scholarly journals Immunization with the MAEBL M2 Domain Protects against Lethal Plasmodium yoelii Infection

2015 ◽  
Vol 83 (10) ◽  
pp. 3781-3792 ◽  
Author(s):  
Juliana A. Leite ◽  
Daniel Y. Bargieri ◽  
Bruna O. Carvalho ◽  
Letusa Albrecht ◽  
Stefanie C. P. Lopes ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Plasmodium Yoelii ◽  
Parasite Development ◽  
Effective Vaccine ◽  
Dependent Manner ◽  
Content Type ◽  
Apical Membrane Antigen 1 ◽  
Erythrocyte Invasion ◽  
Erythrocyte Binding ◽  
Native Antigen

Malaria remains a world-threatening disease largely because of the lack of a long-lasting and fully effective vaccine. MAEBL is a type 1 transmembrane molecule with a chimeric cysteine-rich ectodomain homologous to regions of the Duffy binding-like erythrocyte binding protein and apical membrane antigen 1 (AMA1) antigens. Although MAEBL does not appear to be essential for the survival of blood-stage forms, ectodomains M1 and M2, homologous to AMA1, seem to be involved in parasite attachment to erythrocytes, especially M2. MAEBL is necessary for sporozoite infection of mosquito salivary glands and is expressed in liver stages. Here, thePlasmodium yoeliiMAEBL-M2 domain was expressed in a prokaryotic vector. C57BL/6J mice were immunized with doses ofP. yoeliirecombinant protein rPyM2-MAEBL. High levels of antibodies, with balanced IgG1 and IgG2c subclasses, were achieved. rPyM2-MAEBL antisera were capable of recognizing the native antigen. Anti-MAEBL antibodies recognized different MAEBL fragments expressed in CHO cells, showing stronger IgM and IgG responses to the M2 domain and repeat region, respectively. After a challenge withP. yoeliiYM (lethal strain)-infected erythrocytes (IE), up to 90% of the immunized animals survived and a reduction of parasitemia was observed. Moreover, splenocytes harvested from immunized animals proliferated in a dose-dependent manner in the presence of rPyM2-MAEBL. Protection was highly dependent on CD4+, but not CD8+, T cells toward Th1. rPyM2-MAEBL antisera were also able to significantly inhibit parasite development, as observed inex vivoP. yoeliierythrocyte invasion assays. Collectively, these findings support the use of MAEBL as a vaccine candidate and open perspectives to understand the mechanisms involved in protection.

scholarly journals Chloroquine Potentiates Primaquine Activity against Active and Latent Hepatic Plasmodia Ex Vivo: Potentials and Pitfalls

2020 ◽  
Vol 65 (1) ◽  
pp. e01416-20
Author(s):  
Laurent Dembélé ◽  
Jean-François Franetich ◽  
Valérie Soulard ◽  
Nadia Amanzougaghene ◽  
Shahin Tajeri ◽  
...  
Keyword(s):  
Cell Line ◽  
Ex Vivo ◽  
Plasmodium Yoelii ◽  
Human Hepatocytes ◽  
Dependent Manner ◽  
Primary Hepatocytes ◽  
Content Type ◽  
Primary Human Hepatocytes ◽  
Hepatocarcinoma Cell ◽  
Hepatocarcinoma Cell Line

ABSTRACTFor a long while, 8-aminoquinoline compounds have been the only therapeutic agents against latent hepatic malaria parasites. These have poor activity against the blood-stage plasmodia causing acute malaria and must be used in conjunction with partner blood schizontocidal agents. We examined the impacts of one such agent, chloroquine, upon the activity of primaquine, an 8-aminoquinoline, against hepatic stages of Plasmodium cynomolgi, Plasmodium yoelii, Plasmodium berghei, and Plasmodium falciparum within several ex vivo systems—primary hepatocytes of Macaca fascicularis, primary human hepatocytes, and stably transformed human hepatocarcinoma cell line HepG2. Primaquine exposures to formed hepatic schizonts and hypnozoites of P. cynomolgi in primary simian hepatocytes exhibited similar 50% inhibitory concentration (IC50) values near 0.4 μM, whereas chloroquine in the same system exhibited no inhibitory activities. Combining chloroquine and primaquine in this system decreased the observed primaquine IC50 for all parasite forms in a chloroquine dose-dependent manner by an average of 18-fold. Chloroquine also decreased the primaquine IC50 against hepatic P. falciparum in primary human hepatocytes, P. berghei in simian primary hepatocytes, and P. yoelii in primary human hepatocytes. Chloroquine had no impact on primaquine IC50 against P. yoelii in HepG2 cells and, likewise, had no impact on the IC50 of atovaquone (hepatic schizontocide) against P. falciparum in human hepatocytes. We describe important sources of variability in the potentiation of primaquine activity by chloroquine in these systems. Chloroquine potentiated primaquine activity against hepatic forms of several plasmodia. We conclude that chloroquine specifically potentiated 8-aminoquinoline activities against active and dormant hepatic-stage plasmodia in normal primary hepatocytes but not in a hepatocarcinoma cell line.

