Chlamydiae secrete more than fifty different Inc proteins. While Incs possess little sequence similarity, they share a common membrane topology with cytoplasmic N- and C-terminal domains, separated by two closely spaced transmembrane regions with a short intra-vacuolar loop (Dehoux et al., 2011; Kostriukova et al., 2008; Li et al., 2008; Lutter et al., 2012; Rockey et al., 2002) (Figure 1A). The cytoplasmic N- and C-terminal sequences of the Inc proteins act to bind and manipulate host cell proteins. Reported examples include the binding of the small GTPase Rab4A by CT229 (Rzomp et al., 2006), Rab11A by Cpn0585 (Cortes et al., 2007), SNARE proteins by IncA (Delevoye et al., 2008), centrosomal and cytoskeletal proteins by Inc850 and inclusion protein acting on microtubules (IPAM) (Dumoux et al., 2015; Mital et al., 2015, 2010), myosin phosphatase by CT228 (Lutter et al., 2013), 14-3-3 and Arf family proteins by IncG and InaC (Kokes et al., 2015; Scidmore and Hackstadt, 2001), and the lipid transfer protein CERT by IncD (Derré et al., 2011; Elwell et al., 2011). Despite these reports, there are no known structures of Inc family members either alone or in complex with host effectors.10.7554/eLife.22311.003Figure 1.SNX5, SNX6 and SNX32 tare recruited to C. trachomatis inclusions.(A) HeLa cells stably expressing the mCherry-Rab25 inclusion membrane marker (red) were infected with C. trachomatis serovar L2 (24 hr) and transfected with myc-tagged SNX expression constructs. The samples were fixed and immunolabeled with anti-myc (green) and anti-chlamydial HtrA antibodies (white) and counterstained with DAPI (blue). Similar experiments using GFP-tagged proteins are shown in Figure 1—figure supplement 1A.DOI: http://dx.doi.org/10.7554/eLife.22311.00310.7554/eLife.22311.004Figure 1—figure supplement 1.SNX5, SNX32 and SNX1 are recruited to C. trachomatis inclusions and membrane tubules.(A) Hela cells were transiently transfected with GFP-tagged SNX and mCherry-Rab25 proteins as indicated, and infected with C. trachomatis serovar L2. Cells were imaged by confocal fluorescence microscopy for GFP-tagged proteins (green), endogenous SNX1 (blue), mCherry-Rab25 (red) and DAPI-stained nuclear material (white). Both GFP-SNX5 and GFP-SNX32 are recruited to inclusion membranes, but the distantly related SNX-BAR protein SNX8 is not. The images are maximum projections. (B) An example of SNX1-decorated tubules (green) often observed emanating from inclusion membranes (mCherry-Rab25 in red; DAPI staining in blue). The image is a maximum projection.DOI: http://dx.doi.org/10.7554/eLife.22311.00410.7554/eLife.22311.005Figure 1—figure supplement 2.Recruitment of SNX1, SNX2 and SNX5 to inclusions is not dependent on 3-phosphoinositides.HeLa cells stably expressing mCherry-Rab25 were infected with C. trachomatis serovar L2 (MOI ~0.5) for 24 hr and imaged by immunofluorescence microscopy using antibodies to SNX1, SNX2 and SNX5. mCherry-Rab25 provides marker for the inclusion membrane. The upper panels show control infections and lower panels show cells treated with wortmannin or Vps34-IN1 with concomitant loss of SNX association with endosomal compartments while inclusion localisation is unaffected. The images are maximum projections. Endosomal compartments are labeled with antibodies to endogenous Rab5 or Vps35 and Pearson’s correlation coefficients used to quantify loss of endosomal recruitment (100 cells per group; error bars, S.D). A movie showing the effect of wortmannin on GFP-SNX5 is shown in Video 1.DOI: http://dx.doi.org/10.7554/eLife.22311.005