scholarly journals Toxoplasma gondii Extends the Life Span of Infected Human Neutrophils by Inducing Cytosolic PCNA and Blocking Activation of Apoptotic Caspases

mBio ◽  
2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Tatiane S. Lima ◽  
Sharmila Mallya ◽  
Allen Jankeel ◽  
Ilhem Messaoudi ◽  
Melissa B. Lodoen
Keyword(s):  
Cell Death ◽  
Toxoplasma Gondii ◽  
Life Span ◽  
Peripheral Blood ◽  
Productive Infection ◽  
Human Neutrophils ◽  
Serum Starvation ◽  
Rna Seq ◽  
Content Type ◽  
Blood Neutrophils

ABSTRACT Toxoplasma gondii is an intracellular protozoan parasite that has the remarkable ability to infect and replicate in neutrophils, immune cells with an arsenal of antimicrobial effector mechanisms. We report that T. gondii infection extends the life span of primary human peripheral blood neutrophils by delaying spontaneous apoptosis, serum starvation-induced apoptosis, and tumor necrosis alpha (TNF-α)-mediated apoptosis. T. gondii blockade of apoptosis was associated with an inhibition of processing and activation of the apoptotic caspases caspase-8 and -3, decreased phosphatidylserine exposure on the plasma membrane, and reduced cell death. We performed a global transcriptome analysis of T. gondii-infected peripheral blood neutrophils using RNA sequencing (RNA-Seq) and identified gene expression changes associated with DNA replication and DNA repair pathways, which in mature neutrophils are indicative of changes in regulators of cell survival. Consistent with the RNA-Seq data, T. gondii infection upregulated transcript and protein expression of PCNA, which is found in the cytosol of human neutrophils, where it functions as a key inhibitor of apoptotic pro-caspases. Infection of neutrophils resulted in increased interaction of PCNA with pro-caspase-3. Inhibition of this interaction with an AlkB homologue 2 PCNA-interacting motif (APIM) peptide reversed the infection-induced delay in cell death. Taken together, these findings indicate a novel strategy by which T. gondii manipulates cell life span in primary human neutrophils, which may allow the parasite to maintain an intracellular replicative niche and avoid immune clearance. IMPORTANCE Toxoplasma gondii is an obligate intracellular parasite that can cause life-threatening disease in immunocompromised individuals and in the developing fetus. Interestingly, T. gondii has evolved strategies to successfully manipulate the host immune system to establish a productive infection and evade host defense mechanisms. Although it is well documented that neutrophils are mobilized during acute T. gondii infection and infiltrate the site of infection, these cells can also be actively infected by T. gondii and serve as a replicative niche for the parasite. However, there is a limited understanding of the molecular processes occurring within T. gondii-infected neutrophils. This study reveals that T. gondii extends the life span of human neutrophils by inducing the expression of PCNA, which prevents activation of apoptotic caspases, thus delaying apoptosis. This strategy may allow the parasite to preserve its replicative intracellular niche.

scholarly journals Loss of the Conserved Alveolate Kinase MAPK2 Decouples Toxoplasma Cell Growth from Cell Division

mBio ◽  
2020 ◽  
Vol 11 (6) ◽  
Author(s):  
Xiaoyu Hu ◽  
William J. O’Shaughnessy ◽  
Tsebaot G. Beraki ◽  
Michael L. Reese
Keyword(s):  
Toxoplasma Gondii ◽  
Protein Kinases ◽  
Daughter Cell ◽  
Protozoan Parasite ◽  
Productive Infection ◽  
Centrosome Duplication ◽  
Loss Of Function ◽  
Content Type ◽  
Mitogen Activated Protein ◽  
Parasite Replication

