Chlamydia trachomatis induces low-frequency, sustained CD4 T cell responses in most women, predominantly targeting chlamydial protease-like activity factor, CPAF.

BACKGROUND: Chlamydia trachomatis (CT) is a globally prevalent sexually transmitted infection (STI) that can result in pelvic inflammatory disease, ectopic pregnancy and infertility in women. Currently, there is no prophylactic vaccine. METHODS: This study examined T cell immunity in a cohort of women recently infected with CT. Participants were screened against peptides spanning 33 of 894 possible CT proteins, either ex vivo or using short-term cell lines (STCL). CT-specific T cells were characterized by IFN-γ ELISpot and flow cytometry. RESULTS: Ex vivo CT-specific T cells were rarely detected; however, following in vitro expanded CT-specific T cells were detected by IFN-γ ELISpot in 90% (27/30) of participants. Notably, over 50% of participants had T cell responses targeting chlamydial protease-like activity factor (CPAF). T cell epitopes were dispersed across the CPAF protein. Flow cytometry analysis of STCL found CT-specific cells, were mainly CD4+, produced IFN-γ and TNF-α and were sustained over 12 months. Ex vivo analysis suggested CT-specific T cells mostly exhibited a central memory phenotype. CONCLUSION: Our results indicate that CT infection elicits low-frequency, persistent CD4 T cell responses in most women and that the secreted protein, CPAF, is an immunoprevalent CT antigen. Altogether, these data support development and testing of CT vaccines that enhance CD4 T cells against CPAF.

Coexistence of Ehlers-Danlos Syndrome with Coronary-Pulmonary Arterial Fistula and Other Multiple Coronary Artery Anomalies.

This case report presents a 34-year-old male with Ehlers-Danlos syndrome, type 2 diabetes mellitus, aortic valve regurgitation, and aortic bulb aneurysm. Following spine surgery for thoracic-lumbar stabilization, the patient underwent assessment for aortic bulb aneurysm and aortic valve replacement surgeries. Five months post spinal surgery, a coronary computed tomography angiography was performed. The coronary computed tomography angiography revealed unique findings, including the absence of the left main coronary artery, right coronary artery dominance, ectopic origin of the left circumflex artery from the right sinus of the valsalva, a coronary-pulmonary arterial fistula originating from the right sinus of the valsalva, and an additional right pulmonary vein. The patient was qualified for surgical treatment for an aortic bulb aneurysm, was informed about the high surgical risk, and is awaiting surgery. This case underscores the rarity of Ehlers-Danlos syndrome coexisting with multiple coronary artery anomalies. The presence of a coronary-pulmonary arterial fistula further emphasizes the need for specialized patient monitoring when Ehlers-Danlos syndrome and coronary anomalies converge.

Successful Surgical Treatment for Ruptured Aneurysm of Coronary-Pulmonary Artery Fistula Complicated With Cardiac Tamponade.

A 74-year-old woman with no past medical history showed cardiac tamponade caused by rupture of a coronary-pulmonary artery fistula-related aneurysm. Preoperative pericardial puncture and multidetector computed tomography imaging enabled patient condition optimization and accurate morphologic evaluation of fistula and aneurysm, leading to complete surgical resection of the aneurysm. (Level of Difficulty: Advanced.).

Case report: Conquer a complex variant: Coronary-pulmonary artery fistulas, atrial septal defect and bicuspid pulmonary valve, under beating heart surgery.

