Cryo-electron microscopy offers a promising avenue for elucidating the high-resolution structure of EhPrx, which could reveal critical insights into its biological functions and inter- or intramolecular interactions.
Abstract
ABSTRACT Amoebiasis is a critical global parasitic disease caused by Entamoeba histolytica. It includes amoebic colitis and extraintestinal abscesses. E. histolytica peroxiredoxin (EhPrx) is a crucial antioxidant enzyme that maintains redox homeostasis and participates in counteracting oxidative stress, supporting trophozoite survival and pathogenicity in the host. We observed the EhPrx structure to be a decameric ring formed through the polymerization of five dimers. EhPrx had a unique α-helical structure at its N-terminus. Protein-protein docking and surface plasmon resonance (SPR) assay results suggest that EhPrx may bind to myeloid differentiation factor 2 (MD2), the ligand protein of toll-like receptor 4 (TLR4), rather than directly to TLR4. Furthermore, EhPrx was crucial in the induction of cellular ferroptosis by E. histolytica trophozoites and regulated ferroptosis in host cells through the TLR4/MD2 pathway. These findings may hold a solid structural basis and promising concepts for investigating the pathogenic mechanisms and drug targets of E. histolytica. IMPORTANCE Entamoeba histolytica is the causative agent of amoebiasis, a significant global health concern. Within this parasite, E. histolytica peroxiredoxin (EhPrx) plays a pivotal role in mitigating oxidative stress, a critical survival mechanism in the hostile environment of the human host. EhPrx belongs to a family of antioxidant enzymes responsible for detoxifying peroxides, which are deleterious byproducts of cellular metabolism and host-derived immune responses. By neutralizing reactive oxygen species (ROS), EhPrx safeguards the parasite's cellular integrity, ensuring its survival, proliferation, and pathogenicity. Despite its functional importance, the precise structural details of EhPrx remain elusive, hindering a comprehensive understanding of its molecular mechanisms. Cryo-electron microscopy (cryo-EM) offers a promising avenue for elucidating the high-resolution structure of EhPrx, which could reveal critical insights into its biological functions and inter- or intramolecular interactions. Such structural characterization may be indispensable for advancing our knowledge of E. histolytica biology and identifying novel diagnostic markers to combat amoebiasis effectively. Entamoeba histolytica is the causative agent of amoebiasis, a significant global health concern. Within this parasite, E. histolytica peroxiredoxin (EhPrx) plays a pivotal role in mitigating oxidative stress, a critical survival mechanism in the hostile environment of the human host. EhPrx belongs to a family of antioxidant enzymes responsible for detoxifying peroxides, which are deleterious byproducts of cellular metabolism and host-derived immune responses. By neutralizing reactive oxygen species (ROS), EhPrx safeguards the parasite's cellular integrity, ensuring its survival, proliferation, and pathogenicity. Despite its functional importance, the precise structural details of EhPrx remain elusive, hindering a comprehensive understanding of its molecular mechanisms. Cryo-electron microscopy (cryo-EM) offers a promising avenue for elucidating the high-resolution structure of EhPrx, which could reveal critical insights into its biological functions and inter- or intramolecular interactions. Such structural characterization may be indispensable for advancing our knowledge of E. histolytica biology and identifying novel diagnostic markers to combat amoebiasis effectively.