ABSTRACT Mitogen-activated protein kinases (MAPKs) are a conserved family of protein kinases that regulate signal transduction, proliferation, and development throughout eukaryotes. The apicomplexan parasite Toxoplasma gondii expresses three MAPKs. Two of these, extracellular signal-regulated kinase 7 (ERK7) and MAPKL1, have been implicated in the regulation of conoid biogenesis and centrosome duplication, respectively. The third kinase, MAPK2, is specific to and conserved throughout the Alveolata, although its function is unknown. We used the auxin-inducible degron system to determine phenotypes associated with MAPK2 loss of function in Toxoplasma. We observed that parasites lacking MAPK2 failed to duplicate their centrosomes and therefore did not initiate daughter cell budding, which ultimately led to parasite death. MAPK2-deficient parasites initiated but did not complete DNA replication and arrested prior to mitosis. Surprisingly, the parasites continued to grow and replicate their Golgi apparatus, mitochondria, and apicoplasts. We found that the failure in centrosome duplication is distinct from the phenotype caused by the depletion of MAPKL1. As we did not observe MAPK2 localization at the centrosome at any point in the cell cycle, our data suggest that MAPK2 regulates a process at a distal site that is required for the completion of centrosome duplication and the initiation of parasite mitosis. IMPORTANCE Toxoplasma gondii is a ubiquitous intracellular protozoan parasite that can cause severe and fatal disease in immunocompromised patients and the developing fetus. Rapid parasite replication is critical for establishing a productive infection. Here, we demonstrate that a Toxoplasma protein kinase called MAPK2 is conserved throughout the Alveolata and essential for parasite replication. We found that parasites lacking MAPK2 protein were defective in the initiation of daughter cell budding and were rendered inviable. Specifically, T. gondii MAPK2 (TgMAPK2) appears to be required for centrosome replication at the basal end of the nucleus, and its loss causes arrest early in parasite division. MAPK2 is unique to the Alveolata and not found in metazoa and likely is a critical component of an essential parasite-specific signaling network.

scholarly journals Enhanced migration and adhesion of peripheral blood neutrophils from SAPHO patients revealed by RNA-Seq

2019 ◽  
Vol 14 (1) ◽  
Author(s):  
Yuxiu Sun ◽  
Chen Li ◽  
Mengmeng Zhu ◽  
Shen Zhang ◽  
Yihan Cao ◽  
...  
Keyword(s):  
Peripheral Blood ◽  
Rna Seq ◽  
Blood Neutrophils

scholarly journals Novel Insights into the Molecular Events Linking to Cell Death Induced by Tetracycline in the Amitochondriate Protozoan Trichomonas vaginalis

2015 ◽  
Vol 59 (11) ◽  
pp. 6891-6903 ◽  
Author(s):  
Kuo-Yang Huang ◽  
Fu-Man Ku ◽  
Wei-Hung Cheng ◽  
Chi-Ching Lee ◽  
Po-Jung Huang ◽  
...  
Keyword(s):  
Cell Death ◽  
Trichomonas Vaginalis ◽  
Transmitted Disease ◽  
Chemotherapeutic Agents ◽  
Public Health Issue ◽  
Rna Seq ◽  
Content Type ◽  
Sexually Transmitted ◽  
Molecular Events

ABSTRACTTrichomonas vaginaliscolonizes the human urogenital tract and causes trichomoniasis, the most common nonviral sexually transmitted disease. Currently, 5-nitroimidazoles are the only recommended drugs for treating trichomoniasis. However, increased resistance of the parasite to 5-nitroimidazoles has emerged as a highly problematic public health issue. Hence, it is essential to identify alternative chemotherapeutic agents against refractory trichomoniasis. Tetracycline (TET) is a broad-spectrum antibiotic with activity against several protozoan parasites, but the mode of action of TET in parasites remains poorly understood. Thein vitroeffect of TET on the growth ofT. vaginaliswas examined, and the mode of cell death was verified by various apoptosis-related assays. Next-generation sequencing-based RNA sequencing (RNA-seq) was employed to elucidate the transcriptome ofT. vaginalisin response to TET. We show that TET has a cytotoxic effect on both metronidazole (MTZ)-sensitive and -resistantT. vaginalisisolates, inducing some features resembling apoptosis. RNA-seq data reveal that TET significantly alters the transcriptome via activation of specific pathways, such as aminoacyl-tRNA synthetases and carbohydrate metabolism. Functional analyses demonstrate that TET disrupts the hydrogenosomal membrane potential and antioxidant system, which concomitantly elicits a metabolic shift toward glycolysis, suggesting that the hydrogenosomal function is impaired and triggers cell death. Collectively, we providein vitroevidence that TET is a potential alternative therapeutic choice for treating MTZ-resistantT. vaginalis. The in-depth transcriptomic signatures inT. vaginalisupon TET treatment presented here will shed light on the signaling pathways linking to cell death in amitochondriate organisms.

