UNASSIGNED: Acanthamoeba infection is a serious public health concern, necessitating the development of effective and safe anti-Acanthamoeba chemotherapies. Poly (ADP-ribose) polymerases (PARPs) govern a colossal amount of biological processes, such as DNA damage repair, protein degradation and apoptosis. Multiple PARP-targeted compounds have been approved for cancer treatment. However, repurposing of PARP inhibitors to treat Acanthamoeba is poorly understood.
UNASSIGNED: In the present study, we attempted to fill these knowledge gaps by performing anti-Acanthamoeba efficacy assays, cell biology experiments, bioinformatics, and transcriptomic analyses.
UNASSIGNED: Using a homology model of Acanthamoeba poly (ADP-ribose) polymerases (PARPs), molecular docking of approved drugs revealed three potential inhibitory compounds: olaparib, venadaparib and AZ9482. In particular, venadaparib exhibited superior docking scores (-13.71) and favorable predicted binding free energy (-89.28 kcal/mol), followed by AZ9482, which showed a docking score of -13.20 and a binding free energy of -92.13 kcal/mol. Notably, the positively charged cyclopropylamine in venadaparib established a salt bridge (through E535) and a hydrogen bond (via N531) within the binding pocket. For comparison, AZ9482 was well stacked by the surrounding aromatic residues including H625, Y652, Y659 and Y670. In an assessment of trophozoites viability, AZ9482 exhibited a dose-and time-dependent anti-trophozoite effect by suppressing Acanthamoeba PARP activity, unlike olaparib and venadaparib. An Annexin V-fluorescein isothiocyanate/propidium iodide apoptosis assay revealed AZ9482 induced trophozoite necrotic cell death rather than apoptosis. Transcriptomics analyses conducted on Acanthamoeba trophozoites treated with AZ9482 demonstrated an atlas of differentially regulated proteins and genes, and found that AZ9482 rapidly upregulates a multitude of DNA damage repair pathways in trophozoites, and intriguingly downregulates several virulent genes. Analyzing gene expression related to DNA damage repair pathway and the rate of apurinic/apyrimidinic (AP) sites indicated DNA damage efficacy and repair modulation in Acanthamoeba trophozoites following AZ9482 treatment.
UNASSIGNED: Collectively, these findings highlight AZ9482, as a structurally unique PARP inhibitor, provides a promising prototype for advancing anti-Acanthamoeba drug research.

In humans, Entamoeba histolytica is the main pathogen causing various amoebiases, while E. moshkovskii falls between being a pathogen and non-pathogen. The two species have similar behavior patterns but differ significantly in pathogenicity, with previous studies and clinical data indicating that E. moshkovskii has a low level of pathogenicity. Meaningfully, the biological characteristics of E. moshkovskii make it a potential model organism and a protein display platform for studying the functions of important Entamoeba proteins. Here, an Amoeba-pcDNA3.1 vector capable of overexpressing E. histolytica-sourced Igl-C protein was constructed and successfully transfected into E. moshkovskii. High levels of expression of the Igl-C, EGFP, and NeoR genes were identified in Igl-C-transfected trophozoites using qRT-PCR, and they were subsequently confirmed using immunoblotting. Transfection of Igl-C protein improved the adherence and phagocytosis of E. moshkovskii, demonstrating that E. histolytica Igl mediated amoebic adhesion. Moreover, as a manifestation of protein virulence, the ability of post-transfected trophozoites to induce inflammation in host macrophages was also enhanced. In conclusion, this study utilizing the characteristics of E. moshkovskii confirmed its potential to serve as a model organism. E. moshkovskii could replace E. histolytica as the target of gene editing, allowing more efficient study of amoebic pathogenicity.