scholarly journals Ex VivoMaturation Assay for Testing Antimalarial Sensitivity of Rodent Malaria Parasites

2016 ◽  
Vol 60 (11) ◽  
pp. 6859-6866 ◽  
Author(s):  
Zi Wei Chang ◽  
Benoit Malleret ◽  
Bruce Russell ◽  
Laurent Rénia ◽  
Carla Claser
Keyword(s):  
Ex Vivo ◽  
Single Step ◽  
Parasite Development ◽  
Ring Stage ◽  
Malaria Parasites ◽  
Rodent Malaria ◽  
Content Type ◽  
Parasite Biology

ABSTRACTEx vivoassay systems provide a powerful approach to studying human malaria parasite biology and to testing antimalarials. For rodent malaria parasites, short-termin vitroculture andex vivoantimalarial susceptibility assays are relatively cumbersome, relying onin vivopassage for synchronization, since ring-stage parasites are an essential starting material. Here, we describe a new approach based on the enrichment of ring-stagePlasmodium berghei,P. yoelii, andP. vinckei vinckeiusing a single-step Percoll gradient. Importantly, we demonstrate that the enriched ring-stage parasites develop synchronously regardless of the parasite strain or species used. Using a flow cytometry assay with Hoechst and ethidium or MitoTracker dye, we show that parasite development is easily and rapidly monitored. Finally, we demonstrate that this approach can be used to screen antimalarial drugs.

scholarly journals Klebsiella pneumoniae Expressing VIM-1 Metallo-β-Lactamase Is Resensitized to Cefotaxime via Thiol-Mediated Zinc Chelation

2019 ◽  
Vol 88 (1) ◽  
Author(s):  
Harpa Karadottir ◽  
Maarten Coorens ◽  
Zhihai Liu ◽  
Yang Wang ◽  
Birgitta Agerberth ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Ex Vivo ◽  
Reductase Activity ◽  
Urine Samples ◽  
Dependent Manner ◽  
Content Type ◽  
Endogenous Factors ◽  
Reducing Ability ◽  
Zinc Chelation ◽  
Ht 29

ABSTRACT Antibiotic-resistant Klebsiella pneumoniae isolates constitute a great clinical challenge. One important resistance mechanism in K. pneumoniae is the metallo-β-lactamases (MBLs), which require zinc for their function. Thus, zinc chelation could be a strategy to resensitize K. pneumoniae to β-lactams. However, the potential role for endogenous zinc chelators for this purpose remains to be explored. The aim was to search for endogenous factors that could resensitize MBL-expressing K. pneumoniae to cefotaxime (CTX). Clinical K. pneumoniae isolates expressing different MBLs were screened for sensitivity to CTX in supernatants from human HT-29 colonic epithelial cells. Factors influencing CTX susceptibility were isolated and identified with chromatographic and biochemical methods. Free zinc was measured with a Zinquin assay, the thiol content was assessed with a fluorometric thiol assay, and the reducing ability of the supernatant was measured with a fluorescent l-cystine probe. Urine samples from healthy volunteers were used to validate findings ex vivo. VIM-1-expressing K. pneumoniae regained susceptibility to CTX when grown in supernatants from HT-29 cells. This effect was mediated via free thiols in the supernatant, including l-cysteine, and could be prevented by inhibiting thioredoxin reductase activity in the supernatant. Free thiols in urine samples appeared to have a similar function in restoring CTX activity against VIM-1-expressing K. pneumoniae in a zinc-dependent manner. We have identified l-cysteine as an endogenous zinc chelator resulting in the resensitization of VIM-1-expressing K. pneumoniae to CTX. These results suggest that natural zinc chelators in combination with conventional antibiotics could be used to treat infections caused by VIM-1-expressing pathogens.

