Parasite-infected Cells Research Articles

Proteome profile of Leishmania donovani Centrin1−/− parasite-infected human macrophage cell line and its implications in determining possible mechanisms of protective immunity

Our developed cell division-specific ‘centrin’ gene deleted Leishmania donovani (LdCen1−/−) the causative parasite of the fatal visceral-leishmaniasis (VL), exhibits a selective growth arrest at the intracellular stage and is anticipated as a live attenuated vaccine candidate against VL. LdCen1−/− immunization in animals has shown increased IFN-γ secreting CD4+ and CD8+ T cells along with protection conferred by a protective proinflammatory immune response. A label-free proteomics approach has been employed to understand the physiology of infection and predict disease interceptors during Leishmania-host interactions. Proteomic modulation after infection of human macrophage cell lines suggested elevated annexin A6, implying involvement in various biological processes such as membrane repair, transport, actin dynamics, cell proliferation, survival, differentiation, and inflammation, thereby potentiating its immunological protective capacity. Additionally, S100A8 and S100A9 proteins, known for maintaining homeostatic balance in regulating the inflammatory response, have been upregulated after infection. The inhibitory clade of serpins, known to inhibit cysteine proteases (CPs), was upregulated in host cells after 48 h of infection. This is reflected in the diminished expression of CPs in the parasites during infection. Such proteome analysis confirms LdCen1−/− efficacy as a vaccine candidate and predicts potential markers in future vaccine development strategies against infectious diseases.

Leishmanicidal Activity of Moringa oleifera (L.) Leaf Protein Extract on the Indian Strain of Leishmania donovani: An In Vitro Study

IntroductionRecurring incidents of drug resistance and the health hazards associated with the chemotherapeutic treatment of visceral leishmaniasis necessitate the search for safer yet equally effective biologically available alternate therapeutics. Plant products are being emphasised in this regard. Thus, Moringa oleifera leaf was chosen here, and an in vitro study was undertaken to determine its antileishmanial activity against visceral leishmaniasis. MethodsThe protein in M. oleifera leaf extract (Mo-EX) was identified by denaturing polyacrylamide gel electrophoresis. The antipromastigote activity and cytotoxicity of the proteinaceous Mo-EX were determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Phase contrast microscopy and scanning electron microscopy analyses confirmed the morphological alterations of the promastigotes. The anti-amastigote activity of Mo-EX was determined by microscopic intracellular counting. Apoptosis, reactive oxygen species (ROS) generation, and thiol depletion were assessed by flow cytometric analysis, and nitric oxide production was determined by colorimetric assays. ResultsM. oleifera leaf extract displayed 50% inhibitory concentration valuesagainst Leishmania donovani promastigotes and intracellular amastigotes of 11.85 ± 0.08 and 8.86 ± 0.03 μg/ml, respectively, with a 50% lethal concentration in RAW 264.7 macrophages of 2 036.25 ± 0.04 μg/ml. Mo-EX reduced thiol production, significantly increased ROS generation, and induced apoptosis in L. donovani promastigotes while altering their morphology. Mo-EX evoked ROS and nitric oxide generation in parasite-infected host cells ex vivo. ConclusionsThe findings of the present study elucidate the leishmanicidal activity of protein-rich Mo-EX in L. donovani (MHOM/IN/1983/AG83) promastigotes and amastigotes, and Mo-EX was found to be moderately safe in RAW 264.7 host macrophages in vitro.