BACKGROUND: Giardia lamblia (syn. G. intestinalis, G. duodenalis) is a primitive opportunistic protozoon, and one of the earliest differentiated eukaryotes. Despite its primitive nature, G. lamblia has a sophisticated cytoskeleton system, which is closely related to its proliferation and pathogenicity. Meanwhile, α giardin is a G. lamblia-specific cytoskeleton protein, which belongs to the annexin superfamily. Interestingly, G. lamblia has 21 annexin-like α giardins, i.e., more than higher eukaryotes. The functional differences among α giardin members are not fully understood.
METHODS: We took α-4 giardin, a member of α giardin family, as a research object. A morpholino-mediated knockdown experiment was performed to identify the effect of α-4 giardin on G. lamblia trophozoites biological traits. A yeast two-hybrid cDNA library of G. lamblia strain C2 trophozoites was screened for interaction partners of α-4 giardin. Co-immunoprecipitation and fluorescent colocalization confirmed the relationship between G. lamblia EB1 (gEB1) and α-4 giardin.
RESULTS: α-4 Giardin could inhibit the proliferation and adhesion of G. lamblia trophozoites. In addition, it interacted with G. lamblia EB1 (gEB1).
CONCLUSIONS: α-4 Giardin was involved in proliferation and adhesion in G. lamblia trophozoites, and EB1, a crucial roles in mitosis, was an interacting partner of α-4 giardin.

During asexual growth and replication cycles inside red blood cells, the malaria parasite Plasmodium falciparum primarily relies on glycolysis for energy supply, as its single mitochondrion performs little or no oxidative phosphorylation. Post merozoite invasion of a host red blood cell, the ring stage lasts approximately 20 hours and was traditionally thought to be metabolically quiescent. However, recent studies have shown that the ring stage is active in several energy-costly processes, including gene transcription, protein translation, protein export, and movement inside the host cell. It has remained unclear whether a low glycolytic flux alone can meet the energy demand of the ring stage over a long period post invasion. Here, we demonstrate that the metabolic by-product pyrophosphate (PPi) is a critical energy source for the development of the ring stage and its transition to the trophozoite stage. During early phases of the asexual development, the parasite utilizes Plasmodium falciparum vacuolar pyrophosphatase 1 (PfVP1), an ancient pyrophosphate-driven proton pump, to export protons across the parasite plasma membrane. Conditional deletion of PfVP1 leads to a delayed ring stage that lasts nearly 48 hours and a complete blockage of the ring-to-trophozoite transition before the onset of parasite death. This developmental arrest can be partially rescued by an orthologous vacuolar pyrophosphatase from Arabidopsis thaliana, but not by the soluble pyrophosphatase from Saccharomyces cerevisiae, which lacks proton pumping activities. Since proton-pumping pyrophosphatases have been evolutionarily lost in human hosts, the essentiality of PfVP1 suggests its potential as an antimalarial drug target. A drug target of the ring stage is highly desired, as current antimalarials have limited efficacy against this stage.

Trichomonas vaginalis (T. vaginalis) is a widespread and important sexually transmitted pathogen. Adherence to the surface of the host cell is the precondition forthis parasite\'s parasitism and pathogenicity. Adhesion protein 65 (TvAP65) plays a key role in the process of adhesion. However, how TvAP65 mediates the adhesion and pathogenicity of T. vaginalis to host cellsis unclear. In this study, we knocked down the expression of TvAP65 in trophozoites by small RNA interference. The number of T. vaginalis trophozoites adhering to VK2/E6E7 cells was decreased significantly, and the inhibition of VK2/E6E7 cells proliferation and VK2/E6E7 cells apoptosis and death induced by T. vaginalis were reduced, after the expression of TvAP65 was knocked down. Animal challenge experiments showed that the pathogenicity of trophozoites was decreased by passive immunization with anti-rTvAP65 PcAbs or blocking the TvAP65 protein. Immunofluorescence analysis showed that TvAP65 could bind to VK2/E6E7 cells. In order to screen the molecules interacting with TvAP65 on the host cells, we successfully constructed the cDNA library of VK2/E6E7 cells, and thirteen protein molecules interacting with TvAP65 were screened by yeast two-hybrid system. The interaction between TvAP65 and BNIP3 was further confirmed by coimmunoprecipitation and colocalization. When both TvAP65 and BNIP3 were knocked down by small RNA interference, the number of T. vaginalis adhering to VK2/E6E7 cells and the inhibition of VK2/E6E7 cells proliferation were significantly lower than those of the group with knockdown of TvAP65 or BNIP3 alone. Therefore, the interaction of TvAP65 and BNIP3 in the pathogenesis of T. vaginalis infecting host cells is not unique and involves other molecules. Our study elucidated that the interaction between TvAP65 and BNIP3 mediated the adhesion and pathogenicity of T. vaginalis to host cells, provided a basis for searching for the drug targets of anti-T. vaginalis, and afforded new ideas for the prevention and treatment of trichomoniasis.

