Development of a Proximity Labeling System to Map the Chlamydia trachomatis Inclusion Membrane

Rucks, Elizabeth A.; Olson, Macy G.; Jorgenson, Lisa M; Srinivasan, Rekha R.; Ouellette, Scot P.

doi:10.3389/fcimb.2017.00040

Cited by 29 publications

(59 citation statements)

References 71 publications

(116 reference statements)

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“…In support of this, we were unable to isolate a clonal population of purified ClpP1(S92A) transformants, as we routinely observed small bacterial populations susceptible to antibiotic selection even after multiple passages. This observation indicates that leaky expression may drive plasmid loss, as has been observed in other chlamydial transformation systems (36,37).…”

Section: Figsupporting

confidence: 66%

Initial Characterization of the Two ClpP Paralogs of Chlamydia trachomatis Suggests Unique Functionality for Each

et al. 2019

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Members of Chlamydia are obligate intracellular bacteria that differentiate between two distinct functional and morphological forms during their developmental cycle, elementary bodies (EBs) and reticulate bodies (RBs). EBs are nondividing small electron-dense forms that infect host cells. RBs are larger noninfectious replicative forms that develop within a membrane-bound vesicle, termed an inclusion. Given the unique properties of each developmental form of this bacterium, we hypothesized that the Clp protease system plays an integral role in proteomic turnover by degrading specific proteins from one developmental form or the other. Chlamydia spp. have five uncharacterized clp genes, clpX, clpC, two clpP paralogs, and clpB. In other bacteria, ClpC and ClpX are ATPases that unfold and feed proteins into the ClpP protease to be degraded, and ClpB is a deaggregase. Here, we focused on characterizing the ClpP paralogs. Transcriptional analyses and immunoblotting determined that these genes are expressed midcycle. Bioinformatic analyses of these proteins identified key residues important for activity. Overexpression of inactive clpP mutants in Chlamydia spp. suggested independent function of each ClpP paralog. To further probe these differences, we determined interactions between the ClpP proteins using bacterial two-hybrid assays and native gel analysis of recombinant proteins. Homotypic interactions of the ClpP proteins, but not heterotypic interactions between the ClpP paralogs, were detected. Interestingly, protease activity of ClpP2, but not ClpP1, was detected in vitro. This activity was stimulated by antibiotics known to activate ClpP, which also blocked chlamydial growth. Our data suggest the chlamydial ClpP paralogs likely serve distinct and critical roles in this important pathogen. IMPORTANCE Chlamydia trachomatis is the leading cause of preventable infectious blindness and of bacterial sexually transmitted infections worldwide. Chlamydiae are developmentally regulated obligate intracellular pathogens that alternate between two functional and morphologic forms, with distinct repertoires of proteins. We hypothesize that protein degradation is a critical aspect to the developmental cycle. A key system involved in protein turnover in bacteria is the Clp protease system. Here, we characterized the two chlamydial ClpP paralogs by examining their expression in Chlamydia spp., their ability to oligomerize, and their proteolytic activity. This work will help understand the evolutionarily diverse Clp proteases in the context of intracellular organisms, which may aid in the study of other clinically relevant intracellular bacteria.

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Section: Figsupporting

confidence: 66%

Initial Characterization of the Two ClpP Paralogs of Chlamydia trachomatis Suggests Unique Functionality for Each

et al. 2019

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“…to transform Chlamydia trachomatis with a shuttle vector having been developed by the Clarke lab (41), we sought to take advantage of this, along with an inducible expression system developed by the Hefty lab (42), to determine what effect overexpression of Rho would have on chlamydial growth. Reports in the literature indicate that overexpression of nonessential proteins, such as green fluorescent protein (GFP) or IncA, in C. trachomatis have no biologically significant effect on chlamydial growth whereas overexpression of essential proteins, such as the cell division protein FtsQ, leads to aberrant bacterial morphologies and stalled growth (43,44). We constructed a shuttle vector encoding an anhydrotetracycline (aTc)-inducible 6ϫHis-tagged chlamydial Rho (Rho-6xH) and used this to transform C. trachomatis L2.…”

