1,721,075 research outputs found
Investigating the diversity, adaptations and potential roles of Blastocystis in the gut
Full text linkBlastocystis is ubiquitously distributed coloniser of the gastrointestinal tract. To date, 17 subtypes and other isolates have been characterised from a range of invertebrate and vertebrate hosts, including humans. Despite current knowledge regarding its prevalence, diversity and associations with the gut microbiota, there is still uncertainty about its role as a pathogen. Herein, I aimed to explore the diversity of Blastocystis and investigate its role(s) in the gut using a multiphasic approach, combining culturomics, with molecular biology, phylogenetics, metagenomics, transcriptomics, and metabolomics. This allowed for an investigation of not only the prevalence of Blastocystis, but aided in the exploration of its in vivo and in vitro function.Results from my thesis demonstrate that Blastocystis had a high incidence in asymptomatic captive animals, with an excess of 40% in the species sampled. Additionally, numerous novel hosts were identified, while showing that co-infection with other microbial eukaryotes was relatively frequent. Bacterial community profiling of Blastocystis positive animals demonstrated no overall changes to bacterial diversity, but highlighted a number of taxa that were associated with Blastocystis colonisation. In vitro and in vivo NMR metabolomics further revealed a distinct metabolome in positive individuals. Here, a number of metabolites linked to eubiosis were identified including l-arginine, l-glutamine and propanoate. Lastly, treatment of Blastocystis with oxygen revealed a number of pathways possibly implicated in oxygen stress responses.As a result, my Ph.D work provides a steppingstone to understand the prevalence of Blastocystis and the role of this questionable gut "parasite" in health and disease in both animals and humans
Investigating the cellular localization and functional role of iron-sulphur cluster assembly machinery in Naegleria gruberi
No full textNaegleria gruberi is a eukaryotic microbe that belongs to the group of excavates. It is a free-living eukaryotic unicellular amoeboflagellate, with the capacity to change life forms (amoeba, flagellate, cyst) and shift from aerobic to anaerobic respiration. N. gruberi is an important organism to study for many reasons. Firstly,because of its close relationship with the brain-eating amoeba Naegleria fowleri, which causes Primary Amoebic Meningoencephalitis (PAM). Also, because of its unique internal and external traits. It has the capacity of transitioning to different life forms such as amoeba, flagellate, and cyst, in accordance with its external conditions. Additionally, it holds a crucial position to the phylogenetic tree and possesses similarities to the Last Eukaryotic Common Ancestor (LECA), reflecting its evolution. Amongst organisms there are various cellular and biochemical pathways that have been conserved. Some of them are the Iron- Sulphur (Fe-S) cluster biosynthetic pathways, which are of great importance. The Fe-S cluster mechanisms have been found in all the organisms studied to date and they are crucial for their survival. Eukaryotic cells typically harbour two of these mechanisms. These mechanisms are the Iron-Sulphur cluster assembly mechanism (ISC) and the Cytosolic Iron-Sulphur cluster assembly mechanism (CIA). The ISC is found in both eukaryotic and prokaryotic organisms, and it is responsible for the biosynthesis of Fe-S clusters and their transportation and incorporation of a variety of proteins. It therefore plays an important part in electron transport, enzyme activity, and overall mitochondrial function. On the other hand, the CIA mechanism is found exclusively in eukaryotic organisms, and it involves the maturation of Fe-S clusters, and their incorporation into nuclear and cytosolic proteins. Therefore, it is important for several cellular processes, such as DNA repair. Other Fe-S cluster mechanisms include the Sulphur Mobilization (SUF) mechanism and the Nitrogen Fixation (NIF) mechanism. These mechanisms are usually found in prokaryotes. The SUF mechanism is usually activated under extreme conditions, such as oxidative stress, while the NIF mechanism is responsible for the production of Fe-S cluster for the proteins involved in nitrogen fixation.
