Towards discovering novel aspects of nuclear biology in the malaria parasite "Plasmodium falciparum"

The apicomplexan parasite P. falciparum continues to cause morbidity and mortality imposing a significant health and economic burden on human society. In light of antimalarial drug resistance and the lack of an effective vaccine there is an urgent need to understand the basic biology of Plasmodium parasites in much greater detail. In particular, basic nuclear processes such as those remain surprisingly unsought despite their importance in parasite survival and life cycle progression. Thus, identification and localisation of novel parasite proteins to areas of the nucleus is an important first step towards giving new insights into nuclear architecture and function. The main aim of this thesis was to compile an inventory of the nuclear proteome across the intra-erythrocytic cell cycle using high accuracy mass spectrometry coupled with bioinformatics and in vivo localisation experiments. The dataset was analysed for accuracy and retention of true nuclear proteins revealing a final list of 802 potential nuclear proteins with an estimated precision of 76%. Interestingly, the informational pool of this study was able to identify a large number of novel nuclear components including novel protein domains possibly involved in gene regulation, members of the nuclear pores, the nucleolus and the proteasome (chapter 2). Several transgenic parasite lines used for the experimental validation part of the nuclear core proteome were further investigated in more detail. One of these transgenic cell lines expresses the C-terminally tagged bromo-domain protein PF10_0328 and was investigated by co-immuoprecipitation experiments followed by LC-MS/MS to identify interacting proteins. Bromodomain proteins bind specifically to acetylated lysine residues in histone tails and are important regulators of transcription. Our results suggest that PF10_0328 acts in concert with two additional bromo-domain proteins in regulating transcription in P. falciparum (chapter 3). Further characterisation on the functional level of these three important regulators is currently ongoing in a collaborative effort. Characterisation of bromo-domain proteins could establish new intervention strategies against malaria as the recognition of acetylated histone tails by bromo-domains can be selectively prevented by small molecules. Furthermore, several proteins residing in the nuclear pores and the nucleolus of P. falciparum were used to visualise these structures in relation to chromosome end clusters based on fluorescence microscopy. We show that both structures, involved in nuclear-cytoplasmic trafficking and ribosomal biogenesis, respectively, do not appear to ‘cross-talk’ with silenced chromosome ends at the nuclear periphery of P. falciparum (chapter 4). In conclusion, I believe that my work about several aspects of gene regulation and nuclear architecture increases the understanding of the biology of this medically important pathogen and could have potential to identify new avenues for interventions against malaria

Highlights from the Student Council Symposium 2011 at the International Conference on Intelligent Systems for Molecular Biology and European Conference on Computational Biology

The Student Council (SC) of the International Society for Computational Biology (ISCB) organized their annual symposium in conjunction with the Intelligent Systems for Molecular Biology (ISMB) conference

Characterisation of Trypanosomal Type III and Type IV Hsp40 proteins

The heat shock protein-70 (Hsp70) family of molecular chaperones are ubiquitous highly conserved proteins that are critical for the viability of cellular homeostasis. The ATPase activity of Hsp70 proteins is critical to their function as the affinity of a given Hsp70 for non-native substrate is modulated by ATP binding and hydrolysis. When bound to ATP, Hsp70s possess a low affinity for a given substrate protein, while the hydrolysis of ATP to ADP causes a conformational change that results in a high affinity for substrate proteins. The basal ATPase activity of Hsp70s is too low to facilitate their function in vivo, and co-chaperones are essential to modulate the efficient protein folding by Hsp70. Heat shock protein-40 (Hsp40) heat shock proteins are essential for the in vivo function of Hsp70s by stimulating the ATPase activity of these proteins and facilitating transfer of substrates. The Type III class of Hsp40 proteins have not been well characterised due to their poor levels of conservation at the primary sequence level. This is due to the fact that Type III Hsp40s only contain a J-domain and a poorly conserved C-terminal region. The newly identified Type IV class of Hsp40s, contain an abrogated HPD tripeptide motif in the J-domain and have also not been extensively studied. Trypanosoma brucei (T. brucei) is a unicellular flagellated protozoan parasite. It is the causative agent of Human African Trypansomiasis (HAT) which results in thousands of deaths and devastating agricultural losses in many parts of Africa. T. brucei undergoes a complex lifecycle that is characterised by the transition from an insect vector to a mammalian host in markedly different conditions of temperature, pH, nutrient availability and respiratory requirements. It has been proposed that molecular chaperones may enhance the survival of these parasites due to their cytoprotective effect in combating cellular stress. Due to the fact that T. brucei infection is invariably fatal if left untreated, and that no novel treatment regimens have been developed recently, the identification of potential novel drug targets among proteins essential to the parasite’s survival in the host organism is an attractive aspect of T. brucei research. Because Type III Hsp40s are poorly conserved with respect to Hsp40s found in the human host, the identification of any of these proteins found to be essential to T. brucei survival in humans could potentially make attractive novel drug targets. An in depth in silico investigation into the Type III Hsp40 complement as well as partner Hsp70 proteins in T.brucei was performed. T. brucei possesses 65 Hsp40 proteins, of which 47 were classed as Type III and 6 of which were identified as being putative Type IV Hsp40s. A small but significant number (5) of Type III TbHsp40s contained tetratricopeptide (TPR) domains in addition to the J-domain. The J-domains of the Type III TbHsp40 complement were found to be conserved with respect to those of canonical Hsp40 proteins, although the mutation of certain residues that play a key role in Hsp40-Hsp70 interaction was noted. Potential partnerships of these proteins in the parasite was also investigated. The coding regions of three previously uncharacterised TbHsp40s were successfully amplified from T. brucei TREU927 genomic DNA and cloned into an expression vector. Tbj1, a Tcj1 ortholog, was selected for further study and successfully expressed and biochemically characterised. Tbj1 expressed in E. coli was found to be insoluble, but large amounts were recovered with the aid of a denaturing purification followed by refolding elution strategies, and the bulk of the protein recovered was in compact monomeric form as determined by size-exclusion chromatography fast protein liquid chromatography (SEC-FPLC). The addition of Tbj1 to a thermally aggregated substrate resulted in increased levels of aggregation, although Tbj1 was able to assist two Hsp70 proteins in the suppression of aggregation. Tbj1 proved unable to stimulate the ATPase activity of these same Hsp70s, and could not rescue temperature sensitive cells when replacing E.coli DnaJ and CbpA. It was concluded that Tbj1 does not possess independent chaperone activity, but could display Hsp40 co-chaperone properties under certain circumstances. This could allude to a specialised function in the T. brucei parasite. The lack of human orthologues to Tbj1 could result in the attractiveness of this protein as a novel drug target

