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Eukaryotic SNARE VAMP3 Dynamically Interacts with Multiple Chlamydial Inclusion Membrane Proteins.

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.

SUBMITTER: Bui DC

PROVIDER: S-EPMC7822134 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The obligate intracellular bacterial pathogen, Chlamydia trachomatis, develops within a membrane-bound vacuole termed the inclusion. Affinity purification-mass spectrometry (AP-MS) experiments to study the interactions that occur at the chlamydial inclusion membrane have been performed and, more recently, combined with advances in C. trachomatis genetics. However, each of the four AP-MS published reports used either different experimental approaches or statistical tools to identify proteins that localize at the inclusion. We critically analyzed each experimental approach and performed a meta-analysis of the reported statistically significant proteins for each study, finding that only a few eukaryotic proteins were commonly identified between all four experimental approaches. The two similarly conducted in vivo labeling studies were compared using the same statistical analysis tool, Significance Analysis of INTeractome (SAINT), which revealed a disparity in the number of significant proteins identified by the original analysis. We further examined methods to identify potential background contaminant proteins that remain after statistical analysis. Overall, this meta-analysis highlights the importance of carefully controlling and analyzing the AP-MS data so that pertinent information can be obtained from these various AP-MS experimental approaches. This study provides important guidelines and considerations for using this methodology to study intracellular pathogens residing within a membrane-bound compartment. SIGNIFICANCE: Chlamydia trachomatis, an obligate intracellular pathogen, grows within a membrane-bound vacuole termed the inclusion. The inclusion is studded with bacterial membrane proteins that likely orchestrate numerous interactions with the host cell. Although maintenance of the intracellular niche is vital, an understanding of the host-pathogen interactions that occur at the inclusion membrane is limited by the difficulty in purifying membrane protein fractions from infected host cells. The experimental procedures necessary to solubilize hydrophobic proteins fail to maintain transient protein-protein interactions. Advances in C. trachomatis genetics has allowed us and others to use various experimental approaches in combination with affinity purification mass spectrometry (AP-MS) to study the interactions that occur at the chlamydial vacuolar, or inclusion, membrane. For the first time, two groups have published AP-MS studies using the same tool, the ascorbate peroxidase proximity labeling system (APEX2), which overcomes past experimental limitations because membrane protein interactions are labeled in vivo in the context of infection. The utility of this system is highlighted by its ability to study chlamydial type III secreted inclusion membrane protein (Inc) interactions. Incs act as the mediators of host-pathogen interactions at the inclusion during C. trachomatis infection. When carefully controlled and analyzed, the data obtained can yield copious amounts of useful information. Here, we critically analyzed four previously published studies, including statistical analysis of AP-MS datasets related to Chlamydia-host interactions, to contextualize the data and to identify the best practices in interpreting these types of complex outputs.

Project description:Pmps (Polymorphic Membrane Proteins) are a group of membrane bound surface exposed chlamydial proteins that have been characterized as autotransporter adhesins and are important in the initial phase of chlamydial infection. These proteins all contain conserved GGA (I, L, V) and FxxN tetrapeptide motifs in the N-terminal portion of each protein. All chlamydial species express Pmps. Even in the chlamydia-related bacteria Waddlia chondrophila, a Pmp-like adhesin has been identified, demonstrating the importance of Pmps in Chlamydiales biology. Chlamydial species vary in the number of pmp genes and their differentially regulated expression during the infectious cycle or in response to stress. Studies have also demonstrated that Pmps are able to induce innate immune functional responses in infected cells, including production of IL-8, IL-6 and MCP-1, by activating the transcription factor NF-κB. Human serum studies have indicated that although anti-Pmp specific antibodies are produced in response to a chlamydial infection, the response is variable depending on the Pmp protein. In C. trachomatis, PmpB, PmpC, PmpD and PmpI were the proteins eliciting the strongest immune response among adolescents with and without pelvic inflammatory disease (PID). In contrast, PmpA and PmpE elicited the weakest antibody response. Interestingly, there seems to be a gender bias for Pmp recognition with a stronger anti-Pmp reactivity in male patients. Furthermore, anti-PmpA antibodies might contribute to adverse pregnancy outcomes, at least among women with PID. In vitro studies indicated that dendritic cells infected with C. muridarum were able to present PmpG and PmpF on their MHC class II receptors and T cells were able to recognize the MHC class-II bound peptides. In addition, vaccination with PmpEFGH and Major Outer Membrane Protein (MOMP) significantly protected mice against a genital tract C. muridarum infection, suggesting that Pmps may be an important component of a multi-subunit chlamydial vaccine. Thus, Pmps might be important not only for the pathogenesis of chlamydial infection, but also as potential candidate vaccine proteins.

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Eukaryotic SNARE VAMP3 Dynamically Interacts with Multiple Chlamydial Inclusion Membrane Proteins.

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