Portrait of Dr Anastasios Tsaousis

Dr Anastasios Tsaousis

Senior Lecturer in Molecular and Evolutionary Parasitology
Examinations Officer

About

As an undergraduate student Anastasios (Tasos) studied Biology at University of Crete, Greece (1999-2003). There, he had the opportunity to complete his final year project on "Studying mitochondrial DNA recombination in the mussel Mytilus galloprovincialis". On completion of his undergraduate studies he started working at the Cyprus Institute of Neurology and Genetics as a Research Technician where he was involved in different projects in the field of human and cancer genetics. In parallel, he was also working on a project in collaboration with different laboratories, in an attempt to discover possible recombination in the mitochondrial DNA of animals from already published sequences.
During his PhD studies (2004-2007), he sought to understand the purpose and diversity of mitochondria in microbial eukaryotes. For this reason, he joined the group of Prof. T. Martin Embley and Prof. Robert P. Hirt at the Newcastle University. There, he studied the evolution and function of the mitochondrion-related organelles of microsporidia. His studies presented the first experimental evidence for the existence of a remnant mitochondrion (mitosome) in the microsporidian Encephalitozoon cuniculi. His research also demonstrated the first experimental evidence for the localization and function of a non-mitochondrial ATP transporter in the microsporidian mitosome, the presence of which potentially solves the conundrum of how the mitosome acquires its energy. A second set of data from his PhD studies demonstrated that a functional role of the microsporidian mitosome is an essential eukaryotic pathway- iron/sulphur (Fe/S) cluster biosynthesis; this pathway is believed to be the reason for the existence of mitochondria and related organelles.
As a postdoctoral researcher Anastasios moved to Dalhousie University in Halifax, Nova Scotia in Canada (2008-2012), where he joined Prof. Andrew J. Roger's group. There he was involved in several investigations on the characterization of mitochondrial pathways in anaerobic protists and how lateral gene transfer (LGT) affects their adaptation to their unique lifestyles. In 2012, he moved to the Charles University in Prague, Czech Republic to join Prof. Jan Tachezy's group (as part of his Marie Curie fellowship), where he initiated several studies on the biochemistry and protein composition of mitochondria in anaerobic microbial eukaryotes.
In July 2013, Anastasios joined the School of Biosciences at the University of Kent as a Lecturer. He is now a Senior Lecturer in Molecular and Evolutionary Parasitology. Not only does he teach on various modules both in undergraduate and postgraduate level, he was also an Outreach Officer of the School (2014-2018), a Director of MSc by Research (2017-2018), GMO officer (2014-2018) and he initiated the Resistance Pathogenicity of Infectious Diseases (RAPID group) within the School of Biosciences. He is currently the Chief examiner of the School of Biosciences. Outside the University of Kent, Anastasios has been actively involved with many national and international societies. He has been member of the Eukaryotic Division of the Microbiology Society since 2015 (organised various sessions in the 2015, 2017 & 2018 annual meetings), he is currently the Vice President of the Protistology-UK society (since 2018) and has been elected five times as a member of the executive committee of the International Society for Evolutionary Protistology (since 2010). In 2018, he successfully organised the 22nd meeting of the International Society for Evolutionary Protistology in Paphos, Cyprus.
Anastasios is the Principal Investigator of the Laboratory of Molecular and Evolutionary Parasitology at the University of Kent. The current research of his laboratory is focused on the investigations of the adaptations of microbial eukaryotic organisms (e.g. Cryptosporidium, Blastocystis, Naegleria, Gregarines, ciliates), and their course in parasitic evolution and diversity. To accomplish this, his laboratory is combining detailed bioinformatics analyses of newly generated genomic/transcriptomic/metabolomic results with field, cell biological and biochemical methods to investigate the parasitic and free-living microbial eukaryotes living in diverse and extreme environments. He has currently obtained funding from the Biotechnology and Biological Sciences Research Council, the Bill and Melinda Gates Foundation, the Gordon and Betty Moore Foundation and the Royal Society. 

ORCID ID: 0000-0002-5424-1905

Research interests

Cryptosporidium and cryptosporidiosis Cryptosporidiosis is a diarrheal disease caused by Cryptosporidium, a pathogen of great medical importance, which has appeared in the headlines several times in the past decades. An important fact about cryptosporidiosis is the lack of medical treatment in the form of drugs or vaccines. The parasite is mainly affecting children of a young age (below five), but people with impaired immune systems are also at great risk. In some cases, infected individuals have to deal with unpleasant diarrhoea lasting for several weeks, leading to dehydration that could potentially be deadly. Information on the infection patterns of the parasite and its interactions with the host is very limited, due to the lack of a laboratory system that will enable us to monitor the infection and replication processes of Cryptosporidium within a cell. Our laboratory has managed to overcome this difficulty by infecting with Cryptosporidium different types of cancer cells in a laboratory setting and testing whether they could successfully allow the parasite to grow and replicate. Our laboratory is currently interested to investigate which metabolites the parasite steals from the host cell and how it manipulates the molecular mechanisms of the host for its benefit. In addition, we are currently examining the cellular geography of Cryptosporidium inside and outside its host and the evolutionary adaptations that make it such a successful parasite. Our work will demonstrate what the molecular interactions between Cryptosporidium and its host are, will provide a better understanding of how complex the life-cycle of the parasite is and will generate essential knowledge about this medically important pathogen and will provide new targets for anti-parasitic drug development. 

Establishing Naegleria as a model system to investigate adaptations to eukaryotic cellular adaptations 
This project aims to develop tools and use them to study an organism that is neither animal, plant, algae, nor parasite. It is a single-celled creature living in soils and freshwater around the world. This creature, Naegleria gruberi, possesses nearly all of the cellular features found in animal and plant cells, but evolved away from them nearly 1.5 billion years ago. It is a uniquely placed sampling point from which to collect information about how cells work and gain a global perspective applicable to all eukaryotic cells. Our laboratory is currently developing a state-of-the-art genome editing system based on CRISPR/Cas9 based methodology. This will enable the systematic interrogation of Naegleria genome function by making this organism amenable to the full array of CRISPR-based approaches currently established for other organisms, including knockout, knockdown and activation screens of various genes. We will aim to develop this approach into a powerful high-throughput functional genomics toolbox, thereby enabling understanding of the function(s) of the 15,727 protein-coding genes that are present in Naegleria’s nuclear genome. The overall outcome of this project is to produce a set of protocols, plasmids and tools to be used by the scientific community to address diverse scientific questions, using Naegleria gruberi as a model system. 

