Molecular interactions and signaling mechanisms in malaria pathogenesis and protective immunity development
Malaria is a complex and devastating disease characterized by many systemic clinical manifestations and severe organ-related pathologies. The protozoan parasites of the Plasmodium family are the causative agents and malaria presents enormous health burden in most tropical regions of the world. Nearly half of the world population is at risk of exposure to malaria infection with ~0.5 billion clinical cases and ~1 million deaths occurring annually. In addition, the malaria mortality enormously hinders the socioeconomic development of people in endemic countries. Several Plasmodium species infect humans. Of these, P. falciparum is responsible for the majority of malaria deaths. Currently, drug resistance in malaria parasites is widespread in many endemic regions, posing increased risk of causing severe disease. Moreover, despite extensive efforts for decades in many laboratories, an effective vaccine remains elusive. Therefore, development of new drugs and/or an effective vaccine is needed.
Malaria pathogenesis is for the most part a host immune-mediated process. Production of pro-inflammatory cytokines and other inflammatory mediators function as the first line of defense against malaria by controlling parasite growth. Pro-inflammatory cytokines also promote efficient Th1 development and shape cell-mediated and humoral immune responses for the eventual clearance of infection. However, excessive and dysregulated pro-inflammatory responses lead to pathogenesis. Therefore, a balanced production of pro- and anti-inflammatory responses and appropriate regulation of cell-mediated and humoral responses is required for effective clearance of infection avoiding pathogenesis.
Dendritic cells (DCs) and macrophages are the major cells that initiate pro-inflammatory cytokine responses to malaria infection (Figure 1). Although several parasite factors that initiate innate immune responses by macrophages and DCs have been identified, our knowledge on host-parasite interactions is still incomplete. Moreover, only limited information is known about the signaling mechanisms that regulate innate immune responses and the subsequent molecular and cellular interactions involved in malaria pathogenesis and acquisition of protective immunity. Detailed knowledge of these processes is essential for the development of an effective vaccine and/or immunotherapeutics for malaria.
A key virulent factor of P. falciparum that contributes substantially to the severity of malaria is the expression of a family of 200-400 kDa, antigenically variant, adhesive proteins called P. falciparum erythrocyte membrane protein 1 (PfEMP1) on the surface of infected red blood cells (IRBCs). This protein confers the parasite an ability to sequester in the microvascular capillaries of organs such as brain, kidney and lungs, and in the blood space of placenta by binding to host receptors such as CD36, ICAM-1, thrombospondin on endothelia, and chondritin 4-sulfate (C4S) in the placenta. IRBC adherence leads to vascular obstruction, accumulation of toxic metabolites, inflammation, reciprocal enhancement of inflammatory responses and IRBC adherence, leading to endothelial damage and causing cerebral malaria and liver, kidney and organ dysfunction and failure.
Our laboratory is focused to understand the malaria parasite Plasmodium falciparum-host interactions and signaling mechanisms involved in the development of protective immunity to malaria and in malaria pathogenesis. We are specifically interested in studying parasite immunostimulatory factors and host receptors that initiate innate immune responses and the how innate immunity to malaria regulates the development of protective immunity. Our other interest is to again in-depth understanding of mechanisms involved in the adherence of P. falciparum IRBCs in the placenta that causes placental malaria.
Project on Immunity to Malaria
Previously, our laboratory determined the structures of P. falciparum glycosylphosphatidyl-inositols (GPIs), an immunostimulatory glycolipids and studied their structure-activity relationship. Further, we demonstrated that: (i) the GPI biosynthesis pathway is an effective drug target; (ii) GPI moieties contribute to the immunogenicity of GPI-anchored proteins; (iii) the presence of anti-GPI antibodies in people in endemic areas; (iv) GPIs activate macrophages and dendritic cells by interacting mainly with TLR2 and to certain extent with TLR4, leading to the activation of MAPK and NF-?B signaling pathways with downstream production of pro-inflammatory cytokine responses; (v) MAPK-activated protein kinase 2 (MK2) signaling molecule differentially regulate GPI-induced TNF-a and IL-12 production; (vi) the role of nuclear factor I?B? in the GPI-induced activation of macrophages and production of inflammatory responses. Recently, in efforts to identify the parasite factors that induce fever and other clinical symptoms that coincides with the timing of schizont burst, we tested different components of P. falciparum and showed that merozoites are the major immunostimulatory components and that they activate dendritic cells through TLR9. Subsequently, we showed that the activity of merozoites confines to nucleosomes. Ongoing studies include (i) identification of a specific IL-4-inducing factor of P. falciparum; (ii) study the role of IL-4 in the enhanced parasite clearance of parasite; (iii) roles of TLRs, MyD88, and CD36 in the regulation of cell-mediated and adaptive protective immunity to malaria; (iv) regulation of IL-12 and IL-18 production by T cells in response to malaria infection; Roles of TPL2 and MK2 signaling molecules in malaria-induced organ injury.
Placental malaria project
Sequestration of P. falciparum IRBCs in the placenta causes placental malaria, which is characterized by multiple pathologies, including poor birth weight, spontaneous abortion, still birth, maternal anemia and death. The binding of C4S chains of placental chondroitin sulfate proteoglycan (CSPG) present in the intervillous space and on the syncytiotrophoblast cell lining to VAR2CSA (a specific PfEMP1) expressed on the surface of IRBCs is the underlying mechanism.
Our laboratory has made important contributions toward understanding of the molecular interactions involved in P. falciparum sequestration in the placenta. We identified and characterized the placental CSPG and showed that it is uniquely low sulfated, present mainly in the intervillous space and it is of fetal origin and is the major receptor for IRBC adherence. Low sulfated CSPGs on the syncytiotrophoblast cell surface also function as minor receptors. We have also determined many of the structural features of C4S that interact with IRBCs that include the findings that: (i) a dodecasaccharide (12-mer) motif is the minimum C4S chain length that binds IRBCs; (ii) partially but not fully sulfated C4S supports high affinity binding; (iii) two sulfate groups per 12-mer motif is sufficient for optimal binding; (iv) optimal binding also requires two N-acetylgalactosamine not substituted at O-4, (v) the equatorial orientation of the carboxyl groups at the non-reducing terminus is critical, (vi) the presence of a sulfate group at, or proximal to, the non-reducing end is essential; (vii) the N-acetyl groups and the C-2 and C-3 -OH groups of glucuronic acid are not required; (viii) the reducing end sugar residue of 12-mer motif is not required. Based on these results we developed C4S 12-mer-based photoactivable probes for the identification of parasite adhesive proteins and demonstrated its usefulness. More recent studies have focused on studying the structures of VAR2CSA. A group at NIH in collaboration with us determined the structure of DBL3x domain of VAR2CSA by X-ray crystallography and showed that this is the key domain involved in IRBC binding (Figure 6). NMR studies further showed that the binding motifs of DBL3x confine to S3 subdomain. More recently, we found that the parasite protein called CLAG9, an important component of VAR2CSA multiprotein complex, plays a key role in the transport of VAR2CSA to IRBC surface. Ongoing studies are aimed at understanding VAR2CSA structure and its structural features that interact with C4S 12 mer motifs of placental CSPG and the role of CLAG6 in VAR2CSA transport.