scholarly journals RIPK3 Facilitates Host Resistance to Oral Toxoplasma gondii Infection

2021 ◽  
Vol 89 (5) ◽  
Author(s):  
Patrick W. Cervantes ◽  
Bruno Martorelli Di Genova ◽  
Billy Joel Erazo Flores ◽  
Laura J. Knoll
Keyword(s):  
Cell Death ◽  
Toxoplasma Gondii ◽  
Immune Cell ◽  
Inflammatory Responses ◽  
Parasite Burden ◽  
Kinase Domain ◽  
Content Type ◽  
Protection Mechanism ◽  
Cell Death Pathway ◽  
Toxoplasma Gondii Infection

ABSTRACT Toxoplasma gondii infection activates pattern recognition receptor (PRR) pathways that drive innate inflammatory responses to control infection. Necroptosis is a proinflammatory cell death pathway apart from the innate immune response that has evolved to control pathogenic infection. In this study, we further defined the role of Z-DNA binding protein 1 (ZBP1) as a PRR and assessed its contribution to necroptosis as a host protection mechanism to T. gondii infection. We found that ZBP1 does not induce proinflammatory necroptosis cell death, and ZBP1 null mice have reduced survival after oral T. gondii infection. In contrast, mice deleted in receptor-interacting serine/threonine-protein kinase 3 (RIPK3−/−), a central mediator of necroptosis, have significantly improved survival after oral T. gondii infection without a reduction in parasite burden. The physiological consequences of RIPK3 activity did not show any differences in intestine villus immunopathology, but RIPK3−/− mice showed higher immune cell infiltration and edema in the lamina propria. The contribution of necroptosis to host survival was clarified with mixed-lineage kinase domain-like pseudokinase null (MLKL−/−) mice. We found MLKL−/− mice succumbed to oral T. gondii infection the same as wild-type mice, indicating necroptosis-independent RIPK3 activity impacts host survival. These results provide new insights on the impacts of proinflammatory cell death pathways as a mechanism of host defense to oral T. gondii infection.

scholarly journals HIV-1 Vpr- and Reverse Transcription-Induced Apoptosis in Resting Peripheral Blood CD4 T Cells and Protection by Common Gamma-Chain Cytokines

2015 ◽  
Vol 90 (2) ◽  
pp. 904-916 ◽  
Author(s):  
Benjamin Trinité ◽  
Chi N. Chan ◽  
Caroline S. Lee ◽  
David N. Levy
Keyword(s):  
T Cells ◽  
Cell Death ◽  
T Cell ◽  
Reverse Transcription ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Productive Infection ◽  
Hiv 1

