Effects of DNA damage and vesicular exchange in P. falciparum

Plasmodium falciparum causes human malaria and is a global leading cause of mortality from parasitic infections. While decades of concerted effort has yielded significant result in reducing the endemicity, there is evidence of a recent resurgence in transmission intensity. Notably, Plasmodium falciparum parasites are master adapter to various environmental pressures, including stresses mounted by the host immune system as well as assaults from an arsenal of anti-malarial drugs. Intuitively, this adept adaptability constitutes a major impedance to a continued success in the control program. The mechanisms that confer such a high adaptability to parasite is currently unclear, but the plastic AT-rich genome, a versatile but enigmatic transcription network as well as the recently characterized cell-cell communication module via extracellular vesicles are likely contributors to such cause. Through experimental designs that sought to investigate the dynamics of these aforementioned molecular and cellular programs on both population and single-cell level, the works presented in this thesis aim to further the current understandings on how the parasites respond and adapt to various environmental pressures. In paper I, we investigated the short-term effect of anti-malarial drugs on the genomic plasticity of parasite. We revealed that all the tested anti-malarial drugs can cause acute DNA damages and trigger a robust response to the damages by the upregulation of specific DNA repair pathways. We further demonstrated that the DNA damages elicited by the drug action can be erroneously repaired to incorporate random mutations. Therefore, due to the random nature of DNA damages and the subsequent error-prone repair, this can serve as a mechanism to rapidly diversify the genetic pools of the exposed parasite population and pre-deposit it to a rapid selection of resistant genotypes. In paper II, we described the role of extra-cellular vesicles (EVs) during malaria infection. Infected RBCs are previously characterized for their enhanced capacity to generate EVs. In this study, we discovered that EVs originated from infected cell contain a subset of host miRNAs and the Ago2 proteins. These EVs are readily internalized by endothelial cells and that the miRNAs trafficked within these EVs can accumulate in the endothelial cells and exert a global effect on the post-transcriptional gene regulatory network. We show that this unilateral cellular communication can contribute to vascular dysfunction, local and systemic immunological modulation as well as endothelial activation. In particular, we note that endothelial activation can promote sequestration of infected RBCs and, thereby, serve to avoid splenic clearance. In paper III, we developed and detailed a technical platform that enables single-cell transcriptomic analysis of individual Plasmodium falciparum parasites. We then utilized the method to decipher the transcriptional cascade underlying the process of gametogenesis, which is triggered by yet undetermined environmental cues. Interestingly, we revealed huge heterogeneity even within a highly synchronous parasite population, supporting the presence of a versatile transcriptional network. Moreover, we identified a distinct gene signature that is associated with sexually committed and differentiating parasites. This work has generated important knowledge that can be exploited for the design of transmission blocking drugs

Profiling the Essential Nature of Lipid Metabolism in Asexual Blood and Gametocyte Stages of Plasmodium falciparum

SummaryDuring its life cycle, Plasmodium falciparum undergoes rapid proliferation fueled by de novo synthesis and acquisition of host cell lipids. Consistent with this essential role, Plasmodium lipid synthesis enzymes are emerging as potential drug targets. To explore their broader potential for therapeutic interventions, we assayed the global lipid landscape during P. falciparum sexual and asexual blood stage (ABS) development. Using liquid chromatography-mass spectrometry, we analyzed 304 lipids constituting 24 classes in ABS parasites, infected red blood cell (RBC)-derived microvesicles, gametocytes, and uninfected RBCs. Ten lipid classes were previously uncharacterized in P. falciparum, and 70%–75% of the lipid classes exhibited changes in abundance during ABS and gametocyte development. Utilizing compounds that target lipid metabolism, we affirmed the essentiality of major classes, including triacylglycerols. These studies highlight the interplay between host and parasite lipid metabolism and provide a comprehensive analysis of P. falciparum lipids with candidate pathways for drug discovery efforts

Improving Pancreatic Islet Engraftment after Islet Transplantation through Administration of Gamma-Secretase Inhibitor DAPT

