RESUMO
Nonmuscle myosin IIB (NMIIB) is considered a primary force generator during cell motility. Yet many cell types, including motile cells, do not necessarily express NMIIB. Given the potential of cell engineering for the next wave of technologies, adding back NMIIB could be a strategy for creating supercells with strategically altered cell morphology and motility. However, we wondered what unforeseen consequences could arise from such an approach. Here, we leveraged pancreatic cancer cells, which do not express NMIIB. We generated a series of cells where we added back NMIIB and strategic mutants that increase the ADP-bound time or alter the phosphorylation control of bipolar filament assembly. We characterized the cellular phenotypes and conducted RNA-seq analysis. The addition of NMIIB and the different mutants all have specific consequences for cell morphology, metabolism, cortical tension, mechanoresponsiveness, and gene expression. Major modes of ATP production are shifted, including alterations in spare respiratory capacity and the dependence on glycolysis or oxidative phosphorylation. Several metabolic and growth pathways undergo significant changes in gene expression. This work demonstrates that NMIIB is highly integrated with many cellular systems and simple cell engineering has a profound impact that extends beyond the primary contractile activity presumably being added to the cells.
Assuntos
Miosina não Muscular Tipo IIA , Miosina não Muscular Tipo IIB , Miosina não Muscular Tipo IIB/metabolismo , Reprogramação Celular , Citoesqueleto/metabolismo , Contração Muscular , Fosforilação , Miosina não Muscular Tipo IIA/metabolismoRESUMO
BACKGROUND: The Plasmodium falciparum histidine-rich protein II (PfHRP2) is a common biomarker used in malaria rapid diagnostic tests (RDTs), but can persist in the blood for up to 40 days following curative treatment. The persistence of PfHRP2 presents a false positive limitation to diagnostic interpretation. However, the in vivo dynamics and compartmentalization underlying PfHRP2 persistence have not been fully characterized in the plasma and erythrocyte (RBC) fraction of the whole blood. METHODS: The kinetics and persistence of PfHRP2 in the plasma and RBC fractions of the whole blood were investigated post-treatment in human clinical samples and samples isolated from BALB/c mice infected with a novel transgenic Plasmodium berghei parasite engineered to express PfHRP2 (PbPfHRP2). RESULTS: PfHRP2 levels in human RBCs were consistently 20-40 times greater than plasma levels, even post-parasite clearance. PfHRP2 positive, DNA negative, once-infected RBCs were identified in patients that comprised 0.1-1% of total RBCs for 6 and 12 days post-treatment, even post-atovaquone-proguanil regimens. Transgenic PbPfHRP2 parasites in BALB/c mice produced and exported tgPfHRP2 to the RBC cytosol similar to P. falciparum. As in humans, tgPfHRP2 levels were found to be approximately 20-fold higher within the RBC fraction than the plasma post-treatment. RBC localized tgPfHRP2 persisted longer than tgPfHRP2 in the plasma after curative treatment. tgPfHRP2 positive, but DNA negative once-infected RBCs were also detected in mouse peripheral blood for 7-9 days after curative treatment. CONCLUSIONS: The data suggest that persistence of PfHRP2 is due to slower clearance of protein from the RBC fraction of the whole blood. This appears to be a result of the presence PfHRP2 in previously infected, pitted cells, as opposed to PfHRP2 binding naïve RBCs in circulation post-treatment. The results thus confirm that the extended duration of RDT positivity after parasite clearance is likely due to pitted, once-infected RBCs that remain positive for PfHRP2.