Exploring the anaerobic and other unique adaptations of Blastocystis Blastocystis is an obligate anaerobic parasite also found in patients with irritable bowel syndrome. The actual pathogenicity of Blastocystis is still questionable, since currently there is no direct link between the parasite and the disease caused. As an anaerobic organism, Blastocystis harbor peculiar Mitochondrion-related organelles (MROs), which are considered to be an intermediate form between a typical mitochondrion and a hydrogenosome. Another interesting fact about Blastocystis, concerns the presence of peculiar proteins encoded from its genome: it seems that Blastocystis is a "lateral gene transfer magnet" since several genes have been acquired from diverse eukaryotes and prokaryotes in order to assemble a kind of mixed genome. Using a combination of bioinformatics along with cellular and biochemical techniques, our laboratory aims to investigate these “novel” functions in Blastocystis and its closely relatives (e.g. Proteromonas) and attempt to understand their evolutionary history and the reason for their existence.

Teaching

  • BI505 Infection and Immunity  
  • BI606 Pathogens and Pathogenicity 
  • BI855 Advances in Parasitology 
  • DP1625 Advances in eukaryotic diversity and evolution

Supervision

MSc-R projects available for 2019/20

Exploring Cryptosporidium transportome and how it affects the intracellular interactions with its host Co-Supervisor: Dr Christopher Mulligan Cryptosporidium is an obligate intracellular intestinal parasite of various animals that causes cryptosporidiosis, a diarrheal disease that is most common in young children and is severe in immunosuppressed humans. Its genome is highly reduced, encoding for 4800 proteins, 151 of which are carrier proteins. We hypothesise that these transporters play significant role in the adaptations of the parasite in an intracellular lifestyle and its interactions/communication with the host. The purpose of this project is to explore the localisation and function(s) of these transporters, identify potential links associated with the adaptation to parasitic lifestyle, while exploring probable candidates for tackling cryptosporidiosis.
Additional research costs: £2000

Exploring the eukaryotic gut microbiome among animals While there have been numerous studies exploring the gut microbiome of different animals, these were mainly focused on identifying the bacterial residents of the gut microflora. In addition, not much is known about the eukaryotic residents of the gut and their contribution to the health and disease. This project will be in collaboration with local conservation parks in the Kent region, where the student will use multidisciplinary approaches to investigate the eukaryotic residents of the gut microflora from different animals, either living in the wild or in captivity (including farms). To tackle these aims, the student will use an integrative approach combining culturomics, microscopy, molecular and phylogenetic methods.
Additional research costs: £2000

Establishing and developing an advance culturing platform for Cryptosporidium Cryptosporidium is an obligate intracellular intestinal parasite of various animals that causes cryptosporidiosis, a diarrheal disease that is most common in young children and is severe in immunosuppressed humans. The goal of this project is to develop a more advance culturing platform for Cryptosporidium parasite. The student will make use of the newly develop culturing system from our lab, not only to test it, but also to explore 3D culturing methods and organoids to increase the in vitro production of the parasite.
Additional research costs: £2000  