Coronary artery to pulmonary artery fistula (CPAF) is a congenital or acquired abnormal channel between arteries, with a left-to-right cardiac shunting, which may lead to myocardial ischemia, arrhythmia, thrombotic complications, and heart failure. CPAF is usually detected by coronary angiography but few reports have used beating-heart surgery as a detection method. The patient in this case report is a 39-year-old male diagnosed with atrial septal defect (ASD), bicuspid pulmonary valve, and moderate tricuspid regurgitation (TR). He is asymptomatic. In preoperative evaluation, significant CPAF was suspected using echocardiography. The patient refused coronary angiography due to allergic history. Therefore, the cardiac team designed and performed on-pump beating-heart surgery (OPBHS) to detect and repair these disorders, and suggested OPBHS as a myocardial protection strategy for the patient at low surgical risk. A rare and complex cardiovascular case with CPAFs from two branches of the left anterior descending coronary (LAD) artery to the main pulmonary artery (MPA) with ASD, bicuspid pulmonary valve, and moderate TR has not yet been reported in the literature, and its embryological hypothesis has been further analyzed in this report.

Bioprospecting by Phage Display of Mimetic Peptides of Chlamydia trachomatis for Use in Laboratory Diagnosis.

Background: Chlamydia trachomatis infection is a major public health problem and the most common sexually transmitted infection in the world. Although highly prevalent, 70% to 80% of cases are asymptomatic and undiagnosed. Purpose: To overcome some limitations in terms of rapid diagnosis, phage display technology was used to bioprospect peptide mimetics of C. trachomatis immunoreactive and immunogenic antigens to be selected for the production of synthetic peptides. Methods: Initially, IgG from 22 individuals with C. trachomatis and 30 negative controls was coupled to G protein magnetic beads. The phage display technique consisted of biopanning, genetic sequencing, bioinformatics analysis and phage ELISA. Results: Clones G1, H5, C6 and H7 were selected for testing with individual samples positive and negative for C. trachomatis. Reactions were statistically significant (p < 0.05), with a sensitivity of 90.91, a specificity of 54.55, and AUC values >0.8. One-dimensional analysis with C. trachomatis components indicated that the G1 clone aligned with cell wall-associated hydrolase domain-containing protein, the H5 clone aligned with glycerol-3-phosphate acyltransferase PlsX protein, the C6 clone aligned with a transposase and inactivated derivatives, and the H7 clone aligned with GTP-binding protein. Molecular modeling and three-dimensional analysis indicated the best fit of the four clones with a protein known as chlamydial protease/proteasome-like activity factor (CPAF), an important virulence factor of the bacterium. Conclusion: The peptides produced by phage display are related to the metabolic pathways of C. trachomatis, indicating that they can be used to understand the pathogenesis of the infection. Because of their high sensitivity and AUC values, the peptides present considerable potential for use in platforms for screening C. trachomatis infections.

Transcatheter Occlusion of Coronary-Pulmonary Fistula With a Liquid Embolic Agent After Evaluation by FFR.

A 58-year-old man was admitted for stable angina. The coronary angiogram revealed a coronary-pulmonary fistula with a nonsignificant atheroma. We decided to perform percutaneous embolization of the fistula in view of the symptoms and the hemodynamic assessment findings. Embolization was performed using a liquid embolic agent with no residual flow. (Level of Difficulty: Intermediate.).

Coronary artery-to-pulmonary artery fistula in adults: evaluation with 320-row detector computed tomography coronary angiography.