scholarly journals Regulation of Plasmodium yoelii Oocyst Development by Strain- and Stage-Specific Small-Subunit rRNA

mBio ◽  
2015 ◽  
Vol 6 (2) ◽  
Author(s):  
Yanwei Qi ◽  
Feng Zhu ◽  
Richard T. Eastman ◽  
Young Fu ◽  
Martine Zilversmit ◽  
...  
Keyword(s):  
Developmental Stages ◽  
Small Subunit ◽  
Plasmodium Yoelii ◽  
Rrna Genes ◽  
Parasite Development ◽  
Rrna Gene ◽  
Small Subunit Rrna ◽  
Malaria Parasites ◽  
Content Type ◽  
Small Subunit Rrna Gene

ABSTRACT One unique feature of malaria parasites is the differential transcription of structurally distinct rRNA (rRNA) genes at different developmental stages: the A-type genes are transcribed mainly in asexual stages, whereas the S-type genes are expressed mostly in sexual or mosquito stages. Conclusive functional evidence of different rRNAs in regulating stage-specific parasite development, however, is still absent. Here we performed genetic crosses of Plasmodium yoelii parasites with one parent having an oocyst development defect (ODD) phenotype and another producing normal oocysts to identify the gene(s) contributing to the ODD. The parent with ODD—characterized as having small oocysts and lacking infective sporozoites—was obtained after introduction of a plasmid with a green fluorescent protein gene into the parasite genome and subsequent passages in mice. Quantitative trait locus analysis of genome-wide microsatellite genotypes of 48 progeny from the crosses linked an ~200-kb segment on chromosome 6 containing one of the S-type genes (D-type small subunit rRNA gene [D-ssu]) to the ODD. Fine mapping of the plasmid integration site, gene expression pattern, and gene knockout experiments demonstrated that disruption of the D-ssu gene caused the ODD phenotype. Interestingly, introduction of the D-ssu gene into the same parasite strain (self), but not into a different subspecies, significantly affected or completely ablated oocyst development, suggesting a stage- and subspecies (strain)-specific regulation of oocyst development by D-ssu. This study demonstrates that P. yoelii D-ssu is essential for normal oocyst and sporozoite development and that variation in the D-ssu sequence can have dramatic effects on parasite development. IMPORTANCE Malaria parasites are the only known organisms that express structurally distinct rRNA genes at different developmental stages. The differential expression of these genes suggests that they play unique roles during the complex life cycle of the parasites. Conclusive functional proof of different rRNAs in regulating parasite development, however, is still absent or controversial. Here we functionally demonstrate for the first time that a stage-specifically expressed D-type small-subunit rRNA gene (D-ssu) is essential for oocyst development of the malaria parasite Plasmodium yoelii in the mosquito. This study also shows that variations in D-ssu sequence and/or the timing of transcription may have profound effects on parasite oocyst development. The results show that in addition to protein translation, rRNAs of malaria parasites also regulate parasite development and differentiation in a strain-specific manner, which can be explored for controlling parasite transmission.

scholarly journals A Plasmodium yoelii Mei2-Like RNA Binding Protein Is Essential for Completion of Liver Stage Schizogony

2016 ◽  
Vol 84 (5) ◽  
pp. 1336-1345 ◽  
Author(s):  
Dorender A. Dankwa ◽  
Marshall J. Davis ◽  
Stefan H. I. Kappe ◽  
Ashley M. Vaughan
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Plasmodium Yoelii ◽  
Parasite Development ◽  
Mammalian Host ◽  
Mosquito Vector ◽  
Wild Type ◽  
Liver Stage ◽  
Content Type ◽  
Stage Development

Plasmodiumparasites employ posttranscriptional regulatory mechanisms as their life cycle transitions between host cell invasion and replication within both the mosquito vector and mammalian host. RNA binding proteins (RBPs) provide one mechanism for modulation of RNA function. To explore the role ofPlasmodiumRBPs during parasite replication, we searched for RBPs that might play a role during liver stage development, the parasite stage that exhibits the most extensive growth and replication. We identified a parasite ortholog of theMei2(Meiosisinhibited 2) RBP that is conserved amongPlasmodiumspecies (PlasMei2) and exclusively transcribed in liver stage parasites. Epitope-taggedPlasmodium yoeliiPlasMei2 was expressed only during liver stage schizogony and showed an apparent granular cytoplasmic location. Knockout ofPlasMei2(plasmei2−) inP. yoeliionly affected late liver stage development. TheP. yoeliiplasmei2−liver stage size increased progressively until late in development, similar to wild-type parasite development. However,P. yoeliiplasmei2−liver stage schizonts exhibited an abnormal DNA segregation phenotype and failed to form exoerythrocytic merozoites. Consequently the cellular integrity ofP. yoeliiplasmei2−liver stages became increasingly compromised late in development and the majority ofP. yoeliiplasmei2−underwent cell death by the time wild-type liver stages mature and release merozoites. This resulted in a complete block ofP. yoeliiplasmei2−transition from liver stage to blood stage infection in mice. Our results show for the first time the importance of aPlasmodiumRBP in the coordinated progression of late liver stage schizogony and maturation of new invasive forms.