Publications

Article

  • Bones, A. et al. (2019). Past and future trends of Cryptosporidium in vitro research. Experimental Parasitology [Online] 196:28-37. Available at: 10.1016/j.exppara.2018.12.001.
    Cryptosporidium is a genus of single celled parasites capable of infecting a wide range of animals including humans. Cryptosporidium species are members of the phylum apicomplexa, which includes well-known genera such as Plasmodium and Toxoplasma. Cryptosporidium parasites cause a severe gastro-intestinal disease known as cryptosporidiosis. They are one of the most common causes of childhood diarrhoea worldwide, and infection can have prolonged detrimental effects on the development of children, but also can be life threatening to HIV/AIDS patients and transplant recipients. A variety of hosts can act as reservoirs, and Cryptosporidium can persist in the environment for prolonged times as oocysts. While there has been substantial interest in these parasites, there is very little progress in terms of treatment development and understanding the majority of the life cycle of this unusual organism. In this review, we will provide an overview on the existing knowledge of the biology of the parasite and the current progress in developing in vitro cultivation systems. We will then describe a synopsis of current and next generation approaches that could spearhead further research in combating the parasite.
  • Jossé, L. et al. (2019). A Cell Culture Platform for the cultivation of Cryptosporidium parvum. Current Protocols in Microbiology [Online]. Available at: https://doi.org/10.1002/cpmc.80.
    Cryptosporidium is a genus of ubiquitous unicellular parasites belonging to the phylum Apicomplexa. Cryptosporidium species are the second largest cause of childhood diarrhoea and are associated with increased morbidity. Accompanying this is the low availability of treatment and lack of vaccines. The major barrier to developing effective treatment is the lack of reliable in vitro culture methods. Recently, our lab has successfully cultivated C. parvum in the oesophageal cancer derived cell line COLO-680N, and can maintain infection for several weeks. The success of this cell line was assessed with a combination of various techniques including fluorescent microscopy and qPCR. In addition, to tackle the issue of long-term oocyst production in vitro, a simple, low cost bioreactor system using the COLO-680N cell line was established, which produced infectious oocysts for four months. This chapter provides details on the methodologies used to culture, maintain and assess Cryptosporidium infection and propagation in COLO-680N.
  • Li, F. et al. (2019). Successful Genetic Transfection of the Colonic Protistan Parasite Blastocystis for Reliable Expression of Ectopic Genes. Scientific Reports [Online] 9. Available at: https://dx.doi.org/10.1038/s41598-019-39094-5.
    The microbial parasite Blastocystis colonizes the large intestines of numerous animal species and increasing evidence has linked Blastocystis infection to enteric diseases with signs and symptoms including abdominal pain, constipation, diarrhea, nausea, vomiting, and flatulence. It has also recently been reported to be an important member of the host intestinal microbiota. Despite significant advances in our understanding of Blastocystis cell biology and host-parasite interactions, a genetic modification tool is absent. In this study, we successfully established a robust gene delivery protocol for Blastocystis subtype 7 (ST7) and ectopic protein expression was further tested using a high sensitivity nano-luciferase (Nluc) reporter system, with promoter regions from several genes. Among them, a strong promoter encompassing a region upstream of the legumain 5? UTR was identified. Using this promoter combined with the legumain 3? UTR, which contains a conserved, precise polyadenylation signal, a robust transient transfection technique was established for the first time in Blastocystis. This system was validated by ectopic expression of proteins harbouring specific localization signals. The establishment of a robust, reproducible gene modification system for Blastocystis is a significant advance for Blastocystis research both in vitro and in vivo. This technique will spearhead further research to understand the parasite’s biology, its role in health and disease, along with novel ways to combat the parasite.
  • Miller, C. et al. (2019). NMR metabolomics reveals effects of Cryptosporidium infections on host cell metabolome. Gut Pathogens [Online] 11. Available at: https://doi.org/10.1186/s13099-019-0293-x.
    Background: Cryptosporidium is an important gut microbe whose contributions towards infant and immunocompromise
    patient mortality rates are steadily increasing. Over the last decade, we have seen the development of
    various tools and methods for studying Cryptosporidium infection and its interactions with their hosts. One area that is
    sorely overlooked is the effect infection has on host metabolic processes.
    Results: Using a 1H nuclear magnetic resonance approach to metabolomics, we have explored the nature of the
    mouse gut metabolome as well as providing the first insight into the metabolome of an infected cell line. Statistical
    analysis and predictive modelling demonstrated new understandings of the effects of a Cryptosporidium infection,
    while verifying the presence of known metabolic changes. Of note is the potential contribution of host derived
    taurine to the diarrhoeal aspects of the disease previously attributed to a solely parasite-based alteration of the gut
    environment, in addition to other metabolites involved with host cell catabolism.
    Conclusion: This approach will spearhead our understanding of the Cryptosporidium-host metabolic exchange and
    provide novel targets for tackling this deadly parasite.
  • Yowang, A. et al. (2018). High diversity of Blastocystis subtypes isolated from asymptomatic adults living in Chiang Rai, Thailand. Infection Genetics and Evolution [Online] 65:270-275. Available at: https://doi.org/10.1016/j.meegid.2018.08.010.
    Blastocystis is a common and broadly distributed microbial
    eukaryote inhabiting the gut of humans and other animals. The genetic
    diversity of Blastocystis is extremely high comprising no less than 17
    subtypes in mammals and birds. Nonetheless, little is known about the
    prevalence and distribution of Blastocystis subtypes colonising humans in
    Thailand. Molecular surveys of Blastocystis remain extremely limited and
    usually focus on the central, urban part of the country. To address this
    knowledge gap, we collected stool samples from a population of Thai
    adults (n=178) residing in Chiang Rai Province. The barcoding region of
    the small subunit ribosomal RNA was employed to screen for Blastocystis
    and identify the subtype. Forty-one stool samples (23%) were identified
    as Blastocystis positive. Six of the nine subtypes that colonise humans
    were detected with subtype (ST) three being the most common (68%),
    followed by ST1 (17%) and ST7 (7%). Comparison of subtype prevalence
    across Thailand using all publicly available sequences showed that
    subtype distribution differs among geographic regions in the country. ST1
    was most commonly encountered in the central region of Thailand, while
    ST3 dominated in the more rural north and northeast regions. ST2 was
    absent in the northeast, while ST7 was not found in the center. Thus,
    this study shows that ST prevalence and distribution differs not only
    among countries, but also among geographic regions within a country.
