Parasite-infected Cells Research Articles

Cytotoxic CD8+ T cells recognize and kill Plasmodium vivax-infected reticulocytes.

Plasmodium vivax causes approximately 100 million clinical malaria cases yearly1,2. The basis of protective immunity is poorly understood and thought to be mediated by antibodies3,4. Cytotoxic CD8+ T cells (CTLs) protect against other intracellular parasites by detecting parasite peptides presented by Human Leukocyte Antigen Class I (HLA-I) on host cells. CTLs kill parasite-infected mammalian cells and intracellular parasites by releasing their cytotoxic granules5,6. Perforin (PFN) delivers the antimicrobial peptide granulysin (GNLY) and death-inducing granzymes (Gzm) into the host cell, and GNLY then delivers Gzms into the parasite. CTLs were thought to have no role against Plasmodium spp. blood stages because red blood cells (RBCs) generally do not express HLA-I7. However, P. vivax infects reticulocytes (Retics) that retain the protein translation machinery. Here we show that P. vivax-infected Retics (iRetic) express HLA-I. Infected patient circulating CD8+ T cells highly express cytotoxic proteins and recognize and form immunological synapses with iRetics in an HLA-dependent manner, releasing their cytotoxic granules to kill both host cell and intracellular parasite, preventing reinvasion. iRetic and parasite killing is PFN-independent, but depends on GNLY, which generally efficiently forms pores only in microbial membranes8. We find that P. vivax depletes cholesterol from the iRetic cell membrane, rendering it GNLY-susceptible. This unexpected T cell defense might be mobilized to improve P. vivax vaccine efficacy.

A novel nanoluciferase-based system to monitor Trypanosoma cruzi infection in mice by bioluminescence imaging.

Chagas disease, caused by the intracellular protozoan Trypanosoma cruzi, affects 8–10 million people worldwide and represents a major public health challenge. There is no effective treatment or vaccine to control the disease that is characterized by a mild acute phase followed by a chronic life-long infection. Approximately 30% of chronically infected individuals develop cardiac and/or digestive pathologies. T. cruzi can invade a wide variety of nucleated cells, but only persists at specific tissues in the host. However, the mechanisms that determine tissue tropism and the progression of the infection have not been fully described. Identification of infection niches in animal models has been difficult due to the limited quantity of parasite-infected cells and their focal distribution in tissues during the chronic phase. To better understand the course of chronic infections and parasite dissemination, we developed a bioluminescence imaging system based on the use of transgenic T. cruzi Colombiana strain parasites expressing nanoluciferase. Swiss Webster mice were infected with luminescent trypomastigotes and monitored for 126 days. Whole animal in vivo imaging showed parasites predominantly distributed in the abdominal cavity and surrounding areas throughout the infection. Bioluminescence signal reached a peak between 14 to 21 days post infection (dpi) and decreased progressively over time. Total animal luminescence could still be measured 126 dpi while parasites remained undetectable in blood by microscopy in most animals. Ex vivo imaging of specific tissues and organs dissected post-mortem at 126 dpi revealed a widespread parasite distribution in the skeletal muscle, heart, intestines and mesenteric fat. Parasites were also detected in lungs and liver. This noninvasive imaging model represents a novel tool to study host-parasite interactions and to identify parasite reservoirs of chronic Chagas Disease.

