixed with prey flagellates, and the surrounding medium was collected and subjected to pull-down purification with curdlan gel. By SDS-PAGE analysis and mass spectrometry, not only RAC MVPs but also several other proteins (including other minor components of the vault complex such as poly ADP-ribose polymerase

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