Section: Resultsmentioning

confidence: 99%

Characterization of Chlamydial Rho and the Role of Rho-Mediated Transcriptional Polarity during Interferon Gamma-Mediated Tryptophan Limitation

et al. 2018

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As an obligate intracellular, developmentally regulated bacterium, is sensitive to amino acid fluctuations within its host cell. When human epithelial cells are treated with the cytokine interferon gamma (IFN-γ), the tryptophan (Trp)-degrading enzyme, indoleamine-2,3-dioxygenase, is induced. Chlamydiae within such cells are starved for Trp and enter a state of so-called persistence. lacks the stringent response used by many eubacteria to respond to this stress. Unusually, chlamydial transcription is globally elevated during Trp starvation with transcripts for Trp codon-containing genes disproportionately increased. Yet, the presence of Trp codons destabilized 3' ends of transcripts in operons or large genes. We initially hypothesized that ribosome stalling on Trp codons rendered the 3' ends sensitive to RNase activity. The half-life of chlamydial transcripts containing different numbers of Trp codons was thus measured in untreated and IFN-γ-treated infected cells to determine whether Trp codons influenced the stability of transcripts. However, no effect of Trp codon content was detected. Therefore, we investigated whether Rho-dependent transcription termination could play a role in mediating transcript instability. Rho is expressed as a midcycle gene product, interacts with itself as predicted, and is present in all chlamydial species. Inhibition of Rho via the Rho-specific antibiotic, bicyclomycin, and overexpression of Rho are detrimental to chlamydiae. Finally, when we measured transcript abundance 3' to Trp codons in the presence of bicyclomycin, we observed that transcript abundance increased. These data are the first to demonstrate the importance of Rho in and the role of Rho-dependent transcription polarity during persistence.

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“…Leveraging this advance, we infected cells with a C. trachomatis L2/434 strain engineered to express Inc-APEX fusion proteins, to enable the capture of inclusion membrane interacting proteins in live cells, at multiple times during infection. The APEX system is highly sensitive, has defined the proteomes of cellular compartments refractory to other techniques, and APEX enzymatic function was shown to remain intact when tagged to a chlamydial Inc protein [17,23,24].…”

Section: Resultsmentioning

confidence: 99%

“…In addition to IncB, we have tagged additional Chlamydia type III secreted effectors such as CT694, Tarp, IncA, IncC, InaC, and CT223. Previous work has demonstrated the efficacy of tagging IncF with APEX [24], and we urge the field to exploit this system to accelerate our understanding of the molecular functions of chlamydial type III secreted proteins.…”

Section: Establishment and Maintenance Of The Inclusion Is Critical Tomentioning

confidence: 99%

Proximity-dependent proteomics of theChlamydia trachomatisinclusion membrane reveals functional interactions with endoplasmic reticulum exit sites

Dickinson

Anderson

Webb‐Robertson

et al. 2018

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Chlamydia trachomatis is the most common bacterial sexually transmitted infection, responsible for millions of infections each year. Despite this high prevalence, the elucidation of the molecular mechanisms of Chlamydia pathogenesis has been difficult due to limitations in genetic tools and its intracellular developmental cycle. Within a host epithelial cell, chlamydiae replicate within a vacuole called the inclusion. Many Chlamydia-host interactions are thought to be mediated by the Inc family of type III secreted proteins that are anchored in the inclusion membrane, but their array of host targets are largely unknown. To investigate how the inclusion membrane proteome changes over the course of an infected cell, we have adapted the APEX system of proximity-dependent biotinylation. APEX is capable of specifically labeling proteins within a 20 nm radius in living cells. We transformed C. trachomatis to express the enzyme APEX fused to known inclusion membrane proteins, allowing biotinylation and pull-down of inclusion-associated proteins. Using quantitative mass spectrometry against APEX labeled samples, we identified over 400 proteins associated with the inclusion membrane at early, middle, and late stages of epithelial cell infection. This system was sensitive enough to detect inclusion interacting proteins early in the developmental cycle, at 8 hours post infection, a previously intractable time point. Mass spectrometry analysis revealed a novel, early association between C. trachomatis inclusions and endoplasmic reticulum exit sites (ERES), functional regions of the ER where COPII-coated vesicles originate. Pharmacological and genetic disruption of ERES function severely restricted early chlamydial growth and the development of infectious progeny. APEX is therefore a powerful in situ approach for identifying critical protein interactions on the membranes of pathogen-containing vacuoles. Furthermore, the data derived from proteomic mapping of Chlamydia inclusions has illuminated an important functional role for ERES in promoting chlamydial developmental growth.

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Development of a Proximity Labeling System to Map the Chlamydia trachomatis Inclusion Membrane

Cited by 29 publications

References 71 publications

Initial Characterization of the Two ClpP Paralogs of Chlamydia trachomatis Suggests Unique Functionality for Each

Initial Characterization of the Two ClpP Paralogs of Chlamydia trachomatis Suggests Unique Functionality for Each

Characterization of Chlamydial Rho and the Role of Rho-Mediated Transcriptional Polarity during Interferon Gamma-Mediated Tryptophan Limitation

Proximity-dependent proteomics of theChlamydia trachomatisinclusion membrane reveals functional interactions with endoplasmic reticulum exit sites

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