BACKGROUND: To analyze the imaging features of coronary artery-to-pulmonary artery fistula (CPAF) on coronary computed tomography angiography (CCTA). METHODS: This was a retrospective study of 3,975 patients who underwent 320 row detector CCTA examinations in our hospital from May 2015 to July 2020. A total of 22 patients who diagnosed with CPAF were reviewed for CCTA imaging characteristics, including the origin, number, blood volume, opening size, and course of fistula vessels, and the drainage site, size, and imaging features of the fistula. All cases were analyzed for the presence of coronary atherosclerotic plaque and that of deficient left ventricular myocardial perfusion. RESULTS: A total of 22 CPAF cases detected by CCTA were collected (men, 11; women, 11; median age, 59.6±10.1 years). There were 7, 10, and 5 cases detected with 1, 2, and 3 fistula vessels, respectively, among which 4 originated from the left coronary artery, 4 from the right coronary artery, and 14 had bilateral origins. There were 10 cases in which the fistula vessels presented as a worm-like tortuous dilation with (n=5) or without (n=5) aneurysm, while 12 cases showed malformed vascular networks with (n=8) or without (n=4) aneurysm, respectively. The calculated incidence of aneurysm formation was 59.09%, and fistula vessels with an aneurysm had larger blood volume than those without. All fistula showed a single drainage site, with an average diameter of 2.81±1.48 mm where the diameter of fistula with aneurysm was larger than that without. The fistula vessels drained into the left anterolateral and anterior walls of main pulmonary artery and the proximal left inferior PA, respectively. Typical jet sign, smoke sign, and isodensity sign were presented in 22, 14 and 1 case, respectively. For the coexistent abnormalities analyzed in 22 cases, 17 participants with CPAF demonstrated hypoperfusion of the fistula vessels, and 11 demonstrated calcified plaque accompanied with luminal stenosis to different degrees. CONCLUSIONS: The 320-row detector CCTA can comprehensively characterize the morphological features of CPAF, which is an optimal choice for physicians to make an accurate assessment before formulating patient management strategies.

The role of an enzymatically inactive CPAF mutant vaccination in Chlamydia muridarum genital tract infection.

Chlamydia trachomatis urogenital tract infection causes pelvic inflammatory disease and infertility, increases the risk of co-infection with HPV and HIV. Chlamydial vaccination is considered the most promising approach to prevent and control its infection. Among various chlamydial vaccine candidates, chlamydial protease-like activity factor (CPAF) have been reported to provide robust protective immunity against genital chlamydial infection in mice with reduced vaginal shedding and oviduct pathology. However, CPAF is a serine protease which has enzymatical activity to degrade a large number of substrates. In order to increase the safety of CPAF vaccine, in this study, we used a mutant CPAF that is deficient in enzymatical activity to determine whether proteolytic activity of CPAF affect its vaccine efficacy. The wild type or mutant CPAF immunization causes a significant lower chlamydial shedding from the vaginal and resolve the infection as early as day 20, compared to day 28 in adjuvant control mice. More important, reduced upper reproductive tract pathology were also observed in these two groups. The mutant or wild type CPAF immunization induced not only robust splenic IFN-γ and serum IgG2a but also sIgA secretion in the vaginal fluids. Furthermore, neutralization of chlamydia with immune sera did not provide protection against oviduct pathology. However, adoptive transfer of CD4+ splenocytes isolated from the mutant or wild type CPAF immunized mice resulted in a significant and comparable reduced oviduct pathology. Our results indicate mutant CPAF vaccination is as same efficacy as wild type, and the protection relies on CD4+ T cells, which will further promote the development of CPAF as clinical chlamydial vaccine.

A Novel Cleavage Pattern of Complement C5 Induced by Chlamydia trachomatis Infection via the Chlamydial Protease CPAF.

Urogenital Chlamydia trachomatis infection is one of the most common bacterial sexually transmitted diseases globally. Untreated C. trachomatis infections can ascend to the upper genital tract and establish a series of severe complications. Previous studies using C3-/- and C5-/- mice models demonstrated that C3-independent activation of C5 occurred during C. trachomatis infection. However, the mechanism of how chlamydial infection activates C5 in the absence of C3 has yet to be elucidated. To delineate interactions between C5 and chlamydial infection, cleavage products in a co-incubation system containing purified human C5 and C. trachomatis-HeLa229 cell lysates were analyzed, and a novel cleavage pattern of C5 activation induced by C. trachomatis infection was identified. C5 was cleaved efficiently at the previously unidentified site K970, but was cleaved poorly at site R751. C5b was modified to C5bCt, which later formed C5bCt-9, which had enhanced lytic ability compared with C5b-9. The chlamydial serine protease CPAF contributed to C3-independent C5 activation during C. trachomatis infection. Nafamostat mesylate, a serine protease inhibitor with a good safety profile, had a strong inhibitory effect on C5 activation induced by chlamydial infection. These discoveries reveal the mechanism of C3-independent C5 activation induced by chlamydial infection, and furthermore provide a potential therapeutic target and drug for preventing tubal fibrosis caused by chlamydial infection.

