Figure 1.
SNARE-like motifs in the inclusion protein IncA.
A, Localisation of C. trachomatis IncA SNARE-like motifs; TM designates IncA bilobed hydrophobic domain. B, Alignment of IncA SNARE-like motif Cter from 6 Chlamydia species and four host SNAREs. The first line indicates a and d positions in the heptads, conserved hydrophobic residues are shown in green, the conserved Gln or Arg in red. C, Alignment of IncA SNARE-like motif Nter from 6 Chlamydia species. Swissprot ID of IncA-like proteins (in the order of the aligned sequences): INCA1_CHLTR, Q46210_CHLCV, Q9PKR8_CHLMU, Q254Q8_CHLFF, Q45SH9_CHLPS, Q5L5U6_CHLAB.
Figure 2.
Several host SNARE proteins are specifically recruited around the inclusion membrane.
A, Localization of endogenous SNAREs in HeLa cells infected for 24 h with C. trachomatis serovar D was assessed using specific antibodies. Vamp3 and Vamp7 encircle the inclusion (arrows), while Sec22 does not (arrowheads). SNAREs are shown on the left, host and bacterial DNA in the second column, and merged images in the third column. Distribution of the SNAREs in uninfected cells is shown on the right. B, Cells were transfected with different GFP-SNARE constructs 8 h before infection. The inclusion was labelled with anti-IncA antibodies followed with TRITC-coupled secondary antibodies. GFP-Vamp8 or GFP-Vamp7 encircle the inclusion membrane (arrows), while GFP, GFP-Vamp4 or GFP-Vamp7 deleted from its SNARE domain (GFP-Longin) do not (arrowheads). GFP-tagged SNAREs are shown on the left, IncA in the second column, and merged images in the third column. Distribution of the SNAREs in uninfected cells is shown on the right. C, Quantification of SNARE recruitment to the inclusion. Cells were scored as positive when the entire circumference of the inclusion was surrounded by the SNARE protein. Means and standard deviations of at least two independent experiments are shown.
Table 1.
Distribution and known functions of host SNAREs used in this study.
Figure 3.
GFP-Vamp8 is located in compartments around the inclusion and in the inclusion membrane.
A, Distribution of GFP was observed in fields with an inclusion, in cells transfected with GFP-Vamp4 (top) or GFP-Vamp8 (bottom), and infected. Two representative panels are shown for each construct. Arrowheads point to gold particles less than 50 nm from the inclusion membrane. I, inclusion, B, bacteria. B, Equivalent numbers of gold particles were counted in both sets of images and their localization relative to the inclusion membrane was assessed. Levels of significance are indicated by p-values comparing the distribution of GFP-Vamp4 and GFP-Vamp8 for each category.
Figure 4.
IncA interacts directly with host SNARE proteins.
A, GFP-SNARE proteins (V3 = Vamp3, V4 = Vamp4, V7 = Vamp7, V8 = Vamp8, Longin = Vamp7 without its SNARE motif) were immunoprecipitated from HeLa cells co-expressing IncA or empty vector (pQE). The top left panel (anti-His blot) shows the level of expression of IncA in cell lysates, and the bottom right panel (anti-GFP blot) shows immunoprecipitation of GFP-tagged proteins. IncA co-immunoprecipitated with Vamp3, Vamp7 and Vamp8, and very little with Vamp4 (top right panel, anti-IncA blot). B, Purified IncA-His and purified GST-Vamp3, GST-Vamp4 or GST-Vamp8 were inserted in two populations of liposomes, mixed, and GST-SNAREs were pulled-down using glutathione agarose beads. Pull-down complexes were resolved on SDS-PAGE gels and analyzed after staining with Coomassie.
Figure 5.
IncA plays a predominant role in SNAREs recruitment to the inclusion.
A, The recruitment of transfected GFP-Vamp8 (left) around the inclusion membrane (labelled with antibodies against the inclusion protein Cap1, middle) is reduced in cells infected with a IncA defective mutant strain (Ds5058, bottom panels) compared to wild type (top panels). B, The percentage of cells (n>100) in which the indicated GFP-SNARE proteins encircle the inclusions is reduced in cells infected with the IncA defective mutant strain (grey) compared to wild type (black). Coverslips were analyzed in duplicate by different investigators, and the means and standard deviations of the two resulting counts are shown. C, The percentage of cells (n>100) in which the indicated endogenous SNAREs encircle the inclusions in control cells (black), cells overexpresing C. caviae IncA (dark grey) or Δ75CtrIncA (light grey) is shown. The means and standard deviations of two experiments are shown.
Figure 6.
CT813 can interact with host SNAREs.
GFP-SNARE proteins (V3 = Vamp3, V4 = Vamp4, V7 = Vamp7, V8 = Vamp8, Longin = Vamp7 without its SNARE motif) were immunoprecipitated from HeLa cells co-expressing CT813-His* or empty vector (pQE). The top left panel (anti-His blot) shows the level of expression of CT813-His* in cell lysates, and the bottom right panel (anti-GFP blot) shows immunoprecipitation of GFP-tagged proteins. CT813-His* co-immunoprecipitated with Vamp7 and Vamp8 (top right panel, anti-His blot), and not with Vamp3, Vamp4, GFP or GFP-Longin. Absence of immunoprecipitation with Vamp3 might be due to insufficient expression level of both CT813-His* and GFP-Vamp3, which were consistently low in these experiments.
Figure 7.
IncA SNARE-like motifs fit in the structure of the SNARE complex.
Side view of the complex of three motifs Cter from C. trachomatis IncA with the SNARE motif of Vamp8 (left) and of the endosomal SNARE complex [23] (right), with top view of the central layer for both complexes. The backbone of the helices is shown in orange for IncA and yellow for Vamp8. Dashed lines indicate favorable electrostatic interactions. Acidic residues (Asp, Glu) are shown in red, basic residues (Arg, Lys, His) in blue, and other polar residues (Asn, Glu) in green; in the top views hydrophobic residues are shown in white, and all other residues are left out for clarity. The energetic profiles at the bottom are estimates of the total contribution of a residue to the stability of the complex, averaged over the four helices (see Materials and Methods). The vertical line indicates the position of the central layer. Negative energy indicates that a residue is predicted to stabilize the complex, positive energy that it destabilizes the complex.
Figure 8.
Effect of point mutations in the central layer of IncA SNARE-like motifs.
A, GFP-SNARE proteins were immunoprecipitated from HeLa cells co-expressing IncA wild-type, mutated in motif Nter (T126R) or in both SNARE-like motifs (TRQR), or empty vector (pQE) as control. The top left panel (anti-IncA blot) shows the level of expression of IncA in cell lysates, and the bottom right panel (anti-GFP blot) shows immunoprecipitation of GFP-tagged proteins. IncA wild-type and T126R immunopecipitated with the SNAREs, and the double mutant did not (anti-IncA blot,top right panel). B, Cells transfected for 18 hrs with the indicated His-tagged IncA constructs were infected for 20 h with C. trachomatis serovar D before fixation. Coverslips were stained with anti-His antibodies and anti-EfTu antibodies to label the bacteria. Histograms show the percentage of cells (n>100) with a normal inclusion for each population of transfected cells, in one experiment representative of three (means and standard deviations of two independent counts are shown). Only inclusions that looked intact were scored; in cells transfected with IncA wild-type and IncAT126R, and, to a lesser extent, IncAQ244R and IncAT126RQ244R (TRQR), many disrupted inclusions were also observed.