3D reconstructions of parasite development and the intracellular niche of the microsporidian pathogen Encephalitozoon intestinalis

Microsporidia are an early-diverging group of fungal pathogens with a wide host range. Several microsporidian species cause opportunistic infections in humans that can be fatal. As obligate intracellular parasites with highly reduced genomes, microsporidia are dependent on host metabolites for successful replication and development. Our knowledge of microsporidian intracellular development remains rudimentary, and our understanding of the intracellular niche occupied by microsporidia has relied on 2D TEM images and light microscopy. Here, we use serial block-face scanning electron microscopy (SBF-SEM) to capture 3D snapshots of the human-infecting species, Encephalitozoon intestinalis, within host cells. We track E. intestinalis development through its life cycle, which allows us to propose a model for how its infection organelle, the polar tube, is assembled de novo in developing spores. 3D reconstructions of parasite-infected cells provide insights into the physical interactions between host cell organelles and parasitophorous vacuoles, which contain the developing parasites. The host cell mitochondrial network is substantially remodeled during E. intestinalis infection, leading to mitochondrial fragmentation. SBF-SEM analysis shows changes in mitochondrial morphology in infected cells, and live-cell imaging provides insights into mitochondrial dynamics during infection. Our data provide insights into parasite development, polar tube assembly, and microsporidia-induced host mitochondria remodeling.

Selection of genotypes harbouring mutations in the cytochrome b gene of Theileria annulata is associated with resistance to buparvaquone.

Buparvaquone remains the only effective therapeutic agent for the treatment of tropical theileriosis caused by Theileria annulata. However, an increase in the rate of buparvaquone treatment failures has been observed in recent years, raising the possibility that resistance to this drug is associated with the selection of T. annulata genotypes bearing mutation(s) in the cytochrome b gene (Cyto b). The aim of the present study was: (1) to demonstrate whether there is an association between mutations in the T. annulata Cyto b gene and selection of parasite-infected cells resistant to buparvaquone and (2) to determine the frequency of these mutations in parasites derived from infected cattle in the Aydın region of Türkiye. Susceptibility to buparvaquone was assessed by comparing the proliferative index of schizont-infected cells obtained from cattle with theileriosis before and/or after treatment with various doses of buparvaquone, using the 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colourimetric assay. The DNA sequence of the parasite Cyto b gene from cell lines identified as resistant or susceptible was determined. A total of six nonsynonymous and six synonymous mutations were identified. Two of the nonsynonymous mutations resulted in the substitutions V135A and P253S which are located at the putative buparvaquone binding regions of cytochrome b. Allele-specific PCR (AS-PCR) analyses detected the V135A and P253S mutations at a frequency of 3.90% and 3.57% respectively in a regional study population and revealed an increase in the frequency of both mutations over the years. The A53P mutation of TaPIN1 of T. annulata, previously suggested as being involved in buparvaquone resistance, was not detected in any of the clonal cell lines examined in the present study. The observed data strongly suggested that the genetic mutations resulting in V135A and P253S detected at the putative binding sites of buparvaquone in cytochrome b play a significant role in conferring, and promoting selection of, T. annulata genotypes resistant to buparvaquone, whereas the role of mutations in TaPIN1 is more equivocal.

Single-Cell Mass Spectrometry Enables Insight into Heterogeneity in Infectious Disease.

Cellular heterogeneity is generally overlooked in infectious diseases. In this study, we investigated host cell heterogeneity during infection with Trypanosoma cruzi (T. cruzi) parasites, causative agents of Chagas disease (CD). In chronic-stage CD, only a few host cells are infected with a large load of parasites and symptoms may appear at sites distal to parasite colonization. Furthermore, recent work has revealed T. cruzi heterogeneity with regard to replication rates and drug susceptibility. However, the role of cellular-level metabolic heterogeneity in these processes has yet to be assessed. To fill this knowledge gap, we developed a Single-probe SCMS (single-cell mass spectrometry) method compatible with biosafety protocols, to acquire metabolomics data from individual cells during T. cruzi infection. This study revealed heterogeneity in the metabolic response of the host cells to T. cruzi infection in vitro. Our results showed that parasite-infected cells possessed divergent metabolism compared to control cells. Strikingly, some uninfected cells adjacent to infected cells showed metabolic impacts as well. Specific metabolic changes include increases in glycerophospholipids with infection. These results provide novel insight into the pathogenesis of CD. Furthermore, they represent the first application of bioanalytical SCMS to the study of mammalian-infectious agents, with the potential for broad applications to study infectious diseases.

