Biology subjects

Ouellette, S. P.

Publications and source records attributed to Ouellette, S. P..

23 records · Page 2Linked to original sources

bioRxiv2019

The AAA+ ATPase ClpX Is Critical for Growth and Development of Chlamydia trachomatis

Abstract:Chlamydia trachomatis (Ctr) is an obligate intracellular bacterium that undergoes a complex developmental cycle in which the bacterium differentiates between two functionally and morphologically distinct forms, the EB and RB, each of which expresses its own specialized repertoire of proteins. Both primary (EB to RB) and secondary (RB to EB) differentiation require protein turnover, and we hypothesize that proteases are critical for mediating differentiation. The Clp protease system is well conserved in bacteria and important for protein turnover. Minimally, the system relies on a serine protease subunit, ClpP, and a AAA+ ATPase, such as ClpX, that recognizes and unfolds substrates for ClpP degradation. In Chlamydia, ClpX is encoded within an operon 3 to clpP2. We present evidence that the chlamydial ClpX and ClpP2 orthologs are essential to organism viability and development. We demonstrate here that chlamydial ClpX is a functional ATPase and forms the expected homohexamer in vitro. Overexpression of a ClpX mutant lacking ATPase activity had a limited impact on DNA replication or secondary differentiation but, nonetheless, reduced EB viability with observable defects in EB morphology noted. Conversely, the overexpression of a catalytically inactive ClpP2 mutant significantly impacted developmental cycle progression by reducing the overall number of organisms. Blocking clpP2X transcription using CRISPR interference led to a decrease in bacterial growth, and this effect was complemented in trans by a plasmid copy of clpP2. Taken together, our data indicate that ClpX and the associated ClpP2 serve distinct functions in chlamydial developmental cycle progression and differentiation. Importance Chlamydia trachomatis is the leading cause of infectious blindness globally and the most reported bacterial sexually transmitted infection both domestically and internationally. Given the economic burden, the lack of an approved vaccine, and the use of broad-spectrum antibiotics for treatment of infections, an understanding of chlamydial growth and development is critical for the advancement of novel, targeted antibiotics. The Clp proteins comprise an important and conserved protease system in bacteria. Our work highlights the importance of the chlamydial Clp proteins to this clinically important bacterium. Additionally, our study implicates the Clp system playing an integral role in chlamydial developmental cycle progression, which may help establish models of how Chlamydia spp. and other bacteria progress through their respective developmental cycles. Our work also contributes to a growing body of Clp-specific research that underscores the importance and versatility of this system throughout bacterial evolution and further validates Clp proteins as drug targets.

microbiology↗

bioRxiv2019

Inhibition of tRNA Synthetases Induces Persistence in Chlamydia

Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections, and C. pneumoniae causes community-acquired respiratory infections. In vivo, the host immune system will release interferon-gamma (IFN{gamma}) to combat infection. IFN{gamma} activates human cells to produce the tryptophan (trp) catabolizing enzyme, IDO. Consequently, there is a reduction in cytosolic trp in IFN{gamma}-activated host cells. In evolving to obligate intracellular dependence, Chlamydia has significantly reduced its genome size and content as it relies on the host cell for various nutrients. Importantly, C. trachomatis and C. pneumoniae are trp auxotrophs and are starved for this essential nutrient when the human host cell is exposed to IFN{gamma}. To survive this, chlamydiae enter an alternative growth state referred to as persistence. Chlamydial persistence is characterized by a halt in the division cycle, aberrant morphology, and, in the case of IFN{gamma}-induced persistence, trp codon-dependent changes in transcription. We hypothesize that these changes in transcription are dependent on the particular amino acid starvation state. To investigate the chlamydial response mechanisms acting when other amino acids become limiting, we tested the efficacy of prokaryotic specific tRNA synthetase inhibitors, indolmycin and AN3365, to mimic starvation of trp and leucine, respectively. We show that these drugs block chlamydial growth and induce changes in morphology and transcription consistent with persistence. Importantly, growth inhibition was reversed when the compounds were removed from the medium. With these data, we find that indolmycin and AN3365 are valid tools that can be used to mimic the persistent state independently of IFN{gamma}.\n\nImportanceThe obligate intracellular pathogen Chlamydia trachomatis, although treatable, remains a major public health concern due to rising infection rates. The asymptomatic nature of most Chlamydia infections is hypothesized to be a product of its ability to transition into a slow-growing state referred to as persistence. The most physiologically relevant inducer of persistence is the immune cytokine IFN{gamma}, which in humans activates an enzyme that degrades tryptophan, an essential amino acid that Chlamydia scavenges from the host cell. Unfortunately, the exact timing at which Chlamydia is starved after IFN{gamma} treatment is inexact. To mechanistically study persistence using genetic tools, an experimental model where amino acid starvation can be induced at specific times is needed. Here, we demonstrate the capability of tRNA synthetase inhibitors, indolmycin and AN3365, to model persistence independently from the use of IFN{gamma}. These tools will also allow comparisons between amino acid stress responses in this unique bacterium.

microbiology↗

bioRxiv2019

Critical Role for the Unique N-Terminus of Chlamydial MreB in Directing Its Membrane Association and Interaction with Elements of the Divisome