Characterisation of Trypanosomal Type III and Type IV Hsp40 proteins

The heat shock protein-70 (Hsp70) family of molecular chaperones are ubiquitous highly conserved proteins that are critical for the viability of cellular homeostasis. The ATPase activity of Hsp70 proteins is critical to their function as the affinity of a given Hsp70 for non-native substrate is modulated by ATP binding and hydrolysis. When bound to ATP, Hsp70s possess a low affinity for a given substrate protein, while the hydrolysis of ATP to ADP causes a conformational change that results in a high affinity for substrate proteins. The basal ATPase activity of Hsp70s is too low to facilitate their function in vivo, and co-chaperones are essential to modulate the efficient protein folding by Hsp70. Heat shock protein-40 (Hsp40) heat shock proteins are essential for the in vivo function of Hsp70s by stimulating the ATPase activity of these proteins and facilitating transfer of substrates. The Type III class of Hsp40 proteins have not been well characterised due to their poor levels of conservation at the primary sequence level. This is due to the fact that Type III Hsp40s only contain a J-domain and a poorly conserved C-terminal region. The newly identified Type IV class of Hsp40s, contain an abrogated HPD tripeptide motif in the J-domain and have also not been extensively studied. Trypanosoma brucei (T. brucei) is a unicellular flagellated protozoan parasite. It is the causative agent of Human African Trypansomiasis (HAT) which results in thousands of deaths and devastating agricultural losses in many parts of Africa. T. brucei undergoes a complex lifecycle that is characterised by the transition from an insect vector to a mammalian host in markedly different conditions of temperature, pH, nutrient availability and respiratory requirements. It has been proposed that molecular chaperones may enhance the survival of these parasites due to their cytoprotective effect in combating cellular stress. Due to the fact that T. brucei infection is invariably fatal if left untreated, and that no novel treatment regimens have been developed recently, the identification of potential novel drug targets among proteins essential to the parasite’s survival in the host organism is an attractive aspect of T. brucei research. Because Type III Hsp40s are poorly conserved with respect to Hsp40s found in the human host, the identification of any of these proteins found to be essential to T. brucei survival in humans could potentially make attractive novel drug targets. An in depth in silico investigation into the Type III Hsp40 complement as well as partner Hsp70 proteins in T.brucei was performed. T. brucei possesses 65 Hsp40 proteins, of which 47 were classed as Type III and 6 of which were identified as being putative Type IV Hsp40s. A small but significant number (5) of Type III TbHsp40s contained tetratricopeptide (TPR) domains in addition to the J-domain. The J-domains of the Type III TbHsp40 complement were found to be conserved with respect to those of canonical Hsp40 proteins, although the mutation of certain residues that play a key role in Hsp40-Hsp70 interaction was noted. Potential partnerships of these proteins in the parasite was also investigated. The coding regions of three previously uncharacterised TbHsp40s were successfully amplified from T. brucei TREU927 genomic DNA and cloned into an expression vector. Tbj1, a Tcj1 ortholog, was selected for further study and successfully expressed and biochemically characterised. Tbj1 expressed in E. coli was found to be insoluble, but large amounts were recovered with the aid of a denaturing purification followed by refolding elution strategies, and the bulk of the protein recovered was in compact monomeric form as determined by size-exclusion chromatography fast protein liquid chromatography (SEC-FPLC). The addition of Tbj1 to a thermally aggregated substrate resulted in increased levels of aggregation, although Tbj1 was able to assist two Hsp70 proteins in the suppression of aggregation. Tbj1 proved unable to stimulate the ATPase activity of these same Hsp70s, and could not rescue temperature sensitive cells when replacing E.coli DnaJ and CbpA. It was concluded that Tbj1 does not possess independent chaperone activity, but could display Hsp40 co-chaperone properties under certain circumstances. This could allude to a specialised function in the T. brucei parasite. The lack of human orthologues to Tbj1 could result in the attractiveness of this protein as a novel drug target

