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First published online 4 March 2008
doi: 10.1242/jcs.022350

Journal of Cell Science 121, 947-956 (2008)
Published by The Company of Biologists 2008
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Microneme protein 8 – a new essential invasion factor in Toxoplasma gondii

Henning Kessler1, Angelika Herm-Götz1, Stephan Hegge1, Manuel Rauch1, Dominique Soldati-Favre2, Friedrich Frischknecht1 and Markus Meissner1,*

1 Hygieneinstitute, Department of Parasitology, University Hospital Heidelberg, Im Neuenheimer Feld 324, D-69120 Heidelberg, Germany
2 Department of Microbiology and Molecular Medicine, University of Geneva, CMU, 1, rue Michel-Servet 1211, Geneva 4, Switzerland


Figure 1
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  		Fig. 1. Establishment of a conditional knockout for MIC8. (A) Regulation of mic8Ty expression by ATc in three independent parasite strains: T7S4-1, T7S4-5 and T7S1-4. Parasites were grown with (+) or without (–) ATc for 48 hours, fixed and stained with antibodies against Ty, or against MIC6 as a control. Pictures were taken under identical exposure conditions. (B) Immunofluorescence analysis of parasite strains T7S4-1 and T7S4-5 grown for 48 hours in the absence of ATc, as shown in A. Parasites were stained with the indicated antibodies. Strong overexpression of MIC8Ty results in partial accumulation of MIC8 within the secretory pathway (arrows). Other micronemal proteins, such as MIC6, appear not to be affected by overexpression of MIC8. (C) Homologous recombination of endogenous mic8 with a knockout construct in which a CAT-expression cassette is flanked by 1.6 kb (5'FS) and 2.5 kb (3'FS) results in a knockout locus that can be identified using analytical PCR with specific oligonucleotides, as indicated by the arrows. (D) Analytical PCR analysis to verify homologous recombination of the knockout construct in the recipient strains T7S1-4 and T7S4-1. Numbers on top of each lane indicate the respective oligonucleotide combination (see C) used in each PCR reaction. Scale bars: 25 µm (A); 5 µm (B).

 

Figure 2
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  		Fig. 2. MIC8 is not required for trafficking of MIC3 to the micronemes. (A) Immunofluorescence analysis of MIC8KOi grown for 48 hours in the presence (+) or absence (–) of ATc prior to fixation. Staining was performed with alpha-MIC8, or with alpha-MIC2 as a control (same exposure time for all micrographs). (B) Immunoblot analysis of MIC8KOi and RH parasites grown in the presence or absence of ATc for 48 hours and probed with the indicated antibodies. No significant difference in the expression levels of MIC2, TUB1 or MIC3 could be detected in MIC8KOi cells under the two conditions. (C) Localisation of MIC3 in MIC8KOi parasites grown in the presence of ATc for 48 hours. Parasites were stained with the indicated antibodies. Merged images are shown on the right. Blue stain indicates DAPI (nuclei). (D) Co-localisation studies in the parasite strain T7S4-5, which overexpresses MIC8Ty (see also Fig. 1B). Upper panels: co-localisation study of MIC8Ty and GRASP-RFP (Pfluger et al., 2005Go) in the parasite strain T7S4-5 transiently transfected with GRASP-RFP demonstrates accumulation of MIC8 in a post-Golgi compartment. Middle panels: co-localisation of MIC8Ty and the propeptide of M2AP (Harper et al., 2006Go) in early endosomes. Lower panels: co-localisation study of MIC8Ty and MIC3 shows that MIC3 is not localised in early endosomes because of overexpression of MIC8Ty. Right: enlargement of boxed parasite(s) in the merged pictures. Scale bars, 10 µm.

 

Figure 3
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  		Fig. 3. MIC8 is required for parasite survival and is essential during the invasion of the host cell. (A) Upper panels: growth of MIC8KOi parasites was compared to RHwt parasites using plaque assay. Parasites were inoculated on HFF cells in the presence or absence of ATc for 7 days prior to GIEMSA staining. Lower panels: same experiment as above. Parasites were analysed 72 hours post-infection. Note that in the case of MIC8KOi grown in the presence of ATc, freshly egressed parasites did not invade neighbouring host cells. (B) Analysis of non-induced egress by quantification of intact initially infected host cells. HFF cells were inoculated with MIC8KOi-GFP parasites in the presence or absence of ATc and the decrease in initially infected host cells was analysed over time. The depicted quantification is representative of the results derived from three independent experiments. (C) Attachment assay of freshly released parasites grown in the presence or absence of ATc to fixed cells. Equal numbers of parasites were allowed to attach on host cells at 37°C for 15 minutes before non-attached parasites were removed by consecutive washing steps with cold PBS. Data are mean values of three independent experiments ± s.d. (D) Invasion assay of freshly egressed parasites grown in the presence or absence of ATc as in C. After the indicated time, non-invaded parasites were removed by several washing steps. Intracellular parasites were further incubated for 12 hours and the number of parasitophorous vacuoles was compared. ON, over night. Data shown are mean values of three independent experiments ± s.d. (E) Re-expression of MIC8 in extracellular parasites restores invasion capability. Invasion assay was performed similar to as in D. After MIC8KOi grown in the presence or absence of ATc were allowed to attach for 10 minutes, non-attached parasites were removed. Attached parasites were further incubated in the presence or absence of ATc (–/–: parasites constantly kept in the absence of ATc; +/+: parasites constantly kept in the presence of ATc; +/–: parasites kept in the presence of ATc and further incubated in media without ATc). Data represent mean values of three independent experiments ± s.d.