Malaria parasite classification framework using a novel channel squeezed and boosted CNN.

Malaria is a life-threatening infection that infects the red blood cells and gradually grows throughout the body. The plasmodium parasite is transmitted by a female Anopheles mosquito bite and severely affects numerous individuals within the world every year. Therefore, early detection tests are required to identify parasite-infected cells. The proposed technique exploits the learning capability of deep convolutional neural network (CNN) to distinguish the parasite-infected patients from healthy individuals using thin blood smear. In this regard, the detection is accomplished using a novel STM-SB-RENet block-based CNN that employs the idea of split-transform-merge (STM) and channel squeezing-boosting (SB) in a modified fashion. In this connection, a new convolutional block-based STM is developed, which systematically implements region and edge operations to explore the parasitic infection pattern of malaria related to region homogeneity, structural obstruction and boundary-defining features. Moreover, the diverse boosted feature maps are achieved by incorporating the new channel SB and transfer learning (TL) idea in each STM block at abstract, intermediate and target levels to capture minor contrast and texture variation between parasite-infected and normal artifacts. The malaria input images for the proposed models are initially transformed using discrete wavelet transform to generate enhanced and reduced feature space. The proposed architectures are validated using hold-out cross-validation on the National Institute of Health Malaria dataset. The proposed methods outperform training from scratch and TL-based fine-tuned existing techniques. The considerable performance (accuracy: 97.98%, sensitivity: 0.988, F-score: 0.980 and area under the curve: 0.996) of STM-SB-RENet suggests that it can be utilized to screen malaria-parasite-infected patients. Graphical Abstract.

Leishmania infantum Defective in Lipophosphoglycan Biosynthesis Interferes With Activation of Human Neutrophils

Visceral leishmaniasis (VL) is often associated with hematologic manifestations that may interfere with neutrophil response. Lipophosphoglycan (LPG) is a major molecule on the surface of Leishmania promastigotes, which has been associated with several aspects of the parasite–vector–host interplay. Here, we investigated how LPG from Leishmania (L.) infantum, the principal etiological agent of VL in the New World, influences the initial establishment of infection during interaction with human neutrophils in an experimental setting in vitro. Human neutrophils obtained from peripheral blood samples were infected with either the wild-type L. infantum (WT) strain or LPG-deficient mutant (∆lpg1). In this setting, ∆lpg1 parasites displayed reduced viability compared to WT L. infantum; such finding was reverted in the complemented ∆lpg1+LPG1 parasites at 3- and 6-h post-infection. Confocal microscopy experiments indicated that this decreased survival was related to enhanced lysosomal fusion. In fact, LPG-deficient L. infantum parasites more frequently died inside neutrophil acidic compartments, a phenomenon that was reverted when host cells were treated with Wortmannin. We also observed an increase in the secretion of the neutrophil collagenase matrix metalloproteinase-8 (MMP-8) by cells infected with ∆lpg1 L. infantum compared to those that were infected with WT parasites. Furthermore, collagen I matrix degradation was found to be significantly increased in ∆lpg1 parasite-infected cells but not in WT-infected controls. Flow cytometry analysis revealed a substantial boost in production of reactive oxygen species (ROS) during infection with either WT or ∆lpg1 L. infantum. In addition, killing of ∆lpg1 parasites was shown to be more dependent on the ROS production than that of WT L. infantum. Notably, inhibition of the oxidative stress with Apocynin potentially fueled ∆lpg1 L. infantum fitness as it increased the intracellular parasite viability. Thus, our observations demonstrate that LPG may be a critical molecule fostering parasite survival in human neutrophils through a mechanism that involves cellular activation and generation of free radicals.