Assuntos
Antígenos de Protozoários/sangue , Antimaláricos/administração & dosagem , Eritrócitos/química , Malária Falciparum/tratamento farmacológico , Malária Falciparum/patologia , Plasma/química , Proteínas de Protozoários/sangue , Animais , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Fatores de TempoRESUMO
Plasmodium sporozoites are injected into the skin as mosquitoes probe for blood. From here, they migrate through the dermis to find blood vessels which they enter in order to be rapidly carried to the liver, where they invade hepatocytes and develop into the next life cycle stage, the exoerythrocytic stage. Once sporozoites enter the blood circulation, they are found in hepatocytes within minutes. In contrast, sporozoite exit from the inoculation site resembles a slow trickle and occurs over several hours. Thus, sporozoites spend the majority of their extracellular time at the inoculation site, raising the hypothesis that this is when the malarial parasite is most vulnerable to antibody-mediated destruction. Here, we investigate this hypothesis and demonstrate that the neutralizing capacity of circulating antibodies is greater at the inoculation site than in the blood circulation. Furthermore, these antibodies are working, at least in part, by impacting sporozoite motility at the inoculation site. Using actively and passively immunized mice, we found that most parasites are either immobilized at the site of injection or display reduced motility, particularly in their net displacement. We also found that antibodies severely impair the entry of sporozoites into the bloodstream. Overall, our data suggest that antibodies targeting the migratory sporozoite exert a large proportion of their protective effect at the inoculation site.IMPORTANCE Studies in experimental animal models and humans have shown that antibodies against Plasmodium sporozoites abolish parasite infectivity and provide sterile immunity. While it is well documented that these antibodies can be induced after immunization with attenuated parasites or subunit vaccines, the mechanisms by and location in which they neutralize parasites have not been fully elucidated. Here, we report studies indicating that these antibodies display a significant portion of their protective effect in the skin after injection of sporozoites and that one mechanism by which they work is by impairing sporozoite motility, thus diminishing their ability to reach blood vessels. These results suggest that immune protection against malaria begins at the earliest stages of parasite infection and emphasize the need of performing parasite challenge in the skin for the evaluation of protective immunity.
Assuntos
Anticorpos Antiprotozoários/imunologia , Vasos Sanguíneos/parasitologia , Derme/imunologia , Derme/parasitologia , Esporozoítos/imunologia , Animais , Anopheles/parasitologia , Anticorpos Neutralizantes/imunologia , Feminino , Hepatócitos/parasitologia , Imunização , Imunização Passiva , Malária/sangue , Malária/parasitologia , Camundongos Endogâmicos C57BL , Plasmodium berghei/imunologiaRESUMO
The ability to monitor micropipette injections with a high-resolution fluorescent microscope has utility for a variety of applications. Herein, different approaches were tested for creating broad-band fluorescently labelled glass micropipettes including: UV cured glass glues, baked glass enamel containing fluorescent dyes as well as nanodiamonds attached during pipette formation in the microforge. The most robust and simplest approach was to use labelled baked enamel on the exterior of the pipette. This approach was tested using pipettes designed to mimic a mosquito proboscis for the injection of the malaria parasite, Plasmodium spp., into the dermis of a living mouse ear. The pipette (â¼30 micron diameter) was easily detected in the microscopy field of view and tolerated multiple insertions through the skin. This simple inexpensive approach to fluorescently labelling micropipettes will aid in the development of procedures under the fluorescent microscope.
Assuntos
Culicidae/parasitologia , Malária/transmissão , Microscopia de Fluorescência/métodos , Plasmodium/citologia , Coloração e Rotulagem/métodos , Animais , Culicidae/fisiologia , Camundongos , Modelos TeóricosRESUMO
A devastating complication of Plasmodium falciparum infection is cerebral malaria, in which vascular leakage and cerebral swelling lead to coma and often death. P. falciparum produces a protein called histidine-rich protein II (HRPII) that accumulates to high levels in the bloodstream of patients and serves as a diagnostic and prognostic marker for falciparum malaria. Using a human cerebral microvascular endothelial barrier model, we previously found that HRPII activates the endothelial cell inflammasome, resulting in decreased integrity of tight junctions and increased endothelial barrier permeability. Here, we report that intravenous administration of HRPII induced blood-brain barrier leakage in uninfected mice. Furthermore, HRPII infusion in P. berghei-infected mice increased early mortality from experimental cerebral malaria. These data support the hypothesis that HRPII is a virulence factor that contributes to cerebral malaria by compromising the integrity of the blood-brain barrier.
