Critical Role for the Extended N Terminus of Chlamydial MreB in Directing Its Membrane Association and Potential Interaction with Divisome Proteins.

Chlamydiae lack the conserved central coordinator protein of cell division FtsZ, a tubulin-like homolog. Current evidence indicates that Chlamydia uses the actin-like homolog, MreB, to substitute for the role of FtsZ in a polarized division mechanism. 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, harboring predicted amphipathicity, as well as the conserved amphipathic helix found in other bacterial MreBs. The conserved amphipathic helix-directed green fluorescent protein (GFP) to label the membrane uniformly in Escherichia coli but the extended N-terminal region did not. However, the extended N-terminal region together with the conserved amphipathic region directed GFP to restrict the membrane label to the cell poles. In Chlamydia, the extended N-terminal region was sufficient to direct GFP to the membrane, and this localization was independent of an association with endogenous MreB. Importantly, mutating the extended N-terminal region to reduce its amphipathicity resulted in the accumulation of GFP in the cytosol of the chlamydiae and not in the membrane. The N-terminal domain of MreB was not required for homotypic interactions but was necessary for interactions with cell division components RodZ and FtsK. Our data provide mechanistic support for chlamydial MreB to serve as a substitute for FtsZ by forming a ringlike structure at the site of polarized division.IMPORTANCEChlamydia trachomatis is an obligate intracellular pathogen, causing sexually transmitted diseases and trachoma. The study of chlamydial physiology is important for developing novel therapeutic strategies for these diseases. Chlamydiae divide by a unique MreB-dependent polarized cell division process. In this study, we investigated unique properties of chlamydial MreB and observed that chlamydial species harbor an extended N-terminal region possessing amphipathicity. MreB formed a ring at the septum, like FtsZ in Escherichia coli, and its localization was dependent upon the amphipathic nature of its extended N terminus. Furthermore, this region is crucial for the interaction of MreB with cell division proteins. Given these results, chlamydial MreB likely functions at the septum as a scaffold for divisome proteins to regulate cell division in this organism.

A new metabolic cell-wall labelling method reveals peptidoglycan in Chlamydia trachomatis.

Peptidoglycan (PG), an essential structure in the cell walls of the vast majority of bacteria, is critical for division and maintaining cell shape and hydrostatic pressure. Bacteria comprising the Chlamydiales were thought to be one of the few exceptions. Chlamydia harbour genes for PG biosynthesis and exhibit susceptibility to 'anti-PG' antibiotics, yet attempts to detect PG in any chlamydial species have proven unsuccessful (the 'chlamydial anomaly'). We used a novel approach to metabolically label chlamydial PG using d-amino acid dipeptide probes and click chemistry. Replicating Chlamydia trachomatis were labelled with these probes throughout their biphasic developmental life cycle, and the results of differential probe incorporation experiments conducted in the presence of ampicillin are consistent with the presence of chlamydial PG-modifying enzymes. These findings culminate 50 years of speculation and debate concerning the chlamydial anomaly and are the strongest evidence so far that chlamydial species possess functional PG.

Purification of Tag-Free Chlamydia trachomatis Scc4 for Structural Studies Using Sarkosyl-Assisted on-Column Complex Dissociation.

Chlamydia trachomatis is an obligate intracellular bacterial pathogen that causes the most common sexually transmitted bacterial disease in the world. The bacterium has a unique biphasic developmental cycle with a type III secretion system (T3SS) to invade host cells. Scc4 is a class I T3SS chaperone forming a heterodimer complex with Scc1 to chaperone the essential virulence effector, CopN. Scc4 also functions as an RNA polymerase binding protein to regulate σ66-dependent transcription. Aggregation and low solubility of 6X-histidine-tagged Scc4 and the insolubility of 6X-histidine and FLAG-tagged Scc1 expressed in Escherichia coli have hindered the high-resolution nuclear magnetic resonance (NMR) structure determination of these proteins and motivated the development of an on-column complex dissociation method to produce tag-free Scc4 and soluble FLAG-tagged Scc1. By utilizing a 6X-histidine-tag on one protein, the coexpressed Scc4-Scc1 complex was captured on nickel-charged immobilized metal affinity chromatography resin, and the nondenaturing detergent, sodium N-lauroylsarcosine (sarkosyl), was used to dissociate and elute the non-6X-histidine-tagged protein. Tag-free Scc4 was produced in a higher yield and had better NMR spectral characteristics compared to 6X-histidine-tagged Scc4, and soluble FLAG-tagged Scc1 was purified for the first time in a high yield. The backbone structure of Scc4 after exposure to sarkosyl was validated using NMR spectroscopy, demonstrating the usefulness of the method to produce proteins for structural and functional studies. The sarkosyl-assisted on-column complex dissociation method is generally applicable to protein complexes with high affinity and is particularly useful when affinity tags alter the protein's biophysical properties or when coexpression is necessary for solubility.

