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Effect of chlamydial infection on caspase 3 processing, PARP cleavage, and DNA fragmentation. HeLa cells with (lanes 3 and 4) or without (lanes 1 and 2) chlamydial infection and with (lanes 2 and 4) or without (lanes 1 and 3) staurosporine (1 μM) treatment were lysed for Western blot analysis using antibodies against caspase 3 (top) or PARP (middle). The caspase 3 and PARP antibody staining was developed with a secondary antibody conjugated to horseradish peroxidase followed by visualization using an ECL as described in Materials and Methods. A 3% agarose gel was used for the DNA ladder assay and ethidium bromide was used to visualize the DNA bands (bottom).
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We report that chlamydiae, which are obligate intracellular bacterial pathogens, possess a novel antiapoptotic mechanism. Chlamydia-infected host cells are profoundly resistant to apoptosis induced by a wide spectrum of proapoptotic stimuli including the kinase inhibitor staurosporine, the DNA-damaging agent etoposide, and several immunological apo...
Citations
... By activating both apoptotic-and non-apoptotic death pathways, granzymes extensively contribute to the elimination of harmful damaged cells, reducing their dissemination, which highlights how the effectiveness of the immune system deeply relies on their regulated and effective release (16,49). In the context of pathogen-infected cells, the ability of viruses, bacteria, and parasites to modify the cellular environment poses a serious limitation for the initiation of suicidal pathways and thus the activation of caspases (50)(51)(52)(53)(54)(55)(56)(57). Therefore, the ability of granzymes to directly kill intracellular pathogens within infected cells in addition to killing the infected target cells is particularly important in the immune response to infection (58)(59)(60). ...
Granzymes are a family of serine proteases, composed of five human members: GA, B, H, M and K. They were first discovered in the 1980s within cytotoxic granules released during NK cell- and T cell-mediated killing. Through their various proteolytic activities, granzymes can trigger different pathways within cells, all of which ultimately lead to the same result, cell death. Over the years, the initial consideration of granzymes as mere cytotoxic mediators has changed due to surprising findings demonstrating their expression in cells other than immune effectors as well as new intracellular and extracellular activities. Additional roles have been identified in the extracellular milieu, following granzyme escape from the immunological synapse or their release by specific cell types. Outside the cell, granzyme activities mediate extracellular matrix alteration via the degradation of matrix proteins or surface receptors. In certain contexts, these processes are essential for tissue homeostasis; in others, excessive matrix degradation and extensive cell death contribute to the onset of chronic diseases, inflammation, and autoimmunity. Here, we provide an overview of both the physiological and pathological roles of granzymes, highlighting their utility while also recognizing how their unregulated presence can trigger the development and/or worsening of diseases.
... The genomic DNA of tomato leaves was extracted by the CTAB method (Gawel and Jarret, 1991), and the quality and amount of DNA was visually estimated after separation of 10 µg of DNA extraction in a 2 % agarose gel, staining with ethidium bromide, and visualizing the sample with a UV transluminator (Fan et al., 1998). The DNA laddering was recorded in accordance with a gel imaging system. ...
High temperature is a significant abiotic stress that affects growth and development of plants. BAG (Bcl-2 associated athanogene) protein family members act as co-chaperones and apoptosis inhibitors in multiple cellular processes. BAG, MAPK (Mitogen-activated protein kinase), and programmed cell death (PCD) play critical roles in plant growth and development, stress response, and disease resistance. In this study, we investigated the interaction of BAG, and MAPK as well as their putative role in PCD under either short or long-term heat stress. We constructed mutants of bag2 and mapk2 in tomato using CRISPR/Cas9. Our results revealed that tomato BAG2 and MAPK2 interacted positively both in vivo and in vitro. In addition, after 3 h of heat stress, the activities of Caspase 3 and antioxidant enzymes, expression levels of Caspase 3 and Caspase 9, and contents of H 2 O 2 in bag2 and mapk2 mutant plants were lower than those in WT (wild type) plants. Moreover, under short-term (3 h) heat stress, the DNA fragmentation phenomena and trypan blue coloration in both mutants were less severe than in WT plants; however, DNA integrities were broken, and the number of dead cells was higher under long-term (24 h) heat stress. Additionally, the electrolyte leakage was increased, but the Fv/Fm (maximum photochemical efficiency) value was decreased in mutants following exposure to heat stress. These results suggested that tomato BAG2 and MAPK2 suppressed short-time heat-induced PCD while promoting long-time heat-induced PCD.
