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Journal ArticleDOI

Plasmodium Cysteine Repeat Modular Proteins 3 and 4 are essential for malaria parasite transmission from the mosquito to the host

TL;DR: PCRMP3 and 4 play multiple roles during the Plasmodium life cycle; they are essential for the establishment of sporozoite infection in the mosquito salivary gland, and subsequently for development in hepatocytes and immunization with live sporozoites does not induce protective immune responses.
Abstract: Background: The Plasmodium Cysteine Repeat Modular Proteins (PCRMP) are a family of four conserved proteins of malaria parasites, that contain a number of motifs implicated in host-parasite interactions. Analysis of mutants of the rodent parasite Plasmodium berghei lacking expression of PCRMP1 or 2 showed that these proteins are essential for targeting of P. berghei sporozoites to the mosquito salivary gland and, hence, for transmission from the mosquito to the mouse. Methods: In this work, the role of the remaining PCRMP family members, PCRMP3 and 4, has been investigated throughout the Plasmodium life cycle by generation and analysis of P. berghei gene deletion mutants, Δpcrmp 3a ndΔpcrmp4. The role of PCRMP members during the transmission and hepatic stages of the Plasmodium lifecycle has been evaluated by light- and electron microscopy and by analysis of liver stage development in HEPG2 cells in vitro and by infecting mice with mutant sporozoites. In addition, mice were immunized with live Δpcrmp 3a ndΔpcrmp4 sporozoites to evaluate their immunization potential as a genetically-attenuated parasite-based vaccine. Results: Disruption of pcrmp3 and pcrmp4 in P. berghei revealed that they are also essential for transmission of the parasite through the mosquito vector, although acting in a distinct way to pbcrmp1 and 2. Mutants lacking expression of PCRMP3 or PCRMP4 show normal blood stage development and oocyst formation in the mosquito and develop into morphologically normal sporozoites, but these have a defect in egress from oocysts and do not enter the salivary glands. Sporozoites extracted from oocysts perform gliding motility and invade and infect hepatocytes but do not undergo further development and proliferation. Furthermore, the study shows that immunization with Δcrmp3 and Δcrmp4 sporozoites does not confer protective immunity upon subsequent challenge. Conclusions: PCRMP3 and 4 play multiple roles during the Plasmodium life cycle; they are essential for the establishment of sporozoite infection in the mosquito salivary gland, and subsequently for development in hepatocytes. However, although Δpcrmp3 and Δpcrmp4 parasites are completely growth-impaired in the liver, immunization with live sporozoites does not induce the protective immune responses that have been shown for other genetically-attenuated parasites.

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Citations
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01 Jan 2008
TL;DR: In this paper, the authors demonstrate that a single Plasmodium gene disruption can generate virulence-attenuated parasites that do not induce cerebral complications and, moreover, stimulate strong protective immunity against subsequent challenge with wild-type parasites.
Abstract: Plasmodium parasites lacking plasmepsin 4 (PM4), an aspartic protease that functions in the lysosomal compartment and contributes to hemoglobin digestion, have only a modest decrease in the asexual blood-stage growth rate; however, PM4 deficiency in the rodent malaria parasite Plasmodium berghei results in significantly less virulence than that for the parental parasite. P. berghei Deltapm4 parasites failed to induce experimental cerebral malaria (ECM) in ECM-susceptible mice, and ECM-resistant mice were able to clear infections. Furthermore, after a single infection, all convalescent mice were protected against subsequent parasite challenge for at least 1 year. Real-time in vivo parasite imaging and splenectomy experiments demonstrated that protective immunity acted through antibody-mediated parasite clearance in the spleen. This work demonstrates, for the first time, that a single Plasmodium gene disruption can generate virulence-attenuated parasites that do not induce cerebral complications and, moreover, are able to stimulate strong protective immunity against subsequent challenge with wild-type parasites. Parasite blood-stage attenuation should help identify protective immune responses against malaria, unravel parasite-derived factors involved in malarial pathologies, such as cerebral malaria, and potentially pave the way for blood-stage whole organism vaccines.

