Drug resistance is one of the major impediments to control Plasmodium falciparum malaria worldwide. Heat shock protein 90 (Hsp90) is an essential component of the buffering capacity of eukaryotic cells as a part of the stress response. P. falciparum is no different and requires Hsp90 to chaperone proteins essential for cell cycle progression and drug resistance. Inhibition of P. falciparum Hsp90 (PfHsp90) may be able to not only cripple the parasite but also serve as an adjunctive antimalarial by circumventing drug resistance. The results presented in this thesis identify novel Hsp90 inhibitors that synergize with conventional antimicrobials, such as chloroquine (CQ), when used in combination. The objectives were to identify specific malaria Hsp90 inhibitors, the mechanism of the synergistic phenotype, and whether the strategy translates in vivo. To this end, the antimalarial activity of the purine analog PU-H71, and novel PfHsp90 inhibitors was tested. PU-H71 and the novel inhibitors APPA, harmine, and acrisorcin exhibited antimalarial activity in the nanomolar range and displayed synergistic activity with CQ. PU-H71 was able to reverse CQ resistance in a cell-based assay using the CQ-resistant strain W2. PU-H71 caused ring-stage arrest during the intra-erythrocytic cycle. Co-immunoprecipitation studies revealed that PfHsp90 interacts directly with the CQ resistance transporter (PfCRT). In the P. berghei mouse model of malaria, PU-H71 and harmine were able to reduce parasitemia and synergize with CQ. The interaction of PfHsp90 with PfCRT may underlie the synergistic phenotype. We conclude that PU-H71 and harmine are effective adjunctive antimalarial drugs that may be useful in combination therapies.

Drug resistance is one of the major impediments to control Plasmodium falciparum malaria worldwide. Heat shock protein 90 (Hsp90) is an essential component of the buffering capacity of eukaryotic cells as a part of the stress response. P. falciparum is no different and requires Hsp90 to chaperone proteins essential for cell cycle progression and drug resistance. Inhibition of P. falciparum Hsp90 (PfHsp90) may be able to not only cripple the parasite but also serve as an adjunctive antimalarial by circumventing drug resistance. The results presented in this thesis identify novel Hsp90 inhibitors that synergize with conventional antimicrobials, such as chloroquine (CQ), when used in combination. The objectives were to identify specific malaria Hsp90 inhibitors, the mechanism of the synergistic phenotype, and whether the strategy translates in vivo. To this end, the antimalarial activity of the purine analog PU-H71, and novel PfHsp90 inhibitors was tested. PU-H71 and the novel inhibitors APPA, harmine, and acrisorcin exhibited antimalarial activity in the nanomolar range and displayed synergistic activity with CQ. PU-H71 was able to reverse CQ resistance in a cell-based assay using the CQ-resistant strain W2. PU-H71 caused ring-stage arrest during the intra-erythrocytic cycle. Co-immunoprecipitation studies revealed that PfHsp90 interacts directly with the CQ resistance transporter (PfCRT). In the P. berghei mouse model of malaria, PU-H71 and harmine were able to reduce parasitemia and synergize with CQ. The interaction of PfHsp90 with PfCRT may underlie the synergistic phenotype. We conclude that PU-H71 and harmine are effective adjunctive antimalarial drugs that may be useful in combination therapies.

Severe malaria infections cause almost 1 million deaths annually, mostly among non-immune African children. The pathogenesis of severe malaria is poorly understood. It is increasingly appreciated that while host innate immune responses such as inflammation and phagocytosis are critical for control of parasite replication, they can become dysregulated and contribute to severe disease. The goals of this work were: (1) to characterize inflammatory responses to malaria by defining their relationship to phagocytosis and identifying novel molecular mediators, and (2) to evaluate the utility of biomarkers of inflammation and other host responses for predicting outcome in severe malaria infection. Using an in vitro model of the malaria-macrophage interaction, inflammatory and phagocytic responses to Plasmodium falciparum were found to be partially coupled. Activation of Toll-like receptors (TLRs) by purified parasite components increased internalization of parasitized erythrocytes, but uptake of parasitized erythrocytes did not require TLRs, nor did it trigger cytokine production via TLRs or other receptors. Two candidate molecules – Triggering receptor expressed on myeloid cells-1 (TREM-1) and Chitinase-3 like-1 (CHI3L1) – did not appear to critically modulate inflammation to malaria in vitro or in murine models. However, exogenous TREM-1 activation enhanced the pro- inflammatory nature of the response to P. falciparum, with potential implications for malarial-bacterial co-infection. CHI3L1-deficient mice showed a trend towards earlier death in experimental cerebral malaria, suggesting that CHI3L1 may protect against severe malaria; however, further investigation in more informative models is required. Admission levels of plasma TREM-1, CHI3L1, and other biomarkers of inflammation and endothelial activation were increased in Ugandan children with severe malaria. Simple combinations of these biomarkers predicted mortality among severe malaria patients with high accuracy, warranting larger validation studies. Taken together, these findings identify host responses as putative targets for adjunctive therapies, and suggest the utility of host biomarker combinations as prognostic tests for severe malaria.