The recent advances of chemical biology in artemisinin target and mechanism

Artemisinin is a natural compound that is extracted from the plant Artemisia annua. While it was used to treat fever and pain in the past, it is currently approved by World Health Organization (WHO) to be used in combating malaria. It is an effective anti-malarial drug because of its high efficacy even towards multi-drug resistant Plasmodium strains and exhibits no significant side effects in humans. However, artemisinin has poor solubility in water or oil, poor bioavailability and a short half-life in vivo . Therefore, derivatives of artemisinin consisting of semi-synthetic and fully synthetic compounds with improved properties have been synthesized. Semi-synthetic derivatives include artesunate, artemether and dihydroartemisinin. Artemisinin and its derivatives are sesquiterpene lactones that contain a unique endoperoxide bond in its trioxane ring, which is essential for their anti-malarial activity. The importance of this bond is shown by the findings of studies that the abrogation of the endoperoxide bond led to a significant reduction in toxicity of the drugs. For example, the removal of one O-atom in the endoperoxide moiety was associated with a complete loss of anti-malarial activity. The activation mechanism of artemisinin is hypothesized to involve the reduction of the endoperoxide bond by Fe2+, which is a catalyst that can generate free radicals from peroxidic structures. The selective toxicity of the drug towards parasite-infected cells as compared to non-infected cells is attributed to the higher heme level in parasite-infected cells. In malarial parasite-infected red blood cells, the parasites break down hemoglobin in the food vacuole to gain amino acids for synthesis of their parasite-specific proteins. As a result, heme is released, which can be further degraded into iron in the vacuole. Therefore, hemoglobin degradation gives rise in an increase in both heme and free iron level in infected erythrocytes. As a result, whether heme or iron is the crucial activator of the drug is yet to be elucidated. One of the pleiotropic effects of the drug is that the reduced endoperoxide bond forms cytotoxic carbon-centered radicals that can alkylate target proteins and affect their functions. Other effects include the alkylation of heme, generation of reactive oxygen species (ROS) and inhibition of PfATP6, which is the P. falciparum ortholog of the endoplasmic reticulum Ca2+ pump. Some studies have also found that artemisinin builds up within neutral lipids and damage the parasite membrane. The combination of these toxic effects of artemisinin and its derivatives result in parasitic death. Artemisinin is used to relieve pain and fever in the older days. Today, artemisinin is widely used as an anti-malarial drug as our first line of defense to combat the emergence of drug resistance malaria parasite. Besides its anti-malarial properties, artemisinin is being investigated in other diseases. It is found to possess a wide spectrum of pharmacological activities, including anticancer, anti-asthma etc. However, its mechanism of action (MOA) is still not fully understood. Recently, “omics” approach has been used in identify the targets and unravel the mechanism of action of artemisinin. This review will summarize the recent progress in artemisinin target and mechanism study.

Histone deacetylase inhibitor MS-275 augments expression of a subset of IFN-γ-regulated genes in Toxoplasma gondii-infected macrophages but does not improve parasite control

Toxoplasma gondii is a ubiquitous apicomplexan parasite of mammals and birds and an important pathogen of humans. IFN-γ is the major mediator of host resistance against T. gondii but intriguingly, parasite-infected host cells including macrophages are severely impaired to respond to IFN-γ due to defective transcriptional activation of target genes. Here, we tested the possibility that the impaired responsiveness of T. gondii-infected macrophages to IFN-γ can be restored by inhibiting histone deacetylases (HDACs) using the class I-specific inhibitor MS-275. Treatment of RAW264.7 cells with MS-275 indeed increased MHC class II surface expression in infected and non-infected cells and largely abolished the inhibition of IFN-γ-regulated MHC class II expression exerted by T. gondii. Genome-wide transcriptome profiling revealed that MS-275 increased mean mRNA levels of IFN-γ-regulated genes particularly in non-infected macrophages. Transcript levels of 33% of IFN-γ secondary response genes but only those of a few primary response genes were also increased by MS-275 in T. gondii-infected cells. Importantly, the unresponsiveness of parasite-infected cells to IFN-γ was however not abolished by MS-275. Furthermore, MS-275 also up-regulated several anti-inflammatory cytokines or signaling molecules in T. gondii-infected macrophages. It additionally regulated expression of more than 2500 genes in non-infected macrophages expression of which was surprisingly counteracted by prior infection with T. gondii. FACS analysis and immunofluorescence microscopy revealed that MS-275 did not considerably diminish the number of parasite-positive cells or the intracellular replication in macrophages stimulated or not with IFN-γ. Thus, a supportive therapy using MS-275 appears inappropriate for treatment of toxoplasmosis.