ABSTRACTHIV-1 infection leads to the progressive depletion of the CD4 T cell compartment by various known and unknown mechanisms.In vivo, HIV-1 infects both activated and resting CD4 T cells, butin vitro, in the absence of any stimuli, resting CD4 T cells from peripheral blood are resistant to infection. This resistance is generally attributed to an intracellular environment that does not efficiently support processes such as reverse transcription (RT), resulting in abortive infection. Here, we show thatin vitroHIV-1 infection of resting CD4 T cells induces substantial cell death, leading to abortive infection.In vivo, however, various microenvironmental stimuli in lymphoid and mucosal tissues provide support for HIV-1 replication. For example, common gamma-chain cytokines (CGCC), such as interleukin-7 (IL-7), render resting CD4 T cells permissible to HIV-1 infection without inducing T cell activation. Here, we find that CGCC primarily allow productive infection by preventing HIV-1 triggering of apoptosis, as evidenced by early release of cytochromecand caspase 3/7 activation. Cell death is triggered both by products of reverse transcription and by virion-borne Vpr protein, and CGCC block both mechanisms. When HIV-1 RT efficiency was enhanced by SIVmac239 Vpx protein, cell death was still observed, indicating that the speed of reverse transcription and the efficiency of its completion contributed little to HIV-1-induced cell death in this system. These results show that a major restriction on HIV-1 infection in resting CD4 T cells resides in the capacity of these cells to survive the early steps of HIV-1 infection.IMPORTANCEA major consequence of HIV-1 infection is the destruction of CD4 T cells. Here, we show that delivery of virion-associated Vpr protein and the process of reverse transcription are each sufficient to trigger apoptosis of resting CD4 T cells isolated from peripheral blood. While these 2 mechanisms have been previously described in various cell types, we show for the first time their concerted effect in inducing resting CD4 T cell depletion. Importantly, we found that cytokines such as IL-7 and IL-4, which are particularly active in sites of HIV-1 replication, protect resting CD4 T cells from these cytopathic effects and, primarily through this protection, rather than through enhancement of specific replicative steps, they promote productive infection. This study provides important new insights for the understanding of the early steps of HIV-1 infection and T cell depletion.

scholarly journals N-Formyl-Perosamine Surface Homopolysaccharides Hinder the Recognition of Brucella abortus by Mouse Neutrophils

2016 ◽  
Vol 84 (6) ◽  
pp. 1712-1721 ◽  
Author(s):  
Ricardo Mora-Cartín ◽  
Carlos Chacón-Díaz ◽  
Cristina Gutiérrez-Jiménez ◽  
Stephany Gurdián-Murillo ◽  
Bruno Lomonte ◽  
...  
Keyword(s):  
Cell Death ◽  
Dendritic Cells ◽  
Brucella Abortus ◽  
Normal Serum ◽  
Mouse Serum ◽  
Human Neutrophils ◽  
Content Type ◽  
Animal System ◽  
Surface Polysaccharide ◽  
Killing Action

Brucella abortusis an intracellular pathogen of monocytes, macrophages, dendritic cells, and placental trophoblasts. This bacterium causes a chronic disease in bovines and in humans. In these hosts, the bacterium also invades neutrophils; however, it fails to replicate and just resists the killing action of these leukocytes without inducing significant activation or neutrophilia. Moreover,B. abortuscauses the premature cell death of human neutrophils. In the murine model, the bacterium is found within macrophages and dendritic cells at early times of infection but seldom in neutrophils. Based on this observation, we explored the interaction of mouse neutrophils withB. abortus. In contrast to human, dog, and bovine neutrophils, naive mouse neutrophils fail to recognize smoothB. abortusbacteria at early stages of infection. Murine normal serum components do not opsonize smoothBrucellastrains, and neutrophil phagocytosis is achieved only after the appearance of antibodies. Alternatively, mouse normal serum is capable of opsonizing roughBrucellamutants. Despite this, neutrophils still fail to killBrucella, and the bacterium induces cell death of murine leukocytes. In addition, mouse serum does not opsonizeYersinia enterocoliticaO:9, a bacterium displaying the same surface polysaccharide antigen as smoothB. abortus. Therefore, the lack of murine serum opsonization and absence of murine neutrophil recognition are specific, and the molecules responsible for theBrucellacamouflage areN-formyl-perosamine surface homopolysaccharides. Although the mouse is a valuable model for understanding the immunobiology of brucellosis, direct extrapolation from one animal system to another has to be undertaken with caution.

scholarly journals Enzymatic Dysfunction of Mitochondrial Complex I of the Candida albicansgoa1Mutant Is Associated with Increased Reactive Oxidants and Cell Death