Edwardsiella piscicida is an enteric intracellular bacterial pathogen. It has been reported that this bacterium produces two types of hemolysins: the secreted, pore-forming toxin HlyA and the cell-associated toxin EthA. EthA has been reported to enter epithelial cells by binding to outer membrane vesicles (OMVs) and that once inside the host cells, it releases lipopolysaccharide from the OMVs to induce pyroptosis. We report here the existence of another pathway by which EthA is secreted and delivered into host cells: the type III secretion system (T3SS) of E. piscicida. The ΔFur box-PethB strain that highly expresses EthA was constructed by deleting the binding motif of Fur in front of the ethB-ethA operon. The ΔFur box-PethB strain induces PANoptosis (a combination of pyroptosis, apoptosis and necroptosis) in a T3SS-dependent manner in murine macrophages. The highly expressed EthA stimulates pyroptosis partially via the NLRP3 inflammasome and concomittantly induces apoptosis and necroptosis, as evidenced by elevated ratios of positive cells in TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) staining, elevated protein levels of cleaved caspase-3 and phosphorylated MLKL. PANoptosis is characterized by cell swelling, membrane rupture and cell blebbing. Additionally, the highly expressed EthA inhibits the phosphorylation of the serine/threonine kinase TAK1 and transcription factor p65, preventing the nuclear translocation of phosphorylated p65 into murine macrophages. Overall, EthA has been identified as a novel T3SS substrate in E. piscicida. Its high expression inhibits the phosphorylation of TAK1, thereby increasing PANoptosis and blocking the inflammatory pathway.
P. Tang, Xiulan Jiang, Xinyi Zhou et al.· Veterinary Research· 0 citations
A novel role is identified for FlhF, a GTPase essential for proper flagellar assembly, in promoting resistance to hydrogen peroxide (H2O2) in C. jejuni, offering new insights into flagella-associated defense mechanisms in this pathogen.
Trichomonas vaginalis (Tv) is a eukaryotic parasite that causes the most common sexually transmitted infection, trichomoniasis. Increased inflammation during Tv infection is predicted to drive comorbidities like cervical cancer, but the underlying inflammatory mechanism at play is unclear. Pyroptosis is a form of cell death that releases proinflammatory cytokines. It involves caspase-1 cleavage of Interleukin-1β (IL-1β) cytokine and gasdermin D (GSDMD), which generates a gasdermin N-terminal fragment (GSDMD-NT). GSDMD-NT oligomerization forms pores on the cell membrane, prompting cytokine release and cellular rupture. We hypothesize that Tv damages ectocervical epithelial cells (Ect1) via GSDMD-mediated pyroptosis.
To test whether Tv induces GSDMD-mediated pyroptosis, we generated Ect1 GSDMD-knockout (GSDMD KO) cells and measured cytolysis via lactate dehydrogenase assay. Furthermore, we assayed Ect1 GSDMD KO and WT cell supernatants for differential release of proinflammatory cytokines via enzyme-linked immunosorbent assay (ELISA). In parallel, we sought to identify additional microenvironment-altering proteins released via GSDMD pores during Tv-induced pyroptosis using quantitative proteomics and western blot analysis.
Upon Tv infection, we noted a striking 25% decrease in cytolysis of GSDMD KO cells compared to WT. Furthermore, cell supernatant analysis via ELISA showed a dramatic 53% decrease in IL-1β and a 25% decrease in IL-18 cytokine release in GSDMD KO cells compared to WT. Finally, quantitative proteomics demonstrated the selective release of potential microenvironment-altering proteins via GSDMD-NT pores. We further validated the differential release of these proteins via western blots.
Collectively, we show that Tv triggers GSDMD-mediated pyroptosis in Ect1 cells. To our knowledge, this is the first evidence showing that Tv triggers pyroptosis in Ect1 cells and that female reproductive tract cells, not just immune cells, can undergo robust pyroptosis.
Rees-Stealy Research Foundation Fellowship
Microbial, Parasitic, and Fungal Immunology (MPF)
Johann Tailor, Maryam Howayer, Bryn Baxter et al.· Journal of Immunology· 0 citations
The Hom family and canilysin are defined as helicolysins, a previously uncharacterized metzincin subfamily distinguished by a conserved Thr-turn and an accessory ND, and implicates these proteins in host-pathogen interactions, adhesion, and immunomodulation.
