A central CRMP complex essential for invasion in Toxoplasma gondii

Abstract

Apicomplexa are obligate intracellular parasites. While most species are restricted to specific hosts and cell types, Toxoplasma gondii can invade every nucleated cell derived from warm-blooded animals. This broad host range suggests that this parasite can recognize multiple host cell ligands or structures, leading to the activation of a central protein complex, which should be conserved in all apicomplexans. During invasion, the unique secretory organelles (micronemes and rhoptries) are sequentially released and several micronemal proteins have been suggested to be required for host cell recognition and invasion. However, to date, only few micronemal proteins have been demonstrated to be essential for invasion, suggesting functional redundancy that might allow such a broad host range. Cysteine Repeat Modular Proteins (CRMPs) are a family of apicomplexan-specific proteins. In T. gondii, two CRMPs are present in the genome, CRMPA (TGGT1_261080) and CRMPB (TGGT1_292020). Here, we demonstrate that both proteins form a complex that contains the additional proteins MIC15 and the thrombospondin type 1 domain-containing protein (TSP1). Disruption of this complex results in a block of rhoptry secretion and parasites being unable to invade the host cell. In conclusion, this complex is a central invasion complex conserved in all apicomplexans.

An apical membrane complex for triggering rhoptry exocytosis and invasion in Toxoplasma

“…However, CRMPs behavior strongly argues for a function specifically at the time of rhoptry exocytosis. CRMPs and their partners Tg247195 and Tg277910 seem to not be part of the previously described Nd/NdP exocytic complex, also confirmed by a parallel study (preprint: Singer et al , 2022). However, we cannot exclude the existence of a dynamic/transient complex formed by CRMPs and Nd proteins at the time of rhoptry exocytosis.…”

“…Tg277910 and Tg247195 possess one and three thrombospondin type 1 (TSP‐1) domains, respectively (Fig 3C), known to participate in cell adhesion (Adams & Tucker, 2000). In addition, Tg247195 possesses an H‐type lectin domain (Pietrzyk‐Brzezinska & Bujacz, 2020) and, interestingly, has a role in invasion (preprint: Singer et al , 2022; Possenti et al , 2022) and rhoptry secretion (Possenti et al , 2022). To determine the function of Tg277910, we generated an inducible knockdown HA 3 ‐tagged line (Tg277910_iKD; Fig EV3E–G).…”

The claudin-like apicomplexan microneme protein is required for gliding motility and infectivity of Plasmodium sporozoites

“…While we cannot formally exclude a redistribution of the protein during sample preparation for U-ExM, this localization is reminiscent of the recently described Nd6 and Cysteine Repeat Modular Proteins (CRMPs) in Toxoplasma , which localize to secretory vesicles throughout the cytoplasm and additionally accumulate at the apex of extracellular tachyzoites [ 51 , 52 ]. Like CLAMP, Nd6 and CRMPs are required for rhoptry discharge and host cell invasion in Toxoplasma , but their molecular function is currently unknown [ 51 – 53 ]. Interestingly, immuno-EM documented the localization of Nd6 to an apical vesicle at the site of rhoptry discharge [ 51 ].…”

A conserved complex of microneme proteins mediates rhoptry discharge in Toxoplasma

“…Some microneme proteins have dedicated roles during invasion; the T. gondii microneme protein MIC8 and the P. falciparum proteins EBA175, Ripr, and CyRPA are species‐specific microneme proteins required for rhoptry effector secretion, but not parasite motility (Kessler et al , 2008; Singh et al , 2010; Volz et al , 2016; Knuepfer et al , 2019). Other proteins, like the microneme‐associate CRMP complex are required for rhoptry secretion and found across apicomplexans and even in free‐living ciliate secretion systems (Sparvoli et al , 2022; Singer et al , 2023). After secretion, some rhoptry effectors rewire host cell signaling to suppress innate immune responses, while other effectors, notably the components of the rhoptry neck (RON) complex, prepare the site of rhoptry secretion for apicomplexan invasion.…”

“…Apicomplexans, therefore, appear to have adapted this conserved secretion machinery to their parasitic lifestyles. The CRMP proteins—required for rhoptry discharge but not the assembly of the apical vesicle or RSA—are broadly conserved among Alveolata much like the Nd proteins (Sparvoli et al , 2022) and bind additional microneme proteins in T. gondii (Sparvoli et al , 2022; Singer et al , 2023). MIC8, a microneme protein required for rhoptry discharge in T. gondii , is limited to coccidians (Kessler et al , 2008) suggesting apicomplexans have clade‐specific determinants of rhoptry discharge that potentially tune the process to their respective niches.…”