... 15 In contrast to mitochondrial fragmentation induced by several effectors, an effector secreted by Chlamydia trachomatis into the cytosol of host cells inhibits Drp1 recruitment to the mitochondria, stabilizes the mitochondrial fusion network, and degrades proapoptotic proteins, ultimately suppressing apoptosis for bacterial intracellular survival. [16][17][18] S. Tm, an important gram-negative intracellular vacuolar bacterium, can cause gastroenteritis in humans and typhoid-like systemic illnesses in mice. 19 S. Tm is acquired from contaminated food and water via the fecal -oral route. ...
Mitochondrial dynamics are critical in cellular energy production, metabolism, apoptosis, and immune responses. Pathogenic bacteria have evolved sophisticated mechanisms to manipulate host cells’ mitochondrial functions, facilitating their proliferation and dissemination. Salmonella enterica serovar Typhimurium (S. Tm), an intracellular foodborne pathogen, causes diarrhea and exploits host macrophages for survival and replication. However, S. Tm-associated mitochondrial dynamics during macrophage infection remain poorly understood. In this study, we showed that within macrophages, S. Tm remodeled mitochondrial fragmentation to facilitate intracellular proliferation mediated by Salmonella invasion protein A (SipA), a type III secretion system effector encoded by Salmonella pathogenicity island 1. SipA directly targeted mitochondria via its N-terminal mitochondrial targeting sequence, preventing excessive fragmentation and the associated increase in mitochondrial reactive oxygen species, loss of mitochondrial membrane potential, and release of mitochondrial DNA and cytochrome c into the cytosol. Macrophage replication assays and animal experiments showed that mitochondria and SipA interact to facilitate intracellular replication and pathogenicity of S. Tm. Furthermore, we showed that SipA delayed mitochondrial fragmentation by indirectly inhibiting the recruitment of cytosolic dynamin-related protein 1, which mediates mitochondrial fragmentation. This study revealed a novel mechanism through which S. Tm manipulates host mitochondrial dynamics, providing insights into the molecular interplay that facilitates S. Tm adaptation within host macrophages.
... It has been observed that C. trachomatis disrupts N-Cadherin-dependent cell-cell junctions through the involvement of Ca2+ ions and a reorganization of the actin cytoskeleton and thus increases the exposure of basal cells to HPV [66,67]. C. trachomatis may have antiapoptotic effects; the antiapoptotic activity could depend on encoded protein factors that interrupt many different host cell apoptotic pathways [68]. Furthermore, it has been observed that C. trachomatis can increase the risk of infection with HPV and its persistence through inhibition of the expression of the γ-inducible interferon of the major complex histocompatibility class II (MHC-II) [69]. ...
In recent years, the relationship between the microbiota and various aspects of health has become a focal point of scientific investigation. Although the most studied microbiota concern the gastrointestinal tract, recently, the interest has also been extended to other body districts. Female genital tract dysbiosis and its possible impact on pathologies such as endometriosis, polycystic ovary syndrome (PCOS), pelvic inflammatory disease (PID), and gynecological cancers have been unveiled. The incursion of pathogenic microbes alters the ecological equilibrium of the vagina, triggering inflammation and compromising immune defense, potentially fostering an environment conducive to cancer development. The most common types of gynecological cancer include cervical, endometrial, and ovarian cancer, which occur in women of any age but especially in postmenopausal women. Several studies highlighted that a low presence of lactobacilli at the vaginal level, and consequently, in related areas (such as the endometrium and ovary), correlates with a higher risk of gynecological pathology and likely contributes to increased incidence and worse prognosis of gynecological cancers. The complex interplay between microbial communities and the development, progression, and treatment of gynecologic malignancies is a burgeoning field not yet fully understood. The intricate crosstalk between the gut microbiota and systemic inflammation introduces a new dimension to our understanding of gynecologic cancers. The objective of this review is to focus attention on the association between vaginal microbiota and gynecological malignancies and provide detailed knowledge for future diagnostic and therapeutic strategies.