Evaluation of protective and immune responses following vaccination with recombinant MIP and CPAF from Chlamydia abortus as novel vaccines for enzootic abortion of ewes.

MIP and CPAF from Chlamydia have been shown to be effective in inducing immune responses important in clearing chlamydial infections. This study evaluates the protection conferred by MIP and CPAF as novel vaccines in pregnant C. abortus challenged ewes. Fifty C. abortus sero-negative sheep were randomly allocated into 5 groups of 10 according to the treatment they were to receive (1) 100 µg of MBP-MIP (2) 100 µg CPAF (3) 50 µg MBP-MIP and 50 µg CPAF (4) Tris-buffer (negative control) (5) Enzovax (positive control). Booster inoculations were administered 3 weeks after primary inoculations. Blood samples were taken pre-vaccination and weekly for 5 weeks. Five months after vaccination the ewes were mated. Pregnant ewes were then challenged on day 90 of gestation. Blood samples taken at four time-points post challenge were analysed for IFNγ levels, TNFα and IL-10 expression and anti-chlamydial antibody levels. Vaginal swabs, placental and foetal tissue and bacterial shedding were analysed using qPCR to quantify levels of C. abortus. Enzovax was 100% effective with no abortions occurring. The MIP/CPAF combined vaccine offered the greatest protection of the novel vaccines with 67% of ewes giving birth to one or more live lambs equating to a 50% vaccine efficacy rate. MIP and CPAF administered singly did not confer protection. Enzovax and MIP/CPAF vaccinated ewes had longer gestations and lambs with higher birth weights than negative control ewes. Aborting ewes shed higher numbers of C. abortus than ewes that had live lambs, all vaccinated ewes demonstrated lower levels of bacterial shedding than negative control ewes with Enzovax ewes shedding significantly fewer bacteria. Ewes that went on to abort had significantly higher levels of IFNγ and IL-10 at day 35 post challenge and significantly higher levels of anti-chlamydial antibodies at 24 h post lambing compared to ewes that had live lambs.

Exercise-induced ventricular arrhythmia due to bilateral coronary to pulmonary artery fistulas.

Ischemic polymorphic ventricular ectopy was documented during exercise testing in a 65-year-old Caucasian male patient. Coronary angiogram revealed four coronary to pulmonary artery fistulas (CPAFs) originating from the right and left coronary artery, leading to myocardial ischemia due to steal phenomenon. The three dominant fistulas were coiled percutaneously, while one small fistula was left untreated. During follow-up, no significant residual ventricular arrhythmia was detected.

CPAF, HSP60 and MOMP antigens elicit pro-inflammatory cytokines production in the peripheral blood mononuclear cells from genital Chlamydia trachomatis-infected patients.

BACKGROUND: Persistent inflammation caused by Chlamydia trachomatis in the female genital compartment represents one of the major causes of pelvic inflammatory disease (PID), ectopic pregnancy and infertility in females. Here, we examined the pro-inflammatory cytokine response following stimulation with three different types of C. trachomatis antigens, viz. chlamydial protease-like factor (CPAF), heat shock protein 60 (HSP60) and major outer membrane protein (MOMP). METHODS: A total of 19 patients with genital C. trachomatis infection and 10 age-matched healthy controls were recruited for the study. Peripheral blood mononuclear cells (PBMCs) isolated from genital C. trachomatis-infected females were cultured in the presence of CPAF, HSP60 and MOMP antigens, and cytokines were measured by ELISA assay. RESULTS: We reported that pro-inflammatory cytokines (TNF-α, IL-1ß and IL-6) were robustly secreted following antigenic exposure. Notably, CPAP and MOMP were more potent in triggering IL-1ß, as compared to HSP60. Elevated levels of the proinflammatory cytokines were also noted in the samples infected with plasmid-bearing C. trachomatis as compared to those infected with plasmid-free strains. CONCLUSIONS: Our study highlights distinct ability of chlamydial antigens in triggering pro-inflammatory response in the host immune cells.

