Chlamydial Protein Research Articles

Discordance in the Epithelial Cell-Dendritic Cell Major Histocompatibility Complex Class II Immunoproteome: Implications for Chlamydia Vaccine Development.

Chlamydia trachomatis and Chlamydia muridarum are intracellular bacterial pathogens of mucosal epithelial cells. CD4 T cells and major histocompatibility complex (MHC) class II molecules are essential for protective immunity against them. Antigens presented by dendritic cells (DCs) expand naive pathogen-specific T cells (inductive phase), whereas antigens presented by epithelial cells identify infected epithelial cells as targets during the effector phase. We previously showed that DCs infected by C trachomatis or C muridarum present epitopes from a limited spectrum of chlamydial proteins recognized by Chlamydia-specific CD4 T cells from immune mice. We hypothesized that Chlamydia-infected DCs and epithelial cells present overlapping sets of Chlamydia-MHC class II epitopes to link inductive and effector phases to generate protective immunity. We tested that hypothesis by infecting an oviductal epithelial cell line with C muridarum, followed by immunoaffinity isolation and sequencing of MHC class I- and II-bound peptides. We identified 26 class I-bound and 4 class II-bound Chlamydia-derived peptides from infected epithelial cells. We were surprised to find that none of the epithelial cell class I- and class II-bound chlamydial peptides overlapped with peptides presented by DCs. We suggest the discordance between the DC and epithelial cell immunoproteomes has implications for delayed clearance of Chlamydia and design of a Chlamydia vaccine.

Detection of chlamydia infection within human testicular biopsies.

STUDY QUESTIONCan Chlamydia be found in the testes of infertile men?SUMMARY ANSWERChlamydia can be found in 16.7% of fresh testicular biopsies and 45.3% of fixed testicular biopsies taken from a selection of infertile men.WHAT IS KNOWN ALREADYMale chlamydial infection has been understudied despite male and female infections occurring at similar rates. This is particularly true of asymptomatic infections, which occur in 50% of cases. Chlamydial infection has also been associated with increased sperm DNA damage and reduced male fertility.STUDY DESIGN, SIZE, DURATIONWe collected diagnostic (fixed, n = 100) and therapeutic (fresh, n = 18) human testicular biopsies during sperm recovery procedures from moderately to severely infertile men in a cross-sectional approach to sampling.PARTICIPANTS/MATERIALS, SETTING, METHODSThe diagnostic and therapeutic biopsies were tested for Chlamydia-specific DNA and protein, using real-time PCR and immunohistochemical approaches, respectively. Serum samples matched to the fresh biopsies were also assayed for the presence of Chlamydia-specific antibodies using immunoblotting techniques.MAIN RESULTS AND THE ROLE OF CHANCEChlamydial major outer membrane protein was detected in fixed biopsies at a rate of 45.3%. This was confirmed by detection of chlamydial DNA and TC0500 protein (replication marker). C. trachomatis DNA was detected in fresh biopsies at a rate of 16.7%, and the sera from each of these three positive patients contained C. trachomatis-specific antibodies. Overall, C. trachomatis-specific antibodies were detected in 72.2% of the serum samples from the patients providing fresh biopsies, although none of the patients were symptomatic nor had they reported a previous sexually transmitted infection diagnosis including Chlamydia.LIMITATIONS, REASONS FOR CAUTIONNo reproductively healthy male testicular biopsies were tested for the presence of Chlamydia DNA or proteins or Chlamydia-specific antibodies due to the unavailability of these samples.WIDER IMPLICATIONS FOR THE FINDINGSApplication of Chlamydia-specific PCR and immunohistochemistry in this human male infertility context of testicular biopsies reveals evidence of a high prevalence of previously unrecognised infection, which may potentially have a pathogenic role in spermatogenic failure.STUDY FUNDING/COMPETING INTEREST(S)Funding for this project was provided by the Australian NHMRC under project grant number APP1062198. We also acknowledge assistance from the Monash IVF Group and Queensland Fertility Group in the collection of fresh biopsies, and the Monash Health and co-author McLachlan (declared equity interest) in retrieval and sectioning of fixed biopsies. E.M. declares an equity interest in the study due to financing of fixed biopsy sectioning. All other authors declare no conflicts of interest.TRIAL REGISTRATION NUMBERN/A