... CMUT3.G5 was further used as a recipient strain for transformation with a plasmid that carries a premature stop codon in the pgp3 gene to produce the pGP3-deficient clone CMpGP3S. The CMpGP3S organisms were grown in HeLa cells (human cervical carcinoma epithelial cells; ATCC# CCL-2) and purified as elementary bodies (EBs) using discontinuous density centrifugation as previously described (59,60). The purified EBs were stored in aliquots in sucrose-phosphate-glutamic acid (SPG) buffer (0.2 M sucrose, 20 mM sodium phosphate at pH 7.4, and 5 mM glutamic acid) at −80°C. ...
Following an oral inoculation, Chlamydia muridarum descends to the mouse large intestine for long-lasting colonization. However, a mutant C. muridarum that lacks the plasmid-encoded protein pGP3 due to an engineered premature stop codon (designated as CMpGP3S) failed to do so even following an intrajejunal inoculation. This was because a CD4⁺ T cell-dependent immunity prevented the spread of CMpGP3S from the small intestine to the large intestine. In the current study, we found that mice deficient in IL-22 (IL-22−/−) allowed CMpGP3S to spread from the small intestine to the large intestine on day 3 after intrajejunal inoculation, indicating a critical role of IL-22 in regulating the chlamydial spread. The responsible IL-22 is produced by CD4⁺ T cells since IL-22−/− mice were rescued to block the CMpGP3S spread by donor CD4⁺ T cells from C57BL/6J mice. Consistently, CD4⁺ T cells lacking IL-22 failed to block the spread of CMpGP3S in Rag2−/− mice, while IL-22-competent CD4⁺ T cells did block. Furthermore, mice deficient in cathelicidin-related antimicrobial peptide (CRAMP) permitted the CMpGP3S spread, but donor CD4⁺ T cells from CRAMP−/− mice were still sufficient for preventing the CMpGP3S spread in Rag2−/− mice, indicating a critical role of CRAMP in regulating chlamydial spreading, and the responsible CRAMP is not produced by CD4⁺ T cells. Thus, the IL-22-producing CD4⁺ T cell-dependent regulation of chlamydial spreading correlated with CRAMP produced by non-CD4⁺ T cells. These findings provide a platform for further characterizing the subset(s) of CD4⁺ T cells responsible for regulating bacterial spreading in the intestine.
... A ncRNA has anti-apoptotic effects in C. trachomatis-infected cells. The ability of C. trachomatis to inhibit host cell apoptosis was reported > 20 years ago but has been insufficiently understood mec hanisticall y (Fan et al. 1998 ). ZEB1-AS1, a long ncRNA that was upregulated in HeLa cells in response to persistent C. trachomatis infection, targets miR-1224-5p, leading to the upregulation of mitogen-activ ated pr otein kinase 4 (MAP4K4) and increasing the resistance of infected cells to apoptosis, a strategy to ensure the pathogen's successful intracellular lifestyle (Luo et al. 2022 ). ...
Chlamydia trachomatis, C. pneumoniae, and C. psittaci, the three Chlamydia species known to cause human disease, have been collectively linked to several pathologies, including conjunctivitis, trachoma, respiratory disease, acute and chronic urogenital infections and their complications, and psittacosis. In vitro, animal, and human studies also established additional correlations, such as between C. pneumoniae and atherosclerosis and between C. trachomatis and ovarian cancer. As part of their survival and pathogenesis strategies as obligate intracellular bacteria, Chlamydia spp. modulate all three major types of epigenetic changes, which include DNA methylation, histone post-translational modifications, and microRNA-mediated gene silencing. Some of these epigenetic changes may be implicated in key aspects of pathogenesis, such as the ability of the Chlamydia spp. to induce epithelial-to-mesenchymal transition, interfere with DNA damage repair, suppress cholesterol efflux from infected macrophages, act as a co-factor in HPV-mediated cervical cancer, prevent apoptosis, and preserve the integrity of mitochondrial networks in infected host cells. A better understanding of the individual and collective contribution of epigenetic changes to pathogenesis will enhance our knowledge about the biology of Chlamydia spp. and facilitate the development of novel therapies and biomarkers.
... After washing and blocking, the cell samples were labeled with Hoechst (blue, Sigma) for visualizing DNA and a rabbit anti-chlamydial organisms (unpublished data) plus a goat anti-rabbit IgG conjugated with Cy2 (green; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) for visualizing chlamydial inclusions. The immuno-labeled cell samples were quantitated as described above and used for image acquisition with an Olympus AX-70 fluorescence microscope equipped with multiple filter sets (Olympus, Melville, NY, USA) as described previously (66,72). ...
