Rab GTPases Are Recruited to Chlamydial Inclusions in Both a Species-Dependent and Species-Independent Manner

Abstract

Chlamydiae are obligate intracellular bacteria that replicate within an inclusion that is trafficked to the periGolgi region where it fuses with exocytic vesicles. The host and chlamydial proteins that regulate the trafficking of the inclusion have not been identified. Since Rab GTPases are key regulators of membrane trafficking, we examined the intracellular localization of several green fluorescent protein (GFP)-tagged Rab GTPases in chlamydia-infected HeLa cells. GFP-Rab4 and GFP-Rab11, which function in receptor recycling, and GFPRab1, which functions in endoplasmic reticulum (ER)-to-Golgi trafficking, are recruited to Chlamydia trachomatis, Chlamydia muridarum, and Chlamydia pneumoniae inclusions, whereas GFP-Rab5, GFP-Rab7, and GFPRab9, markers of early and late endosomes, are not. In contrast, GFP-Rab6, which functions in Golgi-to-ER and endosome-to-Golgi trafficking, is associated with C. trachomatis inclusions but not with C. pneumoniae or C. muridarum inclusions, while the opposite was observed for the Golgi-localized GFP-Rab10. Colocalization studies between transferrin and GFP-Rab11 demonstrate that a portion of GFP-Rab11 that localizes to inclusions does not colocalize with transferrin, which suggests that GFP-Rab11's association with the inclusion is not mediated solely through Rab11's association with transferrin-containing recycling endosomes. Finally, GFP-Rab GTPases remain associated with the inclusion even after disassembly of microtubules, which disperses recycling endosomes and the Golgi apparatus within the cytoplasm, suggesting a specific interaction with the inclusion membrane. Consistent with this, GFP-Rab11 colocalizes with C. trachomatis IncG at the inclusion membrane. Therefore, chlamydiae recruit key regulators of membrane trafficking to the inclusion, which may function to regulate the trafficking or fusogenic properties of the inclusion.Chlamydiae are major bacterial pathogens of ocular, urogenital, and pulmonary mucosal surfaces (51). Infections caused by Chlamydia trachomatis are the leading cause of bacterially acquired sexually transmitted disease (10), as well as of preventable blindness worldwide (64). In addition, Chlamydia pneumoniae infections are major causes of upper respiratory tract infections and have recently been linked to chronic heart disease (24, 25). Chlamydiae are obligate intracellular bacteria that replicate within a nonacidified vacuole termed an inclusion (26). Within the inclusion, chlamydiae undergo a biphasic developmental cycle that alternates between the infectious metabolically inactive elementary body (EB) and the noninfectious metabolically active reticulate body (40). Although chlamydiae enter nonprofessional phagocytes by multiple mechanisms (reviewed in reference 26), once the chlamydiae are internalized, they actively modify the properties of the nascent vacuole during the first 2 h postinfection, resulting in trafficking of the inclusion to the peri-Golgi region, fusion of the inclusion with a subset of Golgi-derived exocytic vesicles, and avoi...

Interaction of the Salmonella-containing Vacuole with the Endocytic Recycling System

“…EGFP-Rab4 and EGFP-Rab11 constructs have been previously described (45). EGFP-Rab11S25N was constructed using QuikChange (Stratagene) using EGFP-Rab11A as a template with the mutagenic 5Ј oligonucleotide (5Ј-GGTGTTGGAAAGAATAATCTCCTGTCTCG-3Ј) and the mutagenic 3Ј oligonucleotide (5Ј-CGAGACAGGAGATTATTCTTTCCAA-CACC-3Ј).…”

Chlamydial Intracellular Survival Strategies

“…A subset of these are Rab GTPases including Rabs 4, 11, and 14, which are associated with recycling endosomes, and Rabs 1, 6, and 10, which are associated with ER-Golgi traffic (Rzomp et al 2006;Brumell and Scidmore 2007;Moorhead et al 2007Moorhead et al , 2010Capmany and Damiani 2010). Rabs 6 and 10 are recruited to inclusions in a species-specific manner, whereas recruitment of Rab1, 4, and 11 appear to be ubiquitous to all Chlamydiae (Rzomp et al 2003). The C. trachomatis Inc protein CT229 has been identified as a Rab4-interacting protein, although it is not conserved in other strains or serovars that recruit Rab4, suggesting redundancy in Inc protein function (Rzomp et al 2006).…”

“…The recruitment of Rabs and their associated proteins likely promotes selective interaction/ fusion with host vesicles containing essential nutrients. Rab6, 11, and 14 play a role in the acquisition of sphingomyelin by the inclusion (Rejman Capmany and Damiani 2010), whereas Rab4 and Rab11 participate in iron acquisition by regulating a slow transferrin recycling pathway intercepted by the inclusion (Rzomp et al 2003(Rzomp et al , 2006Ouellette and Carabeo 2010). Rab6 and Rab11 facilitate sphingomyelin transport to the inclusion by regulating fragmentation of the Golgi into ministacks during infection Rejman Lipinski et al 2009).…”

Cytoplasmic lipid droplets are translocated into the lumen of the Chlamydia trachomatis parasitophorous vacuole

“…To test this, we isolated LDs from infected and uninfected HeLa cells grown in 100 M oleic acid for 12 h to obtain enough material for reliable biochemical analysis. Because Rab and 14-3-3 proteins have been reported to bind to inclusion membranes (21,22) and copurify with LDs (18), we tested whether these proteins displayed differential association with LDs during infection. Rab1 and Rab11 cofractionated with LDs, but this association was independent of Chlamydia infection (Fig.…”