Detection of Chlamydia Developmental Forms and Secreted Effectors by Expansion Microscopy

Expansion microscopy (ExM) is a novel tool to improve the resolution of fluorescence-based microscopy that has not yet been used to visualize intracellular pathogens. Here we show the expansion of the intracellular pathogen Chlamydia trachomatis, enabling to differentiate its two distinct forms, catabolic active reticulate bodies (RB) and infectious elementary bodies (EB), on a conventional confocal microscope. We show that ExM enables the possibility to precisely locate chlamydial effector proteins, such as CPAF or Cdu1, within and outside of the chlamydial inclusion. Thus, we claim that ExM offers the possibility to address a broad range of questions and may be useful for further research on various intracellular pathogens.

Microscopy Techniques Used to Visualize Immune-Labeled Chlamydial Inclusion Membrane Proteins in 2D and 3D

Microscopy Techniques Used to Visualize Immune-Labeled Chlamydial Inclusion Membrane Proteins in 2D and 3D

Insight into microtubule nucleation from tubulin-capping proteins

Nucleation is one of the least understood steps of microtubule dynamics. It is a kinetically unfavorable process that is templated in the cell by the γ-tubulin ring complex or by preexisting microtubules; it also occurs in vitro from pure tubulin. Here we study the nucleation inhibition potency of natural or artificial proteins in connection with their binding mode to the longitudinal surface of α- or β-tubulin. The structure of tubulin-bound CopN, a Chlamydia protein that delays nucleation, suggests that this protein may interfere with two protofilaments at the (+) end of a nucleus. Designed ankyrin repeat proteins that share a binding mode similar to that of CopN also impede nucleation, whereas those that target only one protofilament do not. In addition, an αRep protein predicted to target two protofilaments at the (-) end does not delay nucleation, pointing to different behaviors at both ends of the nucleus. Our results link the interference with protofilaments at the (+) end and the inhibition of nucleation.

Therapeutic effect of a Chlamydia pecorum recombinant major outer membrane protein vaccine on ocular disease in koalas (Phascolarctos cinereus).

Chlamydia pecorum is responsible for causing ocular infection and disease which can lead to blindness in koalas (Phascolarctos cinereus). Antibiotics are the current treatment for chlamydial infection and disease in koalas, however, they can be detrimental for the koala’s gastrointestinal tract microbiota and in severe cases, can lead to dysbiosis and death. In this study, we evaluated the therapeutic effects provided by a recombinant chlamydial major outer membrane protein (MOMP) vaccine on ocular disease in koalas. Koalas with ocular disease (unilateral or bilateral) were vaccinated and assessed for six weeks, evaluating any changes to the conjunctival tissue and discharge. Samples were collected pre- and post-vaccination to evaluate both humoral and cell-mediated immune responses. We further assessed the infecting C. pecorum genotype, host MHC class II alleles and presence of koala retrovirus type (KoRV-B). Our results clearly showed an improvement in the clinical ocular disease state of all seven koalas, post-vaccination. We observed increases in ocular mucosal IgA antibodies to whole C. pecorum elementary bodies, post-vaccination. We found that systemic cell-mediated immune responses to interferon-γ, interleukin-6 and interleukin-17A were not significantly predictive of ocular disease in koalas. Interestingly, one koala did not have as positive a clinical response (in one eye primarily) and this koala was infected with a C. pecorum genotype (E’) that was not used as part of the vaccine formula (MOMP genotypes A, F and G). The predominant MHC class II alleles identified were DAb*19, DAb*21 and DBb*05, with no two koalas identified with the same genetic sequence. Additionally, KoRV-B, which is associated with chlamydial disease outcome, was identified in two (29%) ocular diseased koalas, which still produced vaccine-induced immune responses and clinical ocular improvements post-vaccination. Our findings show promise for the use of a recombinant chlamydial MOMP vaccine for the therapeutic treatment of ocular disease in koalas.

Proximity Labeling of the Chlamydia trachomatis Inclusion Membrane.