To search for subunit vaccine candidates, immunogenic chlamydial antigens identified in humans were evaluated for protection against both infection and pathology in a mouse genital tract infection model under three different immunization regimens. The intramuscular immunization regimen was first used to evaluate 106 chlamydial antigens, which revealed that two antigens significantly reduced while 11 increased genital chlamydial burden. The two infection-reducing antigens failed to prevent pathology and 23 additional antigens even exacerbated pathology. Thus, intranasal mucosal immunization was tested next since intranasal inoculation with live Chlamydia muridarum prevented both genital infection and pathology. Two of the 29 chlamydial antigens evaluated were found to prevent genital infection but not pathology and three exacerbate pathology. To further improve protection efficacy, a combinational regimen (intranasal priming + intramuscular boosting + a third intraperitoneal/subcutaneous boost) was tested. This regimen identified four infection-reducing antigens, but only one of them prevented pathology. Unfortunately, this protective antigen was not advanced further due to its amino acid sequence homology with several human molecules. Two pathology-exacerbating antigens were also found. Nevertheless, intranasal mucosal priming with viable C. muridarum in control groups consistently prevented both genital infection and pathology regardless of the subsequent boosters. Thus, screening 140 different chlamydial antigens with 21 repeated multiple times in 17 experiments failed to identify a subunit vaccine candidate but demonstrated the superiority of viable chlamydial organisms in inducing immunity against both genital infection and pathology, laying the foundation for developing a live-attenuated Chlamydia vaccine.
... The immunofluorescence assay for visualizing and counting chlamydial inclusions in the Chlamydia-infected HeLa culture was described previously (96). Briefly, infected HeLa cells grown on coverslips were fixed with paraformaldehyde (Sigma, St. Louis, MO) and permeabilized with saponin (Sigma). ...
An IFNγ-susceptible mutant of Chlamydia muridarum is attenuated in pathogenicity in the genital tract and was recently licensed as an intr acellular O ral vaccine v ector or intrOv. Oral delivery of intrOv induces transmucosal protection in the genital tract, but intrOv itself is cleared from the gut (without shedding any infectious particles externally) by IFNγ from group 3-like innate lymphoid cells (ILC3s). We further characterized the intrOv interactions with ILC3s in the current study, since the interactions may impact both the safety and efficacy of intrOv as an oral Chlamydia vaccine. Intracolonic inoculation with intrOv induced IFNγ that in return inhibited intrOv. The intrOv-IFNγ interactions were dependent on RORγt, a signature transcriptional factor of ILC3s. Consistently, the transfer of oral intrOv-induced ILC3s from RORγt-GFP reporter mice to IFNγ-deficient mice rescued the inhibition of intrOv. Thus, IFNγ produced by intrOv-induced ILC3s is likely responsible for inhibiting intrOv, which is further supported by the observation that oral intrOv did induce significant levels of IFNγ-producing LC3s (IFNγ ⁺ ILC3s). Interestingly, IL-23 receptor knockout (IL-23R −/− ) mice no longer inhibited intrOv, which was accompanied by reduced colonic IFNγ. Transfer of oral intrOv-induced ILC3s rescued the IL-23R −/− mice to inhibit intrOv, validating the dependence of ILC3s on IL-23R signaling for inhibiting intrOv. Clearly, intrOv induces intestinal IFNγ ⁺ ILC3s for its own inhibition in the gut, which is facilitated by IL-23R signaling. These findings have provided a mechanism for ensuring the safety of intrOv as an oral Chlamydia vaccine and a platform for investigating how oral intrOv induces transmucosal protection in the genital tract.
... Once inside the cell, the EB differentiates into an RB and replicates in a membrane bound vacuole called the inclusion. Throughout the developmental cycle, chlamydiae secrete many bacterial effectors into the host cell cytosol to modulate host cell processes including vesicular trafficking (Moore et al., 2008;Capmany and Damiani, 2010;Sixt et al., 2017;Weber et al., 2017;Faris et al., 2019;Pais et al., 2019;Auer et al., 2020), pathogen sensing and inflammatory signaling Sixt et al., 2017), and cell death (Fan et al., 1998;Fischer et al., 2004;Waguia Kontchou et al., 2016;Fischer et al., 2017;Sixt et al., 2017). Near the end of the developmental cycle, RBs convert back into EBs in an asynchronous manner. ...