Severe malaria infections cause almost 1 million deaths annually, mostly among non-immune African children. The pathogenesis of severe malaria is poorly understood. It is increasingly appreciated that while host innate immune responses such as inflammation and phagocytosis are critical for control of parasite replication, they can become dysregulated and contribute to severe disease. The goals of this work were: (1) to characterize inflammatory responses to malaria by defining their relationship to phagocytosis and identifying novel molecular mediators, and (2) to evaluate the utility of biomarkers of inflammation and other host responses for predicting outcome in severe malaria infection. Using an in vitro model of the malaria-macrophage interaction, inflammatory and phagocytic responses to Plasmodium falciparum were found to be partially coupled. Activation of Toll-like receptors (TLRs) by purified parasite components increased internalization of parasitized erythrocytes, but uptake of parasitized erythrocytes did not require TLRs, nor did it trigger cytokine production via TLRs or other receptors. Two candidate molecules – Triggering receptor expressed on myeloid cells-1 (TREM-1) and Chitinase-3 like-1 (CHI3L1) – did not appear to critically modulate inflammation to malaria in vitro or in murine models. However, exogenous TREM-1 activation enhanced the pro- inflammatory nature of the response to P. falciparum, with potential implications for malarial-bacterial co-infection. CHI3L1-deficient mice showed a trend towards earlier death in experimental cerebral malaria, suggesting that CHI3L1 may protect against severe malaria; however, further investigation in more informative models is required. Admission levels of plasma TREM-1, CHI3L1, and other biomarkers of inflammation and endothelial activation were increased in Ugandan children with severe malaria. Simple combinations of these biomarkers predicted mortality among severe malaria patients with high accuracy, warranting larger validation studies. Taken together, these findings identify host responses as putative targets for adjunctive therapies, and suggest the utility of host biomarker combinations as prognostic tests for severe malaria.

Biomarkers measured in the blood can provide information about disease pathophysiology, diagnosis and prognosis. Pronounced proinflammatory responses are characteristic of severe malaria, and excessive activation of the immune system is central to the pathophysiology of both cerebral malaria and placental malaria. Severe malaria is characterized by cytoadherence of parasitized erythrocytes to the microvasculature; impaired tissue perfusion; dysregulated inflammatory responses; and activation of the complement system, mononuclear cells, and endothelium. Despite the availability of effective antimalarial drugs, the mortality rate in severe malaria remains unacceptably high. To glean further insight into malaria pathophysiology, we investigated host biomarkers of immune activation in the blood of subjects with different manifestations of severe disease. C5 has been identified as being necessary and sufficient for the development of experimental cerebral malaria. We hypothesized that C5a (a terminal component of the complement cascade with potent inflammatory properties) may mediate its action by inducing and exacerbating inflammatory processes in severe malaria, leading to endothelial activation and dysregulated angiogenesis. I tested this hypothesis in vitro, and found that C5a interacted with malaria toxin PfGPI to drive deleterious inflammatory and anti-angiogenic responses. As C5a and anti-angiogenic factor sFlt-1 have been implicated in models of pathologic pregnancies, we asked whether increased levels of C5a in subjects with placental malaria were associated with altered angiogenesis and poor birth outcomes. Our results suggest that C5a impairs angiogenic remodelling in placental malaria leading to vascular insufficiency and fetal growth restriction. Further, altered profiles of inflammatory and angiogenic biomarkers in the periphery may identify occult placental malaria infections. We extended these observations to cerebral malaria where similar pathogenic pathways contribute to disease pathophysiology. In adults and children with cerebral malaria, altered profiles of angiogenic proteins were associated with disease severity and mortality and represent putative diagnostic and prognostic biomarkers in severe malaria.

Malaria has exerted a major selective pressure for red blood cell (RBC) polymorphisms that confer protection to severe disease. There is a predominance of blood type O in malaria endemic regions, and several lines of evidence suggest that the outcome of Plasmodium falciparum infection may be influenced by ABO blood type antigens. Based on observations that enhanced phagocytosis of infected polymorphic RBCs is associated with protection to malaria in other red cell disorders, we hypothesized that infected type O RBCs may be more efficiently cleared by the innate immune system than infected type A and B RBCs. The present work demonstrates human macrophages in vitro and murine monocytes in vivo phagocytosed P. falciparum infected O RBCs more avidly than infected A and B RBCs independent of macrophage donor blood type. This difference in clearance may confer relative resistance to severe malaria in individuals with blood type O.

