Chlamydia trachomatis is an intracellular bacterial pathogen that resides and replicates within a membrane-bound compartment, termed ‚inclusion‘. During the developmental cycle, C. trachomatis inserts inclusion membrane proteins into the inclusion membrane. In addition, C. trachomatis selectively recruits host cellular lipids and proteins. Among those proteins are sorting nexins (SNX) of the human retromer. The SNX-BAR retromer is a multi-protein complex comprising two subcomplexes, a trimer of VPS proteins and a dimer of a PX and a BAR domain bearing SNX proteins. SNX proteins localised on the inclusion in a rim-like pattern at mid-infection whereas VPS35 localised adjacent to the inclusion. Moreover, we observed tubular-like structures emanating from the inclusion. These structures are positive for bacterial Inc proteins IncA, IncE as well as host cellular SNX1, 2, 5 and 6. However, little is known about the mechanism and functional consequences of SNX-BAR recruitment. To unravel spatio-temporal dynamics of SNX recruitment, we analysed the localisation of SNX-BAR proteins, VPS35 and one of retromer’s cargo CI-MPR over time. At 8 h p.i., SNX-BAR proteins and VPS35 accumulated proximally to C. trachomatis at the MTOC indicating a rearrangement of retromer components at the early infection stage while the localisation of CI-MPR was not affected. Knockout of both, SNX5 and SNX6 resulted in reduced trafficking of C. trachomatis towards the MTOC. At mid-infection stage, the chlamydial inclusion is decorated with SNX-BAR proteins but not with VPS35. The functional domains of SNX-BAR fusion proteins revealed a different localisation pattern: While the PX domain of SNX5 and SNX6 localised on the inclusion, the opposite was true for SNX1 and SNX2 whose BAR domains localised on the inclusion. Functional analyses of SNX proteins using knockout cell lines revealed increased chlamydial primary infection, genome copy number and infectious progeny formation in SNX5/SNX6 knockout cells suggesting that SNX5/SNX6 restrict C. trachomatis at mid-infection stage. Finally, we identified host-cellular proteins associated with SNX1 by using a proximity-dependent biotinylation assay (BioID) in which a promiscuous biotin ligase is targeted to a definite subcellular location by fusion to SNX1 as SNX1 was recruited to the inclusion. Subsequent nLC-MS/MS analysis of affinity-captured biotinylated proteins close to SNX1 identified IncE, SNX5 and SNX6 suggesting interaction of these proteins with SNX1 at the cytosolic site of the inclusion. Furthermore, we identified RPL13a of the large ribosomal subunit. At mid-infection time points, RPL13a localised on the inclusion suggesting specific recruitment. Depletion of RPL13a resulted in increased genome copy number and infectious progeny formation. In addition, bacterial protein levels were elevated and the inclusion enlarged. As some ribosomal proteins are involved in ribosome heterogeneity, we considered the hypothesis that C. trachomatis co-opts ribosomal proteins such as RPL13a to regulate the host cellular translation machinery. While proteins exhibited similar absorption profiles of C. trachomatis and uninfected cells, ribosomal absorption profiles differed suggesting possible heterogeneous ribosome RNA composition. Taken together, these data highlight how C. trachomatis interfere with host cellular trafficking pathways by recruiting SNX proteins of the human retromer. Moreover, the recruitment of RPL13a to the inclusion may exhibit an interesting example of C. trachomatis interfering in the translation machinery of the host cell.
