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    • Here we show that CT recruits multiple

    2022-08-11

    Here, we show that CT229 recruits multiple Rab GTPases and Rab effector proteins to the inclusion; the CT229 Inc protein redirects and intercepts host clathrin-coated vesicles (CCVs). CT229 is required for recruitment of transferrin (Tfn) positive CCV’s from the recycling pathway to the periphery of the inclusion and is required to redirect cation-independent mannose-6-phoshate receptor (CI-M6PR)-containing vesicles to the vicinity of the inclusion. These findings highlight CT229 as a potential master regulator of host CCV trafficking and provide the first conceptual insights as to how C. trachomatis subverts intracellular trafficking pathways to acquire essential nutrients.
    Results
    Discussion Like many obligate intracellular pathogens, chlamydiae possess a substantially reduced genome (1.04 Mb, 895 ORFs) that lacks many metabolic enzymes. As such, the bacteria must scavenge crucial nutrients, such as lipids, iron, and amino acids, from the host cell to proliferate and cause disease (Elwell et al., 2016). To obtain key nutritional substrates from the host, C. trachomatis employs CT229 as a keystone effector protein to redirect host vesicular traffic toward the chlamydial inclusion. Through interactions with a plethora of Rab GTPases, CT229 reroutes Rab-containing vesicles to the inclusion. Notably, CT229 redirects Tfn from the recycling pathway and M6PR-containing vesicles to the periphery of the inclusion. Strikingly, the absence of CT229 results in defects in homotypic fusion and premature inclusion lysis, presumably due to its inability to acquire essential substrates for incorporation into the bacterial cell and inclusion membrane (Figure 7). Several studies have noted the importance of the Inc protein CT229 for productive chlamydial infection; however, the overarching physiological impact of this Inc on host processes during infection has remained elusive. CT229 has been shown to interact with multiple Rab GTPases (Mirrashidi et al., 2015, Rzomp et al., 2006, Sixt et al., 2017), but until now, whether these Rab-containing vesicles are recruited to vicinity of the chlamydial inclusion during infection, and their role in heme oxygenase infection was unknown. Here, we show that CT229 directly binds and recruits Rab GTPases, perhaps associated with vesicles, that are involved in anterograde transport (Rab1), retrograde transport (Rab6), post-Golgi transport (Rab8, 10, 14), intra-Golgi transport (Rab34), and protein recycling (Rab4, 35). Although CT229 binds and recruits a striking number of Rab GTPases, Rabs, such as Rab5, 7, and 9, are not recruited to the chlamydial inclusion (Rzomp et al., 2003). On the other hand, other Rabs, such as Rab11, are recruited independent of CT229. The selective recruitment or exclusion of specific Rab GTPases allows chlamydiae to tightly control interactions with the host cell, allowing for avoidance of the endocytic/lysosomal pathway while promoting interactions with the recycling pathway. The diversity of Rabs that CT229 binds suggests it may be involved in manipulation of multiple host vesicular trafficking pathways, potentially to acquire key biosynthetic precursors and essential nutrients, such as lipids and iron. Alternatively, CT229 may recruit a plethora of Rab GTPases to camouflage the inclusion as a secretory vesicle or recycling endosome to avoid targeting to the lysosome. To carry out their effector function, GTP-bound Rab GTPases interact with specific Rab effector proteins. Previous studies have shown that Rab effectors, such as BICD1 and OCRL, localize to the inclusion (Moorhead et al., 2007, Moorhead et al., 2010). Here, we show that Rab effector proteins, such as OCRL and RUFY1, localize in close proximity to the chlamydial inclusion in a CT229-dependent manner. This suggests that localization of specific Rab effectors proximal to the inclusion is mediated through interactions with their cognate GTPase and that they themselves may not be directly recruited. The association of Rab effectors with Rab GTPases near the inclusion would allow for essential functions, such as vesicle uncoating and tethering to occur and formation of this complex at the surface of the inclusion membrane would allow for more efficient hijacking of CCV trafficking by the bacterium. In contrast to CT229, the T4SS effector protein LidA from Legionella pneumophila binds to Rab1 and prevents its inactivation by host GTPase activating proteins (GAPs), thus keeping it in an activate conformation (Neunuebel et al., 2012). Surprisingly, binding of LidA to Rab1 prevents association with Rab effector proteins, which may represent a unique way for L. pneumophila to restrict Rab interactions to a specific set of Rab ligands (Neunuebel et al., 2012). We hypothesize that similar to LidA, CT229 interacts with and recruits GTP-bound Rab GTPases to the inclusion and perpetuates signaling by maintaining the Rab GTPase in an active conformation. However, in contrast to LidA, our data suggest that CT229 allows the Rab GTPase to interact with Rab effector proteins. Whether CT229 directly affects the GTP-bound state of the GTPase or simply prevents its inactivation warrants further study.