The aim of this study was to understand the evolution, location, and characteristics of the ISC mechanism within N. gruberi. For this a combination of bioinformatics and wet-lab experimental approaches were used. The bioinformatic analyses includes the alignment of the N. gruberi proteins to the same proteins of other organisms as well as the construction of phylogenetic trees. Those tests point out the conservation of the ISC proteins amongst different species, displaying that some proteins were more conserved than others, in addition to their place in the phylogenetic tree and an initial view to their evolutionary path. All the above showed that the N. gruberi proteins we targeted are closer to those of prokaryotic organisms. Lastly, bioinformatic analysis gave an initial idea of the localization of the proteins within the cell, which places some of them in the mitochondria and others in the cytosol. Additionally, through bioinformatics, the presence of proteins from the other Fe-S cluster mechanisms within the cell was tested. Proteins of the NIF system seem to be absent from the cell. Some proteins were similar to those of the SUF machinery, but further research is important to confirm or reject this hypothesis. Further experimental techniques including immunolocalization placed the proteins of the ISC machinery in both the mitochondria and the cytosol of the organism, proving that the pathway can be translocated outside the mitochondria if necessary
Monitoring initial effects of the addition of biochar to cattle feed and its impact on cattle gut and soil microbiomes, with a focus on the impact to methanogens.
No full textCurrent agricultural practices have a significant impact on climate change primarily through greenhouse gas emissions. Ruminants are responsible for 96% of the methane emissions in agriculture making them a key target for emission reduction efforts. Biochar has been identified as a feed additive with the potential to reduce ruminant methane emissions. This thesis focuses on methanogens (methane producing archaea), this group of archaea have been chosen as the focus of this pilot study due to biochar's potential to mitigate their activity.
Biochar is a carbon rich substance produced through pyrolysis of organic materials. In this study, the biochar used was derived from agricultural waste. Biochar is extremely carbon rich, with a porous structure containing many minerals. Biochar's structure and chemical makeup can have various positive impacts on soil, such as increasing carbon sequestration (and thereby increasing soil organic carbon levels), which can enhance soil structure, water retention, and nutrient availability, it also produces a favourable environment for beneficial microbes. Biochar is also being studied as a feed additive for cattle. The impact of biochar on the gastrointestinal tract is the subject of ongoing research, however several benefits have been suggested. It is hypothesised that with in the gastrointestinal tract of cattle and other ruminants' biochar can act as an electron acceptor reducing the reliance of carbon dioxide which plays a vital role in the production of methane.
This thesis is a preliminary study examining the impact of biochar on methanogens, as a reduction in methanogen populations has the potential to result in a decrease in methane emissions over time. Ruminant gastrointestinal as well as soil methanogen populations are to be monitored via monthly sampling over a four month time period as part of a broader longitudinal study spanning several years. Monitoring will be done by extracting DNA from the faecal and soil samples for them to be Sanger and Illumina sequenced to discover the prevalent genus, as well as to discover how these genus populations change over time.
This thesis outlines and has established the protocols to be used in order to identify changes that may occur to the genus' and abundance of methanogens found in cattle stool and soil samples. The information gathered within this thesis will be the baseline used to continuously monitor changes that have occurred due to the continuous addition of biochar over the longitudinal study.
Here it was discovered that the dominant species of methanogen found across all timepoints for both the stool and soil samples is Methanobrevibacter. Whilst Methanobrevibacter was the dominant species of methanogen found in stool samples across all timepoints using the data gathered from NCBI BLAST we see that a reduction of this species does occur. The Methanobrevibacter sp. accounted for 60% of the species observed in the T0 samples, down to 37.8% at T3. Despite of this, due to the short time frame of this pilot study it is still hard to draw conclusions from this data however, samples will be closely monitored over the upcoming years to produce a more reliable timeframe
Establishing Cryptosporidium parvum as a model organism
Full text linkCryptosporidium parvum is among the most common parasites in the known world and represents one of the leading causes of death among the immunocompromised. As an apicomplexan, C. parvum has many similarities to other globally important parasites such as Plasmodium falciparum and Toxoplasma gondii. Among these similarities are a complex life cycle and the ability to invade host cells. However, unlike most other apicomplexans, the cryptosporidia appear to have lost their namesake organelle, the apicoplast, and drastically reduced the size of their genome. For decades this caused issues in classifying the cryptosporidia. This has been potentially resolved, however, by recent phylogenetic studies that revealed a strong relationship between the cryptosporidia and the gregarines. The gregarines were parasites exclusively of invertebrates, until the reclassification to include the cryptosporidia. Though research into apicomplexan evolution and biology is still a nascent field, even less is known about the invertebrate portion. This is largely due to the lack of molecular tools and culturing techniques that are required to explore any organism beyond basic phylogenetics, in addition to their medical irrelevance prior to the inclusion of Cryptosporidium. Therefore, C. parvum represents a potential model organism for the gregarines and the evolutionary adaptations of apicomplexans from invertebrate to vertebrate hosts. It was the purpose of this thesis, therefore, to establish the tools and methodologies that would be required to begin developing C. parvum as such. To achieve this, first I successfully developed the world's first long-term culturing system of C. parvum, capable of maintain a live parasite culture for 60 days. Additionally, I developed novel methods of detecting and characterising the infection, including NMR based characterisation of infection metabolomes which also revealed a potentially more involved role for Taurine in the pathology of the infection. Furthermore, to demonstrate the power and applicability of this new system I produced the first experimental evidence for a functional ISC system within C. parvum. This also adds to a now growing list of non-canonical mitochondria containing organisms that still maintain an active mitochondrial Fe/S cluster biosynthetic pathway. In conclusion, this thesis represents a large step forward for both the C. parvum and gregarine fields and establishes many of the necessary techniques required for a new push in understanding these apicomplexans and their organelles
Exploring the diversity of mitochondrion related organelles in Cryptosporidium species
Full text linkCryptosporidium is an apicomplexan parasite that causes widespread diarrhoeal disease in both humans and animals. It is a parasite that is responsible for large waterborne outbreaks of Cryptosporidiosis. Humans are thought to acquire the parasite by the ingestion of oocysts, which are shed in the stool of infected animals or humans. Research has been done on this parasite, however up till now there has not been a treatment against this.