In the study of intracellular bacteria that reside within a membrane-bound vacuole, there are many questions related to how prokaryotic or eukaryotic transmembrane or membrane-associated proteins are organized and function within the membranes of these pathogen-containing vacuoles. Yet this host-pathogen interaction interface has proven difficult to experimentally resolve. For example, one method to begin to understand protein function is to determine the protein-binding partners; however, examining protein-protein interactions of hydrophobic transmembrane proteins is not widely successful using standard immunoprecipitation or coimmunoprecipitation techniques. In these scenarios, the lysis conditions that maintain protein-protein interactions are not compatible with solubilizing hydrophobic membrane proteins. In this chapter, we outline two proximity labeling systems to circumvent these issues to study (1) eukaryotic proteins that localize to the membrane-bound inclusion formed by Chlamydia trachomatis using BioID, and (2) chlamydial proteins that are inserted into the inclusion membrane using APEX2. BioID is a promiscuous biotin ligase to tag proximal proteins with biotin. APEX2 is an ascorbate peroxidase that creates biotin-phenoxyl radicals to label proximal proteins with biotin or 3,3'-diaminobenzidine intermediates for examination of APEX2 labeling of subcellular structures using transmission electron microscopy. We present how these methods were originally conceptualized and developed, so that the user can understand the strengths and limitations of each proximity labeling system. We discuss important considerations regarding experimental design, which include careful consideration of background conditions and statistical analysis of mass spectrometry results. When applied in the appropriate context with adequate controls, these methods can be powerful tools toward understanding membrane interfaces between intracellular pathogens and their hosts.

The molecular mechanism of induction of unfolded protein response by Chlamydia

The unfolded protein response (UPR) contributes to chlamydial pathogenesis, as a source of lipids and ATP during replication, and for establishing the initial anti-apoptotic state of host cell that ensures successful inclusion development. The molecular mechanism(s) of UPR induction by Chlamydia is unknown. Chlamydia use type III secretion system (T3SS) effector proteins (e.g, the Translocated Actin-Recruiting Phosphoprotein (Tarp) to stimulate host cell's cytoskeletal reorganization that facilitates invasion and inclusion development. We investigated the hypothesis that T3SS effector-mediated assembly of myosin-II complex produces activated non-muscle myosin heavy chain II (NMMHC-II), which then binds the UPR master regulator (BiP) and/or transducers to induce UPR. Our results revealed the interaction of the chlamydial effector proteins (CT228 and Tarp) with components of the myosin II complex and UPR regulator and transducer during infection. These interactions caused the activation and binding of NMMHC-II to BiP and IRE1α leading to UPR induction. In addition, specific inhibitors of myosin light chain kinase, Tarp oligomerization and myosin ATPase significantly reduced UPR activation and Chlamydia replication. Thus, Chlamydia induce UPR through T3SS effector-mediated activation of NMMHC-II components of the myosin complex to facilitate infectivity. The finding provides greater insights into chlamydial pathogenesis with the potential to identify therapeutic targets and formulations.

Spectroscopic analysis of chlamydial major outer membrane protein in support of structure elucidation.

Chlamydial major outer membrane protein (MOMP) is the major protein constituent of the bacterial pathogen Chlamydia trachomatis. Chlamydia trachomatis Serovars D–K are the leading cause of genital tract infections which can lead to infertility or ectopic pregnancies. A vaccine against Chlamydia is highly desirable but currently not available. MOMP accounts for ~ 60% of the chlamydial protein mass and is considered to be one of the lead vaccine candidates against C. trachomatis. We report on the spectroscopic analysis of C. trachomatis native MOMP Serovars D, E, F, and J as well as C. muridarum MOMP by size exclusion chromatography multi angle light scattering (SEC MALS), circular dichroism (CD) and attenuated total reflectance Fourier transform infrared spectroscopy (ATR‐FTIR). MOMP was purified from the native bacterium grown in either adherent HeLa cells or in different suspension cell lines. Our results confirm that MOMP forms homo‐trimers in detergent micelles. The secondary structure composition of C. trachomatis MOMP was conserved across serovars, but different from composition of C. muridarum MOMP with a 13% (CD) to 18% (ATR‐FTIR) reduction in β‐sheet conformation for C. trachomatis MOMP. When Serovar E MOMP was isolated from suspension cell lines the α‐helix content increased by 7% (CD) to 13% (ATIR‐FTIR). Maintenance of a native‐like tertiary and quaternary structure in subunit vaccines is important for the generation of protective antibodies. This biophysical characterization of MOMP presented here serves, in the absence of functional assays, as a method for monitoring the structural integrity of MOMP.

Pivotal Pathogenic and Biomarker Role of Chlamydia Pneumoniae in Neurovascular Diseases.

