The GTPase Rab4 Interacts withChlamydia trachomatisInclusion Membrane Protein CT229

Abstract

Chlamydiae, which are obligate intracellular bacteria, replicate in a nonlysosomal vacuole, termed an inclusion. Although neither the host nor the chlamydial proteins that mediate the intracellular trafficking of the inclusion have been clearly identified, several enhanced green fluorescent protein (GFP)-tagged Rab GTPases, including Rab4A, are recruited to chlamydial inclusions. GFP-Rab4A associates with inclusions in a species-independent fashion by 2 h postinfection by mechanisms that have not yet been elucidated. To test whether chlamydial inclusion membrane proteins (Incs) recruit Rab4 to the inclusion, we screened a collection of chlamydial Incs for their ability to interact with Rab4A by using a yeast two-hybrid assay. From our analysis, we identified a specific interaction between Rab4A and Chlamydia trachomatis Inc CT229, which is expressed during the initial stages of infection. CT229 interacts with only wild-type Rab4A and the constitutively active GTPase-deficient Rab4AQ67L but not with the dominant-negative GDP-restricted Rab4AS22N mutant. To confirm the interaction between CT229 and Rab4A, we demonstrated that DsRed-CT229 colocalized with GFP-Rab4A in HeLa cells and more importantly wild-type and constitutively active GFP-Rab4A colocalized with CT229 at the inclusion membrane in C. trachomatis serovar L2-infected HeLa cells. Taken together, these data suggest that CT229 interacts with and recruits Rab4A to the inclusion membrane and therefore may play a role in regulating the intracellular trafficking or fusogenicity of the chlamydial inclusion.

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 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).…”

“…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). In the case of C. pneumoniae, Cpn0585 is thought to be responsible for the selective recruitment of Rab1, 10, and 11 (Cortes et al 2007).…”

Manipulation of Rab GTPase Function by Intracellular Bacterial Pathogens

“…on May 8, 2018 by guest http://mmbr.asm.org/ variety of different guanine nucleotide mutants, including constitutively active GTPase-defective, dominant negative, GDPrestrictive, non-nucleotide-binding, and effector binding Rab mutants (125,160). The fact that the expression of any single guanine nucleotide binding mutant fails to inhibit inclusion development suggests that, similar to Legionella (51), chlamydiae may target multiple functionally related trafficking pathways and that the inhibition of a single pathway or single step in a pathway may not alter chlamydial development or inclusion formation.…”

Cloning, Expression, and Biophysical Investigations of Truncated Inclusion Protein A (IncA) from Chlamydia trachomatis

“…28 CT229 has been shown to recruit host Rab4A GTPase to the inclusion membrane. 30 IncA from C. trachomatis, has been identified as a fusion promoting protein and is the focus of this thesis.…”