Fast processing of microscopic images using object-based extended depth of field.

BackgroundMicroscopic analysis requires that foreground objects of interest, e.g. cells, are in focus. In a typical microscopic specimen, the foreground objects may lie on different depths of field necessitating capture of multiple images taken at different focal planes. The extended depth of field (EDoF) technique is a computational method for merging images from different depths of field into a composite image with all foreground objects in focus. Composite images generated by EDoF can be applied in automated image processing and pattern recognition systems. However, current algorithms for EDoF are computationally intensive and impractical, especially for applications such as medical diagnosis where rapid sample turnaround is important. Since foreground objects typically constitute a minor part of an image, the EDoF technique could be made to work much faster if only foreground regions are processed to make the composite image. We propose a novel algorithm called object-based extended depths of field (OEDoF) to address this issue.MethodsThe OEDoF algorithm consists of four major modules: 1) color conversion, 2) object region identification, 3) good contrast pixel identification and 4) detail merging. First, the algorithm employs color conversion to enhance contrast followed by identification of foreground pixels. A composite image is constructed using only these foreground pixels, which dramatically reduces the computational time.ResultsWe used 250 images obtained from 45 specimens of confirmed malaria infections to test our proposed algorithm. The resulting composite images with all in-focus objects were produced using the proposed OEDoF algorithm. We measured the performance of OEDoF in terms of image clarity (quality) and processing time. The features of interest selected by the OEDoF algorithm are comparable in quality with equivalent regions in images processed by the state-of-the-art complex wavelet EDoF algorithm; however, OEDoF required four times less processing time.ConclusionsThis work presents a modification of the extended depth of field approach for efficiently enhancing microscopic images. This selective object processing scheme used in OEDoF can significantly reduce the overall processing time while maintaining the clarity of important image features. The empirical results from parasite-infected red cell images revealed that our proposed method efficiently and effectively produced in-focus composite images. With the speed improvement of OEDoF, this proposed algorithm is suitable for processing large numbers of microscope images, e.g., as required for medical diagnosis.

A Plasmodium falciparum S33 proline aminopeptidase is associated with changes in erythrocyte deformability

Infection with the apicomplexan parasite Plasmodium falciparum is a major cause of morbidity and mortality worldwide. One of the striking features of this parasite is its ability to remodel and decrease the deformability of host red blood cells, a process that contributes to disease. To further understand the virulence of Pf we investigated the biochemistry and function of a putative Pf S33 proline aminopeptidase (PfPAP). Unlike other P. falciparum aminopeptidases, PfPAP contains a predicted protein export element that is non-syntenic with other human infecting Plasmodium species. Characterization of PfPAP demonstrated that it is exported into the host red blood cell and that it is a prolyl aminopeptidase with a preference for N-terminal proline substrates. In addition genetic deletion of this exopeptidase was shown to lead to an increase in the deformability of parasite-infected red cells and in reduced adherence to the endothelial cell receptor CD36 under flow conditions. Our studies suggest that PfPAP plays a role in the rigidification and adhesion of infected red blood cells to endothelial surface receptors, a role that may make this protein a novel target for anti-disease interventions strategies.

The Gametocytes of Leucocytozoon sabrazesi Infect Chicken Thrombocytes, Not Other Blood Cells.