Chlamydia trachomatis infection of ocular conjunctiva can lead to blindness, while infection of the female genital tract can lead to chronic pelvic pain, ectopic pregnancy, and/or infertility. Conjunctival and fallopian tube inflammation and the resulting disease sequelae are attributed to immune responses induced by chlamydial infection at these mucosal sites. The conserved chlamydial plasmid has been implicated in enhancing infection, via improved host cell entry and exit, and accelerating innate inflammatory responses that lead to tissue damage. The chlamydial plasmid encodes eight open reading frames, three of which have been associated with virulence: a secreted protein, Pgp3, and putative transcriptional regulators, Pgp4 and Pgp5. Although Pgp3 is an important plasmid-encoded virulence factor, recent studies suggest that chlamydial plasmid-mediated virulence extends beyond the expression of Pgp3. In this review, we discuss studies of genital, ocular, and gastrointestinal infection with C. trachomatis or C. muridarum that shed light on the role of the plasmid in disease development, and the potential for tissue and species-specific differences in plasmid-mediated pathogenesis. We also review evidence that plasmid-associated inflammation can be independent of bacterial burden. The functions of each of the plasmid-encoded proteins and potential molecular mechanisms for their role(s) in chlamydial virulence are discussed. Although the understanding of plasmid-associated virulence has expanded within the last decade, many questions related to how and to what extent the plasmid influences chlamydial infectivity and inflammation remain unknown, particularly with respect to human infections. Elucidating the answers to these questions could improve our understanding of how chlamydia augment infection and inflammation to cause disease.
... Preventing apoptosis is critical for obligate intracellular bacteria since maintaining a replicative niche is essential to complete the infection cycle. Obligate intracellular pathogens, including Rickettsia, Anaplasma, Mycobacterium, Chlamydia, and others, have evolved multiple regulatory mechanisms to inhibit host cell apoptosis, including regulation of mitochondria-mediated intrinsic apoptosis (58)(59)(60)(61)(62)(63)(64)(65). Additionally, intracellular bacteria regulate the BCL-2 family of proteins to stabi lize mitochondria and promote host cell survival. ...
Ehrlichia chaffeensis TRP120 effector has evolved short linear motif (SLiM) ligand mimicry to repurpose multiple evolutionarily conserved cellular signaling pathways, including Wnt, Notch, and Hedgehog. In this investigation, we demonstrate that E. chaffeensis and recombinant TRP120 deactivate Hippo signaling, resulting in the activation of Hippo transcription coactivator Yes-associated protein (Yap). Moreover, a homologous 6 amino acid (QDVASH) SLiM shared by TRP120 and Wnt3a/5a ligands phenocopied Yap and β-catenin activation induced by E. chaffeensis, rTRP120, and Wnt5a. Similar Hippo gene expression profiles were also stimulated by E. chaffeensis, rTRP120, SLiM, and Wnt5a. Single siRNA knockdown of Hippo transcription co-activator/factors, Yap, and transcriptional enhanced associate domain (TEAD) significantly decreased E. chaffeensis infection. Yap activation was abolished in THP-1 Wnt Frizzled-5 (Fzd5) receptor knockout cells (KO), demonstrating Fzd5 receptor dependence. In addition, the TRP120-Wnt-SLiM antibody blocked Hippo deactivation (Yap activation). Expression of anti-apoptotic Hippo target gene SLC2A1 (encodes glucose transporter 1; GLUT1) was upregulated by E. chaffeensis and corresponded to increased levels of GLUT1. Conversely, siRNA knockdown of SLC2A1 significantly inhibited infection. Higher GLUT1 levels correlated with increased B cell lymphoma-extra large (BCL-xL) and decreased BCL2-associated X, apoptosis regulator (Bax) levels. Moreover, blocking Yap activation with the inhibitor Verteporfin induced apoptosis that corresponded to significant reductions in GLUT1 and BCL-xL levels and activation of Bax and Caspase-3 and -9. This study identifies a novel shared Wnt/Hippo SLiM ligand mimic and demonstrates that E. chaffeensis deactivates the Hippo pathway to engage the anti-apoptotic Yap-GLUT1-BCL-xL axis.