MMV560185 from pathogen box induces extrinsic pathway of apoptosis in Theileria annulata infected bovine leucocytes

Tropical theileriosis is a lymphoproliferative disease caused by the intracellular schizonts of Theileria annulata, an apicomplexan parasite. It causes severe infection in cattle and the untreated cattle would possibly die within 3–4 weeks of infection. The chemotherapy for this disease is largely dependent on the use of hydroxynaphthoquinone, namely buparvaquone. There have been reports recently of the development of resistance against this drug in T. annulata. Hence, identification of new drug molecule(s) or repurposing of existing drug molecule(s) against T. annulata is quite important. Here, we present the screening of 400 compounds included in the open-access Pathogen box from Medicine for Malaria Venture (MMV) to discover the novel compounds with potential inhibitory activity against T. annulata infected bovine leucocytes. We identified two compounds, MMV000062 and MMV560185, with IC50 values of 2.97 μM and 3.07 μM, respectively. MMV000062 and MMV560185 were found non-toxic to BoMac cells with CC50 values 34 μM and > 100 μM, respectively. The therapeutic indices of these compounds, MMV000062 and MMV560185, were calculated as more than 33 and 11, respectively. Further, it was observed that the parasite-infected cells under long-term culture were unable to recover with these compounds. We further deciphered that MMV560185 kills the infected cell by activation of TNFR-1 mediated extrinsic pathway of the apoptosis. The phenotypic characteristics of apoptosis were confirmed by Transmission Electron Microscopy. Our results suggest that it may be possible to develop MMV560185 further for chemotherapeutics of tropical theilerosis.

Spatial Point Pattern Analysis Identifies Mechanisms Shaping the Skin Parasite Landscape in Leishmania donovani Infection.

Increasing evidence suggests that in hosts infected with parasites of the Leishmania donovani complex, transmission of infection to the sand fly vector is linked to parasite repositories in the host skin. However, a detailed understanding of the dispersal (the mechanism of spread) and dispersion (the observed state of spread) of these obligatory-intracellular parasites and their host phagocytes in the skin is lacking. Using endogenously fluorescent parasites as a proxy, we apply image analysis combined with spatial point pattern models borrowed from ecology to characterize dispersion of parasitized myeloid cells (including ManR+ and CD11c+ cells) and predict dispersal mechanisms in a previously described immunodeficient model of L. donovani infection. Our results suggest that after initial seeding of infection in the skin, heavily parasite-infected myeloid cells are found in patches that resemble innate granulomas. Spread of parasites from these initial patches subsequently occurs through infection of recruited myeloid cells, ultimately leading to self-propagating networks of patch clusters. This combination of imaging and ecological pattern analysis to identify mechanisms driving the skin parasite landscape offers new perspectives on myeloid cell behavior following parasitism by L. donovani and may also be applicable to elucidating the behavior of other intracellular tissue-resident pathogens and their host cells.

Proteomic identification of the contents of small extracellular vesicles from in vivo Plasmodium yoelii infection

Small extracellular vesicles, including exosomes, are formed by the endocytic pathway and contain genetic and protein material which reflect the contents of their cells of origin. These contents have a role in vesicle-mediated information transfer, as well as physiological and pathological functions. Thus, these vesicles are of great interest as therapeutic targets, or as vehicles for immunomodulatory control. In Plasmodium spp. infections, vesicles derived from the parasite or parasite-infected cells have been shown to induce the expression of pro-inflammatory elements, which have been correlated with manifestations of clinical disease. Herein, we characterised the protein cargo of naturally occurring sEVs in the plasma of P. yoelii-infected mice. After in vivo infections, extracellular vesicles in the size range of exosomes were collected by sequential centrifugation/ultracentrifugation followed by isopycnic gradient separation. Analysis of the vesicles was performed by transmission electron microscopy, dynamic light scattering, SDS–PAGE and flow cytometry. LC-MS analysis followed by bioinformatics analysis predicted parasite protein cargo associated with exosomes. Within these small extracellular vesicles, we identified proteins of interest as vaccine candidates, uncharacterized proteins which may be targets of T cell immunoreactivity, and proteins involved in metabolic processes, regulation, homeostasis and immunity. Importantly, the small extracellular vesicles studied in our work were obtained from in vivo infection rather than from the supernatant of in vitro cultures. These findings add to the growing interest in parasite small extracellular vesicles, further our understanding of the interactions between host and parasite, and identify novel proteins which may represent potential targets for vaccination against malaria.

