Blocking palmitoylation of Toxoplasma gondii myosin light chain 1 disrupts glideosome composition but has little impact on parasite motility
Preprint posted on 14 August 2020 https://www.biorxiv.org/content/10.1101/2020.08.13.250399v1
Article now published in mSphere at http://dx.doi.org/10.1128/msphere.00823-20
Toxoplasma gondii is an apicomplexan parasite that causes toxoplasmosis- a widespread infection that is common among humans. Unfortunately, Toxoplasma gondii causes severe disease in immunocompromised individuals, and in the developing foetus if it infects pregnant women. T. gondii uses gliding motility to invade host cells, and disseminate within the host body throughout infection. The linear model of gliding motility (reviewed in 1) establishes that T. gondii MyoA (a class XIVa myosin), and its associated light chains TgMLC1 and either TgELC1 or TgELC2, are anchored to the parasite’s inner membrane complex (IMC) via TgGAP45, which in turn binds TgGAP40 and TgGAP50 – both transmembrane proteins. TgGAP50 is thought to serve as a fixed anchor against which the motor complex can generate force. This entire complex, is known as the glideosome.
TgMLC1 is thought to play two key roles within the glideosome: a) amplifying small motions at the myosin active site into larger movements capable of displacing actin filaments due to binding to the C-terminal tail of TgMyoA to reinforce the motor’s lever arm; and b) the interaction of the N-terminal portion of TgMLC1 at the C-terminal portion of TgGAP45 is thought to be the critical link that tethers the motor to the IMC. Ultimately, TgMLC1 has been shown to be essential for 3D motility, invasion, and host cell egress.
Despite the linear model having dominated the field for a decade, several phenotypic observations are hard to reconcile with the linear model, suggesting alternative motility mechanisms not explained by the model, might exist. In their current work, Rompikuntal et al (2) investigated the phenotypic consequences of mutations that block TgMLC1 palmitoylation.
Key findings and developments
Palmitoylation is a widespread post-translational modification thought to play an important role in the biology of T. gondii. S-palmitoylation of proteins mediates membrane association and can regulate phenomena such as subcellular localization, trafficking, structure, stability and various aspects of protein function. Various recent proteomic studies have identified several hundreds of putatively palmitoylated proteins, including all components of the glideosome.
C8 and C11 are likely palmitoylation sites on TgMLC1. Moreover, mutations blocking TgMLC1 palmitoylation do not alter its subcellular localization.
Two of the five cysteine residues of TgMLC1 have been predicted as potential palmitoylation sites, namely C8 and C11. Both sites are located in the N-terminal extension of TgMLC1- the region that binds to TgGAP45. The authors began by experimentally confirming whether C8 and/or C11 are sites of palmitoylation, by replacing the endogenous TgMLC1 gene with mutant alleles that produced either single or double cysteine-to-serine mutations, rendering these sites non-palmitoylatable. FLAG-tagged lines of the single and double mutants, as well as a control line, were grown in the palmitic acid analogue 17-octadecynoic acid (17-ODYA). Using a combination of assays including TgMLC1 immunoprecipitation, rhodamine-azide tagging of 17-ODYA, and fluorescence scanning, the authors measured the amount of rhodamine fluorescence associated with TgMLC1. They found fluorescence was significantly reduced in the C8S and C11S mutants compared to WT, while in the double mutant no fluorescence was detected, demonstrating that the mutations partially or fully block 17-ODYA labelling. This altogether suggests that C8 and C11 are indeed, very likely sites of palmitoylation on TgLMC1.
Exploring TgMLC1 localization.
The authors then went on to determine the subcellular localization of non-palmitoylatable TgMLC1. While in WT parasites TgMLC1 localizes uniformly to the parasite periphery, the authors found that in the single and double C8 and/or C11 cysteine-to-serine mutants, TgMLC1 localization remains unaltered. Therefore, blocking palmitoylation does not alter TgLMC1 sub-cellular localization.
The authors then tested whether blocking palmitoylation of TgMLC1 alters its phase partitioning in Triton X-114. Triton X-114 allows separation into aqueous and detergent phases, respectively enriched in hydrophilic and integral membrane proteins. The WT and single C8 and C11 mutants of TgMLC1 partitioned roughly equally into both phases, but the double C8/C11 mutant was found almost entirely in the aqueous phase. This suggests a lack of direct membrane association in the absence of palmitoylation.
Effects of TgMLC1 palmitoylation on the composition of the glideosome
It has been suggested that most, if not all components of the glideosome are palmitoylated. The authors analysed FLAG pulldowns from the 17-ODYA experiments (described above), from the C8/C11 single and double mutants, and performed Western blots to determine the presence of glideosome components TgGAP45, TgELC1 and TgMyoA. They concluded that blocking TgMLC1 palmitoylation seems to block its ability to interact with TgGAP45, while simultaneously increasing its interaction with TgMyoA and TgELC1.
Blocking TgMLC1 palmitoylation has no effect on parasite motility
Given the previous results demonstrating that blocking palmitoylation affects interactions between glideosome components, motility defects in the mutants were expected. However, the authors found that the double mutants parasites’ motility was indistinguishable from the WT TgMLC1 line, in terms of motility initiation, mean displacement, mean speed, and maximum speed, while showing little effects on track length. This was unexpected, since the linear motor model would predict that disruption of the interaction between TgMLC1 and TgGAP45 should result in incapacity to generate force required for movement.
The authors investigated whether the mutants could undergo changes in the composition of the glideosome that could functionally compensate for the lack of TgMLC1-TgGAP45 interactions. Investigation of whether TgGAP45 associates with any new proteins in the absence of its normal interaction with TgMLC1, showed that TgGAP45 does not appear to interact with any myosins or myosin light chains when its interaction with TgMLC1 is disrupted. Investigation of whether TgMyoA associates with other proteins in the mutants, which might serve to anchor TgMyoA to the IMC in the absence of TgMLC1-TgGAP45 interactions, resulted in a similar conclusion. Finally, the authors tested whether TgMLC1 itself might interact with other IMC-anchored proteins in the absence of its interaction with TgGAP45. The authors ultimately concluded that the near normal motility seen in the mutant parasites is not explained by the binding of either TgGAP45 or components of the motor to alternative proteins that could compensate for the lack of interactions between TgMLC1 and TgGAP45.
What I like about this preprint
I find the topic of parasite motility very interesting and I think the authors address an important gap in knowledge, clearly identifying that we do not yet fully understand gliding motility in Apicomplexan parasites, and doing so is important to understand their pathogenicity within human hosts. I enjoyed reading the manuscript as it is very succinct and clear from beginning to end.
- Frenal K, et al, 2017,Gliding motility powers invasion and egress in Apicomplexa, Nature Rev Microbiol, 15: 645-660.
- Rompikuntal PK, et al, 2020, Blocking palmitoylation of Toxoplasma gondii myosin light chain 1 disrupts glideosome composition but has little impact on parasite motility, bioRxiv.
Posted on: 8 December 2020Read preprint
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