96 citations

Journal ArticleDOI
TL;DR: A comprehensive analysis of heterochromatin landscapes across different Plasmodium species, strains, and life cycle stages reveals conserved and specialized features of epigenetic control across the genus Plas modium.

95 citations


Cites background from "Plasmodium Cysteine Repeat Modular ..."

  • ...…(Gilberger et al., 2003), RBC remodeling in gametocytes (geco) (Morahan et al., 2011), mosquito midgut invasion (warp) (Yuda et al., 2001), sporozoite maturation or egress (ccp1, crmp4) (Simon et al., 2009; Douradinha et al., 2011), or liver stage development (lsa1) (Mikolajczak et al., 2011)....

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  • ..., 2001), sporozoite maturation or egress (ccp1, crmp4) (Simon et al., 2009; Douradinha et al., 2011), or liver stage development (lsa1) (Mikolajczak et al....

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Journal ArticleDOI
TL;DR: It is found that malaria parasites use two overlapping, extensive, and independent programs of translational repression across sporozoite maturation to temporally regulate protein expression, thus promoting their successful development and vector-to-host transition.
Abstract: Plasmodium sporozoites are transmitted from infected mosquitoes to mammals, and must navigate the host skin and vasculature to infect the liver. This journey requires distinct proteomes. Here, we report the dynamic transcriptomes and proteomes of both oocyst sporozoites and salivary gland sporozoites in both rodent-infectious Plasmodium yoelii parasites and human-infectious Plasmodium falciparum parasites. The data robustly define mRNAs and proteins that are upregulated in oocyst sporozoites (UOS) or upregulated in infectious sporozoites (UIS) within the salivary glands, including many that are essential for sporozoite functions in the vector and host. Moreover, we find that malaria parasites use two overlapping, extensive, and independent programs of translational repression across sporozoite maturation to temporally regulate protein expression. Together with gene-specific validation experiments, these data indicate that two waves of translational repression are implemented and relieved at different times during sporozoite maturation, migration and infection, thus promoting their successful development and vector-to-host transition.

88 citations

Journal ArticleDOI
TL;DR: Understanding the molecular and cellular mechanisms that mediate the remarkable sporozoite journey in the invertebrate vector and the vertebrate host can inform evidence-based next-generation drug development programs and immune intervention strategies.
Abstract: Plasmodium sporozoite transmission is a critical population bottleneck in parasite life-cycle progression and, hence, a target for prophylactic drugs and vaccines. The recent progress of a candidate antisporozoite subunit vaccine formulation to licensure highlights the importance of sporozoite transmission intervention in the malaria control portfolio. Sporozoites colonize mosquito salivary glands, migrate through the skin, penetrate blood vessels, breach the liver sinusoid, and invade hepatocytes. Understanding the molecular and cellular mechanisms that mediate the remarkable sporozoite journey in the invertebrate vector and the vertebrate host can inform evidence-based next-generation drug development programs and immune intervention strategies.

71 citations


Cites background from "Plasmodium Cysteine Repeat Modular ..."

  • ...…TRAP family of adhesins, including SIAP-1/ag17/S5, S23/ SSP3, PCRMP3, and PCRMP4, play critical roles in sporozoite transmission (Engelmann et al. 2009; Douradinha et al. 2011; Harupa et al. 2014), indicative of a complex and nonredundant protein network that mediates sporozoite–host interactions....

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  • ...Several Plasmodium-specific membrane proteins that do not belong to the TRAP family of adhesins, including SIAP-1/ag17/S5, S23/ SSP3, PCRMP3, and PCRMP4, play critical roles in sporozoite transmission (Engelmann et al. 2009; Douradinha et al. 2011; Harupa et al. 2014), indicative of a complex and nonredundant protein network that mediates sporozoite–host interactions....