Inflammation driven tumor-like signaling in prostatic epithelial cells by sexually transmitted Trichomonas vaginalis.

To identify the sequence of inflammation-driven signaling cascades and other molecular events that might cause tumor-like transformation of prostatic cells. Cytokine array analysis, Reactome and STRING analysis, immunoblotting, and immunocytochemistry were used to investigate the molecular mechanisms governing inflammation-driven adverse changes in human prostatic cells caused by the sexually transmitted infection, Trichomonas vaginalis, resulting in prostatitis, benign prostatic hyperplasia and prostate cancer. Array analysis showed upregulation of 23 cytokines within 24h of infection of human prostatic epithelial RWPE-1 cells with the parasite, invitro. Reactome and STRING analysis of array data identified interleukin-6, interleukin-8, nuclear factor kappa B, signal transducer and activator of transcription3 and cyclooxygenase2 as chief instigators of prostatic anomaly, which were found to be significantly upregulated by immunofluorescence and western blotting analyses. STRING further connected these instigators with macrophage migration inhibitory factor, PIM-1 and prostate-specific antigen; which was confirmed by their marked stimulation in infected prostatic cells by immunoblotting and immunocytochemistry. Upregulated proliferation markers, such as Ki67, proliferating cell nuclear antigen and B-cell lymphoma2, suggested tumor-like signaling in infected RWPE-1 cells, which was further supported by downregulation of E-cadherin, upregulation of vimentin and activation of focal adhesion kinase. Prostate tumor DU145 cells were more sensitive to parasite invasion, and showed rapid upregulation with nuclear translocation of sensitive parameters, such as nuclear factor kappa B, signal transducer and activator of transcription3, and macrophage migration inhibitory factor. The migration of DU145 cells augmented when incubated in spent media from parasite-infected RWPE-1 cells. The initiation of inflammation driven tumor-like cell signaling in parasite-infected human prostatic epithelial cells is apparent, with the prostate tumor (DU145) cells being more sensitive to T. vaginalis than normal (RWPE-1) prostatic cells.

Naïve CD8 T cell IFNγ responses to a vacuolar antigen are regulated by an inflammasome-independent NLRP3 pathway and Toxoplasma gondii ROP5.

Host resistance to Toxoplasma gondii relies on CD8 T cell IFNγ responses, which if modulated by the host or parasite could influence chronic infection and parasite transmission between hosts. Since host-parasite interactions that govern this response are not fully elucidated, we investigated requirements for eliciting naïve CD8 T cell IFNγ responses to a vacuolar resident antigen of T. gondii, TGD057. Naïve TGD057 antigen-specific CD8 T cells (T57) were isolated from transnuclear mice and responded to parasite-infected bone marrow-derived macrophages (BMDMs) in an antigen-dependent manner, first by producing IL-2 and then IFNγ. T57 IFNγ responses to TGD057 were independent of the parasite’s protein export machinery ASP5 and MYR1. Instead, host immunity pathways downstream of the regulatory Immunity-Related GTPases (IRG), including partial dependence on Guanylate-Binding Proteins, are required. Multiple T. gondii ROP5 isoforms and allele types, including ‘avirulent’ ROP5A from clade A and D parasite strains, were able to suppress CD8 T cell IFNγ responses to parasite-infected BMDMs. Phenotypic variance between clades B, C, D, F, and A strains suggest T57 IFNγ differentiation occurs independently of parasite virulence or any known IRG-ROP5 interaction. Consistent with this, removal of ROP5 is not enough to elicit maximal CD8 T cell IFNγ production to parasite-infected cells. Instead, macrophage expression of the pathogen sensors, NLRP3 and to a large extent NLRP1, were absolute requirements. Other members of the conventional inflammasome cascade are only partially required, as revealed by decreased but not abrogated T57 IFNγ responses to parasite-infected ASC, caspase-1/11, and gasdermin D deficient cells. Moreover, IFNγ production was only partially reduced in the absence of IL-12, IL-18 or IL-1R signaling. In summary, T. gondii effectors and host machinery that modulate parasitophorous vacuolar membranes, as well as NLR-dependent but inflammasome-independent pathways, determine the full commitment of CD8 T cells IFNγ responses to a vacuolar antigen.