Peptidase Inhibitor 15 (PI15) Regulates Chlamydial CPAF Activity.

Obligate intracellular pathogenic Chlamydia trachomatis express several serine proteases whose roles in chlamydial development and pathogenicity are not completely understood. The chlamydial protease CPAF is expressed during the replicative phase of the chlamydial developmental cycle and is secreted into the lumen of the Chlamydia-containing vacuole called inclusion. How the secreted protease is activated in the inclusion lumen is currently not fully understood. We have identified human serine peptidase inhibitor PI15 as a potential host factor involved in the regulation of CPAF activation. Silencing expression as well as over expression of PI15 affected normal development of Chlamydia. PI15 was transported into the chlamydial inclusion lumen where it co-localized with CPAF aggregates. We show that PI15 binds to the CPAF zymogen and potentially induces CPAF protease activity at low concentrations. However, at high concentrations PI15 inhibits CPAF activity possibly by blocking its protease domain. Our findings shed light on a new aspect of chlamydial host co-evolution which involves the recruitment of host cell proteins into the inclusion to control the activation of bacterial proteases like CPAF that are important for the normal development of Chlamydia.

CPAF selectively degrades chlamydial T cell antigens for inhibiting antigen presentation.

INTRODUCTION: Chlamydia trachomatis is the leading cause of sexually transmitted bacterial disease, which may cause significant threats, such as pelvic inflammatory disease and tubal factor infertility, to women if untreated. The pathological mechanisms of chlamydia-induced disease remain largely unknown, but it has been proposed that CPAF, a chlamydia-secreted serine protease, may play major roles in aiding chlamydial infection and contribute to chlamydia pathogenesis during in vivo infection. According to previous results, CPAF targets host immunity by degrading antimicrobial peptides and neutralizing complement activity; however, whether CPAF is involved in chlamydial antigen presentation has never been reported. METHODOLOGY: Antigen presentation assay was used to monitor the effects of CPAF on OT1-, OT2-, and chlamydia T cell antigen-mediated antigen presentation. In vitro cell-free degradation assay was used to detect CPAF processing of chlamydia T cell antigens. RESULTS: We found that CPAF preferably inhibits OT2- but not OT1-mediated antigen presentation. CPAF inhibits OT2 antigen presentation by direct proteolytic cleavage in the wild type CPAF, but not enzymatic mutants. Importantly, several previously identified chlamydial T cell antigens were selectively degraded by CPAF when co-incubated in vitro. In addition, specific inhibition T cell antigen presentation by CPAF was correlated with T cell antigen cleavage by CPAF in vitro assay. CONCLUSIONS: Our experiments demonstrated that CPAF selectively and specifically degrades chlamydial T cell antigens, which chlamydia may utilize as a novel mechanism for evading host immune responses to promote chlamydia survival.

Neutralizing antichlamydial activity of complement by chlamydia-secreted protease CPAF.

Ascending infection by sexually transmitted Chlamydia trachomatis is required for chlamydial induction of tubal pathology. To achieve ascension, the C. trachomatis organisms may have to spread from cell to cell, which inevitably exposes the organisms to extracellular mucosal effectors such as complement factors that are known to possess strong antichlamydial activities. Here, we report that the chlamydia-secreted protease CPAF efficiently neutralized complement factor C3-dependent antichlamydial activity. The neutralization was dependent on the proteolytic activity of CPAF and correlated with the CPAF-mediated degradation of complement factor C3 and factor B. As a result, CPAF preferentially inhibited the alternative complement activation pathway. The significance and limitation of these observations were discussed.