Abstract: Rapid and effective revascularization of transplanted pancreatic islets is vital for the survival and function of the islet graft. Insufficient vascularization after islet transplantation may be one causative factor to the failure of islet grafts in clinical transplantation. The aim of this study was to investigate if N-{N-[2-(3,5-Difluorophenyl)acetyl]-(S)-alanyl}- (S)-phenylglycine- tert-butyl ester (DAPT) administration can improve engraftment of transplanted islets. DAPT is a dipeptidic gamma-secretase inhibitor which inhibits Notch signaling. Notch signaling is involved in angiogenesis and inhibition may result in excessive formation of new blood vessels. Excessive vasculature may be beneficial in the immediate posttransplantation period since the transplanted islets are dependent on diffusion of oxygen and nutrients before revascularization. Islets isolated from C57BL/6 mice were transplanted beneath the renal capsule of C57BL/6 mice. After islet transplantation DAPT or vehicle was administered subcutaneously for three days. Mice treated with DAPT had an increased vascular density when compared to control mice two days and one month posttransplantation. Moreover, mice treated with DAPT showed 54±8.2 % functional blood vessels compared to 40±6.7 % in control mice two days posttransplantation. After one month, the fraction of functional blood vessels increased to 86±2.8 % in DAPT treated mice compared to 61±9.4 % in control mice. Our findings demonstrated that administration of DAPT may be a feasible strategy to improve engraftment of transplanted islets

Interleukin-35 Prevents Development of Autoimmune Diabetes Possibly by Maintaining the Phenotype of Regulatory B Cells

The anti-inflammatory role of regulatory B cells (Breg cells) has been associated with IL-35 based on studies of experimental autoimmune uveitis and encephalitis. The role of Breg cells and IL-35(+) Breg cells for type 1 diabetes (T1D) remains to be investigated. We studied PBMCs from T1D subjects and healthy controls (HC) and found lowered proportions of Breg cells and IL-35(+) Breg cells in T1D. To elucidate the role of Breg cells, the lymphoid organs of two mouse models of T1D were examined. Lower proportions of Breg cells and IL-35(+) Breg cells were found in the animal models of T1D compared with control mice. In addition, the systemic administration of recombinant mouse IL-35 prevented hyperglycemia after multiple low dose streptozotocin (MLDSTZ) injections and increased the proportions of Breg cells and IL-35(+) Breg cells. A higher proportion of IFN-gamma(+) cells among Breg cells were found in the PBMCs of the T1D subjects. In the MLDSTZ mice, IL-35 administration decreased the proportions of IFN-gamma(+) cells among the Breg cells. Our data illustrate that Breg cells may play an important role in the development of T1D and that IL-35 treatment prevents the development of hyperglycemia by maintaining the phenotype of the Breg cells under an experimental T1D condition.De två sista författarna delar sistaförfattarskapet.</p

Anti-Peroxyl Radical Quality and Antibacterial Properties of Rooibos Infusions and Their Pure Glycosylated Polyphenolic Constituents

The anti-peroxyl radical quality of two aqueous rooibos infusions and solutions of their most abundant glycosylated polyphenols was evaluated using pyrogallol red and fluorescein-based oxygen radical absorbance ratios. It was observed that the artificial infusions, prepared using only the most abundant polyphenols present in rooibos and at concentrations similar to those found in the natural infusions, showed greater antioxidant quality than the latter infusions, reaching values close to those reported for tea infusions. Additionally, the antimicrobial activity of the natural and artificial infusions was assessed against three species of bacteria: Gram (+) Staphylococus epidermidis and Staphylococcus aureus and Gram (−) Escherichia coli. When compared to the natural infusions the artificial beverages did not demonstrate any bacterostatic/cidal activity, suggesting that the antibacterial activity of rooibos is related to compounds other than the glycosylated polyphenols employed in our study

Shared elements of host-targeting pathways among apicomplexan parasites of differing lifestyles

Apicomplexans are a diverse group of obligate parasites occupying different intracellular niches that require modification to meet the needs of the parasite. To efficiently manipulate their environment, apicomplexans translocate numerous parasite proteins into the host cell. Whereas some parasites remain contained within a parasitophorous vacuole membrane (PVM) throughout their developmental cycle, others do not, a difference that affects the machinery needed for protein export. A signal-mediated pathway for protein export into the host cell has been characterized in Plasmodium parasites, which maintain the PVM. Here, we functionally demonstrate an analogous host-targeting pathway involving organellar staging prior to secretion in the related bovine parasite, Babesia bovis, a parasite that destroys the PVM shortly after invasion. Taking into account recent identification of a similar signal-mediated pathway in the coccidian parasite Toxoplasma gondii, we suggest a model in which this conserved pathway has evolved in multiple steps from signal-mediated trafficking to specific secretory organelles for controlled secretion to a complex protein translocation process across the PVM