It was previously shown that the sorting nexins SNX1, SNX2, SNX5 and SNX6 are recruited to the inclusion membrane in C. trachomatis infected cells (Aeberhard et al., 2015; Mirrashidi et al., 2015). We first confirmed this for myc-tagged SNX1, SNX2 and SNX5 in HeLa cells infected with C. trachomatis serovar L2 (MOI ~0.5) for 18 hr. All three proteins were recruited to the inclusion membrane as assessed by co-localisation with the inclusion marker mCherry-Rab25 (Figure 1B) (Teo et al., 2016), as were GFP-tagged SNX1 and SNX5 but not the more distantly homologous GFP-SNX8 (Figure 1—figure supplement 1A). We also observed localization of the SNX proteins to extensive inclusion-associated membrane tubules in a subset of infected cells as described previously (Figure 1—figure supplement 1B) (Aeberhard et al., 2015; Mirrashidi et al., 2015). Interestingly, when infected cells are treated with wortmannin, a pan-specific inhibitor of phosphoinositide-3-kinase (PI3K) activity, we see a loss of the SNX proteins from punctate endosomes, but not from the inclusion membrane (Figure 1—figure supplement 2; Video 1). A similar result is seen for specific inhibition of PtdIns3P production by Vps34 using Vps34-IN1 (Figure 1—figure supplement 2). This offers two possibilities; that either SNX recruitment to the inclusion occurs via protein-protein interactions, and does not depend on the presence of 3-phosphoinositide lipids that typically recruit SNX proteins to endosomal membranes, or alternatively that PI3Ks are not present at the inclusion and therefore wortmannin treatment has no effect at this particular compartment. Given our structural and mutagenesis studies below we favor the former explanation.Video 1.Movie showing that wortmannin disrupts SNX5 recruitment to endosomes but not the chlamydial inclusion.HeLa cells stably expressing mCherry-Rab25 (red) were transfected transiently with GFP-SNX5 (green) and infected with Chlamydia trachomatis L2 for 24 hr. Time-lapse videomicroscopy was performed using an interval of 1 min on an inverted Nikon Ti-E deconvolution microscope with environmental control at 40 x magnification. 10 min into recording 100 nM wortmannin was added.DOI: http://dx.doi.org/10.7554/eLife.22311.00610.7554/eLife.22311.006