A novel protease inhibitor causes inclusion vacuole reduction and disrupts the intracellular growth of Chlamydia trachomatis.

Chlamydia (C.) trachomatis, characterized by a unique biphasic life cycle, is an obligate intracellular bacterial pathogen which is responsible for the highest number of sexually transmitted bacterial infections globally. However, its pathogenic mechanisms have not been fully elucidated because of its unique developmental cycle and obligate intracellular nature. High temperature requirement (HtrA), a critical protease and chaperone, has been previously demonstrated to be essential for several functions and the replicative phase in the C. trachomatis developmental cycle. In the current study, we designed and synthesized a novel peptidomimetic inhibitor targeting C. trachomatis HtrA (CtHtrA) using homology modeling and chemical synthesis. The inhibitor was tested in chlamydia in the mid-replicative phase and resulted in a significant loss of viable infectious progeny and diminishing inclusion size and number at a relatively low concentration. This finding not only indicates that CtHtrA plays a critical role during the replicative phase of the chlamydial developmental cycle but also reveals a useful target for the design of novel anti-chlamydial agents.

Sensitive detection of DNA from Chlamydia trachomatis by using flap endonuclease-assisted amplification and graphene oxide-based fluorescence signaling.

A simple and sensitive method is described for the determination of DNA. It relies on the use of (a) an invasive reaction that is catalyzed by flap endonuclease 1 (FEN 1), and (b) graphene oxide (GO)-based fluorescence signalling. The presence of target DNA mediates the formation of the invasive structure, and this induces FEN 1 to catalyze multiple cycles of cleavage reaction at the junction, thereby liberating numerous fluorophore-labeled flaps. The released flaps are intentionally designed too short to be adsorbed onto GO. Hence, intense green fluorescence whose maximum emission is observed at 520 nm after excitation at 480 nm is restored even in the presence of GO. The method can be applied to the determination of target DNA from Chlamydia trachomatis, one of the major pathogenic bacteria causing sexually transmitted diseases. The assay is sensitive and specific with the limit of detection of 6.7 pM, and was applied to reliable determination of Chlamydia trachomatis DNA in human serum. Graphical abstract Flap endonuclease 1 (FEN 1)-catalyzed invasive reaction and graphene oxide (GO)-based fluorescence signalling are integrated to develop a novel and sensitive target DNA detection method.

Both the N- and C- terminal regions of the Chlamydial inclusion protein D (IncD) are required for interaction with the pleckstrin homology domain of the ceramide transport protein CERT.

Chlamydia trachomatis is an obligate intracellular bacterium that replicates within a membranous compartment, the inclusion, in host cells. Its intracellular life cycle requires host sphingolipids, which are in part acquired through the ER-Golgi localized ceramide transport protein (CERT). The Chlamydia-encoded inclusion membrane protein IncD is composed of two closely linked long hydrophobic domains with their N- and C-termini exposed to the host cytosol. IncD binds directly to the pleckstrin homology (PH) domain of CERT, likely redirecting ceramide to the inclusion. The precise regions of IncD required for this interaction have not been delineated. Using co-transfection studies together with phylogenetic studies, we demonstrate that both the IncD N- and C-terminal regions are required for binding to the CERT PH domain and define key interaction residues. Native gel electrophoresis analysis demonstrates that the transmembrane region of IncD forms SDS-resistant but dithiothreitol-sensitive homodimers, which in turn can assemble to form higher order oligomers through additional N- and C-terminal domain contacts. IncD oligomerization may facilitate high affinity binding to CERT, allowing C. trachomatis to efficiently redirect host ceramide to the inclusion.

