Functional dissection of molecular mechanisms underlying host invasion and replication in the obligate intracellular pathogen Toxoplasma gondii
Apicomplexans are a large group of obligate intracellular parasites including Plasmodium falciparum, the causative agent of human malaria, and Toxoplasma gondii, an important pathogen of immunocompromised individuals. T. gondii is capable of replicating in a broad range of host cells and establishes its infection by actively invading its host cells. This invasion process is mediated by the rhoptries, unique apical secretory organelles that inject a complex of proteins into the host cell to facilitate penetration through formation of a tight-junction interface between the parasite and host called the moving junction (MJ). During the invasion process, a parasitophorus vacuole is created within which the parasite resides, evading destruction by host lysosomes. Sequestered within this vacuole, the parasite is free to divide by a unique process of internal budding known as endodyogeny wherein two daughter parasites form within a mother cell. Following repeated rounds of replication, the host is disrupted and the parasites egress out to re-invade neighboring cells, beginning this lytic cycle anew. A better understanding of these unique processes of host cell invasion and parasite replication are needed as the pathology caused by T. gondii and other apicomplexans is dependent on these processes.
In the first section of this dissertation, I describe the discovery and functional analysis of a Toxoplasma palmitoyl acyl transferase (TgDHHC7) that localizes to the surface of the rhoptries. Remarkably, conditional disruption of this enzyme results in a loss of apical tethering of the rhoptries and a complete block in their function, allowing for a definitive establishment of their role in invasion but not replication or egress. Palmitoylation by membrane-resident PATs is a well-characterized mechanism for recruiting proteins to target membrane systems. Therefore, it is likely that TgPAT1 facilitates apical tethering of rhoptries by recruiting one or more proteins to the cytosolic face of the rhoptry membrane which then serve to mediate docking. Indeed, knockdown of the palmitoylated rhoptry armadillo-repeat protein TgARO recapitulates loss of TgDHHC7, showing this protein is also required for rhoptry tethering and strongly suggesting it is a target of TgDHHC7.
During invasion, a complex of the rhoptry neck proteins RON2/4/5/8 localizes to the MJ where it is thought to provide a stable anchoring point for host penetration. This complex is also believed to serve as a molecular filter that restricts access of host plasma membrane proteins to the nascent parasitophorus vacuole, protecting it from lysosomal fusion. During the initiation of invasion, the preformed MJ/RON complex is injected into the host cell where RON2 spans the plasma membrane while RON4/5/8 localize to its cytosolic face. While an important interaction between a parasite surface-bound adhesin, AMA1, and RON2 outside of the host cell has been elucidated, little is known about the interactions and role of the MJ/RONs present within the host cytosol. In the second section, I provide a comprehensive analysis of RON5. Using a conditional knockdown approach, I show RON5 is critical for the organization of the MJ RON complex and that disruption of this complex results in a block in rhoptry secretion and host invasion, demonstrating the importance of MJ RONs for host entry. Furthermore, domain analysis of RON5 using functional complementation reveals that a C-terminal region of RON5 is critical for RON2 stability and invasion, defining the first functionally important domain in RON5.
Apicomplexan parasites undergo complex life cycles, employing unique forms of internal budding for their replication. The simplest example of these is the Toxoplasma binary division system known as endodyogeny, wherein two daughter parasites are assembled within an intact mother cell. Internal budding is facilitated through the de novo construction of an inner membrane complex (IMC), a series of flattened vesicles and underlying cytoskeletal features that provides the scaffold for daughter cell assembly within the cytosol. Little is known regarding the molecular mechanisms that orchestrate internal budding. The final section of this work identifies the apicomplexan-specific IMC Sub-compartment Protein (ISP) family of IMC proteins in Toxoplasma. These proteins are organized into distinct sub-compartments within the IMC and this arrangement was found to depend on coordinated myristoylation and palmitoylation of conserved N-terminal residues in each protein as well as a unique hierarchical targeting mechanism. Interestingly, while a replicating parasite typically produces two daughters per round of division, disruption of ISP2 results in the assembly of aberrant numbers of daughters with a corresponding loss in parasite fitness indicating a role for this family in the control of budding. Together, these studies provide valuable new insights into host cell invasion and parasite division in this important opportunistic pathogen.