Chlamydia Pneumoniae (C. Pn) is an obligatory intracellular bacterium that is associated with respiratory tract infections like pneumonia, pharyngitis and bronchitis. It has also been implicated in cerebrovascular (stroke) as well as cardiovascular diseases. The most possible pathway via which C. Pn elicits its pathogenesis could be via activation of Human Vascular Smooth Muscle Cells (VSMCs) proliferation resulting in the stimulation of Toll-Like Receptor-4 (TLR-4) and/or phospho-44/42(p44/p42) Mitogen-Activated Protein Kinases (MAPK). It is also established that tyrosine phosphorylation of IQ domain GTPase-activating protein 1 (IQGAP1) also contributed to C. Pn infection-triggered Vascular Endothelial Cell (VEC) movements via the SRC tyrosine kinase inhibitor PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) resulting in angiogenesis. It is also proven that restricted inflammatory cell infiltrates as well as apoptosis have been linked to C. Pn or C. Pn-specific proteins in atherosclerotic plaques of patients with stroke. It is further an evidence that C. Pn enters the cerebral vasculature during the initial infection and worsen atherosclerosis either directly or indirectly. Chronic, persistent C. Pn infection is also capable of triggering the secretion of Chlamydial Heat Shock Protein 60 (cHSP60) in the vessel wall resulting in augmentation of inflammation. C. Pn also aids in the activation of explicit cell-intermediated immunity within plaques. Macrophages in the carotid plaques co-exist with CD4+ lymphocytes which are capable of triggering the release of pro-inflammatory cytokines resulting in the augmentation of atherogenic development during C. Pn infection. C. Pn actively participated in the modification of both histones H3 and H4 during chromatin analysis via the interleukin 8(IL-8) gene facilitator as well as conscription of nuclear factor kappa-B(NF-κB) or NF- κB/p65 complex and polymerase II (Pol II). This review, therefore, focuses on the crucial involvement of C. Pn in the pathogenesis of cerebrovascular events.

The Chlamydia M278 Major Outer Membrane Peptide Encapsulated in the Poly(lactic acid)-Poly(ethylene glycol) Nanoparticulate Self-Adjuvanting Delivery System Protects Mice Against a Chlamydia muridarum Genital Tract Challenge by Stimulating Robust Systemic and Local Mucosal Immune Responses.

Recently, we reported that our PPM chlamydial nanovaccine [a biodegradable co-polymeric PLA-PEG (poly(lactic acid)-poly(ethylene glycol))-encapsulated M278 peptide (derived from the major outer membrane protein (MOMP) of Chlamydia)] exploits the caveolin-mediated endocytosis pathway for endosomal processing and MHC class II presentation to immune-potentiate Chlamydia-specific CD4+ T-cell immune effector responses. In the present study, we employed the Chlamydia muridarum mouse infection model to evaluate the protective efficacy of PPM against a genital tract challenge. Our results show that mice immunized with PPM were significantly protected against a homologous genital tract challenge evidently by reduced vaginal bacterial loads. Protection of mice correlated with enhanced Chlamydia-specific adaptive immune responses predominated by IFN-γ along with CD4+ T-cells proliferation and their differentiation to CD4+ memory (CD44high CD62Lhigh) and effector (CD44high CD62Llow) T-cell phenotypes. We observed the elevation of M278- and MOMP-specific serum antibodies with high avidity in the ascending order IgG1 > IgG2b > IgG2a. A key finding was the elevated mucosal IgG1 and IgA antibody titers followed by an increase in MOMP-specific IgA after the challenge. The Th1/Th2 antibody titer ratios (IgG2a/IgG1 and IgG2b/IgG1) revealed that PPM evoked a Th2-directed response, which skewed to a Th1-dominated antibody response after the bacterial challenge of mice. In addition, PPM immune sera neutralized the infectivity of C. muridarum in McCoy cells, suggesting the triggering of functional neutralizing antibodies. Herein, we reveal for the first time that subcutaneous immunization with the self-adjuvanting biodegradable co-polymeric PPM nanovaccine immune-potentiated robust CD4+ T cell-mediated immune effector responses; a mixed Th1 and Th2 antibody response and local mucosal IgA to protect mice against a chlamydial genital tract challenge.

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.

