Meng Feng

Amoebiasis is a critical global parasitic disease caused by Entamoeba histolytica . It includes amoebic colitis and extraintestinal abscesses. E. histolytica peroxiredoxin ( Eh Prx) 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 Eh Prx structure to be a decameric ring formed through the polymerization of five dimers. Eh Prx had a unique α-helical structure at its N-terminus. Protein-protein docking and surface plasmon resonance (SPR) assay results suggest that Eh Prx may bind to myeloid differentiation factor 2 (MD2), the ligand protein of toll-like receptor 4 (TLR4), rather than directly to TLR4. Furthermore, Eh Prx 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 ( Eh Prx) plays a pivotal role in mitigating oxidative stress, a critical survival mechanism in the hostile environment of the human host. Eh Prx 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 Eh Prx 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 Eh Prx, 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.

The H1N1 influenza virus poses a major global public health challenge, highlighting an urgent demand for advanced diagnostic technologies that enable rapid and accurate detection. This study established a novel CRISPR-Cas12a2-based diagnostic platform for highly sensitive and rapid detection of influenza A H1N1 virus. Targeting the conserved M gene, the assay combined optimized crRNA design with the unique dual RNA cleavage and collateral ssDNA degradation activities of SuCas12a2, enabling fluorescence-based detection with a sensitivity of 5 copies/μL. Clinical validation of 205 nasopharyngeal swabs demonstrated 91.22% concordance with real-time qPCR (κ = 0.65) and identified 18 additional positive cases, indicating superior sensitivity. The assay achieved a detection limit of 5 RNA copies/μL within 90 min by combining through a reverse transcription PCR with CRISPR detection. Rigorous specificity testing confirmed 100% discrimination against other respiratory pathogens. These results positioned CRISPR-Cas12a2 as a robust diagnostic alternative, offering advantages in speed and sensitivity. This technology represents a significant advancement in molecular diagnostics, meeting the critical needs for rapid and accurate pathogen detection during outbreaks.

The Gal/GalNAc lectin in ‌ Entamoeba histolytica ‌ is pivotal for its pathogenicity, and its intermediate subunit (Igl) is a crucial virulence factor. Here, the interference of eukaryotically expressed Igl on normal metabolic processes within Caco-2 cells was explored. Targeted metabolomic analysis detected 232 metabolites in Caco-2 cells, revealing 63 with significant alterations after Igl treatment. Notably, the lactate content significantly increased, indicating that Igl activates aerobic glycolysis. Metabolic flux analysis demonstrated that Igl could inhibit aerobic respiration and the tricarboxylic acid cycle in host cells. It significantly increased glucose intake, lactate production, and ATP generation, demonstrating its ability to induce a Warburg-like effect. Through single-cell transcriptomic experiments, significant increases in changes were observed in 4 out of 12 individual host cell groups after Igl treatment. Group marker genes identified these cell groups as having inhibited aerobic respiration and activated stress responses, further indicating that Igl stimulation can affect the aerobic respiration and normal metabolic processes of cells. Additionally, RNA interference experiments were performed to verify the effect of Igl on host cell autophagy when PRKAA1 was silenced. Confocal microscopy and western blotting revealed significantly enhanced autophagy signals after Igl treatment, and it was confirmed that mammalian target of rapamycin was involved in this process. These results indicate that Igl induces metabolic reprogramming and a Warburg-like shift in host epithelial cells, thereby activating aerobic glycolysis and regulating host cell autophagy. This represents a novel mechanism of Igl in the pathogenicity of E. histolytica . IMPORTANCE: Entamoeba histolytica causes amoebiasis, an infection manifesting as colitis and extraintestinal abscesses. Multi-omics approaches provide critical insights into the role of the Gal/GalNAc lectin intermediate subunit (Igl) within host cells, offering a foundation for developing effective treatments. Our findings indicate that Igl induces metabolic reprogramming in host epithelial cells. Specifically, Igl can trigger a Warburg-like shift, a phenomenon characterized by the activation of aerobic glycolysis. This shift results in increased glucose intake, lactate production, and ATP generation while inhibiting aerobic respiration and the tricarboxylic acid cycle. Furthermore, Igl regulates host cell autophagy, a process further confirmed through RNA interference experiments targeting PRKAA1 , which revealed the involvement of mammalian target of rapamycin. Taken together, our data suggest that Igl promotes trophozoite virulence through a novel mechanism that involves metabolic reprogramming and a Warburg-like shift in host cells.