Pathogenic Chlamydia Lack a Classical Sacculus but Synthesize a Narrow, Mid-cell Peptidoglycan Ring, Regulated by MreB, for Cell Division.

The peptidoglycan (PG) cell wall is a peptide cross-linked glycan polymer essential for bacterial division and maintenance of cell shape and hydrostatic pressure. Bacteria in the Chlamydiales were long thought to lack PG until recent advances in PG labeling technologies revealed the presence of this critical cell wall component in Chlamydia trachomatis. In this study, we utilize bio-orthogonal D-amino acid dipeptide probes combined with super-resolution microscopy to demonstrate that four pathogenic Chlamydiae species each possess a ≤ 140 nm wide PG ring limited to the division plane during the replicative phase of their developmental cycles. Assembly of this PG ring is rapid, processive, and linked to the bacterial actin-like protein, MreB. Both MreB polymerization and PG biosynthesis occur only in the intracellular form of pathogenic Chlamydia and are required for cell enlargement, division, and transition between the microbe's developmental forms. Our kinetic, molecular, and biochemical analyses suggest that the development of this limited, transient, PG ring structure is the result of pathoadaptation by Chlamydia to an intracellular niche within its vertebrate host.

Structural characterization of muropeptides from Chlamydia trachomatis peptidoglycan by mass spectrometry resolves "chlamydial anomaly".

The "chlamydial anomaly," first coined by James Moulder, describes the inability of researchers to detect or purify peptidoglycan (PG) from pathogenic Chlamydiae despite genetic and biochemical evidence and antibiotic susceptibility data that suggest its existence. We recently detected PG in Chlamydia trachomatis by a new metabolic cell wall labeling method, however efforts to purify PG from pathogenic Chlamydiae have remained unsuccessful. Pathogenic chlamydial species are known to activate nucleotide-binding oligomerization domain-containing protein 2 (NOD2) innate immune receptors by as yet uncharacterized ligands, which are presumed to be PG fragments (muramyl di- and tripeptides). We used the NOD2-dependent activation of NF-κB by C. trachomatis-infected cell lysates as a biomarker for the presence of PG fragments within specific lysate fractions. We designed a new method of muropeptide isolation consisting of a double filtration step coupled with reverse-phase HPLC fractionation of Chlamydia-infected HeLa cell lysates. Fractions that displayed NOD2 activity were analyzed by electrospray ionization mass spectrometry, confirming the presence of muramyl di- and tripeptides in Chlamydia-infected cell lysate fractions. Moreover, the mass spectrometry data of large muropeptide fragments provided evidence that transpeptidation and transglycosylation reactions occur in pathogenic Chlamydiae. These results reveal the composition of chlamydial PG and disprove the "glycanless peptidoglycan" hypothesis.

Functional analysis of three topoisomerases that regulate DNA supercoiling levels in Chlamydia.

Chlamydia is a medically important bacterium that infects eukaryotic cells. Temporal expression of chlamydial genes during the intracellular infection is proposed to be regulated by changes in DNA supercoiling levels. To understand how chlamydial supercoiling levels are regulated, we purified and analyzed three putative Chlamydia trachomatis topoisomerases. As predicted by sequence homology, CT189/190 are the two subunits of DNA gyrase, whereas CT643 is a topoisomerase I. CT660/661 have been predicted to form a second DNA gyrase, but the reconstitute holoenzyme decatenated and relaxed DNA, indicating that the proteins are subunits of topoisomerase IV. Promoter analysis showed that each topoisomerase is transcribed from its own operon by the major chlamydial RNA polymerase. Surprisingly, all three topoisomerase promoters had higher activity from a more supercoiled DNA template. This supercoiling-responsivesness is consistent with negative feedback control of topoisomerase I and topoisomerase IV expression, which is typical of other bacteria. However, activation of the chlamydial gyrase promoter by increased supercoiling is unorthodox compared with the relaxation-induced transcription of gyrase in other bacteria. We present a model in which supercoiling levels during the intracellular chlamydial developmental cycle are regulated by unusual positive feedback control of the gyrase promoter and the temporal expression of three topoisomerases.