Predicting tubal factor infertility by using markers of humoral and cell‐mediated immune response against Chlamydia trachomatis

The accuracy of Chlamydia trachomatis antibody test in predicting tubal factor infertility (TFI) is limited, and more accurate methods are needed. Cell-mediated immune response (CMI) is crucial in the resolution of pathogen, but it may play an important role in the pathogenesis of C trachomatis-associated tubal damage. We studied whether combining the markers of C trachomatis-induced CMI to humoral immune response improves the accuracy of serology in TFI prediction. Our prospective study consists of 258 subfertile women, of whom 22 (8.5%) had TFI. Women with other causes for subfertility served as a reference group. Serum C trachomatis major outer membrane protein (MOMP) and chlamydial heat-shock protein 60 (cHSP60) IgG antibodies were measured by ELISA. CMI was studied by lymphocyte proliferation assay in vitro. Serological markers were more prevalent in women with TFI than in other subfertile women (40.9% vs 12.3% for MOMP IgG and 27.3% vs 10.2% for cHSP60 IgG). The best test combination for TFI was C.trachomatis MOMP and cHSP60 antibody with an accuracy of 90.3%, sensitivity of 22.7% and specificity of 96.6%. Positive post-test probability of this combination was 54.2%, and negative post-test probability was 12.4%. Adding of the markers of CMI did not significantly improve the accuracy of serology in TFI prediction. The accuracy of TFI prediction increases when the combination of C trachomatis MOMP and cHSP60 antibody tests is used. C trachomatis-induced CMI was common in our study population, but the markers of CMI did not predict TFI.

The Role of Chlamydial Colonization of the Gastrointestinal Tract in the Development and Persistence of Chronic Chlamydial Infections

Diseases caused by C. trachomatis have an adverse impact on male and female health and the reproductive function and are among the leading causes of infertility. Even though C. trachomatis is a sexually transmitted pathogen adapted to the epithelial cells of the urogenital tract (UGT), it has a fairly wide tropism and can infect blood cells, conjunctival epithelium, synovial cells, hepatocytes, and enteroendocrine cells. The pathogen is detected in various divisions of the gastrointestinal tract (GIT) in men and women. Studies of the infection in mice showed that chlamydia could spread in the body with lymph or blood flow, colonize various digestive organs, and persist in these organs for a long time without causing a significant pathology, but still inducing chronic pathological inflammation in the reproductive organs. Studies in the C. muridarum model demonstrated the role of the conservative chlamydial protein Pgp3 (encoded by the plasmid sequence) in colonization of the digestive tract. There is accumulating experimental and clinical evidence characterizing the intestine as a favorable niche for the persistence of chlamydia, since the intracellular pathogen сan avoid the competitive impact of the intestinal microflora, and immune homeostasis on the intestinal mucosa weakens the protective action of the immune response. Generalization of the urogenital chlamydial infection and colonization of the digestive tract by chlamydia are probably the main causes for the emergence of a chronic infection and the development of pathology in the reproductive sphere and other organs. If the role of this mechanism in the emergence of a chronic infection is proven, new approaches for the diagnosis and treatment of chlamydiosis will become necessary. The present review addresses these new data.

Capsid protein Vp1 from chlamydiaphage φCPG1 effectively alleviates cytotoxicity induced by Chlamydia trachomatis.

Chlamydia trachomatis is the leading cause of sexually transmitted bacterial infections. C. trachomatis genital infection may lead to pelvic inflammatory disease, ectopic pregnancy and tubal infertility, which are major public health problems. However, the pathogenic mechanisms of this bacterium remain unclear, and the efficacy of clinical therapeutics is unsatisfactory. In the current study, whether Vp1 can alleviate the cytotoxicity induced by Chlamydia trachomatis infection was investigated. C. trachomatis was pre-treated with BSA or purified Vp1 protein and used to infect HeLa cells. It was observed that Vp1 significantly inhibited the infectivity of C. trachomatis in cell cultures. In addition, the Vp1 pretreatment reduced the chlamydial Hsp60 protein levels and decreased the C. trachomatis inclusion number. The Vp1 pretreatment also prevented C. trachomatis-induced cytotoxicity in host cells. Furthermore, the chlamydial suppression of host cell proapoptotic p53 protein and the induction of antiapoptotic cIAP-2 and Mcl-1 gene expression were reversed by the Vp1 pretreatment. These observations suggest that Vp1 has a clear inhibitory effect on C. trachomatis growth in vitro.