Cryptosporidium have lost their mitochondrial genome and therefore various mitochondrial pathways have been conserved in some of these species. Using bioinformatics analysis, we summarized the differences and similarities in the metabolic pathways in the mitosomes of six Cryptosporidium species (C. parvum, C. hominis, C. tyzzeri, C. ubiquitum, C.andersoni and C.muris) . As only C. parvum and C. hominis pathways have been mapped on KEGG Pathway Database, amino acid sequences were extracted for proteins involved in glycolysis/gluconeogenesis (map00010), TCA cycle (map00020), protein import machinery (map03060), pentose phosphate pathway (map00030), pyruvate metabolism (map00620) and oxidative phosphorylation (map00190). Amino acid sequences for the protein coding genes were run in BlastP on CryptoDB using the set parameters. The gene results were analyzed and used to produce diagrams to show the metabolic pathways in the mitosomes of the Cryptosporidium species.In this study we show that the essential TCA cycle enzymes were present in the gastric species C. andersoni and C.muris, whereas C.parvum, C.hominis, C.tyzzeri and C.ubiquitum only retain the membrane bound malate-quinone oxidoreductase (MQO) and pyruvate synthase. All species investigated showed a highly reduced protein import machinery in comparison to P.falciparum, and conserved glycolysis/gluconeogenesis proteins and Fe-S (iron-sulfur) cluster assembly, suggesting that these two pathways are the main ways in which the parasite acquires energy.Our results from the CryptoDB Blast search for the Protein Import Machinery and other pathways investigated contained some putative protein coding genes which needs to be experimentally validated. The Tim 9-10 which is responsible for transporting hydrophobic proteins to the inner mitochondrial membrane was annotated as uncharacterized in three protein coding genes: C.hominis (GY17_00000465), C.parvum (cgd6_4450) and C.tyzzeri (CTYZ_00001648). Further studies such as PCR analysis as well as Western blotting can be used to characterize the protein present and molecular structural analysis carried out to find out the shape and immunofluorescent assays as well as neutralization assays used to assess the function of the protein. The results from this study can be helpful to direct further research in understanding the functions of the mitosomes of these species and could be pivotal in developing new therapeutic drugs that are used to target various pathways in Cryptosporidium species
In-vitro survival and enlargement of Gregarina polymorpha and attempts to generate an invertebrate gut cell line
No full textGregarines are a large group of ancestral Apicomplexan parasites that exclusively infect invertebrate hosts. Their basal phylogenetic positioning means their study could help to elucidate the evolution of obligate parasitism from free-living photosynthetic ancestors. Yet their lack of clinical significance to humans means little research is dedicated to gregarines and the lack of a culturing system for these organisms dramatically impedes that which is. Due to the inclusion of a life cycle stage in most gregarines which necessitates attachment to host cells for development to progress, a culturing system must incorporate appropriate host cells. The present thesis describes attempts to develop a gregarine host gut cell line, to be used as a culturing platform as well as attempts to sustain three gregarine species. For these cultures a variety of conditions were tested, including temperature, culture medium, antimicrobial concentration, and the type of culturing vessel. Microfluidics were even employed for seven gregarine cultures to simulate the environment of an invertebrate gut more accurately. However, none of the 91 primary gut cell cultures that were created displayed growth or replication though most were attached to the culture vessel base. Further, only Gregarina polymorpha, hosted by Tenebrio molitor larvae, survived, and grew in-vitro. Additionally, during attempts to sequence gregarines from freshwater insect larvae many micrographs were obtained depicting various developmental stages of both Pileophalus sinesis hosted by the caddisfly Brachycentrus subnubilis, and Enterocystis ephemerae hosted by the mayfly Ephemera danica