CTL0511 from Chlamydia trachomatis Is a Type 2C Protein Phosphatase with Broad Substrate Specificity.

UNLABELLED: Protein phosphorylation has become increasingly recognized for its role in regulating bacterial physiology and virulence. Chlamydia spp. encode two validated Hanks'-type Ser/Thr protein kinases, which typically function with cognate protein phosphatases and appear capable of global protein phosphorylation. Consequently, we sought to identify a Ser/Thr protein phosphatase partner for the chlamydial kinases. CTL0511 from Chlamydia trachomatis L2 434/Bu, which has homologs in all sequenced Chlamydia spp., is a predicted type 2C Ser/Thr protein phosphatase (PP2C). Recombinant maltose-binding protein (MBP)-tagged CTL0511 (rCTL0511) hydrolyzed p-nitrophenyl phosphate (pNPP), a generic phosphatase substrate, in a MnCl2-dependent manner at physiological pH. Assays using phosphopeptide substrates revealed that rCTL0511 can dephosphorylate phosphorylated serine (P-Ser), P-Thr, and P-Tyr residues using either MnCl2 or MgCl2, indicating that metal usage can alter substrate preference. Phosphatase activity was unaffected by PP1, PP2A, and PP3 phosphatase inhibitors, while mutation of conserved PP2C residues significantly inhibited activity. Finally, phosphatase activity was detected in elementary body (EB) and reticulate body (RB) lysates, supporting a role for protein dephosphorylation in chlamydial development. These findings support that CTL0511 is a metal-dependent protein phosphatase with broad substrate specificity, substantiating a reversible phosphorylation network in C. trachomatis IMPORTANCE: Chlamydia spp. are obligate intracellular bacterial pathogens responsible for a variety of diseases in humans and economically important animal species. Our work demonstrates that Chlamydia spp. produce a PP2C capable of dephosphorylating P-Thr, P-Ser, and P-Tyr and that Chlamydia trachomatis EBs and RBs possess phosphatase activity. In conjunction with the chlamydial Hanks'-type kinases Pkn1 and PknD, validation of CTL0511 fulfills the enzymatic requirements for a reversible phosphoprotein network. As protein phosphorylation regulates important cellular processes, including metabolism, differentiation, and virulence, in other bacterial pathogens, these results set the stage for elucidating the role of global protein phosphorylation in chlamydial physiology and virulence.

An α-helical core encodes the dual functions of the chlamydial protein IncA.

Chlamydia is an intracellular bacterium that establishes residence within parasitophorous compartments (inclusions) inside host cells. Chlamydial inclusions are uncoupled from the endolysosomal pathway and undergo fusion with cellular organelles and with each other. To do so, Chlamydia expresses proteins on the surface of the inclusion using a Type III secretion system. These proteins, termed Incs, are located at the interface between host and pathogen and carry out the functions necessary for Chlamydia survival. Among these Incs, IncA plays a critical role in both protecting the inclusion from lysosomal fusion and inducing the homotypic fusion of inclusions. Within IncA are two regions homologous to eukaryotic SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor) domains referred to as SNARE-like domain 1 (SLD1) and SNARE-like domain 2 (SLD2). Using a multidisciplinary approach, we have discovered the functional core of IncA that retains the ability to both inhibit SNARE-mediated fusion and promote the homotypic fusion of Chlamydia inclusions. Circular dichroism and analytical ultracentrifugation experiments show that this core region is composed almost entirely of α-helices and assembles into stable homodimers in solution. Altogether, we propose that both IncA functions are encoded in a structured core domain that encompasses SLD1 and part of SLD2.

Optimization of candidate proteins for serological screening of Chlamydia trachomatis infection.