Perils and Promises of Pathogenic Protozoan Extracellular Vesicles.

Extracellular vesicles (EVs) are membranous structures formed during biological processes in living organisms. For protozoan parasites, secretion of EVs can occur directly from the parasite organellar compartments and through parasite-infected or antigen-stimulated host cells in response to in vitro and in vivo physiological stressors. These secreted EVs characteristically reflect the biochemical features of their parasitic origin and activating stimuli. Here, we review the species-specific morphology and integrity of parasitic protozoan EVs in concurrence with the origin, functions, and internalization process by recipient cells. The activating stimuli for the secretion of EVs in pathogenic protozoa are discoursed alongside their biomolecules and specific immune cell responses to protozoan parasite-derived EVs. We also present some insights on the intricate functions of EVs in the context of protozoan parasitism.

Intracellular Parasites Toxoplasma gondii and Besnoitia besnoiti, Unveiled in Single Host Cells Using AP-SMALDI MS Imaging

The obligate intracellular apicomplexan parasites Toxoplasma gondii and Besnoitia besnoiti are important causes of disease in both humans and cattle. To date, effective specific treatments are lacking for both infections. To counteract severe symptoms leading to, e.g., disabilities and even abortion in the case of human toxoplasmosis and bovine besnoitiosis, novel targets are required for development of drugs and vaccines. A promising emerging technique for molecular characterization of organisms is high-resolution atmospheric-pressure scanning microprobe matrix-assisted laser desorption/ionization (AP-SMALDI) mass spectrometry imaging (MSI) which enables semiquantitative visualization of metabolite distributions. MSI was here used to trace and characterize lipid metabolites in primary bovine umbilical vein endothelial cells (BUVECs) upon infection with tachyzoites, an early and pathogenic fast-replicating life stage of T. gondii and B. besnoiti. A cell bulk, derived from noninfected controls and parasite-infected cell pellets, was analyzed by AP-SMALDI MSI in technical and biological triplicates. Multivariate statistical analysis including hierarchical clustering and principle component analysis revealed infection-specific metabolites in both positive- and negative-ion mode, identified by combining database search and LC-MS2 experiments. MSI analyses of host cell monolayers were conducted at 5 μm lateral resolution, allowing single apicomplexan-infected cells to be allocated. This is the first mass spectrometry imaging study on intracellular T. gondii and B. besnoiti infections and the first detailed metabolomic characterization of B. besnoiti tachyzoites. MSI was used here as an efficient tool to discriminate infected from noninfected cells at the single-cell level in vitro.

In-depth comparison of cell-based methodological approaches to determine drug susceptibility of visceral Leishmania isolates.

Monitoring the drug susceptibility of Leishmania isolates still largely relies on standard in vitro cell-based susceptibility assays using (patient-isolated) promastigotes for infection. Although this assay is widely used, no fully standardized/harmonized protocol is yet available hence resulting in the application of a wide variety of host cells (primary cells and cell lines), different drug exposure times, detection methods and endpoint criteria. Advocacy for standardization to decrease inter-laboratory variation and improve interpretation of results has already repeatedly been made, unfortunately still with unsatisfactory progress. As a logical next step, it would be useful to reach at least some agreement on the type of host cell and basic experimental design for routine amastigote susceptibility determination. The present laboratory study using different L. infantum strains as a model for visceral leishmaniasis species compared primary cells (mouse peritoneal exudate (PEC), mouse bone marrow derived macrophages and human peripheral blood monocyte derived macrophages) and commercially available cell lines (THP-1, J774, RAW) for either their susceptibility to infection, their role in supporting intracellular amastigote multiplication and overall feasibility/accessibility of experimental assay protocol. The major findings were that primary cells are better than cell lines in supporting infection and intracellular parasite multiplication, with PECs to be preferred for technical reasons. Cell lines require drug exposure of >96h with THP-1 to be preferred but subject to a variable response to PMA stimulation. The fast dividing J774 and RAW cells out-compete parasite-infected cells precluding proper assay read-out. Some findings could possibly also be applicable to cutaneous Leishmania strains, but this still needs cross-checking. Besides inherent limitations in a clinical setting, susceptibility testing of clinical isolates may remain problematic because of the reliance on patient-derived promastigotes which may exhibit variable degrees of metacyclogenesis and infectivity.