2011 ◽  
Vol 10 (5) ◽  
pp. 672-682 ◽  
Author(s):  
Dongmei Li ◽  
Hui Chen ◽  
Abigail Florentino ◽  
Deepu Alex ◽  
Patricia Sikorski ◽  
...  
Keyword(s):  
Cell Death ◽  
Complex I ◽  
Alternative Oxidase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Potassium Cyanide ◽  
Human Neutrophils ◽  
Oxidase Gene ◽  
Content Type ◽  
Chronological Aging

ABSTRACTWe have previously shown that deletion ofGOA1(growth andoxidantadaptation) ofCandida albicansresults in a loss of mitochondrial membrane potential, ATP synthesis, increased sensitivity to oxidants and killing by human neutrophils, and avirulence in a systemic model of candidiasis. We established that translocation of Goa1p to mitochondria occurred during peroxide stress. In this report, we show that thegoa1Δ (GOA31), compared to the wild type (WT) and a gene-reconstituted (GOA32) strain, exhibits sensitivity to inhibitors of the classical respiratory chain (CRC), including especially rotenone (complex I [CI]) and salicylhydroxamic acid (SHAM), an inhibitor of the alternative oxidase pathway (AOX), while potassium cyanide (KCN; CIV) causes a partial inhibition of respiration. In the presence of SHAM, however, GOA31 has an enhanced respiration, which we attribute to the parallel respiratory (PAR) pathway and alternative NADH dehydrogenases. Interestingly, deletion ofGOA1also results in a decrease in transcription of the alternative oxidase geneAOX1in untreated cells as well as negligibleAOX1andAOX2transcription in peroxide-treated cells. To explain the rotenone sensitivity, we measured enzyme activities of complexes I to IV (CI to CIV) and observed a major loss of CI activity in GOA31 but not in control strains. Enzymatic data of CI were supported by blue native polyacrylamide gel electrophoresis (BN-PAGE) experiments which demonstrated less CI protein and reduced enzyme activity. The consequence of a defective CI in GOA31 is an increase in reactive oxidant species (ROS), loss of chronological aging, and programmed cell death ([PCD] apoptosis)in vitrocompared to control strains. The increase in PCD was indicated by an increase in caspase activity and DNA fragmentation in GOA31. Thus,GOA1is required for a functional CI and partially for the AOX pathway; loss ofGOA1compromises cell survival. Further, the loss of chronological aging is new to studies ofCandidaspecies and may offer an insight into therapies to control these pathogens. Our observation of increased ROS production associated with a defective CI and PCD is reminiscent of mitochondrial studies of patients with some types of neurodegenerative diseases where CI and/or CIII dysfunctions lead to increased ROS and apoptosis.

scholarly journals Celastrol attenuates fMLP-induced superoxide anion generation, myeloperoxidase production, and elastase release by human neutrophils

2021 ◽  
Vol 18 (9) ◽  
pp. 1805-1809
Author(s):  
Nipapan Malisorn ◽  
Ammara Chaikan
Keyword(s):  
Peripheral Blood ◽  
Human Peripheral Blood ◽  
Human Neutrophils ◽  
Superoxide Generation ◽  
Xtt Assay ◽  
Dependent Inhibition ◽  
Blood Neutrophils ◽  
Ic50 Values ◽  
Dose Dependent Inhibition ◽  
Anti Inflammatory

Purpose: To investigate the anti-inflammatory effect of celastrol via attenuation of formyl-methionylleucyl-phenylalanine (fMLP)-induced superoxide generation, myeloperoxidase production, and elastase release by peripheral blood neutrophils. Methods: Cytotoxicity of celastrol on human peripheral blood neutrophils was investigated using a 2Htetrazolium hydroxide (XTT) assay. Human neutrophils were stimulated with 100-nM fMLP; the effect of celastrol on superoxide generation was determined via ferricytochrome C reduction, the effect on myeloperoxidase production by tetramethylbenzidine oxidation, and the effect on elastase activity by Boc-Ala-ONp hydrolysis. Results: Treatment of human neutrophils with celastrol showed dose-dependent inhibition of fMLPinduced superoxide generation, myeloperoxidase production, and elastase release with half-maximal inhibitory concentration (IC50) values of 5.9 ± 0.1, 1.9 ± 0.2, and 1.5 ± 0.1 µM, respectively. Conclusion: These results indicate that celastrol possesses anti-inflammatory properties via attenuation of fMLP-induced superoxide generation, myeloperoxidase production, and elastase release by peripheral blood neutrophils.