Regulación de la familia de proteínas BCLl-2 en células infectadas con Chlamydia Trachomatis / Regulation of the bcl-2 to family in cells infected with Chlamydia Trachomatis

El presente artículo revisa los mecanismos inhibitorios de la apoptosis usados por Chlamydia trachomatis frente a la familia de proteínas Bcl-2, para lograr su supervivencia intracelular. Chlamydia trachomatis es una bacteria intracelular obligada Gram negativa responsable de la infección de transmisión sexual más común en el mundo; éste microorganismo es capaz de inhibir la apoptosis de la célula huésped durante su ciclo de desarrollo, obteniendo un refugio seguro para su supervivencia. Frente a estímulos como la infección de la célula por un patógeno, la familia de proteínas Bcl-2 regula la apoptosis por medio de la liberación del citocromo c de la mitocondria para desencadenar la muerte celular programada (MCP). Ct usa diversos mecanismos de regulación antiapoptótica para sobrevivir dentro de la célula huésped; dentro de ellos se observa la secreción de proteínas tales como CPAF y CADD, la escisión de la proteína Bid, el secuestro de Bad y en general el bloqueo de proteínas pertenecientes a la familia Bcl-2 con dominio BH3, que afectan directamente la vía de mitocondrial ocasionando la persistencia, desarrollo y replicación de Ct en la célula huésped.

Chlamydia trachomatis (Ct) is a Gram-negative obligate intracellular bacteria responsible for the infection ofmost common sexually transmitted infection in the world; The organism is able to inhibit host cell apoptosis during development cycle, obtaining a safe survival. To stimuli such as infection of the cell by a pathogen, the Bcl-2 familyregulates cellapoptosisreleasing cytochrome c from mitochondria to trigger programmed cell death (PCD). Ct used various anti-apoptotic regulation mechanisms to survive within the host cell, such as:1. Proteins secretion of CPAF and CADD, 2. The cleavage of the protein Bid, 3.The Bad kidnapping and blockingproteins, belonging to the Bcl-2 family -(BH3 domain). All these mechanisms affect the cell mitochondrial pathway causingpersistence infection, development and Ct replicationwithin the host cell.The objective of this review is to show one of the anti-apoptotic mechanisms, which Ct can survive within the host cell, evading the immune systemresponse.

Chlamydia-secreted protease CPAF degrades host antimicrobial peptides.

Chlamydia trachomatis infection in the lower genital tract, if untreated, can ascend to the upper genital tract, potentially leading to complications such as tubal factor infertility. The ascension involves cell-to-cell spreading, which may require C. trachomatis organisms to overcome mucosal extracellular effectors such as antimicrobial peptides. We found that among the 8 antimicrobial peptides tested, the cathelicidin LL-37 that is produced by both urogenital epithelial cells and the recruited neutrophils possessed a most potent antichlamydial activity. Interestingly, this antichlamydial activity was completely inhibited by CPAF, a C. trachomatis-secreted serine protease. The inhibition was dependent on CPAF's proteolytic activity. CPAF selectively degraded LL-37 and other antimicrobial peptides with an antichlamydial activity. CPAF is known to secrete into and accumulate in the infected host cell cytoplasm at the late stage of chlamydial intracellular growth and may be released to confront the extracellular antimicrobial peptides before the intra-inclusion organisms are exposed to extracellular environments during host cell lysis and chlamydial spreading. Thus, the finding that CPAF selectively targets host antimicrobial peptides that possess antichlamydial activities for proteolysis suggests that CPAF may contribute to C. trachomatis pathogenicity by aiding in ascending infection.

The chlamydial protease CPAF: important or not, important for what?

The protease CPAF is only found in Chlamydiales and in at least most bacteria that share with Chlamydia the biphasic life-style in a cytosolic inclusion. CPAF is intriguing: it appears to be secreted from the inclusion across the inclusion membrane into the cytosol. A bacterial protease ravaging in the cytosol of a human cell may cause a plethora of effects. Curiously, very few are known. The current discussion is bogged down by a focus on experimental artifact, while proposed functions of CPAF remain speculative. I here make the attempt to summarize what we know about CPAF.