The aim of this study was to optimize candidate antigen proteins for serological screening of Chlamydia trachomatis infection. C. trachomatis positive serum and swabs of genital secretions were collected from 50 patients in the Tianjin Medical University General Hospital, as well as from 30 patients negative for C. trachomatis. Samples were assessed by colloidal gold assay in a sexually transmitted disease clinic as follows: serum antibodies for eight kinds of C. trachomatis immunodominant proteins (Pgp3, CPAF, CT143, CT101, CT694, CT875, CT813, and IncA) were detected, and two traditional gold standards, immunofluorescence and C. trachomatis cell culture of genital secretions, were used for comparison in order to determine the antigen protein combinations with the highest sensitivity and specificity. Of the 50 samples that tested positive for C. trachomatis infection by colloidal gold assay, 44 tested positive by micro-immunofluorescence, whereas 6 tested negative. In contrast, 14 samples tested positive by cell culture, whereas 36 tested negative. Serological results of the immunodominant protein combination of Pgp3, CT694, and CT875 shared positive coincidence rates of 97.73 and 92.86% with C. trachomatis micro-immunofluorescence and cell culture, respectively. No antibodies of the three proteins were detected in the 30 C. trachomatis samples that tested negative by colloidal gold assay; these samples also tested negative in C. trachomatis genital secretion culture. Overall, the combination of the three immunodominant proteins Pgp3, CT694, and CT875 had good sensitivity and specificity for serological screening of C. trachomatis infection, and the process was simple and easy to apply.

Novel HLA-B27-restricted epitopes from Chlamydia trachomatis generated upon endogenous processing of bacterial proteins suggest a role of molecular mimicry in reactive arthritis.

Reactive arthritis (ReA) is an HLA-B27-associated spondyloarthropathy that is triggered by diverse bacteria, including Chlamydia trachomatis, a frequent intracellular parasite. HLA-B27-restricted T-cell responses are elicited against this bacterium in ReA patients, but their pathogenetic significance, autoimmune potential, and relevant epitopes are unknown. High resolution and sensitivity mass spectrometry was used to identify HLA-B27 ligands endogenously processed and presented by HLA-B27 from three chlamydial proteins for which T-cell epitopes were predicted. Fusion protein constructs of ClpC, Na(+)-translocating NADH-quinone reductase subunit A, and DNA primase were expressed in HLA-B27(+) cells, and their HLA-B27-bound peptidomes were searched for endogenous bacterial ligands. A non-predicted peptide, distinct from the predicted T-cell epitope, was identified from ClpC. A peptide recognized by T-cells in vitro, NQRA(330-338), was detected from the reductase subunit. This is the second HLA-B27-restricted T-cell epitope from C. trachomatis with relevance in ReA demonstrated to be processed and presented in live cells. A novel peptide from the DNA primase, DNAP(211-223), was also found. This was a larger variant of a known epitope and was highly homologous to a self-derived natural ligand of HLA-B27. All three bacterial peptides showed high homology with human sequences containing the binding motif of HLA-B27. Molecular dynamics simulations further showed a striking conformational similarity between DNAP(211-223) and its homologous and much more flexible human-derived HLA-B27 ligand. The results suggest that molecular mimicry between HLA-B27-restricted bacterial and self-derived epitopes is frequent and may play a role in ReA.

Electronic structural flexibility of heterobimetallic Mn/Fe cofactors: R2lox and R2c proteins.

The electronic structure of the Mn/Fe cofactor identified in a new class of oxidases (R2lox) described by Andersson and Högbom [Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 5633] is reported. The R2lox protein is homologous to the small subunit of class Ic ribonucleotide reductase (R2c) but has a completely different in vivo function. Using multifrequency EPR and related pulse techniques, it is shown that the cofactor of R2lox represents an antiferromagnetically coupled Mn(III)/Fe(III) dimer linked by a µ-hydroxo/bis-µ-carboxylato bridging network. The Mn(III) ion is coordinated by a single water ligand. The R2lox cofactor is photoactive, converting into a second form (R2loxPhoto) upon visible illumination at cryogenic temperatures (77 K) that completely decays upon warming. This second, unstable form of the cofactor more closely resembles the Mn(III)/Fe(III) cofactor seen in R2c. It is shown that the two forms of the R2lox cofactor differ primarily in terms of the local site geometry and electronic state of the Mn(III) ion, as best evidenced by a reorientation of its unique (55)Mn hyperfine axis. Analysis of the metal hyperfine tensors in combination with density functional theory (DFT) calculations suggests that this change is triggered by deprotonation of the µ-hydroxo bridge. These results have important consequences for the mixed-metal R2c cofactor and the divergent chemistry R2lox and R2c perform.