The Human Centrosomal Protein CCDC146 Binds Chlamydia trachomatis Inclusion Membrane Protein CT288 and Is Recruited to the Periphery of the Chlamydia-Containing Vacuole.

Chlamydia trachomatis is an obligate intracellular human pathogen causing mainly ocular and genital infections of significant clinical and public health impact. C. trachomatis multiplies intracellularly in a membrane bound vacuole, known as inclusion. Both extracellularly and from within the inclusion, C. trachomatis uses a type III secretion system to deliver several effector proteins into the cytoplasm of host cells. A large proportion of these effectors, the inclusion membrane (Inc) proteins, are exposed to the host cell cytosol but possess a characteristic hydrophobic domain mediating their insertion in the inclusion membrane. By yeast two-hybrid, we found that C. trachomatis Inc CT288 interacts with the human centrosomal protein CCDC146 (coiled-coil domain-containing protein 146). The interaction was also detected by co-immunoprecipitation in mammalian cells either ectopically expressing CCDC146 and CT288 or ectopically expressing CCDC146 and infected by a C. trachomatis strain expressing epitope-tagged and inclusion membrane-localized CT288. In uninfected mammalian cells, ectopically expressed full-length CCDC146 (955 amino acid residues) localized at the centrosome; but in cells infected by wild-type C. trachomatis, its centrosomal localization was less evident and CCDC146 accumulated around the inclusion. Recruitment of CCDC146 to the inclusion periphery did not require intact host Golgi, microtubules or microfilaments, but was dependent on chlamydial protein synthesis. Full-length CCDC146 also accumulated at the periphery of the inclusion in cells infected by a C. trachomatis ct288 mutant; however, a C-terminal fragment of CCDC146 (residues 692–955), which interacts with CT288, showed differences in localization at the periphery of the inclusion in cells infected by wild-type or ct288 mutant C. trachomatis. This suggests a model in which chlamydial proteins other than CT288 recruit CCDC146 to the periphery of the inclusion, where the CT288-CCDC146 interaction might contribute to modulate the function of this host protein.

Chlamydial Plasmid-Encoded Protein pGP3 Inhibits Development of Psoriasis-Like Lesions in Mice.

BackgroundThe anti-microbial protein cathelicidin LL-37 plays an important role in the pathogenesis of psoriasis by inducing inflammation. Our previous study showed that the chlamydial plasmid-encoded protein pGP3 forms a stable complex with LL-37 to neutralize its pro-inflammatory activity. Here, we explored whether pGP3 can inhibit the development of lesions in mice with imiquimod-induced psoriasis.Material/MethodsThe protein pGP3 was expressed in bacteria and purified using glutathione-conjugated agarose beads and a precision protease. The ability of the purified pGP3 to block chemotaxis mediated by LL-37 was tested in vitro using bone marrow-derived neutrophils. The ability of the protein to inhibit the development of psoriasis-like lesions was tested by topically or subcutaneously administering pGP3 in doses of 10 or 50 μg to mice previously treated with imiquimod. Mouse skin was evaluated using the psoriasis area and severity index (PASI) score and photography. Skin biopsies were taken on day 8 and analyzed histologically.ResultsPurified pGP3 inhibited LL-37-mediated chemotaxis. Mice treated with 50 μg pGP3 showed clinical improvement with less severe erythema, infiltration, and scales; these mice also showed thinner dermis and less hyperkeratosis, parakeratosis, and inflammatory cell infiltration than mice treated with without 10 μg pGP3.ConclusionsPGP3 can inhibit the development of psoriasis-like lesions in mice, possibly through its ability to bind LL-37. Future work should examine the mechanisms underlying this therapeutic effect.

Chlamydia protein Pgp3 studied at high resolution in a new crystal form.