A. Rodríguez-Banqueri, T. Goulas, Marina Girbal-González et al.· Journal of Molecular Biology· 0 citations
Vibrio parahaemolyticus is a major pathogen causing disease outbreaks in marine bivalves, but the mode of cell death induced by infection in clams remains unclear. In this study, we investigated whether V. parahaemolyticus infection triggers ferroptosis-like cell death in the clam Meretrix petechialis. Transmission electron microscopy revealed marked mitochondrial ultrastructural alterations in hepatopancreas tissues, including mitochondrial shrinkage and cristae disruption in infected clams. Vibrio infection also induced Fe2+ accumulation in hemocytes, while reactive oxygen species (ROS) levels and lipid peroxidation were significantly increased in hepatopancreas tissues. Meanwhile, reduced glutathione (GSH) levels and MpGPX4 expression were significantly decreased, indicating impairment of the GSH-GPX4 antioxidant axis. The mRNA expression of ferroptosis-related genes was also significantly altered, with upregulation of MpACSL4, MpSAT1, and MpFerroportin, and downregulation of MpGCL, MpNrf2, and MpFerritin. In addition, Torin 1 treatment reduced hemocyte viability and decreased MpGPX4 expression, suggesting a possible association between mTOR-related signaling and MpGPX4 expression. Lipidomic analysis further showed that Ferrostatin-1 reshaped infection-associated lipid metabolism, particularly phospholipid remodeling and PUFA-related pathways, including glycerophospholipid, linoleic acid, and arachidonic acid metabolism. Collectively, our study integrates a Vibrio infection model, Ferrostatin-1 intervention and mTOR-GPX4-related analysis to provide a multi-level evidence framework for ferroptosis-like responses during V. parahaemolyticus infection in M. petechialis, offering new insights into pathogen-induced cell death mechanisms in marine bivalves.
Shujing Zhang, Fengxia Xu, Mengyao Gao et al.· Fish and Shellfish Immunolog...· 0 citations
OBJECTIVES
Hypervirulent Klebsiella pneumoniae (hvKP) causes pyogenic liver abscess and fulminant sepsis via vascular endothelial dysfunction and injury. This study aimed to elucidate how its type VI secretion system (T6SS) effector hemolysin-coregulated protein (Hcp) compromises vascular integrity by inducing endothelial pyroptosis.
METHODS
We stimulated macrophages with Hcp or hvKP strains (wild-type/ hcp-knockout/ complemented), then applied supernatants to human umbilical vein endothelial cells (HUVECs) to evaluate pyroptosis, cytokine release, and endothelial activation. Mechanistic validation studies employed high-mobility group box 1 (HMGB1)⁻/⁻ macrophages, caspase-1-knockdown (KD) HUVECs, and pharmacological inhibitors, including a receptor for advanced glycation end products (RAGE) inhibitor (FPS-ZM1) and an HMGB1-neutralizing antibody. A murine hvKP liver abscess model assessed survival, HMGB1 dynamics, and vascular pathology.
RESULTS
Hcp stimulated macrophage HMGB1 secretion. The resulting Hcp-HMGB1 complexes were internalized by HUVECs via RAGE, thereby inducing pyroptosis and the release of interleukin (IL)-1β and IL-18 consistent with the canonical pyroptotic pathway. These effects were abolished by HMGB1 knockout, caspase-1 KD, or RAGE inhibition. Additionally, Hcp upregulated endothelial activation markers in an HMGB1-dependent manner. Mice infected with hcp-knockout hvKP showed improved survival rates, lower serum HMGB1 levels, and reduced endothelial injury.
CONCLUSIONS
Thus, the T6SS effector Hcp drives endothelial pyroptosis through macrophage-derived HMGB1- and RAGE-mediated signaling, establishing the Hcp-HMGB1-RAGE-caspase-1 axis as a promising therapeutic target for hvKP-associated endothelial dysfunction.
Bo Ni, Zhaoyu Liu, Z. Chang et al.· International Journal of Ant...· 0 citations
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