Structural basis of Toxoplasma gondii perforin-like protein 1 membrane interaction and activity during egress

Abstract

Intracellular pathogens must egress from the host cell to continue their infectious cycle. Apicomplexans are a phylum of intracellular protozoans that have evolved members of the membrane attack complex and perforin (MACPF) family of pore forming proteins to disrupt cellular membranes for traversing cells during tissue migration or egress from a replicative vacuole following intracellular reproduction. Previous work showed that the apicomplexan Toxoplasma gondii secretes a perforin-like protein (TgPLP1) that contains a C-terminal Domain (CTD) which is necessary for efficient parasite egress. However, the structural basis for CTD membrane binding and egress competency remained unknown. Here, we present evidence that TgPLP1 CTD prefers binding lipids that are abundant in the inner leaflet of the lipid bilayer. Additionally, solving the high-resolution crystal structure of the TgPLP1 APCβ domain within the CTD reveals an unusual double-layered β-prism fold that resembles only one other protein of known structure. Three direct repeat sequences comprise subdomains, with each constituting a wall of the β-prism fold. One subdomain features a protruding hydrophobic loop with an exposed tryptophan at its tip. Spectrophotometric measurements of intrinsic tryptophan fluorescence are consistent with insertion of the hydrophobic loop into a target membrane. Using CRISPR/Cas9 gene editing we show that parasite strains bearing mutations in the hydrophobic loop, including alanine substitution of the tip tryptophan, are equally deficient in egress as a strain lacking TgPLP1 altogether. Taken together our findings suggest a crucial role for the hydrophobic loop in anchoring TgPLP1 to the membrane to support its cytolytic activity and egress function.

Perforin-Like Proteins of Apicomplexan Parasites

“…Recently published crystal structures of purified APC-β domains of TgPLP1 gave insight into the architecture and membrane-binding properties of ApiPLPs (Guerra et al, 2018 ; Ni et al, 2018 ). The studies revealed that the TgPLP1 APC-β domain has an unusual β-prism fold comprising three subdomains.…”

“…The studies revealed that the TgPLP1 APC-β domain has an unusual β-prism fold comprising three subdomains. One of these subdomains exhibits a protruding hydrophobic loop tipped by an exposed tryptophan that is presumably responsible for membrane insertion upon binding (Guerra et al, 2018 ; Ni et al, 2018 ). Accordingly, mutant parasites expressing TgPLP1 with a loop that is either shortened or changed in amino acid identity or hydrophobicity display an impaired egress phenotype recapitulating the TgPLP1-knock out phenotype, including the formation of smaller plaques compared to wildtype parasites, impaired PVM rupture and delayed egress as determined via LDH activity measurement in culture supernatants (see below; Kafsack et al, 2009 ; Guerra et al, 2018 ).…”

“…Of the two PLPs detected in T. gondii , only TgPLP1 hitherto showed a clear involvement in host cell exit (Kafsack et al, 2009 ; Roiko and Carruthers, 2013 ; Guerra et al, 2018 ). TgPLP1 localizes to the micronemes of tachyzoites and is secreted in a calcium-dependent manner during egress similar to other micronemal proteins.…”

“…TgPLP1 activity appears to be further regulated by interaction with specific phospholipids located in the respective membrane, particularly phosphatidylethanolamine (PE) or phosphatidylserine (PS), which are characteristic components of the inner leaflet of the red blood cell membrane. The availability of PE and PS, e.g., during egress, increases TgPLP1 activity, whereas the absence of these preferred phospholipid receptors in the outer leaflet of the target cell, e.g., during cell invasion, strongly limits the activity of TgPLP1 (Guerra et al, 2018 ). In a first model of tachyzoite egress, it has been postulated that TgPLP1 is transported to the PV after secretion, where it interacts with the PVM.…”

“…This interaction triggers the lytic activity of the protein. After dissolution of the PVM, TgPLP1 binds to phospholipids of the HCM and, hence, further lyses this membrane, in consequence facilitating the final exit of the parasite from its host cell (Guerra et al, 2018 ).…”