Surface attachment, promoted by the actomyosin system of Toxoplasma gondii is important for efficient gliding motility and invasion

Abstract

BackgroundApicomplexan parasites employ a unique form of movement, termed gliding motility, in order to invade the host cell. This movement depends on the parasite’s actomyosin system, which is thought to generate the force during gliding. However, recent evidence questions the exact molecular role of this system, since mutants for core components of the gliding machinery, such as parasite actin or subunits of the MyoA-motor complex (the glideosome), remain motile and invasive, albeit at significantly reduced efficiencies. While compensatory mechanisms and unusual polymerisation kinetics of parasite actin have been evoked to explain these findings, the actomyosin system could also play a role distinct from force production during parasite movement.ResultsIn this study, we compared the phenotypes of different mutants for core components of the actomyosin system in Toxoplasma gondii to decipher their exact role during gliding motility and invasion. We found that, while some phenotypes (apicoplast segregation, host cell egress, dense granule motility) appeared early after induction of the act1 knockout and went to completion, a small percentage of the parasites remained capable of motility and invasion well past the point at which actin levels were undetectable. Those act1 conditional knockout (cKO) and mlc1 cKO that continue to move in 3D do so at speeds similar to wildtype parasites. However, these mutants are virtually unable to attach to a collagen-coated substrate under flow conditions, indicating an important role for the actomyosin system of T. gondii in the formation of attachment sites.ConclusionWe demonstrate that parasite actin is essential during the lytic cycle and cannot be compensated by other molecules. Our data suggest a conventional polymerisation mechanism in vivo that depends on a critical concentration of G-actin. Importantly, we demonstrate that the actomyosin system of the parasite functions in attachment to the surface substrate, and not necessarily as force generator.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-016-0343-5) contains supplementary material, which is available to authorized users.

Toxoplasma gondii F-actin forms an extensive filamentous network required for material exchange and parasite maturation

“…By 48 hr onwards, no F-actin structures could be observed and Cb-Halo was completely cytosolic, as expected due to cytosolic expression of this reagent (Figure 6B). The loss of filaments within the act1 cKO over-time follows the down-regulation of ACT1 in act1 cKO, indicating a polymerisation mechanism similar to eukaryotic actins (Whitelaw et al, 2017). To exclude a role of host cell actin in the formation of the inter-parasite connections, we treated infected host cells with the actin-disrupting drug latrunculin A, which specifically inhibits polymerization of host cell, but not parasite, actin (Hegge et al, 2010; Whitelaw et al, 2017).…”

“…A recent study suggested that the polymerization process of apicomplexan actin needs to be reinvestigated, as heterologously expressed apicomplexan actin, the basis for many of the previous studies, is incorrectly folded (Olshina et al, 2016). Furthermore, it was demonstrated that conditional deletion of act1 in T. gondii results in complete abrogation of known actin functions, long before G-actin levels are fully depleted, suggesting that in vivo the formation of F-actin depends on a critical monomer concentration (Whitelaw et al, 2017). This raises concerns about previous studies of actin polymerization kinetics based on mis-folded actin.…”

Gliding motility powers invasion and egress in Apicomplexa

“…The conserved, short single-headed myosin heavy chain myosin A (MYOA), was then identified in T. gondii as the motor that was able to generate the rearward traction force necessary for motility, entry into, and egress from, host cells 48 . The composition and molecular details of the glideosome have been elucidated in more detail and discovered, respectively [49][50][51][52] , and the importance of the glideosome in motility and invasion has been assessed further 23,25,26,53,54 (Supplementary information S6 (table)). …”

“…Remarkably, tachyzoites that lack TgMYOA could still be propagated in culture, although their lytic cycle was severely impaired 23 . A model based on retrograde membrane flow has been proposed to account for the residual motility 25,26 . However, a probable explanation is the functional redundancy and parasite adaptation conferred by the presence of a second myosin heavy chain, TgMYOC 53 .…”

“…4a). The conditional excision of T. gondii actin (TgACT1) severely compromised motility, invasion and egress 23,24 , although the parasites could still be propagated for several days, which raised the possibility of the coexistence of an actin-independent mechanism of motility 25,26 . Nature Reviews | Microbiology .…”