scholarly journals Allergic Airway Inflammation Decreases Lung Bacterial Burden following Acute Klebsiella pneumoniae Infection in a Neutrophil- and CCL8-Dependent Manner

2014 ◽  
Vol 82 (9) ◽  
pp. 3723-3739 ◽  
Author(s):  
Daniel E. Dulek ◽  
Dawn C. Newcomb ◽  
Kasia Goleniewska ◽  
Jaqueline Cephus ◽  
Weisong Zhou ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Airway Inflammation ◽  
Ex Vivo ◽  
Allergic Airway Inflammation ◽  
Dependent Manner ◽  
Content Type ◽  
Th2 Cytokine ◽  
Interleukin 17A ◽  
Th17 Cytokines

ABSTRACTThe Th17 cytokines interleukin-17A (IL-17A), IL-17F, and IL-22 are critical for the lung immune response to a variety of bacterial pathogens, includingKlebsiella pneumoniae. Th2 cytokine expression in the airways is a characteristic feature of asthma and allergic airway inflammation. The Th2 cytokines IL-4 and IL-13 diminishex vivoandin vivoIL-17A protein expression by Th17 cells. To determine the effect of IL-4 and IL-13 on IL-17-dependent lung immune responses to acute bacterial infection, we developed a combined model in which allergic airway inflammation and lung IL-4 and IL-13 expression were induced by ovalbumin sensitization and challenge prior to acute lung infection withK. pneumoniae. We hypothesized that preexisting allergic airway inflammation decreases lung IL-17A expression and airway neutrophil recruitment in response to acuteK. pneumoniaeinfection and thereby increases the lungK. pneumoniaeburden. As hypothesized, we found that allergic airway inflammation decreased the number ofK. pneumoniae-induced airway neutrophils and lung IL-17A, IL-17F, and IL-22 expression. Despite the marked reduction in postinfection airway neutrophilia and lung expression of Th17 cytokines, allergic airway inflammation significantly decreased the lungK. pneumoniaeburden and postinfection mortality. We showed that the decreased lungK. pneumoniaeburden was independent of IL-4, IL-5, and IL-17A and partially dependent on IL-13 and STAT6. Additionally, we demonstrated that the decreased lungK. pneumoniaeburden associated with allergic airway inflammation was both neutrophil and CCL8 dependent. These findings suggest a novel role for CCL8 in lung antibacterial immunity againstK. pneumoniaeand suggest new mechanisms of orchestrating lung antibacterial immunity.

scholarly journals Analysis of Endothelial Adherence of Bartonella henselae and Acinetobacter baumannii Using a Dynamic HumanEx VivoInfection Model

2015 ◽  
Vol 84 (3) ◽  
pp. 711-722 ◽  
Author(s):  
Marko Weidensdorfer ◽  
Ju Ik Chae ◽  
Celestine Makobe ◽  
Julia Stahl ◽  
Beate Averhoff ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Pathogenic Bacteria ◽  
Ex Vivo ◽  
Bartonella Henselae ◽  
Dependent Manner ◽  
Content Type ◽  
Human Pathogenic Bacteria ◽  
Infection Models ◽  
Animal Infection

Bacterial adherence determines the virulence of many human-pathogenic bacteria. Experimental approaches elucidating this early infection event in greater detail have been performed using mainly methods of cellular microbiology. However,in vitroinfections of cell monolayers reflect thein vivosituation only partially, and animal infection models are not available for many human-pathogenic bacteria. Therefore,ex vivoinfection of human organs might represent an attractive method to overcome these limitations. We infected whole human umbilical cordsex vivowithBartonella henselaeorAcinetobacter baumanniiunder dynamic flow conditions mimicking thein vivoinfection situation of human endothelium. For this purpose, methods for quantifying endothelium-adherent wild-type and trimeric autotransporter adhesin (TAA)-deficient bacteria were set up. Data revealed that (i)A. baumanniibinds in a TAA-dependent manner to endothelial cells, (ii) this organ infection model led to highly reproducible adherence rates, and furthermore, (iii) this model allowed to dissect the biological function of TAAs in the natural course of human infections. These findings indicate that infection models usingex vivohuman tissue samples (“organ microbiology”) might be a valuable tool in analyzing bacterial pathogenicity with the capacity to replace animal infection models at least partially.