Leucocytozoon parasites infect a large number of avian hosts, including domestic chicken, and cause significant economical loss to the poultry industry. Although the transmission stages of the parasites were observed in avian blood cells more than a century ago, the specific host cell type(s) that the gametocytes infect remain uncertain. Because all the avian blood cells, including red blood cells (RBCs), are nucleated, and the developing parasites dramatically change the morphology of the infected host cells, it has been difficult to identify Leucocytozoon infected host cell(s). Here we use cell-type specific antibodies to investigate the identities of the host cells infected by Leucocytozoon sabrazesi gametocytes. Anti-RBC antibodies stained RBCs membrane strongly, but not the parasite-infected cells, ruling out the possibility of RBCs being the infected host cells. Antibodies recognizing various leukocytes including heterophils, monocytes, lymphocytes, and macrophages did not stain the infected cells either. Antisera raised against a peptide of the parasite cytochrome B (CYTB) stained parasite-infected cells and some leukocytes, particularly cells with a single round nucleus as well as clear/pale cytoplasm suggestive of thrombocytes. Finally, a monoclonal antibody known to specifically bind chicken thrombocytes also stained the infected cells, confirming that L. sabrazesi gametocytes develop within chicken thrombocytes. The identification of L. sabrazesi infected host cell solves a long unresolved puzzle and provides important information for studying parasite invasion of host cells and for developing reagents to interrupt parasite transmission.

SiRNA Screening Identifies the Host Hexokinase 2 (HK2) Gene as an Important Hypoxia-Inducible Transcription Factor 1 (HIF-1) Target Gene in Toxoplasma gondii-Infected Cells.

ABSTRACTAlthough it is established that oxygen availability regulates cellular metabolism and growth, little is known regarding how intracellular pathogens use host factors to grow at physiological oxygen levels. Therefore, large-scale human small interfering RNA screening was performed to identify host genes important for growth of the intracellular protozoan parasite Toxoplasma gondii at tissue oxygen tensions. Among the genes identified by this screen, we focused on the hexokinase 2 (HK2) gene because its expression is regulated by hypoxia-inducible transcription factor 1 (HIF-1), which is important for Toxoplasma growth. Toxoplasma increases host HK2 transcript and protein levels in a HIF-1-dependent manner. In addition, parasite growth at 3% oxygen is restored in HIF-1-deficient cells transfected with HK2 expression plasmids. Both HIF-1 activation and HK2 expression were accompanied by increases in host glycolytic flux, suggesting that enhanced HK2 expression in parasite-infected cells is functionally significant. Parasite dependence on host HK2 and HIF-1 expression is not restricted to transformed cell lines, as both are required for parasite growth in nontransformed C2C12 myoblasts and HK2 is upregulated in vivo following infection. While HK2 is normally associated with the cytoplasmic face of the outer mitochondrial membrane at physiological O2 levels, HK2 relocalizes to the host cytoplasm following infection, a process that is required for parasite growth at 3% oxygen. Taken together, our findings show that HIF-1-dependent expression and relocalization of HK2 represent a novel mechanism by which Toxoplasma establishes its replicative niche at tissue oxygen tensions.

Platelet Factor 4 Enables Platelets to Suppress Early Parasite Growth in a Mouse Model of Blood Stage Malarial Infection