 

Figure 4
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  		Fig. 4. MIC8 is not involved in gliding motility. (A) Three different forms of movement can be identified by quantitative image analysis: circular gliding (C), helical gliding (H) and twirling (T). These forms give distinct patterns when the acquired images are projected. The projection (proj.) of 100 images acquired at 3-second intervals represents an overall observation time of 5 minutes. All three forms of movements can be detected in the case of RHwt and MIC8KOi grown in the presence or absence of ATc. (B) Comparison of overall motility. Although differences between RH and MIC8KOi parasites can be observed (~30 vs 12% overall gliding motility, respectively), no significant reduction in gliding motility can be observed between MIC8KOi grown in the presence or absence of ATc. (C) Comparison of the relative frequency of each gliding type of motile parasites. A direct comparison between MIC8KOi grown in the presence or absence of ATc did not show any significant differences in the frequency of the three forms of gliding motility. Mean values of three independent experiments ± s.d. are shown.

 

Figure 5
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  		Fig. 5. Complementation analysis of MIC8KOi using different chimeric proteins. (A) Schematic of the complementation strategy. Transfection of MIC8KOi with complementation constructs under ATc selection results in three different integration events: (1) homologous recombination leads to a promoter exchange resulting in constitutive expression of Ty-tagged MIC8 (stained in green); (2) a combination of both homologous and non-homologous recombination leads to constitutive expression of both MIC8Ty (stained in green) and the respective myc-tagged complementation construct (stained in red); (3) only non-homologous recombination leads to exclusive, constitutive expression of the myc-tagged complementation construct. This event indicates functional complementation. The immunofluorescence analysis shown was performed on a representative pool of MIC8KOi parasites complemented with pMIC8MIC8mycTMCTDMIC8 after 6 days under ATc selection. (B) Top: overview of the complementation constructs used in this study. The TMD and CTD of MIC8myc have been substituted as indicated. In the case of MIC2, two constructs were generated (see text). In the case of MIC8mycGPI (GPI), the TMD and CTD of MIC8 was exchanged for the GPI anchoring signal of the major surface antigen SAG1. Bottom: representative immunofluorescence analyses of MIC8KOi transfected with the indicated complementation construct. Stable transfection has been achieved by application of ATc selection as shown in A. Numbers in each image indicate the average percentage of parasites (± s.d.) in which random integration occurred in four independent experiments. Scale bars: 12.5 µm.

 

Figure 6
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  		Fig. 6. MIC8 is essential for the formation of the moving junction. (A) Formation of evacuoles is abolished in parasites depleted of MIC8. MIC8KOi, grown in the presence or absence of ATc, was incubated with host cells in the presence of CD. Formation of evacuoles (green) and an MJ (red) was analysed with monoclonal antibodies against Rop2-5 and polyclonal antibodies against RON4. No evacuoles were observed when MIC8KOi was grown in the presence of ATc. Blue staining indicates DAPI (nuclei). (B) Extracellular RON4 was detected by immunofluorescence analysis on non-permeabilised parasites expressing cytosolic GFP as a viability marker. MIC8KOi parasites were allowed to attach to host cells in the presence or absence of CD for 20 minutes. Red indicates an MJ. (C) Quantification of extracellular RON4, the presence of which indicates the formation of an MJ. Approximately 200 parasites were analysed in each assay (error bars indicate s.d. obtained from five independent assays). The units on the y-axis are percent of parasites with RON4 signal. (D) Depletion of MIC8 does not affect RON4 expression levels. Immunoblots from MIC8KOi and RH parasites, grown for 48 hours in the presence (+) or absence (–) of ATc, show efficient downregulation of MIC8 expression in the presence of ATc. No differences in RON4 expression levels could be detected. Scale bars: 15 µm (A); 20 µm (B).

 

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