Methods for the refinement of genome-scale metabolic networks

More accurate metabolic networks of pathogens and parasites are required to support the identification of important enzymes or transporters that could be potential targets for new drugs. The overall aim of this thesis is to contribute towards a new level of quality for metabolic network reconstruction, through the application of several different approaches. After building a draft metabolic network using an automated method, a large amount of manual curation effort is still necessary before an accurate model can be reached. PathwayBooster, a standalone software package, which I developed in Python, supports the first steps of model curation, providing easy access to enzymatic function information and a visual pathway display to enable the rapid identification of inaccuracies in the model. A major current problem in model refinement is the identification of genes encoding enzymes which are believed to be present but cannot be found using standard methods. Current searches for enzymes are mainly based on strong sequence similarity to proteins of known function, although in some cases it may be appropriate to consider more distant relatives as candidates for filling these pathway holes. With this objective in mind, a protocol was devised to search a proteome for superfamily relatives of a given enzymatic function, returning candidate enzymes to perform this function. Another, related approach tackles the problem of misannotation errors in public gene databases and their influence on metabolic models through the propagation of erroneous annotations. I show that the topological properties of metabolic networks contains useful information about annotation quality and can therefore play a role in methods for gene function assignment. An evolutionary perspective into functional changes within homologous domains opens up the possibility of integrating information from multiple genomes to support the reconstruction of metabolic models. I have therefore developed a methodology to predict functional change within a gene superfamily phylogeny

Identification of Oocyst-Driven Toxoplasma gondii Infections in Humans and Animals through Stage-Specific Serology—Current Status and Future Perspectives

The apicomplexan zoonotic parasite Toxoplasma gondii has three infective stages: sporozoites in sporulated oocysts, which are shed in unsporulated form into the environment by infected felids; tissue cysts containing bradyzoites, and fast replicating tachyzoites that are responsible for acute toxoplasmosis. The contribution of oocysts to infections in both humans and animals is understudied despite being highly relevant. Only a few diagnostic antigens have been described to be capable of discriminating which parasite stage has caused an infection. Here we provide an extensive overview of the antigens and serological assays used to detect oocyst-driven infections in humans and animals according to the literature. In addition, we critically discuss the possibility to exploit the increasing knowledge of the T. gondii genome and the various ‘omics datasets available, by applying predictive algorithms, for the identification of new oocyst-specific proteins for diagnostic purposes. Finally, we propose a workflow for how such antigens and assays based on them should be evaluated to ensure reproducible and robust results.Peer Reviewe

The urothelium and lamina propria as an alternative target for clinical antimuscarinics

Introduction: Overactive bladder is the most common type of bladder dysfunction and involves spontaneous contractions of the urinary bladder during the filling phase. The first-line pharmaceutical therapies for managing this disorder are antimuscarinics (Moro et al., 2011), which have a primary action of blocking the action of acetylcholine in the urothelium and lamina propria (Nardulli et al., 2012). However, more than 70% of patients who are administered these drugs cease their treatment regimen due to lower than expected treatment benefits or adverse side effects (Vouri et al., 2019). The reason for this is unclear, although this does suggest a varied effectiveness or selectivity of antimuscarinics on urinary bladder tissue. Aim: This study aims to find the differences in the abilities to inhibit contractions of the U&LP for commonly prescribed clinical antimuscarinics. Methods: Strips of porcine U&LP were mounted in carbogen-gassed Krebs-bicarbonate solution at 37°C. The tissues were paired with carbachol concentration-response curves performed in the absence or presence of clinically used antimuscarinics. The concentration for each antagonist was chosen at which the inhibited contractions reached a significant, but sub-maximal, extent. pEC50 values for each curve were analysed and estimated affinities calculated. Ethical approval was not required for this study as tissues were sourced from the local abattoir after slaughter for the routine commercial provision of food. Results: The clinical antimuscarinics producing right parallel shifts from the control in the U&LP (concentration; n value; estimated affinity or pkD; paired Student’s two-tailed t-test) included oxybutynin (1µM; 18; 7.08; p<0.001), solifenacin (1µM; 11; 6.88; p<0.001), darifenacin (100nM; 10; 6.48; p<0.001), tolterodine (1µM; 10; 8.00; p<0.001), trospium (100nM; 10; 7.63; p<0.001) and fesoterodine (100nM; 11; 7.40; p<0.001). Propiverine (concentration; n value; paired Student’s two-tailed t-test) did not produce a shift (1µM; 11; p=0.50). Conclusion: The data highlights a variance in the effectiveness of each clinically used antimuscarinic to antagonise the response to muscarinic receptor activation of the U&LP