    Potential explanations for these observations are discussed herewith.
  • Herman, E. et al. (2018). Identification and characterisation of the cryptic Golgi Apparatus in Naegleria gruberi. Journal of Cell Science [Online] 131. Available at: http://dx.doi.org/10.1101/221721.
    Although the Golgi apparatus has a conserved morphology of flattened stacked cisternae in the vast majority of eukaryotes, the organelle has lost the stacked organization in several eukaryotic lineages raising the question of what range of morphologies is possible for the Golgi. In order to understand this range of organellar diversity, it is necessary to characterise the Golgi in many different lineages. Here we identify the Golgi apparatus in Naegleria, the first description of an unstacked Golgi organelle in a non-parasitic eukaryote, other than fungi. We provide a comprehensive list of Golgi-associated membrane trafficking genes encoded in two separate species of Naegleria and transcriptomic support to show that nearly all are expressed in mouse-passaged N. fowleri cells. We then study distribution of the Golgi marker NgCOPB by fluorescence, identifying membranous structures that can be disrupted by Brefeldin A treatment consistent with Golgi localisation. Confocal and immuno-electron microscopy revealed that NgCOPB is localized to membranous structures consistent with tubules. Our data not only identify the Golgi organelle for the first time in this major eukaryotic lineage, but also provide the rare example of a tubular form of the organelle representing an important sampling point for the comparative understanding of Golgi organellar diversity.
  • Miller, C. et al. (2018). A cell culture platform for Cryptosporidium that enables long-term cultivation and new tools for the systematic investigation of its biology. International Journal for Parasitology [Online] 48:197-201. Available at: https://doi.org/10.1016/j.ijpara.2017.10.001.
    Cryptosporidium parasites are a major cause of diarrhoea that pose a particular threat to children in developing areas and immunocompromised individuals. Curative therapies and vaccines are lacking, mainly due to lack of a long-term culturing system of this parasite. Here, we show that COLO-680N cells infected with two different Cryptosporidium parvum strains produce sufficient infectious oocysts to infect subsequent cultures, showing a substantial fold increase in production, depending on the experiment, over the most optimistic HCT-8 models. Oocyst identity was confirmed using a variety of microscopic- and molecular-based methods. This culturing system will accelerate research on Cryptosporidium and the development of anti-Cryptosporidium drugs.
  • Tsaousis, A. et al. (2018). The human gut colonizer Blastocystis respires using Complex II and alternative oxidase to buffer transient oxygen fluctuations in the gut. Frontiers in Cellular and Infection Microbiology [Online]. Available at: http://dx.doi.org/10.3389/fcimb.2018.00371/full.
    Blastocystis is the most common eukaryotic microbe in the human gut. It is linked to irritable bowel syndrome (IBS), but its role in disease has been contested considering its widespread nature. This organism is well adapted to its anoxic niche and lacks typical eukaryotic features such as a cytochrome-driven mitochondrial electron transport. Although generally considered a strict or obligate anaerobe, its genome encodes an alternative oxidase. Alternative oxidases are energetically wasteful enzymes as they are non-protonmotive and energy is liberated in heat, but they are considered to be involved in oxidative stress protective mechanisms. Our results demonstrate that the Blastocystis cells themselves respire oxygen via this alternative oxidase thereby casting doubt on its strict anaerobic nature. Inhibition experiments using alternative oxidase and Complex II specific inhibitors clearly demonstrate their role in cellular respiration. We postulate that the alternative oxidase in Blastocystis is used to buffer transient oxygen fluctuations in the gut and that it likely is a common colonizer of the human gut and not causally involved in IBS. Additionally the alternative oxidase could act as a protective mechanism in a dysbiotic gut and thereby explain the absence of Blastocystis in established IBS environments.
  • Miller, C., Jossé, L. and Tsaousis, A. (2018). Localization of Fe?S Biosynthesis Machinery in Cryptosporidium parvum Mitosome. Journal of Eukaryotic Microbiology [Online]. Available at: https://doi.org/10.1111/jeu.12663.
    Cryptosporidium is a protozoan, apicomplexan, parasite that poses significant risk to humans and animals, as a common cause of potentially fatal diarrhea in immunodeficient hosts. The parasites have evolved a number of unique biological features that allow them to thrive in a highly specialized parasitic lifestyle. For example, the genome of Cryptosporidium parvum is highly reduced, encoding only 3,805 proteins, which is also reflected in its reduced cellular and organellar content and functions. As such, its remnant mitochondrion, dubbed a mitosome, is one of the smallest mitochondria yet found. While numerous studies have attempted to discover the function(s) of the C. parvum mitosome, most of them have been focused on in silico predictions. Here, we have localized components of a biochemical pathway in the C. parvum mitosome, in our investigations into the functions of this peculiar mitochondrial organelle. We have shown that three proteins involved in the mitochondrial iron-sulfur cluster biosynthetic pathway are localized in the organelle, and one of them can functionally replace its yeast homolog. Thus, it seems that the C. parvum mitosome is involved in iron-sulfur cluster biosynthesis, supporting the organellar and cytosolic apoproteins. These results spearhead further research on elucidating the functions of the mitosome and broaden our understanding in the minimalistic adaptations of these organelles.
  • Gentekaki, E. et al. (2017). Extreme genome diversity in the hyper-prevalent parasitic eukaryote Blastocystis. PLOS Biology [Online] 15:e2003769. Available at: http://dx.doi.org/10.1371/journal.pbio.2003769.
    Blastocystis is the most prevalent eukaryotic microbe colonizing the human gut, infecting approximately 1 billion individuals worldwide. Although Blastocystis has been linked to intestinal disorders, its pathogenicity remains controversial because most carriers are asymptomatic. Here, the genome sequence of Blastocystis subtype (ST) 1 is presented and compared to previously published sequences for ST4 and ST7. Despite a conserved core of genes, there is unexpected diversity between these STs in terms of their genome sizes, guanine-cytosine (GC) content, intron numbers, and gene content. ST1 has 6,544 protein-coding genes, which is several hundred more than reported for ST4 and ST7. The percentage of proteins unique to each ST ranges from 6.2% to 20.5%, greatly exceeding the differences observed within parasite genera. Orthologous proteins also display extreme divergence in amino acid sequence identity between STs (i.e., 59%–61%median identity), on par with observations of the most distantly related species pairs of parasite genera. The STs also display substantial variation in gene family distributions and sizes, especially for protein kinase and protease gene families, which could reflect differences in virulence. It remains to be seen to what extent these inter-ST differences persist at the intra-ST level. A full 26% of genes in ST1 have stop codons that are created on the mRNA level by a novel polyadenylation mechanism found only in Blastocystis. Reconstructions of pathways and organellar systems revealed that ST1 has a relatively complete membrane-trafficking system and a near-complete meiotic toolkit, possibly indicating a sexual cycle. Unlike some intestinal protistan parasites, Blastocystis ST1 has near-complete de novo pyrimidine, purine, and thiamine biosynthesis pathways and is unique amongst studied stramenopiles in being able to metabolize ?-glucans rather than ?-glucans. It lacks all genes encoding heme-containing cytochrome P450 proteins. Predictions of the mitochondrion-related organelle (MRO) proteome reveal an expanded repertoire of functions, including lipid, cofactor, and vitamin biosynthesis, as well as proteins that may be involved in regulating mitochondrial morphology and MRO/endoplasmic reticulum (ER) interactions. In sharp contrast, genes for peroxisome-associated functions are absent, suggesting Blastocystis STs lack this organelle. Overall, this study provides an important window into the biology of Blastocystis, showcasing significant differences between STs that can guide future experimental investigations into differences in their virulence and clarifying the roles of these organisms in gut health and disease.