Chlamydiae lack the conserved central coordinator protein of cell division FtsZ, a tubulin-like homolog. Current evidence indicates Chlamydia uses the actin-like homolog, MreB, to substitute for the role of FtsZ. Interestingly, we observed MreB as a ring at the septum in dividing cells of Chlamydia. We hypothesize that MreB, to substitute for FtsZ in Chlamydia, must possess unique properties compared to canonical MreB orthologs. Sequence differences between chlamydial MreB and orthologs in other bacteria revealed that chlamydial MreB possesses an extended N-terminal region and the conserved amphipathic helix found in other bacterial MreBs. The extended N-terminal region was sufficient to restore the localization of a truncated E. coli MreB mutant lacking its amphipathic helix to the membrane and was crucial for interactions with cell division components RodZ and FtsK, though the region was not required for homotypic interactions. Importantly, the N-terminal region was sufficient to direct GFP to the membrane when expressed in Chlamydia. A mutant N-terminal region with reduced amphipathicity was unable to perform these functions. From these data, the extended N-terminal region of chlamydial MreB is critical for localization and interactions of this protein. Our data provide mechanistic support for chlamydial MreB to serve as a substitute for FtsZ. ImportanceChlamydia trachomatis is an obligate intracellular pathogen, causing sexual transmitted diseases and trachoma. Studying chlamydial physiology, especially its cell division mechanism, is important for developing novel therapeutic strategies for the treatment of these diseases. Since chlamydial cell division has unique features, including a polarized cell division process independent of FtsZ, a canonical cell division coordinator, studying the subject is helpful for understanding undefined aspects of chlamydial growth. In this study, we characterized MreB, a substitute for FtsZ, as a cell division coordinator. It forms a filamentous ring at the septum, like FtsZ in E. coli. We show that the localization of MreB is dependent upon the amphipathic nature of its extended N-terminus. Furthermore, this region is crucial for its interaction with other proteins involved in cell division. Given these results, chlamydial MreB may function as a scaffold for cell divisome proteins at the septum and regulate cell division in this organism.

microbiology↗

bioRxiv2019

Proximity Labeling to Map Host-Pathogen Interactions at the Membrane of a Bacteria Containing Vacuole in Chlamydia trachomatis Infected Human Cells

As an obligate intracellular pathogenic bacterium, C. trachomatis develops within a membrane-bound vacuole, termed the inclusion. The inclusion membrane is modified by chlamydial inclusion membrane proteins (Incs), which act as the mediators of host-pathogen interactions. An in vivo understanding of Inc-Inc and Inc-eukaryotic protein interactions and how these contribute to overall host-chlamydial interactions at this unique membrane is lacking. Previous bacterial two-hybrid studies established that certain Incs have the propensity to bind other Incs while others have limited Inc-Inc interactions. We hypothesize some Incs organize the inclusion membrane whereas other Incs bind eukaryotic proteins to promote chlamydial-host interactions. To test this hypothesis, we used the ascorbate peroxidase proximity labeling system (APEX2), which labels proximal proteins with biotin in vivo, and chose to analyze Inc proteins with varying Inc-binding propensities. We inducibly expressed these Incs fused to APEX2 in Chlamydia trachomatis L2, verified their localization and labeling activities by transmission electron microscopy, and used affinity purification-mass spectrometry to identify biotinylated proteins. To analyze our mass spectrometry results for statistical significance, we used Significance Analysis of INTeractome (SAINT), which demonstrated that our Inc-APEX2 constructs labeled Inc proteins as well as known and previously unreported eukaryotic proteins that localize to the inclusion. Our results broadly support two types of Inc interactions: Inc-Inc versus Inc-host. One eukaryotic protein, LRRFIP1 (LRRF1) was found in all of our Inc-APEX2 datasets, which is consistent with previously published AP-MS datasets. For the first time, we demonstrate by confocal and super-resolution microscopy that endogenous LRRF1 localizes to the chlamydial inclusion. We also used bacterial two-hybrid studies and pulldown assays to determine if LRRF1 was identified as a true interacting protein or was proximal to our Inc-APEX2 constructs. Combined, our data highlight the utility of APEX2 to capture the complex in vivo protein-protein interactions at the chlamydial inclusion.\n\nAuthor summaryMany intracellular bacteria, including the obligate intracellular pathogen Chlamydia trachomatis, grow within a membrane-bound \"bacteria containing vacuole\" (BCV) that, in most cases, prevents association with the lysosome. Secreted cytosolic effectors modulate host activity, but an understanding of the host-pathogen interactions that occur at the BCV membrane is limited by the difficulty in purifying membrane fractions from infected host cells. Here, we used the ascorbate peroxidase proximity labeling system (APEX2), which labels proximal proteins with biotin in vivo, to study the interactions that occur at the chlamydial vacuolar, or inclusion, membrane. The inclusion membrane is modified by chlamydial type III secreted inclusion membrane proteins (Incs), which act as the mediators of host-pathogen interactions. Our results broadly support two types of Inc interactions: Inc-Inc versus Inc-host. Our data highlight the utility of APEX2 to capture the complex protein-protein interactions at a membrane site in vivo in the context of infection.

microbiology↗

bioRxiv2018

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

Chlamydia is an obligate intracellular bacterium that differentiates between two distinct functional and morphological forms during its developmental cycle: elementary bodies (EBs) and reticulate bodies (RBs). EBs are non-dividing, small electron dense forms that infect host cells. RBs are larger, non-infectious 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 has 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 these genes are expressed mid-cycle. Bioinformatic analyses of these proteins identified key residues important for activity. Over-expression of inactive clpP mutants in Chlamydia 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, ClpP2, but not ClpP1, protease activity 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.\n\nImportanceChlamydia 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, 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.

microbiology↗