BACKGROUND: Trichomonas vaginalis is a widespread and important sexually transmitted pathogen. Adherence to the surface of the host cell is the precondition for the parasitism and pathogenicity of this parasite. Trichomonas vaginalis adhesion protein 33 (TvAP33) plays a key role in the process of adhesion, but how this protein mediates the adhesion and pathogenicity of T. vaginalis to host cells is unclear.
METHODS: The expression of TvAP33 in trophozoites was knocked down by small interfering RNA. VK2/E6E7 cells and mice infected with T. vaginalis were used to evaluate the pathogenicity of T. vaginalis. We constructed a complementary DNA library of VK2/E6E7 cells and screened the protein molecules interacting with TvAP33 by the yeast two-hybrid system. The interaction between TvAP33 and BNIP3 (Bcl-2 interacting protein 3) was analyzed by co-immunoprecipitation and colocalization.
RESULTS: Following knockdown of TvAP33 expression, the number of T. vaginalis trophozoites adhering to VK2/E6E7 cells decreased significantly, and the inhibition of VK2/E6E7 cell proliferation and VK2/E6E7 cell apoptosis and death induced by T. vaginalis were reduced. Animal challenge experiments showed that the pathogenicity of trophozoites decreased following passive immunization with TvAP33 antiserum or blocking of the TvAP33 protein. Immunofluorescence analysis revealed that TvAP33 could bind to VK2/E6E7 cells. Eighteen protein molecules interacting with TvAP33 were identified by the yeast two-hybrid system. The interaction between TvAP33 and BNIP3 was further confirmed by co-immunoprecipitation and colocalization. When the expression of both TvAP33 and BNIP3 in trophozoites was knocked down by small RNA interference, the number of T. vaginalis adhering to VK2/E6E7 cells and the inhibition of VK2/E6E7 cell proliferation were significantly lower compared to trophozoites with only knockdown of TvAP33 or only BNIP3. Therefore, the interaction of TvAP33 and BNIP3 in the pathogenesis of T. vaginalis infecting host cells is not unique and involves other molecules.
CONCLUSIONS: Our study showed that the interaction between TvAP33 and BNIP3 mediated the adhesion and pathogenicity of T. vaginalis to host cells, providing a basis for searching for drug targets for T. vaginalis as well as new ideas for the prevention and treatment of trichomoniasis.

Carbohydrates are the main source of nutrition for B. coli, supplying energy for cell growth and development. The research aimed at investigating the mechanism of starch on the growth and replication of B. coli. Single-cell separation was used to isolate single trophozoites of B. coli under a stereomicroscope, transcriptomic profiling was conducted based on the SMART-seq2 single-cell RNA-seq method. Comparative genomic analysis was performed on B. coli and eight other ciliates to obtain specific and expanded gene families of B. coli. GO and KEGG enrichment analysis were used to analyze the key genes of B. coli under the action of starch in the present study. The results of single-cell RNA-seq depicts starch affected the growth and replication of B. coli in two ways: (1) the cell cycle was positively promoted by the activation of the cAMP/PKA signaling pathway via glycolysis; (2) the cell autophagy was suppressed through the PI3K/AKT/mTOR pathway. Genes involved in endocytosis, carbohydrate utilization, and the cAMP/PKA signaling pathway were highly enriched in both specific and expanded gene families of B. coli. Starch can be ingested and hydrolyzed into glucose, in turn affecting various biological processes of B. coli. The molecular mechanism of the effect of starch on the growth and proliferation of B. coli by promoting cell cycle and inhibiting the autophagy of trophozoites has been elucidated in our study.

Giardia duodenalis is an important intestinal protozoan parasite, infections of which are frequently seen in cattle and cause intermittent diarrhea and weight loss in young animals around the world. The release of neutrophil extracellular traps (NETs) is an effector mechanism of neutrophils to fight against invading pathogens including parasites. In this present study, we aimed to investigate the effect of Giardia duodenalis trophozoites on bovine NETs formation, and to further examine its basic characteristics and molecular mechanisms. Scanning electron microscopy analyses displayed that Giardia duodenalis trophozoites exposure triggered NET-like filamentary structures released by bovine polymorphonuclear leukocytes (PMNs), and many trophozoites were entrapped within these structures. Immunofluorescence analyses illustrated that these structures were mainly composed of DNA, histones, Myeloperoxidase (MPO) and neutrophil elastase (NE), which confirmed the classical characteristics of NETs. NETs quantification showed that Giardia duodenalis trophozoites significantly increased NETs formation, and it is in a dose-dependent manner from 4:1-1:2 ratio of PMN: trophozoites. Furthermore, pharmacological inhibitory experiment indicated that P2X1 receptor and PAD4 were essential for Giardia duodenalis trophozoites-triggered NETs formation. Additionally, LC3B-based immunostaining analyses revealed that autophagy and NETs formation occurred simultaneously in Giardia duodenalis trophozoites-exposed bovine PMN, imply that autophagy may play a key role in Giardia duodenalis trophozoites-triggered bovine NETs. In summary, these findings suggest that NETs formation might have a crucial role in innate host defense against Giardia duodenalis trophozoites. Hence, we call for future molecular investigations not only on Giardia duodenalis trophozoites-triggered NETs but also on its potential role in giardiasis-related pathology in vivo.