Vibrio cholerae ghosts (VCG) exert immunomodulatory effect on dendritic cells for enhanced antigen presentation and induction of protective immunity.

BACKGROUND: We previously showed that the Vibrio cholerae ghost platform (VCG; empty V. cholerae cell envelopes) is an effective delivery system for vaccine antigens promoting the induction of substantial immunity in the absence of external adjuvants. However, the mechanism by which these cell envelopes enhance immunity and stimulate a predominantly Th1 cellular and humoral immune response has not been elucidated. We hypothesized that the immunostimulatory ability of VCG involves dendritic cell (DC) activation. OBJECTIVE: The aims of this study were: a) to investigate the ability of DCs [using mouse bone marrow-derived DCs (BMDCs) as a model system] to take up and internalize VCGs; b) to evaluate the immunomodulatory effect of internalized VCGs on DC activation and maturation and their functional capacity to present chlamydial antigen to naïve and infection-sensitized CD4+ T cells and; c) to evaluate the ability of VCGs to enhance the protective immunity of a chlamydial antigen. RESULTS: VCGs were efficiently internalized by DCs without affecting their viability and modulated DC-mediated immune responses. VCG-pulsed DCs showed increased secretion of proinflammatory cytokines and expression of co-stimulatory molecules associated with DC maturation in response to stimulation with UV-irradiated chlamydial elementary bodies (UV-EBs). Furthermore, this interaction resulted in effective chlamydial antigen presentation to infection-sensitized but not naïve CD4+ T cells and enhancement of protective immunity. CONCLUSIONS: The present study demonstrated that VCGs activate DCs leading to the surface expression of co-stimulatory molecules associated with DC activation and maturation and enhancement of protective immunity induced by a chlamydial antigen. The results indicate that the immunoenhancing activity of VCG for increased T-cell activation against antigens is mediated, at least in part, through DC triggering. Thus, VCGs could be harnessed as immunomodulators to target antigens to DCs for enhancement of protective immunity against microbial infections.

The chlamydial anomaly clarified?

Getting visible: A new method to label bacterial cell walls shows the presence of functional peptidoglycan in the important pathogen Chlamydia trachomatis. This might clarify the long-standing paradox of the "chlamydial anomaly".

Long-term stability of a vaccine formulated with the amphipol-trapped major outer membrane protein from Chlamydia trachomatis.

Chlamydia trachomatis is a major bacterial pathogen throughout the world. Although antibiotic therapy can be implemented in the case of early detection, a majority of the infections are asymptomatic, requiring the development of preventive measures. Efforts have focused on the production of a vaccine using the C. trachomatis major outer membrane protein (MOMP). MOMP is purified in its native (n) trimeric form using the zwitterionic detergent Z3-14, but its stability in detergent solutions is limited. Amphipols (APols) are synthetic polymers that can stabilize membrane proteins (MPs) in detergent-free aqueous solutions. Preservation of protein structure and optimization of exposure of the most effective antigenic regions can avoid vaccination with misfolded, poorly protective protein. Previously, we showed that APols maintain nMOMP secondary structure and that nMOMP/APol vaccine formulations elicit better protection than formulations using either recombinant or nMOMP solubilized in Z3-14. To achieve a greater understanding of the structural behavior and stability of nMOMP in APols, we have used several spectroscopic techniques to characterize its secondary structure (circular dichroism), tertiary and quaternary structures (immunochemistry and gel electrophoresis) and aggregation state (light scattering) as a function of temperature and time. We have also recorded NMR spectra of (15)N-labeled nMOMP and find that the exposed loops are detectable in APols but not in detergent. Our analyses show that APols protect nMOMP much better than Z3-14 against denaturation due to continuous heating, repeated freeze/thaw cycles, or extended storage at room temperature. These results indicate that APols can help improve MP-based vaccine formulations.

Identification of linear B-cell epitopes within Tarp of Chlamydia trachomatis.

