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

Date Published: December 4, 2018

Publisher: Public Library of Science

Author(s): Alfredo J. Guerra, Ou Zhang, Constance M. E. Bahr, My-Hang Huynh, James DelProposto, William C. Brown, Zdzislaw Wawrzak, Nicole M. Koropatkin, Vern B. Carruthers, Michael J. Blackman.

http://doi.org/10.1371/journal.ppat.1007476

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.

Partial Text

Cellular egress from the host is a crucial step in the infectious cycle of intracellular pathogens. Accordingly, such pathogens have evolved multiple exit strategies, which can be divided into those that leave the host cell intact and those that rupture the host cell. Several bacterial pathogens, including L. monocytogenes, use an actin-based protrusion mechanism that allows a bacterium to enter a neighboring host cell without damaging the original host cell [1]. Other bacteria have developed extrusion or expulsion mechanisms that also leave the host cell intact [2–5]. Also, pyroptotic and apoptotic mechanisms leverage cell-death signaling as a means for intracellular pathogens to exit the host cell [6]. Many apicomplexan parasites, including Toxoplasma gondii, use a cytolytic mechanism of egress that obliterates the infected cell. Cytolytic egress results in direct tissue destruction and indirect collateral damage from the ensuing inflammatory response, a hallmark of acute infection by apicomplexan parasites and a key aspect of disease [7].

This paper provides new insight into the structure and function of APCβ, an apicomplexan specific membrane-binding domain associated with parasite egress and cell traversal. Previous studies established that the TgPLP1 CTD, which includes APCβ, has membrane-binding activity and is crucial for TgPLP1 function in egress [16]; however, the lipid binding specificity and structure of this domain remained unknown. The structure solved herein is essentially identical to the TgPLP1APCβ domain reported recently by Ni et al. [17], but our work additionally defines the lipid binding specificity, provides evidence for insertion of the hydrophobic loop into membranes, and establishes a critical role for this loop in TgPLP1 function during parasite egress.

 

Source:

http://doi.org/10.1371/journal.ppat.1007476

 

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