Furthermore AMA1 was shown to interact with actin via aldolase, thus fulfilling the requirements for generating specific force transmission during host cell penetration. A detailed reverse genetic Diacerein analysis of parasites lacking AMA1 has however, demonstrated that this protein does not play a critical role during force transmission, formation of the TJ, or parasite entry. Furthermore, preliminary results indicated that parasites lacking MyoA are viable, while depletion of Act1 results in apicoplast loss, although parasites still retain some level of invasiveness. These phenotypes suggest that alternative invasion pathways that do not rely on the MyoA-actin motor may operate. These findings suggest several possibilities. First, multiple redundancies can exist that would complement for the deletion of individual genes – in the case of AMA1 this was considered unlikely. Similarly it is hard to imagine a functional redundancy for the single copy gene TgAct1. In contrast, the huge repertoire of apicomplexan myosins might show some redundancies that could complement for the lack of MyoA in the myoA KO. Therefore, a more complex invasion mechanism might be in place that can partially substitute for loss of a functional acto-myoA system. In this case one would expect that mutant parasites do not follow the well described step-wise process that includes formation of the typical TJ, an accepted marker for active parasite entry. To resolve this conundrum we used the DiCre regulation system to engineer parasites lacking proteins of the gliding machinery that are considered crucial in providing functional motor activity. Conditional knockouts for gap40, 45, 50, mlc1, and act1 were established and analysed in depth along with the myoA KO mutant line we previously generated. First we found that parasites without a functional motor complex remained motile, indicating that movement can be generated in the absence of the known myosin motor and parasite actin. Secondly, none of the generated mutants showed a block in host cell invasion and in all cases entry occurred through a normally appearing TJ. Strikingly, a delay in TJ formation was detected that corresponds to the reduced overall invasion rate of the act1 KO. The absence of the respective gene product in the induced population was also confirmed on protein level in immunofluorescence or Western blot analysis. A faint signal could be observed in the induced populations for GAP45 and Act1 in western blot analysis 72 hours after removal of the respective gene. This corresponds to a minority of parasites that have not excised the floxed gene. Accordingly, in IFA analysis two distinct populations could be identified, with only very few YFP negative parasites that were positive for the protein of Yunaconitine interest. In all cases it was straightforward to identify and analyse the respective KO population, since removal of the floxed gene resulted in the activation of YFP expression, leading to YFP positive KO parasites. It was previously suggested that MyoA is the core motor of the invasion machinery and interacts with MLC1, GAP40, GAP45, and GAP50. The phenotypic analysis of the conditional myoA KO, correlates to the phenotype previously described for a tetracycline-inducible KD mutant of myoA, where residual host cell invasion was observed but attributed to leaky myoA expression. When gliding motility was analysed in a trail deposition assay, myoA KO parasites exhibited a residual gliding motility with,15% of parasites moving by mostly circular gliding.