scholarly journals Structural Characterization of the Erythrocyte Binding Domain of the Reticulocyte Binding Protein Homologue Family of Plasmodium yoelii

2011 ◽  
Vol 79 (7) ◽  
pp. 2880-2888 ◽  
Author(s):  
Ardina Grüber ◽  
Karthigayan Gunalan ◽  
Jeya Kumar Ramalingam ◽  
Malathy S. S. Manimekalai ◽  
Gerhard Grüber ◽  
...  
Keyword(s):  
Binding Protein ◽  
Intervention Strategy ◽  
Plasmodium Yoelii ◽  
Binding Domain ◽  
Content Type ◽  
Binding Domains ◽  
X Ray Scattering ◽  
Ligand Interactions ◽  
Erythrocyte Binding

ABSTRACTInvasion of the host cell by the malaria parasite is a key step for parasite survival and the only stage of its life cycle where the parasite is extracellular, and it is therefore a target for an antimalaria intervention strategy. Multiple members of the reticulocyte binding protein homologues (RH) family are found in all plasmodia and have been shown to bind to host red blood cells directly. In the study described here, we delineated the erythrocyte binding domain (EBD) of one member of the RH family, termed Py235, fromPlasmodium yoelii. Moreover, we have obtained the low-resolution structure of the EBD using small-angle X-ray scattering. Comparison of the EDB structure to other characterizedPlasmodiumreceptor binding domains suggests that there may be an overall structural conservation. These findings may help in developing new approaches to target receptor ligand interactions mediated by parasite proteins.

scholarly journals Acquired Antibodies to Merozoite Antigens in Children from Uganda with Uncomplicated or Severe Plasmodium falciparum Malaria

2013 ◽  
Vol 20 (8) ◽  
pp. 1170-1180 ◽  
Author(s):  
Hodan Ahmed Ismail ◽  
Ulf Ribacke ◽  
Linda Reiling ◽  
Johan Normark ◽  
Tom Egwang ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Uncomplicated Malaria ◽  
Severe Disease ◽  
Plasmodium Falciparum Malaria ◽  
Antibody Responses ◽  
Enzyme Linked Immunosorbent Assay ◽  
Content Type ◽  
Apical Membrane Antigen 1 ◽  
Merozoite Antigens ◽  
Erythrocyte Binding

ABSTRACTMalaria can present itself as an uncomplicated or severe disease. We have here studied the quantity and quality of antibody responses against merozoite antigens, as well as multiplicity of infection (MOI), in children from Uganda. We found higher levels of IgG antibodies toward erythrocyte-binding antigen EBA181, MSP2 ofPlasmodium falciparum3D7 and FC27 (MSP2-3D7/FC27), and apical membrane antigen 1 (AMA1) in patients with uncomplicated malaria by enzyme-linked immunosorbent assay (ELISA) but no differences against EBA140, EBA175, MSP1, and reticulocyte-binding protein homologues Rh2 and Rh4 or for IgM against MSP2-3D7/FC27.Patients with uncomplicated malaria were also shown to have higher antibody affinities for AMA1 by surface plasmon resonance (SPR). Decreased invasion of two clinicalP. falciparumisolates in the presence of patient plasma correlated with lower initial parasitemia in the patients, in contrast to comparisons of parasitemia to ELISA values or antibody affinities, which did not show any correlations. Analysis of the heterogeneity of the infections revealed a higher MOI in patients with uncomplicated disease, with theP. falciparumK1 MSP1 (MSP1-K1) and MSP2-3D7 being the most discriminative allelic markers. Higher MOIs also correlated positively with higher antibody levels in several of the ELISAs. In conclusion, certain antibody responses and MOIs were associated with differences between uncomplicated and severe malaria. When different assays were combined, some antibodies, like those against AMA1, seemed particularly discriminative. However, only decreased invasion correlated with initial parasitemia in the patient, signaling the importance of functional assays in understanding development of immunity against malaria and in evaluating vaccine candidates.

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