Platelets are best known for their roles in hemostasis, but there is accumulating evidence for their importance in immunity and host defence against infection. We have shown that platelets are required for host protection during malarial infection and are a major determinant for survival. Platelets control the growth of intraerythrocytic Plasmodium parasites by directly binding to infected erythrocytes and killing the parasite inside 1. We 2 and others 3 have also described the parasite killing mechanism exerted by platelets. Upon binding to parasite-infected red cells, platelets release a protein with dual chemokine and antimicrobial functions called platelet factor 4 (PF4). PF4 binds to the red cell Duffy antigen chemokine receptor, and is then absorbed into the parasite where it lyses the parasite food vacuole and kills the parasite.This work aimed to further investigate the role of PF4 in vivo using mice a PF4 knockout mouse strain (Pf4tm1Mako/tm1Mako; 4) and blood stage infection with a murine malaria model parasite, P. chabaudi. We confirmed that the homozygous mutant mice did not produce any PF4 protein. Following challenge with P. chabaudi (1 x 104 parasites administered intravenously), rates of survival amongst cohorts of age and sex matched wild type and homozygous mice (both on a C57BL/6 background) were not significantly different (approximately 30% for females and 10% for males). However, a deficiency of PF4 resulted in significant differences in parasite growth during the pre-peak parasitemia phase of the infection. Between days 5 and 8 following infection, proportions of parasite-infected red cells in the circulation of PF4-null mice were double those in wild-type mice. This coincided with a 50-60% reduction in the frequency of growth arrested or dead parasites (detected using terminal deoxynucleotidyl transferase dUTP nick end labelling, also known as TUNEL labelling).Together these data indicate that platelet-derived PF4 has an important in vivo malaria-protective role. The protein is necessary for the platelet to control parasite growth during the early stage of erythrocytic malarial infection. However, the highly virulent nature of the P. chabaudi strain used in these experiments precluded detection of any predicted survival disadvantage in the PF4-null mice. 1.McMorran BJ, Marshall VM, de Graaf C, et al. Platelets kill intraerythrocytic malarial parasites and mediate survival to infection. Science. 2009;323(5915):797-800.2.McMorran BJ, Wieczorski L, Drysdale KE, et al. Platelet factor 4 and Duffy antigen required for platelet killing of Plasmodium falciparum. Science. 2012;338(6112):1348-1351.3.Love MS, Millholland MG, Mishra S, et al. Platelet Factor 4 Activity against P. falciparum and Its Translation to Nonpeptidic Mimics as Antimalarials. Cell host & microbe. 2012;12(6):815-823.4.Eslin DE, Zhang C, Samuels KJ, et al. Transgenic mice studies demonstrate a role for platelet factor 4 in thrombosis: dissociation between anticoagulant and antithrombotic effect of heparin. Blood. 2004;104(10):3173-3180. DisclosuresNo relevant conflicts of interest to declare.

Photodynamic effects of zinc phthalocyanines on intracellular amastigotes of Leishmania amazonensis and Leishmania braziliensis.

This study investigated the photoactivity of four zinc phthalocyanines (PcZns) on a murine macrophage cell line infected with Leishmania amazonensis or Leishmania braziliensis. Infected and uninfected cells were incubated with PcZns at different concentrations (1-10μM) for 3h and then exposed to an LED device in continuous wave mode at 660nm with a fluency of 50J/cm(2) (25mV). Enzymatic activity was determined by MTT assay 24h after light treatment. The results demonstrated that all PcZns exhibited high photoactivity, particularly when used at 10μM. The photodynamic effects were different for uninfected cells versus parasite-infected cells and among the four PcZns. Uninfected cells were more sensitive to photoactivity than infected cells. Although PcZns photodynamic therapy provided promising results, further studies are necessary to better understand its mechanism of action in the treatment of leishmaniasis.

Parasite impairment by targeting Plasmodium-infected RBCs using glyceryl-dilaurate nanostructured lipid carriers

Antimalarial therapy is a major contributor to declining malaria morbidity and mortality. However, the high toxicity and low bioavailability of current antimalarials and emerging drug resistance necessitates drug-delivery research. We have previously developed glyceryl-dilaurate nanolipid carriers (GDL-NLCs) for antimalarial drug delivery. Here, we show evidence that GDL-NLCs themselves selectively target Plasmodium-infected red blood cells (iRBCs), and cause severe parasite impairment. The glyceryl-dilaurate lipid-moiety was important in the targeting. GDL-NLCs localized to the parasite mitochondrion and uptake led to mitochondrial-membrane polarization and Ca2+ ion accumulation, ROS release, and stage-specific iRBC lysis. GDL-NLC treatment also resulted in externalization of iRBC-membrane phosphatidylserine and enhanced iRBC clearance by macrophages. GDL-NLC uptake disrupted the parasite-induced tubulovesicular network, which is vital for nutrient import by the parasite. Laser optical trap studies revealed that GDL-NLCs also restored iRBC flexibility. Such restoration of iRBC flexibility may help mitigate the vasculature clogging that can lead to cerebral malaria. We demonstrate the suitability of GDL-NLCs for intravenous delivery of antimalarial combinations artemether–clindamycin and artemether–lumefantrine in the murine model. Complete parasite clearance was achieved at 5−20% of the therapeutic dose of these combinations. Thus, this nanostructured lipid formulation can solubilize lipophilic drugs, selectively target and impair the parasite-infected red cell, and therefore constitutes a potent delivery vehicle for antimalarials.