Unravelling the prevalence and impact of Cryptosporidium parvum on the gut microbiome, metabolome, and health status of neonatal calves
No full textCryptosporidium parvum (C. parvum), colonises and infects the gastrointestinal tract (GIT) of a diverse range of vertebrate hosts, resulting in the development of cryptosporidiosis, a clinical condition characterised by profuse watery diarrhoea. Additional symptoms in infected hosts include dehydration, weight loss, fever, and abdominal pain. This disease has a global distribution, affecting millions of individuals each year and disproportionately impacting children in developing countries, where it stands as a prominent cause of mortality. Furthermore, cryptosporidiosis is notably prevalent among young livestock, particularly neonatal calves, exhibiting symptoms similar to those seen in human infants. This prevalence leads to significant economic losses in the dairy and beef industry. Moreover, infected animals can excrete and disseminate millions of infectious parasites daily into the environment, perpetuating the cycle of infection and posing a continued threat to human health. Addressing the global challenge of cryptosporidiosis necessitates an exploration of the diversity of Cryptosporidium species (spp.), their zoonotic potential, and the dynamics of transmission within dairy farms across various countries. Additionally, it is essential to recognise the pivotal role of the gut microbiota in maintaining gut health, bolstering defences against harmful pathogens, facilitating energy production, and preserving the integrity of the gut barrier. Disruptions in the normal gut microbiota have been linked to various diseases, but the precise molecular and biochemical mechanisms through which C. parvum disrupts these intricate systems remain poorly understood. Therefore, a comprehensive investigation into the interaction between the parasite and the gut microorganisms and metabolites of the host holds the potential to reveal novel therapeutic strategies and identify potential biomarkers for detecting the presence of C. parvum. This research could provide invaluable insights into mitigating the impact of C. parvum on gut health and its associated diseases. To achieve these objectives, I first conducted an extensive analysis of Cryptosporidium spp. prevalence and transmission dynamics in cattle farms spanning a broad geographic area encompassing Belgium, France, the Netherlands, and Cyprus. In addition, I also delved into the genetic variation of C. parvum to evaluate the potential circulation of zoonotic subtypes within dairy farms. This project required the development and implementation of a robust protocol for deoxyribonucleic acid (DNA) extraction from faecal samples. Subsequently, a two-step nested polymerase chain reaction (PCR) was utilised to amplify and sequence specific fragments of the 18S ribosomal rRNA (18S rRNA) and 60-kDa glycoprotein (gp60) genes of the targeted organism. Furthermore, I employed a combination of bioinformatics and phylogenetic analyses to characterise the obtained sequences. The findings of this research shed light on the widespread prevalence of Cryptosporidium spp. within dairy farms across different countries. Notably, the research also raised concerns regarding cattle serving as potential carriers of C. parvum zoonotic subtypes, suggesting a potential threat to human health.
For the second part of my PhD, I pioneered a multiomics approach to understand the impact of C. parvum propagation in the calves GIT. This research aimed to monitor how the parasite infection modulates the gut microbiome and metabolome of the host by first characterising the impact of C. parvum infection on the taxonomic composition of the neonatal calves' gut microbiota and then assess the collective change of metabolites in the gut during C. parvum infection. To accomplish this, a cross-sectional study was conducted on 23 cattle farms in the Lille region of France. A total of 134 faecal samples were collected from neonatal calves aged between four to 21 days.