  • Rueckert, S. and Tsaousis, A. (2017). Report of the 2017 Protistology-UK Spring Meeting. European Journal of Protistology [Online] 61:307-310. Available at: https://doi.org/10.1016/j.ejop.2017.10.003.
  • Betts, E. et al. (2017). Genetic diversity of Blastocystis in non-primate animals. Parasitology [Online] 1. Available at: https://doi.org/10.1017/S0031182017002347.
    Blastocystis is an anaerobic protist, commonly inhabiting the intestinal tract of both humans and other animals. Blastocystis is extremely diverse comprising 17 genetically distinct subtypes in mammals and birds. Pathogenicity of this enteric microbe is currently disputed and knowledge regarding its distribution, diversity and zoonotic potential is fragmentary. Most research has focused on Blastocystis from primates, while sampling from other animals remains limited. Herein, we investigated the prevalence and distribution of Blastocystis in animals held within a conservation park in South East England. A total of 118 samples were collected from 27 vertebrate species. The barcoding region of the small-subunit ribosomal RNA was used for molecular identification and subtyping. Forty one per cent of the species were sequence positive for Blastocystis indicating a high prevalence and wide distribution among the animals in the park. Six subtypes were identified, one of which is potentially novel. Moreover, the majority of animals were asymptomatic carriers, suggesting that Blastocystis is not pathogenic in animals. This study provides a thorough investigation of Blastocystis prevalence within a wildlife park in the UK and can be used as a platform for further investigations on the distribution of other eukaryotic gut microbes.
  • Saintas, E. et al. (2017). Acquired resistance to oxaliplatin is not directly associated with increased resistance to DNA damage in SK-N-ASrOXALI4000, a newly established oxaliplatin-resistant sub-line of the neuroblastoma cell line SK-N-AS. PLoS ONE [Online] 12:e0172140. Available at: http://dx.doi.org/10.1371/journal.pone.0172140.
    The formation of acquired drug resistance is a major reason for the failure of anti-cancer therapies after initial response. Here, we introduce a novel model of acquired oxaliplatin resistance, a sub-line of the non-MYCN-amplified neuroblastoma cell line SK-N-AS that was adapted to growth in the presence of 4000 ng/mL oxaliplatin (SK-N-ASrOXALI4000). SK-N-ASrOXALI4000 cells displayed enhanced chromosomal aberrations compared to SK-N-AS, as indicated by 24-chromosome fluorescence in situ hybridisation. Moreover, SK-N-ASrOXALI4000 cells were resistant not only to oxaliplatin but also to the two other commonly used anti-cancer platinum agents cisplatin and carboplatin. SK-N-ASrOXALI4000 cells exhibited a stable resistance phenotype that was not affected by culturing the cells for 10 weeks in the absence of oxaliplatin. Interestingly, SK-N-ASrOXALI4000 cells showed no cross resistance to gemcitabine and increased sensitivity to doxorubicin and UVC radiation, alternative treatments that like platinum drugs target DNA integrity. Notably, UVC-induced DNA damage is thought to be predominantly repaired by nucleotide excision repair and nucleotide excision repair has been described as the main oxaliplatin-induced DNA damage repair system. SK-N-ASrOXALI4000 cells were also more sensitive to lysis by influenza A virus, a candidate for oncolytic therapy, than SK-N-AS cells. In conclusion, we introduce a novel oxaliplatin resistance model. The oxaliplatin resistance mechanisms in SK-N-ASrOXALI4000 cells appear to be complex and not to directly depend on enhanced DNA repair capacity. Models of oxaliplatin resistance are of particular relevance since research on platinum drugs has so far predominantly focused on cisplatin and carboplatin.
  • Richardson, E. et al. (2015). Evolutionary cell biology: Functional insight from “Endless forms most beautiful”. Molecular Biology of the Cell [Online] 26:4532-4538. Available at: http://www.molbiolcell.org/content/26/25/4532.short.
    In animal and fungal model organisms, the complexities of cell biology have been analyzed in exquisite detail and much is known about how these organisms function at the cellular level. However, the model organisms cell biologists generally use include only a tiny fraction of the true diversity of eukaryotic cellular forms. The divergent cellular processes observed in these more distant lineages are still largely unknown in the general scientific community. Despite the relative obscurity of these organisms, comparative studies of them across eukaryotic diversity have had profound implications for our understanding of fundamental cell biology in all species and have revealed the evolution and origins of previously observed cellular processes. In this Perspective, we will discuss the complexity of cell biology found across the eukaryotic tree, and three specific examples of where studies of divergent cell biology have altered our understanding of key functional aspects of mitochondria, plastids, and membrane trafficking.
  • Tsaousis, A. et al. (2014). Evolution of the Cytosolic Iron/Sulfur cluster Assembly machinery in Blastocystis sp. and other microbial eukaryotes. Eukaryotic Cell [Online] 13:143-153. Available at: http://dx.doi.org/10.1128/EC.00158-13.
    The Cytosolic Iron/Sulfur cluster Assembly (CIA) machinery is responsible for the assembly of cytosolic and nuclear iron/sulfur clusters, cofactors that are vital for all living cells. This machinery is uniquely found in eukaryotes and consists of at least eight proteins in opisthokont lineages such as animals and yeast. We sought to identify and characterize homologues of the CIA system proteins in the anaerobic stramenopile parasite Blastocystis sp. NandII strain. We identified transcripts encoding six of the components - Cia1, Cia2, MMS19, Nbp35, Nar1, and a putative Tah18 - and showed that the last three of them localized to the cytoplasm of the cell using immuno-fluorescence microscopy, immuno-electron microscopy and subcellular fractionation. We then used comparative genomic and phylogenetic approaches to investigate the evolutionary history of these proteins. While most Blastocystis homologues branch with their eukaryotic counterparts, the putative Blastocystis Tah18 seems to have a separate evolutionary origin and therefore possibly a different function. Furthermore, our phylogenomic analyses revealed that all eight CIA components described in opisthokonts originated before the diversification of extant eukaryotic lineages and were likely already present in the Last Eukaryotic Common Ancestor (LECA). Nbp35, Nar1 Cia1 and Cia2 proteins have been conserved during the subsequent evolutionary diversification of eukaryotes and are present in virtually all extant lineages, whereas the other CIA proteins have patchy phylogenetic distributions. Cia2 appears to be homologous to SufT, a component of the prokaryotic SUF system, making this the first reported evolutionary link between the CIA and any other Fe/S biogenesis pathway. All of our results suggest that the CIA machinery is an ubiquitous biosynthetic pathway in eukaryotes, but its apparent plasticity in composition raises questions regarding how it functions in non-model organisms and how it interfaces with various iron/sulfur cluster systems (i.e., ISC, NIF and/or SUF) found in eukaryotic cells.