scholarly journals Cell Death of Gamma Interferon-Stimulated Human Fibroblasts upon Toxoplasma gondii Infection Induces Early Parasite Egress and Limits Parasite Replication

2013 ◽  
Vol 81 (12) ◽  
pp. 4341-4349 ◽  
Author(s):  
Wendy Niedelman ◽  
Joris K. Sprokholt ◽  
Barbara Clough ◽  
Eva-Maria Frickel ◽  
Jeroen P. J. Saeij
Keyword(s):  
Cell Death ◽  
Toxoplasma Gondii ◽  
Human Fibroblasts ◽  
Protozoan Parasite ◽  
Resistance Mechanisms ◽  
Gamma Interferon ◽  
Content Type ◽  
Food Borne Illness ◽  
Ifn Γ ◽  
Toxoplasma Gondii Infection

ABSTRACTThe intracellular protozoan parasiteToxoplasma gondiiis a major food-borne illness and opportunistic infection for the immunosuppressed. Resistance toToxoplasmais dependent on gamma interferon (IFN-γ) activation of both hematopoietic and nonhematopoietic cells. Although IFN-γ-induced innate immunity in nonhematopoietic cells has been extensively studied in mice, it remains unclear what resistance mechanisms are relied on in nonhematopoietic human cells. Here, we report an IFN-γ-induced mechanism of resistance toToxoplasmain primary human foreskin fibroblasts (HFFs) that does not depend on the deprivation of tryptophan or iron. In addition, infection is still controlled in HFFs deficient in the p65 guanylate binding proteins GBP1 or GBP2 and the autophagic protein ATG5. Resistance is coincident with host cell death that is not dependent on the necroptosis mediator RIPK3 or caspases and is correlated with early egress of the parasite before replication. This IFN-γ-induced cell death and early egress limits replication in HFFs and could promote clearance of the parasite by immune cells.

scholarly journals Wheat Germ Agglutinin Induces NADPH-Oxidase Activity in Human Neutrophils by Interaction with Mobilizable Receptors

1999 ◽  
Vol 67 (7) ◽  
pp. 3461-3468 ◽  
Author(s):  
Anna Karlsson
Keyword(s):  
Sialic Acid ◽  
Nadph Oxidase ◽  
Peripheral Blood ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Human Neutrophils ◽  
Extracellular Milieu ◽  
Blood Neutrophils

ABSTRACT Wheat germ agglutinin (WGA), a lectin with specificity forN-acetylglucosamine and sialic acid, was investigated with respect to its ability to activate the NADPH-oxidase of in vivo-exudated neutrophils (obtained from a skin chamber), and the activity was compared to that of peripheral blood neutrophils. The exudate cells responded to WGA, by both releasing reactive oxygen species into the extracellular milieu and producing oxygen metabolites intracellularly. The peripheral blood cells were unresponsive. To mimic the in vivo-exuded neutrophils with regards to receptor exposure, peripheral blood neutrophils were induced to mobilize their granules and vesicles to varying degrees (in vitro priming), prior to challenge with WGA. The oxidative response to WGA increased with increasing levels of granule mobilization, and the receptor(s) could be shown to reside in the secretory vesicles and/or the gelatinase granules in resting neutrophils. Several WGA-binding glycoproteins were detected in subcellular fractions containing these organelles. The extra- and intracellular NADPH-oxidase responses showed differences in sialic acid dependency, indicating that these two responses are mediated by different receptor structures.

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