Chlamydia trachomatis is one of the most prevalent sexually transmitted pathogens. There is currently no commercially available vaccine against C. trachomatis. Chlamydial translocated actin-recruiting phosphoprotein (Tarp) can induce cellular and humoral immune responses in murine models and has been regarded as a potential vaccine candidate. In this report, the amino acid sequence of Tarp was analyzed using computer-assisted techniques to scan B-cell epitopes, and six possible linear B-cell epitopes peptides (aa80-95, aa107-123, aa152-170, aa171-186, aa239-253 and aa497-513) with high predicted antigenicity and high conservation were investigated. Sera from mice immunized with these potential immunodominant peptides was analyzed by ELISA, which showed that epitope 152-170 elicited serum immunoglobulin G (IgG) response and epitope 171-186 elicited both serum IgG and mucosal secretory immunoglobulin A response. The response of immune sera of epitope 171-186 to endogenous Tarp antigen obtained from the Hela229 cells infected with C. trachomatis was confirmed by Western blot and indirect fluorescence assay. In addition, binding of the antibodies against epitope 171-186 to endogenous Tarp was further confirmed by competitive ELISA. Our results demonstrated that the putative epitope (aa171-186) was an immunodominant B-cell epitope of Tarp. If proven protective and safe, this epitope, in combination with other well-documented epitopes, might be included into a candidate epitope-based vaccine against C. trachomatis.

Proteolytic activation of Chlamydia trachomatis HTRA is mediated by PDZ1 domain interactions with protease domain loops L3 and LC and beta strand ß5.

Chlamydia trachomatis is a bacterial pathogen responsible for one of the most prevalent sexually transmitted infections worldwide. Its unique development cycle has limited our understanding of its pathogenic mechanisms. However, CtHtrA has recently been identified as a potential C. trachomatis virulence factor. CtHtrA is a tightly regulated quality control protein with a monomeric structural unit comprised of a chymotrypsin-like protease domain and two PDZ domains. Activation of proteolytic activity relies on the C-terminus of the substrate allosterically binding to the PDZ1 domain, which triggers subsequent conformational change and oligomerization of the protein into 24-mers enabling proteolysis. This activation is mediated by a cascade of precise structural arrangements, but the specific CtHtrA residues and structural elements required to facilitate activation are unknown. Using in vitro analysis guided by homology modeling, we show that the mutation of residues Arg362 and Arg224, predicted to disrupt the interaction between the CtHtrA PDZ1 domain and loop L3, and between loop L3 and loop LD, respectively, are critical for the activation of proteolytic activity. We also demonstrate that mutation to residues Arg299 and Lys160, predicted to disrupt PDZ1 domain interactions with protease loop LC and strand ß5, are also able to influence proteolysis, implying their involvement in the CtHtrA mechanism of activation. This is the first investigation of protease loop LC and strand ß5 with respect to their potential interactions with the PDZ1 domain. Given their high level of conservation in bacterial HtrA, these structural elements may be equally significant in the activation mechanism of DegP and other HtrA family members.

The atypical response regulator protein ChxR has structural characteristics and dimer interface interactions that are unique within the OmpR/PhoB subfamily.

Typically as a result of phosphorylation, OmpR/PhoB response regulators form homodimers through a receiver domain as an integral step in transcriptional activation. Phosphorylation stabilizes the ionic and hydrophobic interactions between monomers. Recent studies have shown that some response regulators retain functional activity in the absence of phosphorylation and are termed atypical response regulators. The two currently available receiver domain structures of atypical response regulators are very similar to their phospho-accepting homologs, and their propensity to form homodimers is generally retained. An atypical response regulator, ChxR, from Chlamydia trachomatis, was previously reported to form homodimers; however, the residues critical to this interaction have not been elucidated. We hypothesize that the intra- and intermolecular interactions involved in forming a transcriptionally competent ChxR are distinct from the canonical phosphorylation (activation) paradigm in the OmpR/PhoB response regulator subfamily. To test this hypothesis, structural and functional studies were performed on the receiver domain of ChxR. Two crystal structures of the receiver domain were solved with the recently developed method using triiodo compound I3C. These structures revealed many characteristics unique to OmpR/PhoB subfamily members: typical or atypical. Included was the absence of two α-helices present in all other OmpR/PhoB response regulators. Functional studies on various dimer interface residues demonstrated that ChxR forms relatively stable homodimers through hydrophobic interactions, and disruption of these can be accomplished with the introduction of a charged residue within the dimer interface. A gel shift study with monomeric ChxR supports that dimerization through the receiver domain is critical for interaction with DNA.