Background: Acanthamoeba is a widely distributed unicellular eukaryotic organism, causing Acanthamoeba keratitis, granulomatous amoebic encephalitis and chronic infectious ulcers. At present, there are no effective drugs available for treating Acanthamoeba infections, highlighting the urgent need for the development of novel anti- Acanthamoeba therapies. Purpose: This study aims to investigate the inhibitory effect of licorice on A. castellanii trophozoites growth and elucidate its molecular mechanisms. Methods: The effect of licorice on trophozoite viability was assessed employing the CellTiter-Glo assay, while the viability of cells was evaluated via CCK-8 assay. Apoptotic cells were identified through Hoechst 33,342/PI double staining and caspase-3 detection. ROS level was measured via flow cytometry utilising DCFH-DA. Mitochondrial dysfunction was analysed with JC-1 and mtSOX Deep Red staining. RNA sequencing and RT-qPCR analysis were conducted to investigate the potential anti- Acanthamoeba mechanism. Results: Our results showed that isoliquiritigenin (ISL) and glabridin (GLA) effectively inhibited A. castellanii trophozoite growth in vitro dose-and time-dependently. ISL and GLA induced several apoptosis features, including Hoechst/PI-positive staining and increased caspase-3 expression. ISL and GLA increased intracellular ROS production, decreased SOD expression and mitochondrial membrane potential and enhanced mitochondrial ROS generation. ISL and GLA effectively prevent host cells from A. castellanii trophozoites invasion. RNA sequencing indicated that ISL may modulate the NAD ⁺ metabolic process, while GLA may influence the sterol metabolic process. ISL reduced the NAD ⁺ /NADH ratio, while GLA lowered the levels of 7-dehydrocholesterol in A. castellanii trophozoites. Conclusion: These findings suggest that ISL and GLA may serve as the active components of licorice for treating A. castellanii trophozoites. Our findings suggest that ISL suppresses trophozoites by regulating NAD ⁺ metabolism, while GLA inhibits trophozoites through the regulation of sterol metabolism. Abstract Figure:

Alzheimer’s disease (AD) is a multifactorial brain disorder and infectious diseases are considered as one of the predisposing factors for AD. Toxoplasma gondii, an obligate intracellular parasitic protozoan, is suspected of being associated with AD. Serum samples were collected from 109 AD patients and 114 age-matched healthy controls. ELISA was performed using recombinant T. gondii cyst wall protein 1 (CST1) to detect T. gondii antibodies. A parallel experiment was performed with Toxoplasma gondii tachyzoites lysate protein. To analyze whether factors associated with the onset of AD included chronic T. gondii infection, a multivariate logistic regression model was applied, further validating the correlation between chronic T. gondii infection and AD. AD patients exhibited significantly higher levels of Toxoplasma-specific antibodies in their serum compared to the control group, with statistically significant differences (p < 0.05). Multivariate logistic regression analysis revealed that Toxoplasma infection is a risk factor for AD (p < 0.01), and the CST1 antigen can significantly improve the model’s performance in predicting the occurrence of AD. The results indicate that chronic infection with Toxoplasma gondii could be one of the risk factors for the development of AD, potentially predisposing individuals with underlying health conditions to the disease. This further validates the correlation between Toxoplasma gondii and AD.

The protozoan parasite Entamoeba histolytica is the causative agent of amebiasis, with clinical outcomes ranging from asymptomatic infections to severe invasive diseases. The innate immune system, particularly macrophages, is of paramount importance in resisting the invasion of host tissues and organs by the trophozoites of E. histolytica. Parasite-derived pathogenic factors, such as lectins, play a pivotal role in the promotion of macrophage polarization phenotypes that have undergone alteration. Nevertheless, the precise mechanisms by which E. histolytica modulates immune polarization remain largely unknown. The current study focused on the immunomodulatory effects of the Igl-C fragment of E. histolytica Gal/GalNAc lectin on macrophage polarization. These results demonstrated that Igl-C could induce the secretion of IL-1β, IL-6, and other cytokines, activating a mixed M1/M2 polarization state. M1 polarization of macrophages occurs in the early stages and gradually transitions to M2 polarization in the later stages, which may contribute to the persistence of the infection. Igl-C induces the macrophage M1 phenotype and causes the release of immune effector molecules, including iNOS and cytokines, by activating the NF-κB p65 and JAK-STAT1 transcription factor signaling pathways. Furthermore, Igl-C supports the macrophage M2 phenotype via JAK-STAT3 and IL-4-STAT6 pathways, which activate arginase expression in later stages, contributing to the tissue regeneration and persistence of the parasite. The involvement of distinct signaling pathways in mediating this response highlights the complex interplay between the parasite and the host immune system. These findings enhance our understanding of the Igl-C-mediated pathogenic mechanisms during E. histolytica infection.

Background: Gut microbiota plays a significant role in the colorectal cancer (CRC) process. Ectopic colonization of multiple oral bacteria is reportedly associated with CRC pathogenesis and progression, but the details remain unclear.Methods: We enrolled a cohort of 50 CRC patients and 52 healthy controls from an East China population. Taxonomic and functional analysis of the fecal microbiota were performed using 16S rDNA (50 + 52 samples) and shotgun metagenomic sequencing (8 + 6 samples), respectively, with particular attention paid to gut‐colonized oral bacteria.Results and Conclusions: The results showed more detected bacterial species but lower species evenness within the samples from CRC patients. To determine the specific bacteria enriched in each group, we analyzed their possible protective, carcinogenic, or opportunistic roles in the CRC process. Among the ectopic oral bacteria, we observed a significant increase in the abundance of Fusobacterium and decreased abundance of Prevotella and Ruminococcus in the CRC group. Main differences in the functional composition of these two groups were related to energy metabolism and biosynthesis, especially the glycolytic pathway. Furthermore, we validated the colonization of Fusobacterium nucleatum subsp. animalis within CRC tissues and studied its impact on the host intestinal epithelium and tumor cells. With high selectivity for cancerous tissues, this subspecies promoted CRC cell proliferation and induced potential DNA damage.

Metabolic reprogramming is considered a hallmark of some infectious diseases. However, in amoebiasis, a neglected tropical disease caused by protozoan parasite E. histolytica , metabolic changes in host cells have yet to be proven.