A rapid sensitive, flow cytometry-based method for the detection of Plasmodium vivax-infected blood cells

BackgroundPlasmodium vivax preferentially infects Duffy-positive reticulocytes and infections typically have few parasite-infected cells in the peripheral circulation. These features complicate detection and quantification by flow cytometry (FC) using standard nucleic acid-based staining methods. A simple antibody-based FC method was developed for rapid parasite detection along with simultaneous detection of other parasite and erythrocyte markers.MethodsClinical samples were collected from patients diagnosed with P. vivax at a district Malaria Clinic in Kanchanaburi, Thailand. One μL of infected blood was washed, fixed, stained with a Plasmodium pan-specific anti-PfBiP antibody conjugated with Alexa Fluor 660, and analysed by FC. Additional primary conjugated antibodies for stage-specific markers of P. vivax for late trophozoite-early schizonts (MSP1-Alexa Fluor 660), late-stage schizonts (DBP-Alexa Fluor 555), and sexual stages (Pvs16) were used to differentiate intra-erythrocytic developmental stages.ResultsThe percentages of P. vivax-infected cells determined by the FC method and manually by microscopic examination of Giemsa-stained thick blood smears were positively correlated by Spearman’s rank correlation coefficient (R 2 = 0.93843) from 0.001 to 1.00% P. vivax-infected reticulocytes.ConclusionsThe FC-based method is a simple, robust, and efficient method for detecting P. vivax-infected reticulocytes.

Chapter 26 - Adaptive fluorescence microscopy by online feedback image analysis

Obtaining sufficient statistics in quantitative fluorescence microscopy is often hampered by the tedious and time-consuming task of manually locating comparable specimen and repeatedly launching the same acquisition protocol. Recent advances in combining fluorescence microscopy with online image analysis tackle this problem by fully integrating the task of identifying and locating the specimen of interest in an automated acquisition workflow. Here, we describe the general requirements and specific microscope control and image analysis software solutions for implementing such automated online feedback microscopy. We demonstrate the power of the method by two selected applications addressing high-throughput 3D imaging of sparsely parasite-infected tissue culture cells and automated fluorescence recovery after photobleaching experiments to quantify the turnover of vesicular coat proteins at ER exit sites.

Immuno-EM Analysis of PF13_0191-GFP Expressing Parasites

This protocol was used to prepare pre-embedding samples of Plasmodium falciparum blood stage parasites that overexpressed the parasite protein PF13_0191 tagged with GFP. Using GFP-specific antibodies and Protein A-Gold the localisation of the overexpressed protein in the infected host cell was determined using standard transmission electron microscopy (EM). Pre-embedding EM is a common method where the antibodies are introduced before embedding and sectioning. This method avoids the problem that antigens are often difficult to detect on EM-sections after embedding. In the method described here antigens in the parasite-infected host cell are detected. Entry of the antibody is made possible through permeabilisation of the host cell with tetanolysin. In principle this method could also be used to detect antigens within the parasite if the sample is appropriately fixed and permeabilised before addition of the relevant antibody. While access of the antibody will avoid the detection problems often seen with post-embedding methods, this procedure will produce comparably poorer morphology.