  • Tsaousis, A. et al. (2014). A nonmitochondrial hydrogen production in Naegleria gruberi. Genome Biology and Evolution [Online] 6:792-9. Available at: http://www.dx.doi.org/10.1093/gbe/evu065.
    Naegleria gruberi is a free-living heterotrophic aerobic amoeba well known for its ability to transform from an amoeba to a flagellate form. The genome of N. gruberi has been recently published, and in silico predictions demonstrated that Naegleria has the capacity for both aerobic respiration and anaerobic biochemistry to produce molecular hydrogen in its mitochondria. This finding was considered to have fundamental implications on the evolution of mitochondrial metabolism and of the last eukaryotic common ancestor. However, no actual experimental data have been shown to support this hypothesis. For this reason, we have decided to investigate the anaerobic metabolism of the mitochondrion of N. gruberi. Using in vivo biochemical assays, we have demonstrated that N. gruberi has indeed a functional [FeFe]-hydrogenase, an enzyme that is attributed to anaerobic organisms. Surprisingly, in contrast to the published predictions, we have demonstrated that hydrogenase is localized exclusively in the cytosol, while no hydrogenase activity was associated with mitochondria of the organism. In addition, cytosolic localization displayed for HydE, a marker component of hydrogenase maturases. Naegleria gruberi, an obligate aerobic organism and one of the earliest eukaryotes, is producing hydrogen, a function that raises questions on the purpose of this pathway for the lifestyle of the organism and potentially on the evolution of eukaryotes.
  • Tsaousis, A. et al. (2012). Evolution of Fe/S cluster biogenesis in the anaerobic parasite Blastocystis. Proceedings of the National Academy of Sciences [Online] 109:10426-10431. Available at: http://dx.doi.org/10.1073/pnas.1116067109.
    Iron/sulfur cluster (ISC)-containing proteins are essential components of cells. In most eukaryotes, Fe/S clusters are synthesized by the mitochondrial ISC machinery, the cytosolic iron/sulfur assembly system, and, in photosynthetic species, a plastid sulfur-mobilization (SUF) system. Here we show that the anaerobic human protozoan parasite Blastocystis, in addition to possessing ISC and iron/sulfur assembly systems, expresses a fused version of the SufC and SufB proteins of prokaryotes that it has acquired by lateral transfer from an archaeon related to the Methanomicrobiales, an important lineage represented in the human gastrointestinal tract microbiome. Although components of the Blastocystis ISC system function within its anaerobic mitochondrion-related organelles and can functionally replace homologues in Trypanosoma brucei, its SufCB protein has similar biochemical properties to its prokaryotic homologues, functions within the parasite's cytosol, and is up-regulated under oxygen stress. Blastocystis is unique among eukaryotic pathogens in having adapted to its parasitic lifestyle by acquiring a SUF system from nonpathogenic Archaea to synthesize Fe/S clusters under oxygen stress.
  • Long, S. et al. (2011). Stage-specific requirement for Isa1 and Isa2 proteins in the mitochondrion of Trypanosoma brucei and heterologous rescue by human and Blastocystis orthologues. Molecular Microbiology [Online] 81:1403-1418. Available at: http://dx.doi.org/10.1111/j.1365-2958.2011.07769.x.
    IscA/Isa proteins function as alternative scaffolds for the assembly of Fe-S clusters and/or provide iron for their assembly in prokaryotes and eukaryotes. Isa are usually non-essential and in most organisms are confined to the mitochondrion. We have studied the function of TbIsa1 and TbIsa2 in Trypanosoma brucei, where the requirement for both of them to sustain cell growth depends on the life cycle stage. The TbIsa proteins are abundant in the procyclic form, which contains an active organelle. Both proteins are indispensable for growth, as they are required for the assembly of Fe-S clusters in mitochondrial aconitase, fumarase and succinate dehydrogenase. Reactive oxygen species but not iron accumulate in the procyclic mitochondrion upon ablation of the TbIsa proteins, but their depletion does not influence the assembly of Fe-S clusters in cytosolic proteins. In the bloodstream form, which has a downregulated mitochondrion, the TbIsa proteins are non-essential. The Isa2 orthologue of the anaerobic protist Blastocystis partially rescued the growth and enzymatic activities of TbIsa1/2 knock-down. Rescues of single knock-downs as well as heterologous rescues with human Isa orthologues partially recovered the activities of aconitase and fumarase. These results show that the Isa1 and Isa2 proteins of diverse eukaryotes have overlapping functions.
  • Tsaousis, A. et al. (2011). A Functional Tom70 in the Human Parasite Blastocystis sp.: Implications for the Evolution of the Mitochondrial Import Apparatus. Molecular Biology and Evolution [Online] 28:781-791. Available at: http://dx.doi.org/10.1093/molbev/msq252.
    Core proteins of mitochondrial protein import are found in all mitochondria, suggesting a common origin of this import machinery. Despite the presence of a universal core import mechanism, there are specific proteins found only in a few groups of organisms. One of these proteins is the translocase of outer membrane 70 (Tom70), a protein that is essential for the import of preproteins with internal targeting sequences into the mitochondrion. Until now, Tom70 has only been found in animals and Fungi. We have identified a tom70 gene in the human parasitic anaerobic stramenopile Blastocystis sp. that is neither an animal nor a fungus. Using a combination of bioinformatics, genetic complementation, and immunofluorescence microscopy analyses, we demonstrate that this protein functions as a typical Tom70 in Blastocystis mitochondrion-related organelles. Additionally, we identified putative tom70 genes in the genomes of other stramenopiles and a haptophyte, that, in phylogenies, form a monophyletic group distinct from the animal and the fungal homologues. The presence of Tom70 in these lineages significantly expands the evolutionary spectrum of eukaryotes that contain this protein and suggests that it may have been part of the core mitochondrial protein import apparatus of the last common ancestral eukaryote.
  • Hjort, K. et al. (2010). Diversity and reductive evolution of mitochondria among microbial eukaryotes. Philosophical Transactions of the Royal Society B: Biological Sciences [Online] 365:713-727. Available at: http://dx.doi.org/10.1098/rstb.2009.0224.