Entamoeba histolytica is an intestinal protozoan parasite with remarkable ability to kill and phagocytose host cells, causing amoebic colitis and extraintestinal abscesses. The intermediate subunit (Igl) of galactose (Gal)- and N-acetyl-d-galactosamine (GalNAc)-specific lectins is considered an important surface antigen involved in the pathogenesis of E. histolytica. Here, we applied mass spectrometry-based quantitative proteomics technology to analyze the protein expression profile changes occurring in host Caco2 cells incubated with E. histolytica trophozoites or stimulated by purified native Igl protein. The expression levels of 1,490 and 489 proteins were significantly altered in the E. histolytica-treated and Igl-treated groups, respectively, among 6,875 proteins totally identified. Intriguingly, central carbon metabolism of host cells was suppressed in both E. histolytica-treated and Igl-treated groups, with evidence of decreased expression levels of several key enzymes, including pyruvate kinase muscle type 2, presenting a Warburg-like effect in host cells. Besides, Igl had potential physical interactions with central carbon metabolism enzymes and the proteolytic degradation family members proteasome subunit alpha and beta, which may be responsible for the degradation of key enzymes in carbon metabolism. These results provided a novel perspective on the pathogenic mechanism of E. histolytica and compelling evidence supporting the important role of Igl in the virulence of E. histolytica. IMPORTANCE 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. In this study, advanced data-independent acquisition mass spectrometry-based quantitative proteomics was applied to investigate the overall host cellular metabolic changes as high-throughput proteomics could measure molecular changes in a cell or tissue with high efficiency. Enrichment analysis of differentially expressed proteins showed biological processes and cellular pathways related to amoeba infection and Igl cytotoxicity. Specifically, central carbon metabolism of host cells was dramatically suppressed in both E. histolytica-treated and Igl-treated groups, indicating the occurrence of a Warburg-like effect induced by trophozoites or Igl from E. histolytica. Distinct differences in ubiquitin-mediated proteolysis, rapamycin (mTOR) signaling pathway, autophagy, endocytosis, and tight junctions provided novel perspectives on the pathogenic mechanism of E. histolytica.

The extracellular protozoan parasite Giardia duodenalis is a well-known and important causative agent of diarrhea on a global scale. Macrophage pyroptosis has been recognized as an important innate immune effector mechanism against intracellular pathogens. Yet, the effects of noninvasive Giardia infection on macrophage pyroptosis and the associated molecular triggers and regulators remain poorly defined. Here we initially observed that NLRP3 inflammasome-mediated pyroptosis was activated in Giardia-treated macrophages, and inhibition of ROS, NLRP3, or caspase-1 could block GSDMD cleavage, IL-1β, IL-18 and LDH release, and the cell viability reduction. We also confirmed that Giardia-induced NLRP3 inflammasome activation was involved in its K63 deubiquitination. Thus, six candidate deubiquitinases were screened, among which A20 was identified as an effective regulator. We then screened TLRs on macrophage membranes and found that upon stimulation TLR4 was tightly correlated to ROS enhancement, A20-mediated NLRP3 deubiquitination, and pyroptotic signaling. In addition, several Giardia-secreted proteins were predicted as trigger factors via secretome analysis, of which peptidyl-prolyl cis-trans isomerase B (PPIB) independently induced macrophage pyroptosis. This was similar to the findings from the trophozoite treatment, and also led to the TLR4-mediated activation of NLRP3 through K63 deubiquitination by A20. Collectively, the results of this study have significant implications for expanding our understanding of host defense mechanisms after infection with G. duodenalis.