The protein Pgp3 is implicated in the sexually transmitted disease chlamydia and comprises an extended complex arrangement of a C-terminal domain (CTD) and an N-terminal domain (NTD) linked by a triple-helix coiled coil (THCC). Here, the X-ray crystal structure of Pgp3 from an LGV1 strain is reported at the highest X-ray diffraction resolution obtained to date for the full protein. The protein was crystallized using a high concentration of potassium bromide, which resulted in a new crystal form with relatively low solvent content that diffracted to a resolution of 1.98 Å. The three-dimensional structure of this new crystal form is described and compared with those of other crystal forms, and the potassium bromide binding sites and the relevance to chlamydia isolates from around the globe are described. The crystal packing is apparently driven by the CTDs. Since the threefold axes of the THCC and NTD are not collinear with the threefold axis of a CTD, this naturally leads to disorder in the THCC and the portion of the NTD that does not directly interact with the CTD via crystal packing. The key avenue to resolving these oddities in the crystal structure analysis was a complete new analysis in space group P1 and determining the space group as P212121. This space-group assignment was that originally determined from the diffraction pattern but was perhaps complicated by translational noncrystallographic symmetry. This crystal structure of a three-domain multi-macromolecular complex with two misaligned threefold axes was a unique challenge and has not been encountered before. It is suggested that a specific intermolecular interaction, possibly of functional significance in receptor binding in chlamydia, might allow the design of a new chemotherapeutic agent against chlamydia.

The Effect of Exogenously added and Endogenously Produced Cytokines on the Regulation of Inflammatory Mediators and SOCS Proteins in Mouse Macrophages Exposed to Chlamydia muridarum Major Outer Membrane Protein

Abstract The major outer membrane protein (MOMP) of Chlamydia is a candidate for vaccine developmental efforts due to its high immunogenic potential. We previously reported that IL-10 can regulate Chlamydia inflammation by inhibiting its associated-induced inflammatory mediators possibly via induction of the Suppressor of Cytokine Signaling (SOCS) proteins. In this study, we determined the effect of exogenously added and endogenously produced cytokines on the regulation of inflammatory mediators and SOCS proteins as induced in mouse J774 macrophages exposed to C. muridarum (Cm) recombinant MOMP. Macrophages were stimulated with rMOMP (10 ug/mL) in the presence and absence of exogenous IL-10, TNF and IL-6 (each at 10 ng/mL). Cytokine and chemokine specific ELISAs revealed that exogenously added IL-6 and TNF did not alter the levels of inflammatory mediators (CCL5, CXCL10, IL-12) induced by rMOMP; whereas IL-10 downregulated all mediators. Neutralization of endogenously produced IL-6 and TNF, respectively by anti-IL-6 or anti-TNF antibodies (each at 25 μg/mL) did not significantly perturb the expression of rMOMP-induced inflammatory mediators. On the contrary, neutralization of IL-10 by an anti-IL-10 antibody increased the production of all mediators, thus confirming that IL-10 regulates Cm-induced inflammation. Removal of endogenously produced IL-10 increased SOCS1 expression, which suggest an important role of IL-10 in the regulation of SOCS1 expression to potentially control Cm-induced inflammatory responses. Collectively, our data sheds more light on the potential roles of these cytokines implicated during chlamydial infections, but more importantly, how IL-10 regulates Chlamydia inflammation via SOCS proteins.

Urine metabolome in women with Chlamydia trachomatis infection.

The aim of this study was to characterize the urine metabolome of women with Chlamydia trachomatis (CT) uro-genital infection (n = 21), comparing it with a group of CT-negative subjects (n = 98). By means of a proton-based nuclear magnetic resonance (1H-NMR) spectroscopy, we detected and quantified the urine metabolites of a cohort of 119 pre-menopausal Caucasian women, attending a STI Outpatients Clinic in Italy. In case of a CT positive result, CT molecular genotyping was performed by omp1 gene semi-nested PCR followed by RFLP analysis. We were able to identify several metabolites whose concentrations were significantly higher in the urine samples of CT-positive subjects, including sucrose, mannitol, pyruvate and lactate. In contrast, higher urinary levels of acetone represented the main feature of CT-negative women.These results were not influenced by the age of patients nor by the CT serovars (D, E, F, G, K) responsible of the urethral infections. Since the presence of sugars can increase the stability of chlamydial proteins, higher levels of sucrose and mannitol in the urethral lumen, related to a higher sugar consumption, could have favoured CT infection acquisition or could have been of aid for the bacterial viability. Peculiar dietary habits of the subjects enrolled, in term of type and amount of food consumed, could probably explain these findings. Lactate and pyruvate could result from CT-induced immunopathology, as a product of the inflammatory microenvironment. Further studies are needed to understand the potential role of these metabolites in the pathogenesis of CT infection, as well as their diagnostic/prognostic use.