    All extant eukaryotes are now considered to possess mitochondria in one form or another. Many parasites or anaerobic protists have highly reduced versions of mitochondria, which have generally lost their genome and the capacity to generate ATP through oxidative phosphorylation. These organelles have been called hydrogenosomes, when they make hydrogen, or remnant mitochondria or mitosomes when their functions were cryptic. More recently, organelles with features blurring the distinction between mitochondria, hydrogenosomes and mitosomes have been identified. These organelles have retained a mitochondrial genome and include the mitochondrial-like organelle of Blastocystis and the hydrogenosome of the anaerobic ciliate Nyctotherus. Studying eukaryotic diversity from the perspective of their mitochondrial variants has yielded important insights into eukaryote molecular cell biology and evolution. These investigations are contributing to understanding the essential functions of mitochondria, defined in the broadest sense, and the limits to which reductive evolution can proceed while maintaining a viable organelle.
  • Gaston, D., Tsaousis, A. and Roger, A. (2009). Chapter 2 Predicting Proteomes of Mitochondria and Related Organelles from Genomic and Expressed Sequence Tag Data. Methods in Enzymology [Online] 457:21-47. Available at: http://dx.doi.org/10.1016/S0076-6879(09)05002-2.
    In eukaryotes, determination of the subcellular location of a novel protein encoded in genomic or transcriptomic data provides useful clues as to its possible function. However, experimental localization studies are expensive and time-consuming. As a result, accurate in silico prediction of subcellular localization from sequence data alone is an extremely important field of study in bioinformatics. This is especially so as genomic studies expand beyond model system organisms to encompass the full diversity of eukaryotes. Here we review some of the more commonly used programs for prediction of proteins that function in mitochondria, or mitochondrion-related organelles in diverse eukaryotic lineages and provide recommendations on how to apply these methods. Furthermore, we compare the predictive performance of these programs on a mixed set of mitochondrial and non-mitochondrial proteins. Although N-terminal targeting peptide prediction programs tend to have the highest accuracy, they cannot be effectively used for partial coding sequences derived from high-throughput expressed sequence tag surveys where data for the N-terminus of the encoded protein is often missing. Therefore methods that do not rely on the presence of an N-terminal targeting sequence alone are extremely useful, especially for expressed sequence tag data. The best strategy for classification of unknown proteins is to use multiple programs, incorporating a variety of prediction strategies, and closely examine the predictions with an understanding of how each of those programs will likely handle the data.
  • Tsaousis, A. et al. (2008). A novel route for ATP acquisition by the remnant mitochondria of Encephalitozoon cuniculi. Nature [Online] 453:553-556. Available at: http://dx.doi.org/10.1038/nature06903.
    Mitochondria use transport proteins of the eukaryotic mitochondrial carrier family (MCF) to mediate the exchange of diverse substrates, including ATP, with the host cell cytosol. According to classical endosymbiosis theory, insertion of a host-nuclear-encoded MCF transporter into the protomitochondrion was the key step that allowed the host cell to harvest ATP from the enslaved endosymbiont. Notably the genome of the microsporidian Encephalitozoon cuniculi has lost all of its genes for MCF proteins. This raises the question of how the recently discovered microsporidian remnant mitochondrion, called a mitosome, acquires ATP to support protein import and other predicted ATP-dependent activities. The E. cuniculi genome does contain four genes for an unrelated type of nucleotide transporter used by plastids and bacterial intracellular parasites, such as Rickettsia and Chlamydia, to import ATP from the cytosol of their eukaryotic host cells. The inference is that E. cuniculi also uses these proteins to steal ATP from its eukaryotic host to sustain its lifestyle as an obligate intracellular parasite. Here we show that, consistent with this hypothesis, all four E. cuniculi transporters can transport ATP, and three of them are expressed on the surface of the parasite when it is living inside host cells. The fourth transporter co-locates with mitochondrial Hsp70 to the E. cuniculi mitosome. Thus, uniquely among eukaryotes, the traditional relationship between mitochondrion and host has been subverted in E. cuniculi, by reductive evolution and analogous gene replacement. Instead of the mitosome providing the parasite cytosol with ATP, the parasite cytosol now seems to provide ATP for the organelle.
  • Goldberg, A. et al. (2008). Localization and functionality of microsporidian iron–sulphur cluster assembly proteins. Nature [Online] 452:624-628. Available at: http://dx.doi.org/10.1038/nature06606.
    Microsporidia are highly specialized obligate intracellular parasites of other eukaryotes (including humans) that show extreme reduction at the molecular, cellular and biochemical level. Although microsporidia have long been considered as early branching eukaryotes that lack mitochondria, they have recently been shown to contain a tiny mitochondrial remnant called a mitosome. The function of the mitosome is unknown, because microsporidians lack the genes for canonical mitochondrial functions, such as aerobic respiration and haem biosynthesis. However, microsporidial genomes encode several components of the mitochondrial iron-sulphur (Fe-S) cluster assembly machinery. Here we provide experimental insights into the metabolic function and localization of these proteins. We cloned, functionally characterized and localized homologues of several central mitochondrial Fe-S cluster assembly components for the microsporidians Encephalitozoon cuniculi and Trachipleistophora hominis. Several microsporidial proteins can functionally replace their yeast counterparts in Fe-S protein biogenesis. In E. cuniculi, the iron (frataxin) and sulphur (cysteine desulphurase, Nfs1) donors and the scaffold protein (Isu1) co-localize with mitochondrial Hsp70 to the mitosome, consistent with it being the functional site for Fe-S cluster biosynthesis. In T. hominis, mitochondrial Hsp70 and the essential sulphur donor (Nfs1) are still in the mitosome, but surprisingly the main pools of Isu1 and frataxin are cytosolic, creating a conundrum of how these key components of Fe-S cluster biosynthesis coordinate their function. Together, our studies identify the essential biosynthetic process of Fe-S protein assembly as a key function of microsporidian mitosomes.
  • Tsaousis, A. et al. (2005). Widespread recombination in published animal mtDNA sequences. Molecular Biology and Evolution [Online] 22:925-33. Available at: http://dx.doi.org/10.1093/molbev/msi084.
    Mitochondrial DNA (mtDNA) recombination has been observed in several animal species, but there are doubts as to whether it is common or only occurs under special circumstances. Animal mtDNA sequences retrieved from public databases were unambiguously aligned and rigorously tested for evidence of recombination. At least 30 recombination events were detected among 186 alignments examined. Recombinant sequences were found in invertebrates and vertebrates, including primates. It appears that mtDNA recombination may occur regularly in the animal cell but rarely produces new haplotypes because of homoplasmy. Common animal mtDNA recombination would necessitate a reexamination of phylogenetic and biohistorical inference based on the assumption of clonal mtDNA transmission. Recombination may also have an important role in producing and purging mtDNA mutations and thus in mtDNA-based diseases and senescence.