In this study, I employed both a nested PCR and qPCR protocol for accurate detection and quantification of the parasite. After molecular detection and faecal scoring, the calves were classified into three groups: non-Cryptosporidium infected (HH), Cryptosporidium infected without diarrhoea (IH), and Cryptosporidium infected with diarrhoea (DH). Out of the total 134 calves, 73 were found to be negative for C. parvum while the remaining 61 calves tested positive for the parasite (IH = 41 and DH = 20). The collected faecal samples were subjected to 16S rRNA gene sequencing, while 102 out of the 134 faecal samples were analysed through One-dimensional proton nuclear magnetic resonance (1D 1H-NMR) metabolomics. The resulting data underwent thorough analysis using various bioinformatics tools. The 16S rRNA analysis, revealed a significant impact of C. parvum on the gut microbiota of infected calves (particularly for the DH group) when compared to the non-infected group (HH). Notably, opportunistic bacterial genera like Fusobacterium_A, Clostridium_P and Gallibacterium exhibited increased relative abundance in the DH group. Conversely, bacterial genera associated with a healthy gut, such as Bifidobacterium_388775 and Parabacteroides_B_862066 had higher relative abundances in the HH group. Metabolomic profiles between these groups also showed marked differences, specifically between the DH versus the HH group. DH calves exhibited a significant decrease in the abundance of five metabolites, with special focus on the short-chain fatty acid (SCFA) Valerate, in contrast with the HH group. Conversely, the DH calves displayed a significant increase in 10 metabolites, including Ethanol, Formate, Lactate and Succinate, which are intermediates for SCFAs. The observed accumulation of these metabolites coupled with the decrease in valerate might be closely related to an increase in harmful bacteria and a decrease in commensal bacteria in the DH group in response to C. parvum infection. The findings from this study underscore the substantial impact of C. parvum infections on the composition of faecal microbiota in calves, leading to changes in faecal metabolites that might compromise the calves' ability to resist C. parvum infection. This research lays a crucial foundation for further investigations into the role of intestinal microbiota and their metabolism in neonatal calf diarrhoea associated with C. parvum, providing insights that can potentially inform future strategies for managing and preventing cryptosporidiosis. Moreover, I've established, for the first time, an NMR-based metabolomics approach to quantify metabolite changes in faeces post-C. parvum infection in calves. This method could be employed for future investigations examining the interplay between gut flora and metabolites in the context of other enteric protozoan infections
Host-Parasite Interactions of Cryptosporidium
No full textCryptosporidium is an obligate intracellular parasite that relies heavily on the host for survival. Upon invasion the parasite manipulates both metabolic and physiological properties of COLO-680N host cells. This project involved a combination of live cell imaging techniques and nuclear magnetic resonance (NMR) spectroscopy to elucidate some of the key manipulations of the host cell resulting from cryptosporidial infection. Here we demonstrate with live cell imaging analysis that the parasite increases the recruitment of actin in infected COLO-680N cultures and that the parasite is localising host cell mitochondria to the site of infection, as well as increasing the mitochondrial membrane potential of infected cells. NMR metabolomics demonstrated that the parasite is potentially manipulating metabolic pathways in the host to create a compartmentalised fatty acid synthesis system to bypass normally the self- limiting fatty acid synthesis pathways of the host in the cytosol and mitochondria. Evidence potentially revealing mechanisms behind apoptosis regulation and osmotic imbalance responsible for the pathogenicity were also uncovered in this study
Investigating the Biochemical and Organellar Adaptations of Proteromonas lacertae
Full text linkProteromonas lacertae is an anaerobic, biflagellated microbial eukaryote belonging to Stramenopiles, one of the largest and most diverse groups of eukaryotes, characterised by the presence of tripartite, hair-like structures on the larger of the two flagella. At least one microbial Stramenopile is known to not possess these characteristic features (e.g. Blastocystis) which does not resemble other organisms from this group in any way. In-spite them being morphologically very different, Proteromonas happens to be the closest-known relative of Blastocystis, both being within the opalines. They are also some of the only Stramenopiles known to colonise larger organisms, Proteromonas is found in the hindgut of lizards and Blastocystis is known to colonise the intestinal tract of a range of animals, yet whether either of them actually cause disease has yet to be confirmed. Not only is their morphology strikingly different, their mitochondria also bear no resemblance to one another. Blastocystis possesses multiple anaerobic mitochondria-related organelles (MROs), whereas Proteromonas has a single, large lobed mitochondrion closely associated with the nucleus. A striking biochemical observation making Blastocystis unique amongst Stramenopiles is the presence of an alternative oxidase, and the absence of complexes III, IV and V of the electron transport chain (ETC). As well as this, it has been predicted to harbour proteins that could establish a reduced/incomplete tricarboxylic acid (TCA) cycle in its MROs. The main focus of this investigation is to explore the mitochondrial protein composition of Proteromonas and compare it to Blastocystis. In addition, we will attempt to characterise some of the biochemical pathways, including the ETC, proteins involved in the TCA cycle and AOX. Preliminary results on these investigations will be presented. This data shows that, biochemically, Proteromonas and Blastocystis are very similar, suggesting that these mitochondrial adaptations occurred prior to the diversification of these two organisms
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