Characterization of the interaction between Toxoplasma gondii rhoptry neck protein 4 and host cellular β-tubulin

Toxoplasma rhoptry neck protein 4 (TgRON4) is a component of the moving junction macromolecular complex that plays a central role during invasion. TgRON4 is exposed on the cytosolic side of the host cell during invasion, but its molecular interactions remain unclear. Here, we identified host cellular β-tubulin as a binding partner of TgRON4, but not Plasmodium RON4. Coimmunoprecipitation studies in mammalian cells demonstrated that the C-terminal 15-kDa region of β-tubulin was sufficient for binding to TgRON4, and that a 17-kDa region in the proximal C-terminus of TgRON4 was required for binding to the C-terminal region of β-tubulin. Analysis of T. gondii-infected lysates from CHO cells expressing the TgRON4-binding region showed that the C-terminal region of β-tubulin interacted with TgRON4 at early invasion step. Our results provide evidence for a parasite-specific interaction between TgRON4 and the host cell cytoskeleton in parasite-infected cells.

Exposure to low doses of chemically attenuated blood stage malaria parasites rapidly activates CD4+ T cells and induces strain and species transcending immunity (P4477)

Abstract There has been a re-appraisal of the role of cell-mediated immunity in protection against malaria. We have developed a vaccine strategy using chemical attenuation of infected red cells to induce protective cellular immunity. Our focus has been on attenuation using a class of chemicals typified by centanamycin, which binds in AT-rich regions of the parasite genome. Chemically attenuated parasites (CAPs) do not grow in immunodeficient mice. We observe that low doses of Plasmodium chabaudi ring stage CAPs induce profound protection against different strains and species of Plasmodium. CAPs prepared using other species of Plasmodium are also effective. CAPs are rapidly removed from the peripheral blood following vaccination and are taken up by dendritic cells and macrophages in the spleen and liver. Vaccination does not induce detectable antibodies but protection is T-cell dependent. Following vaccination there is a rapid increase within the blood of activated CD4+ T cells (defined as CD49d+ CD11a+). Transfused ovalbumin-specific transgenic OTII cells are not activated following vaccination. This novel approach is now being applied to P. falciparum where we plan to vaccinate humans with low doses of P. falciparum CAP, determine their immune responses and then challenge vaccinees with viable parasite-infected human red cells to determine vaccine efficacy.

Enhanced Egress of Intracellular Eimeria tenella Sporozoites by Splenic Lymphocytes from Coccidian-Infected Chickens

Egress, which describes the mechanism that some intracellular parasites use to exit from parasitophorous vacuoles and host cells, plays a very important role in the parasite life cycle and is central to Eimeria propagation and pathogenesis. Despite the importance of egress in the intracellular parasite's life cycle, very little information is known on this process compared to other steps, e.g., invasion. The present study was conducted to investigate the interplay between the host adaptive immune system and Eimeria egression. Splenic lymphocytes or soluble immune factors were incubated with parasite-infected host cells for 3 or 5 h, and the percentage of egress was calculated according to an established formula. Viability of egressed parasites and host cells was tested using trypan blue exclusion and annexin V and propidium iodide staining, respectively. We found that premature egression of sporozoites from Eimeria tenella-infected primary chicken kidney cells or from chicken peripheral blood mononuclear cells occurred when the cells were cocultured in vitro with spleen lymphocytes from E. tenella-infected chickens but not when they were cocultured with splenocytes from uninfected chickens. Eimeria-specific antibodies and cytokines (gamma interferon [IFN-γ], interleukin-2 [IL-2], and IL-15), derived from E. tenella-primed B and T lymphocytes, respectively, were capable of promoting premature egress of sporozoites from infected host cells. Both egressed parasites and host cells were viable, although the latter showed reduced reinvasion ability. These results suggest a novel, immune-mediated mechanism that the host exploits to interrupt the normal Eimeria life cycle in vivo and thereby block the release of mature parasites into the environment.