Book section

  • Tsaousis, A. et al. (2011). The biochemical adaptations of mitochondrion-related organelles of parasitic and free-living microbial eukaryotes to low oxygen environments. in: Altenbach, A. D., Bernhard, J. M. and Seckbach, J. eds. Anoxia: Evidence for Eukaryote Survival and Paleontological Strategies. Netherlands: Springer Netherlands, pp. 51-81. Available at: http://link.springer.com/chapter/10.1007%2F978-94-007-1896-8_4.
    While many multicellular anaerobes possess mitochondria that resemble those of aerobic eukaryotes, microbial eukaryotes that live exclusively in anoxic and low oxygen environments harbor mitochondrion-related organelles (MROs). Currently, these organelles are broadly classified as either hydrogenosomes (anaerobic ATP-producing organelles that produce molecular hydrogen) or mitosomes (organelles that do not generate ATP); however, ongoing studies of diverse microbial lineages are revealing a wider spectrum of functional types. In adaptation to low oxygen conditions, the MROs of anaerobic eukaryotes have acquired unique characteristics, some of which do not appear to derive from the ?-proteobacterium that gave rise to the ancestral mitochondrion. These characteristics include alternative pathways for pyruvate metabolism as well as enzymes such as [FeFe]-hydrogenases that collectively function in anaerobic energy metabolism. In addition to these pathways, the mitochondrial protein import, metabolic exchange, and Fe–S cluster biosynthesis machineries are present in all MROs studied to date; these systems support the protein, solute, and energy requirements of both the organelles and the cells that harbor them. MROs represent a unique class of organelles that have successfully adapted by reduction or alteration of existing pathways as well as by acquisition of novel metabolic machineries that allowed their hosts to thrive in diverse environments without oxygen.
  • Tsaousis, A. et al. (2010). The Blastocystis mitochondrion-like organelles. in: Clark, C. G., Adam, R. D. and Johnson, P. eds. Anaerobic Parasitic Protozoa: Genomics and Molecular Biology. Caister Academic Press. Available at: http://www.horizonpress.com/protozoa.
    The organelles in Blastocystis that resemble mitochondria are an enigma as the organism is a strict anaerobe. Recent sequence analyses of the organelle genome and over 12,000 expressed sequence tags (ESTs) has given us many insights into the role these organelles play in the metabolism of the cell. The genome encodes several subunits of NADH dehydrogenase (complex I) but lacks all trace of genes for cytochrome and ATPase subunits (Complexes III-V). ESTs confirm the presence of complexes I and II, and indicate that this partial electron transport chain may lead to an alternative oxidase. The ESTs also suggest that many other metabolic pathways characteristic of mitochondria are still present in the Blastocystis organelles. However, other findings show that the organelle also has characteristics in common with hydrogenosomes, as a gene encoding [FeFe] hydrogenase is present and the protein has been localised to the organelles. The nuclear genome should clarify many of the remaining questions surrounding these unusual organelles.

Forthcoming

  • Robertson, L. et al. (2019). Are molecular tools clarifying or confusing our understanding of the public health threat from zoonotic enteric protozoa in wildlife? International Journal for Parasitology: Parasites and Wildlife [Online]. Available at: https://doi.org/10.1016/j.ijppaw.2019.01.010.
    Emerging infectious diseases are frequently zoonotic, often originating in wildlife, but enteric protozoa are considered relatively minor contributors. Opinions regarding whether pathogenic enteric protozoa may be transmitted between wildlife and humans have been shaped by our investigation tools, and has led to oscillations regarding whether particular species are zoonotic or have host-adapted life cycles.

    When the only approach for identifying enteric protozoa was morphology, it was assumed that many enteric protozoa colonized multiple hosts and were probably zoonotic. When molecular tools revealed genetic differences in morphologically identical species colonizing humans and other animals, host specificity seemed more likely. Parasites from animals found to be genetically identical - at the few genes investigated - to morphologically indistinguishable parasites from human hosts, were described as having zoonotic potential. More discriminatory molecular tools have now sub-divided some protozoa again. Meanwhile, some infection events indicate that, circumstances permitting, some “host-specific” protozoa, can actually infect various hosts. These repeated changes in our understanding are linked intrinsically to the investigative tools available.

    Here we review how molecular tools have assisted, or sometimes confused, our understanding of the public health threat from nine enteric protozoa and example wildlife hosts (Balantoides coli - wild boar; Blastocystis sp. - wild rodents; Cryptosporidium spp. - wild fish; Encephalitozoon spp. - wild birds; Entamoeba spp. - non-human primates; Enterocytozoon bieneusi - wild cervids; Giardia duodenalis - red foxes; Sarcocystis nesbitti - snakes; Toxoplasma gondii - bobcats).

    Molecular tools have provided evidence that some enteric protozoa in wildlife may infect humans, but due to limited discriminatory power, often only the zoonotic potential of the parasite is indicated. Molecular analyses, which should be as discriminatory as possible, are one, but not the only, component of the toolbox for investigating potential public health impacts from pathogenic enteric protozoa in wildlife.
  • Miller, C. et al. (2017). Effects of Cryptosporidium infections on host cell metabolome and host mitochondrial associations. mSphere [Online]. Available at: https://doi.org/10.1101/145979.
    Cryptosporidium is an important gut microbe whose contributions towards infant and immunocompromise patient mortality rates are steadily increasing. However, current techniques for studying the parasite are few and far between, relying on a combination of in-silico predictions and medical reports. The development of an in-vitro culture system, using COLO-680N cells (derived from an esophogeal squamous cell carcinoma), has provided the Cryptosporidium community with the opportunity to expand its toolkit for investigating this disease. One area in particular that is sorely overlooked is the effect infection has on host metabolic processes, especially those of the host mitochondria, which have been shown anecdotally in previous studies as being in abundance surrounding the sites of infection. Using a 1H Nuclear Magnetic Resonance approach to metabolomics, we have explored the nature of the mouse gut metabolome as well as providing the first insight into the metabolome of an infected cell line. Through a combination of Partial Least Squares Discriminant Analysis and predictive modelling, we demonstrate new understandings of the effects of a Cryptosporidium infection, while verifying the presence of known metabolic changes. Of particular note is the potential contribution of host derived taurine to the diuretic aspects of the disease previously attributed to a solely parasite based alteration of the gut environment. This practical and informative approach can spearhead our understanding of the Cryptosporidium-host metabolic exchange and thus provide novel targets for tackling this deadly parasite.
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