Changes in the Molecular and Functional Phenotype of Bovine Monocytes during Theileria parva Infection.

Theileria parva is the causative agent of East Coast fever (ECF), a tick-borne disease that kills over a million cattle each year in sub-Saharan Africa. Immune protection against T. parva involves a CD8+ cytotoxic T cell response to parasite-infected cells. However, there is currently a paucity of knowledge regarding the role played by innate immune cells in ECF pathogenesis and T. parva control. Here, we demonstrate an increase in intermediate monocytes (CD14++ CD16+) with a concomitant decrease in the classical (CD14++ CD16-) and nonclassical (CD14+ CD16+) subsets at 12 days postinfection (dpi) during lethal infection but not during nonlethal T. parva infection. Ex vivo analyses of monocytes demonstrated upregulation of interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α) mRNA and increased nitric oxide production during T. parva lethal infection compared to nonlethal infection at 10 dpi. Interestingly, no significant differences in peripheral blood parasite loads were observed between lethally and nonlethally infected animals at 12 dpi. In vitro stimulation with T. parva schizont-infected cells or Escherichia coli lipopolysaccharide (LPS) resulted in significant upregulation of IL-1β production by monocytes from lethally infected cattle compared to those from nonlethally infected animals. Strikingly, monocytes from lethally infected animals produced significant amounts of IL-10 mRNA after stimulation with T. parva schizont-infected cells. In conclusion, we demonstrate that T. parva infection leads to alterations in the molecular and functional phenotypes of bovine monocytes. Importantly, since these changes primarily occur in lethal infection, they can serve as biomarkers for ECF progression and severity, thereby aiding in the standardization of protection assessment for T. parva candidate vaccines.

Rhoptry and Dense Granule Secreted Effectors Regulate CD8+ T Cell Recognition of Toxoplasma gondii Infected Host Cells.

Toxoplasma gondii secretes rhoptry (ROP) and dense granule (GRA) effector proteins to evade host immune clearance mediated by interferon gamma (IFN-γ), immunity-related GTPase (IRG) effectors, and CD8+ T cells. Here, we investigated the role of parasite-secreted effectors in regulating host access to parasitophorous vacuole (PV) localized parasite antigens and their presentation to CD8+ T cells by the major histocompatibility class I (MHC-I) pathway. Antigen presentation of PV localized parasite antigens by MHC-I was significantly increased in macrophages and/or dendritic cells infected with mutant parasites that lacked expression of secreted GRA (GRA2, GRA3, GRA4, GRA5, GRA7, GRA12) or ROP (ROP5, ROP18) effectors. The ability of various secreted GRA or ROP effectors to suppress antigen presentation by MHC-I was dependent on cell type, expression of IFN-γ, or host IRG effectors. The suppression of antigen presentation by ROP5, ROP18, and GRA7 correlated with a role for these molecules in preventing PV disruption by IFN-γ-activated host IRG effectors. However, GRA2 mediated suppression of antigen presentation was not correlated with PV disruption. In addition, the GRA2 antigen presentation phenotypes were strictly co-dependent on the expression of the GRA6 protein. These results show that MHC-I antigen presentation of PV localized parasite antigens was controlled by mechanisms that were dependent or independent of IRG effector mediated PV disruption. Our findings suggest that the GRA6 protein underpins an important mechanism that enhances CD8+ T cell recognition of parasite-infected cells with damaged or ruptured PV membranes. However, in intact PVs, parasite secreted effector proteins that associate with the PV membrane or the intravacuolar network membranes play important roles to actively suppress antigen presentation by MHC-I to reduce CD8+ T cell recognition and clearance of Toxoplasma gondii infected host cells.

