Abstract
Chlamydia trachomatis, an obligate intracellular pathogen, undergoes a biphasic developmental cycle within a membrane-bound vacuole called the chlamydial inclusion. To facilitate interactions with the host cell, Chlamydia modifies the inclusion membrane with type III secreted proteins, called Incs. As with all chlamydial proteins, Incs are temporally expressed, modifying the chlamydial inclusion during the early and mid-developmental cycle. VAMP3 and VAMP4 are eukaryotic SNARE proteins that mediate membrane fusion and are recruited to the inclusion to facilitate inclusion expansion. Their recruitment requires de novo chlamydial protein synthesis during the mid-developmental cycle. Thus, we hypothesize that VAMP3 and VAMP4 are recruited by Incs. In chlamydia-infected cells, identifying Inc binding partners for SNARE proteins specifically has been elusive. To date, most studies examining chlamydial Inc and eukaryotic proteins have benefitted from stable interacting partners or a robust interaction at a specific time postinfection. While these types of interactions are the predominant class that have been identified, they are likely the exception to chlamydia-host interactions. Therefore, we applied two separate but complementary experimental systems to identify candidate chlamydial Inc binding partners for VAMPs. Based on these results, we created transformed strains of C. trachomatis serovar L2 to inducibly express a candidate Inc-FLAG protein. In chlamydia-infected cells, we found that five Incs temporally and transiently interact with VAMP3. Further, loss of incA or ct813 expression altered VAMP3 localization to the inclusion. For the first time, our studies demonstrate the transient nature of certain host protein-Inc interactions that contribute to the chlamydial developmental cycle.
Highlights
Chlamydia trachomatis, an obligate intracellular pathogen, undergoes a biphasic developmental cycle within a membrane-bound vacuole called the chlamydial inclusion
To further our understanding of eukaryotic SNARE recruitment to the chlamydial inclusion, we asked if VAMP3 was recruited to the chlamydial inclusion and if the pattern of VAMP3 localization was similar to the localization of VAMP4, which we previously characterized at only a single time point postinfection [39]
Since VAMP3 normally traffics through the Golgi apparatus to the plasma membrane in host cells and the Golgi apparatus becomes fragmented around the inclusion during infection with C. trachomatis serovar L2 [43], we wanted to understand if the Golgi structure played a role in VAMP3 localization at C. trachomatis serovar L2 inclusions
Summary
An obligate intracellular pathogen, undergoes a biphasic developmental cycle within a membrane-bound vacuole called the chlamydial inclusion. To facilitate interactions with the host cell, Chlamydia modifies the inclusion membrane with type III secreted proteins, called Incs. VAMP3 and VAMP4 are eukaryotic SNARE proteins that mediate membrane fusion and are recruited to the inclusion to facilitate inclusion expansion Their recruitment requires de novo chlamydial protein synthesis during the mid-developmental cycle. Most studies examining chlamydial Inc and eukaryotic proteins have benefitted from stable interacting partners or a robust interaction at a specific time postinfection While these types of interactions are the predominant class that have been identified, they are likely the exception to chlamydia-host interactions. The composition of the IM is derived from both the host and the pathogen, as it acquires host lipids like sphingomyelin and cholesterol for eventual incorporation into bacterial membranes [8, 9] and is studded with a class of chlamydial type III secreted effectors called inclusion membrane proteins, or Incs [10,11,12]
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