Tip-Enhanced Raman Scattering (TERS) from Hemozoin Crystals within a Sectioned Erythrocyte

Tip-enhanced Raman scattering (TERS) is a powerful technique to obtain molecular information on a nanometer scale, however, the technique has been limited to cell surfaces, viruses, and isolated molecules. Here we show that TERS can be used to probe hemozoin crystals at less than 20 nm spatial resolution in the digestive vacuole of a sectioned malaria parasite-infected cell. The TERS spectra clearly show characteristic bands of hemozoin that can be correlated to a precise position on the crystal by comparison with the corresponding atomic force microscopy (AFM) image. These are the first recorded AFM images of hemozoin crystals inside malaria-infected cells and clearly show the hemozoin crystals protruding from the embedding medium. TERS spectra recorded of these crystals show spectral features consistent with a five-coordinate high-spin ferric heme complex, which include the electron density marker band ν(4) at 1373 cm(-1) and other porphyrin skeletal and ring breathing modes at approximately 1636, 1557, 1412, 1314, 1123, and 1066 cm(-1). These results demonstrate the potential of the AFM/TERS technique to obtain nanoscale molecular information within a sectioned single cell. We foresee this approach paving the way to a new independent drug screening modality for detection of drugs binding to the hemozoin surface within the digestive vacuole of the malaria trophozoite.

Differential Regulation of Effector- and Central-Memory Responses to Toxoplasma gondii Infection by IL-12 Revealed by Tracking of Tgd057-Specific CD8+ T Cells

Production of the pro-inflammatory cytokine IL-12 by innate phagocytes drives the differentiation of IFN-γ-producing effector T cells during Toxoplasma gondii infection. However, the role of IL-12 in the regulation of memory CD8+ T cell differentiation and function during murine toxoplasmosis is unclear. To track memory CTL development, we identified a novel H-2Kb-restricted CTL population specific for the Toxoplasma antigen tgd057. Tgd057-specific CTLs were induced by both vaccination and natural peroral infection, and were representative of the polyclonal CTL population. Tgd057-specific primary effector cells required IL-12 for the differentiation of KLRG1+ effector subpopulations and IFN-γ production in response to restimulation with parasite-infected cells, but not to restimulation with cognate peptide. The effect of IL-12 deficiency during the primary response was profoundly imprinted on memory CTLs, which continued to show defects in cell numbers, KLRG1+ effector memory subpopulation differentiation, and IFN-γ recall responses. Importantly, isolated CD62Lhi KLRG1- CD8+ T cells differentiated in the absence of IL-12 were enhanced in their ability to generate IFN-γ-producing secondary tgd057-specific effector cells. Our data, for the first time, demonstrate the negative impact of IL-12 signaling on the quality of the central memory CTL compartment. Thus, despite the beneficial role of IL-12 in promoting effector differentiation, excessive exposure to IL-12 during CTL priming may limit the development of long-term protective immunity through the decreased fitness of central memory CTL responses.

Plasmodium berghei-infection induces volume-regulated anion channel-like activity in human hepatoma cells

Parasite infection can lead to alterations in the permeability of host plasma membranes. Presented here is the first demonstration that this phenomenon occurs in Plasmodium-infected liver cells. Using the whole-cell patch-clamp technique, volume-regulated anion channel (VRAC) activity was characterized in Huh-7 cells (a human hepatoma cell line) before and after infection with Plasmodium berghei. Consistent with the presence of VRACs, hypotonic bath solution induced large ion currents in Huh-7 cells that rectified outwardly, reversed close to the equilibrium potential for Cl- and were inhibited by tamoxifen, clomiphene, mefloquine and 5-nitro-2, 3-(phenylpropylamino)-benzoic acid (NPPB), with IC50 values of 4 ± 1, 4 ± 2, 2 ± 1 and 52 ± 12 μM respectively. In isotonic conditions, initial current recordings measured in uninfected and immature (24 h post invasion) parasite-infected Huh-7 cells were similar (with conductances of 14 ± 3 versus 19 ± 5 pS/pF). However, in mature (48–72 h post invasion) parasite-infected Huh-7 cells there was a sevenfold increase in currents (with a conductance of 98 ± 16 pS/pF). The elevated currents observed in the latter are consistent with VRAC-like activity and the possible reasons for their activation are discussed.