Secreted parasite Pin1 isomerase stabilizes host PKM2 to reprogram host cell metabolism

Metabolic reprogramming is an important feature of host–pathogen interactions and a hallmark of tumorigenesis. The intracellular apicomplexa parasite Theileria induces a Warburg-like effect in host leukocytes by hijacking signaling machineries, epigenetic regulators and transcriptional programs to create a transformed cell state. The molecular mechanisms underlying host cell transformation are unclear. Here we show that a parasite-encoded prolyl-isomerase, TaPin1, stabilizes host pyruvate kinase isoform M2 (PKM2) leading to HIF-1α-dependent regulation of metabolic enzymes, glucose uptake and transformed phenotypes in parasite-infected cells. Our results provide a direct molecular link between the secreted parasite TaPin1 protein and host gene expression programs. This study demonstrates the importance of prolyl isomerization in the parasite manipulation of host metabolism.

Alarmin S100A11 initiates a chemokine response to the human pathogen Toxoplasma gondii.

Toxoplasma gondii is a common protozoan parasite that infects up to one-third of the world’s population. Notably, very little is known about innate immune-sensing mechanisms for this obligate intracellular parasite by human cells. Here, by applying an unbiased biochemical screening approach, we have identified that human monocytes recognized the presence of T. gondii infection via detection of the alarmin S100A11 protein, which is released from parasite-infected cells via caspase-1-dependent mechanisms. S100A11 induced a potent chemokine response to T. gondii via engagement of its receptor RAGE and regulated monocyte recruitment in vivo by inducing expression of the chemokine CCL2. Our experiments have revealed a sensing system for T. gondii by human cells that is based on detection infection-mediated release of alarmin S100A11 and RAGE-dependent induction of CCL2, a crucial chemokine required for host resistance to the parasite.

Toxoplasma gondii stabilises tetrameric complexes of tyrosine-phosphorylated signal transducer and activator of transcription-1 and leads to its sustained and promiscuous DNA binding.

Toxoplasma gondii is an obligate intracellular parasite that infects up to 30% of humans worldwide. It can lead to severe diseases particularly in individuals with immature or defective immune responses. Control of T.gondii relies on the IFN-γ-induced signal transducer and activator of transcription-1 (STAT1) pathway. T.gondii, however, largely inactivates STAT1-mediated gene transcription by T.gondii inhibitor of STAT1-dependent transcription (TgIST), a parasite effector protein binding to STAT1. Here, we have analysed requirements of STAT1 to bind TgIST and characterised downstream effects on STAT1 signalling. TgIST bound to STAT1 dimers but more efficiently assembled with STAT1 tetramers, which are essential for effective IFN-γ responsiveness. Such binding was abrogated in N-terminal, but not C-terminal deletion mutants of STAT1. Furthermore, TgIST did not bind to the STAT1F77A substitution mutant that cannot form STAT1 tetramers, resulting in a complete unresponsiveness of parasite-infected STAT1F77A -expressing cells to IFN-γ. Remarkably, binding of TgIST considerably increased the affinity of the aberrant STAT1 tetramers for DNA consensus sequence binding motifs and even enabled binding to nonconsensus sequences. Consistent with the increased DNA binding, STAT1 from parasite-infected cells remained phosphorylated at Tyr701 and Ser727 and was retained within the nucleus in a DNA-bound state. The sustained and promiscuous binding activity particularly of STAT1 tetramers to unspecific DNA sites lacking a consensus STAT1-binding motif is an as yet unrecognised mechanism contributing to the defective IFN-γ-mediated signalling in T.gondii-infected cells.