Assuntos
Antígenos de Protozoários/farmacologia , Barreira Hematoencefálica/efeitos dos fármacos , Edema Encefálico/patologia , Malária Cerebral/patologia , Malária Falciparum/patologia , Proteínas de Protozoários/farmacologia , Animais , Barreira Hematoencefálica/patologia , Modelos Animais de Doenças , Camundongos , Junções Íntimas/patologiaRESUMO
As the Plasmodium parasite transitions between mammalian and mosquito host, it has to adjust quickly to new environments. Palmitoylation, a reversible and dynamic lipid post-translational modification, plays a central role in regulating this process and has been implicated with functions for parasite morphology, motility and host cell invasion. While proteins associated with the gliding motility machinery have been described to be palmitoylated, no palmitoyl transferase responsible for regulating gliding motility has previously been identified. Here, we characterize two palmityol transferases with gene tagging and gene deletion approaches. We identify DHHC3, a palmitoyl transferase, as a mediator of ookinete development, with a crucial role for gliding motility in ookinetes and sporozoites, and we co-localize the protein with a marker for the inner membrane complex in the ookinete stage. Ookinetes and sporozoites lacking DHHC3 are impaired in gliding motility and exhibit a strong phenotype in vivo; with ookinetes being significantly less infectious to their mosquito host and sporozoites being non-infectious to mice. Importantly, genetic complementation of the DHHC3-ko parasite completely restored virulence. We generated parasites lacking both DHHC3, as well as the palmitoyl transferase DHHC9, and found an enhanced phenotype for these double knockout parasites, allowing insights into the functional overlap and compensational nature of the large family of PbDHHCs. These findings contribute to our understanding of the organization and mechanism of the gliding motility machinery, which as is becoming increasingly clear, is mediated by palmitoylation.
Assuntos
Aciltransferases/fisiologia , Anopheles/parasitologia , Fígado/parasitologia , Plasmodium berghei/enzimologia , Proteínas de Protozoários/fisiologia , Animais , Células Hep G2 , Interações Hospedeiro-Parasita , Humanos , Lipoilação , Camundongos , Oocistos/enzimologia , Oocistos/crescimento & desenvolvimento , Plasmodium berghei/fisiologia , Processamento de Proteína Pós-Traducional , Glândulas Salivares/parasitologia , Esporozoítos/enzimologia , Esporozoítos/crescimento & desenvolvimentoRESUMO
The circumsporozoite protein (CSP) is the major surface protein of the sporozoite stage of malaria parasites and has multiple functions as the parasite develops and then migrates from the mosquito midgut to the mammalian liver. The overall structure of CSP is conserved among Plasmodium species, consisting of a species-specific central tandem repeat region flanked by two conserved domains: the NH2-terminus and the thrombospondin repeat (TSR) at the COOH-terminus. Although the central repeat region is an immunodominant B-cell epitope and the basis of the only candidate malaria vaccine in Phase III clinical trials, little is known about its functional role(s). We used the rodent malaria model Plasmodium berghei to investigate the role of the CSP tandem repeat region during sporozoite development. Here we describe two mutant parasite lines, one lacking the tandem repeat region (ΔRep) and the other lacking the NH2-terminus as well as the repeat region (ΔNΔRep). We show that in both mutant lines oocyst formation is unaffected but sporozoite development is defective.
Assuntos
Malária/parasitologia , Plasmodium berghei/crescimento & desenvolvimento , Proteínas de Protozoários/metabolismo , Esporozoítos/crescimento & desenvolvimento , Animais , Plasmodium berghei/química , Plasmodium berghei/genética , Plasmodium berghei/ultraestrutura , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Deleção de Sequência , Esporozoítos/química , Esporozoítos/metabolismo , Esporozoítos/ultraestruturaRESUMO
We used a Biomphalaria glabrata snail model for our studies and investigated the suitability of B. glabrata neonates, reared on a Nostoc sp. diet, for infection with Echinostoma caproni miracidia. We found that neonatal snails could become infected with E. caproni miracidia with 31 ± 11% standard error (SE) of our exposed snails containing rediae infections at 4 wk post-exposure (PE). However, the survival of exposed neonates was significantly (P < 0.05, Student's t-test) less than that of the unexposed controls at 1, 2, 3, and 4 wk PE.