Biomarkers measured in the blood can provide information about disease pathophysiology, diagnosis and prognosis. Pronounced proinflammatory responses are characteristic of severe malaria, and excessive activation of the immune system is central to the pathophysiology of both cerebral malaria and placental malaria. Severe malaria is characterized by cytoadherence of parasitized erythrocytes to the microvasculature; impaired tissue perfusion; dysregulated inflammatory responses; and activation of the complement system, mononuclear cells, and endothelium. Despite the availability of effective antimalarial drugs, the mortality rate in severe malaria remains unacceptably high. To glean further insight into malaria pathophysiology, we investigated host biomarkers of immune activation in the blood of subjects with different manifestations of severe disease. C5 has been identified as being necessary and sufficient for the development of experimental cerebral malaria. We hypothesized that C5a (a terminal component of the complement cascade with potent inflammatory properties) may mediate its action by inducing and exacerbating inflammatory processes in severe malaria, leading to endothelial activation and dysregulated angiogenesis. I tested this hypothesis in vitro, and found that C5a interacted with malaria toxin PfGPI to drive deleterious inflammatory and anti-angiogenic responses. As C5a and anti-angiogenic factor sFlt-1 have been implicated in models of pathologic pregnancies, we asked whether increased levels of C5a in subjects with placental malaria were associated with altered angiogenesis and poor birth outcomes. Our results suggest that C5a impairs angiogenic remodelling in placental malaria leading to vascular insufficiency and fetal growth restriction. Further, altered profiles of inflammatory and angiogenic biomarkers in the periphery may identify occult placental malaria infections. We extended these observations to cerebral malaria where similar pathogenic pathways contribute to disease pathophysiology. In adults and children with cerebral malaria, altered profiles of angiogenic proteins were associated with disease severity and mortality and represent putative diagnostic and prognostic biomarkers in severe malaria.

Malaria has exerted a major selective pressure for red blood cell (RBC) polymorphisms that confer protection to severe disease. There is a predominance of blood type O in malaria endemic regions, and several lines of evidence suggest that the outcome of Plasmodium falciparum infection may be influenced by ABO blood type antigens. Based on observations that enhanced phagocytosis of infected polymorphic RBCs is associated with protection to malaria in other red cell disorders, we hypothesized that infected type O RBCs may be more efficiently cleared by the innate immune system than infected type A and B RBCs. The present work demonstrates human macrophages in vitro and murine monocytes in vivo phagocytosed P. falciparum infected O RBCs more avidly than infected A and B RBCs independent of macrophage donor blood type. This difference in clearance may confer relative resistance to severe malaria in individuals with blood type O.

In response to growing levels of resistance to currently used antimalarials, there is an urgent need to develop drugs that exhibit novel mechanisms to kill Plasmodium parasites. The objective of this study was to examine the antiparasitic activity of newly synthesized compounds based on imidazolium and triazolium rings. According to our structure/activity relationship studies the key components appear to be their positively charged rings and hydrophobic side groups, and bivalent compounds, which incorporate two positively charged rings, show even greater potency than monovalent compounds. Depending on the concentration used, our compounds appear to primarily inhibit intracellular parasite development or invasion into red blood cells. Selected compounds have been tested in vivo using a P. berghei ANKA murine model. Together, our findings demonstrate that small imidazolium- and triazolium-based compounds display both in vitro and in vivo activity through a novel mechanism of action that may involve inhibition of erythrocyte invasion.

The increasing prevalence of apicomplexan parasites such as Plasmodium, Toxoplasma, and Cryptosporidium represents a significant global healthcare burden. Treatment options are increasingly limited due to the emergence of new resistant strains. We postulate that parasites have evolved distinct metabolic strategies critical for growth and survival during human infections, and therefore susceptible to drug targeting using a systematic approach. I developed iCS306, a fully characterized metabolic network reconstruction of the model organism Toxoplasma gondii via extensive curation of available genomic and biochemical data. Using available microarray data, metabolic constraints for six different clinical strains of Toxoplasma were modeled. I conducted various in silico experiments using flux balance analysis in order to identify essential metabolic processes, and to illustrate the differences in metabolic behaviour across Toxoplasma strains. The results elucidate probable explanations for the underlying mechanisms which account for the similarities and differences among strains of Toxoplasma, and among species of Apicomplexa.