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Journal ArticleDOI
24 Jan 2017-eLife
TL;DR: It is found that a thrombospondin-repeat containing sporozoite-specific Protein 1 (TRP1) is important for oocyst egress and salivary gland invasion, and hence for the transmission of malaria.
Abstract: Malaria is caused by a parasite transmitted by certain types of mosquito. The parasite lives in different organs within its vertebrate animal and insect hosts and to cope with these different environments it has a complex life cycle with several highly specialized life stages. To move from an infected mosquito into vertebrates the parasite produces spore-like cells called sporozoites that are able to enter different tissues and move very fast. These cells develop inside parasite-made structures called oocysts, which form at the stomach wall of the mosquito. After emerging from the oocyst, sporozoites float through the mosquito’s circulatory system and eventually enter the salivary glands where they can be transmitted to vertebrates when the mosquito bites. Efforts to develop malaria treatments and vaccines have focused on understanding the parasite’s life cycle and identifying ways to control or eradicate key stages. Most researchers focus on the stage where the parasite is living in the vertebrate and actively causing disease, while the events in the mosquito are less intensely investigated. While several parasite proteins have been shown to be important for the release of sporozoites from oocysts, the molecular events leading to this release have not yet been fully resolved. Klug and Frischknecht used time-lapse microscopy to film the release of the sporozoites of a malaria parasite known as Plasmodium berghei. The experiments show that the sporozoites can leave oocysts in several different ways. Furthermore, Klug and Frischknecht identified a new parasite protein named TRP1 that is essential for the sporozoites to leave oocysts and invade the salivary glands. Sporozoites lacking TRP1 were not able to move and they were unable to leave the oocyst or invade the salivary glands. Klug and Frischknecht propose a new working model of the molecular events that govern sporozoite release in which TRP1 is required for sporozoites to move prior to their exit from oocysts. In the future, using the same techniques to analyze genetically modified parasites will help to reveal more details about sporozoite release.

54 citations


Cites background from "Plasmodium Cysteine Repeat Modular ..."

  • ...Sporozoites lacking SERA5, PCRMP3, PCRMP4, PfCCp2 and PfCCp3 fail to egress from oocysts (Aly and Matuschewski, 2005; Douradinha et al., 2011; Pradel et al., 2004), whereas parasites lacking CSP do not complete sporozoite formation (Ménard et al....

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  • ..., 2002), the Plasmodium cysteine repeat modular proteins PCRMP3 and PCRMP4 (Douradinha et al., 2011) and the LCCL-domain-containing proteins PfCCp2 and PfCCp3 (Pradel et al....

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  • ...…the GPI-anchored circumsporozoite protein, CSP (Wang et al., 2005; Aly and Matuschewski, 2005; Tewari et al., 2002), the Plasmodium cysteine repeat modular proteins PCRMP3 and PCRMP4 (Douradinha et al., 2011) and the LCCL-domain-containing proteins PfCCp2 and PfCCp3 (Pradel et al., 2004) Video 3....

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  • ...Sporozoites lacking SERA5, PCRMP3, PCRMP4, PfCCp2 and PfCCp3 fail to egress from oocysts (Aly and Matuschewski, 2005; Douradinha et al., 2011; Pradel et al., 2004), whereas parasites lacking CSP do not complete sporozoite formation (Ménard et al., 1997)....

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  • ...The factors that are known to date are the serine repeat antigen 5, SERA5 (previously named egress cysteine protease 1; ECP1), the GPI-anchored circumsporozoite protein, CSP (Wang et al., 2005; Aly and Matuschewski, 2005; Tewari et al., 2002), the Plasmodium cysteine repeat modular proteins PCRMP3 and PCRMP4 (Douradinha et al., 2011) and the LCCL-domain-containing proteins PfCCp2 and PfCCp3 (Pradel et al., 2004) Video 3....

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References
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Journal ArticleDOI
14 Oct 1967-Nature
TL;DR: Preliminary results on the production of protective immunity in mice by vaccination with X-irradiated sporozoites of Plasmodium berghei are reported.
Abstract: STUDIES with avian malaria have shown that killed sporozoites as well as sporozoites inactivated with ultraviolet light can produce a partial immunity after injection into birds1,2. On the other hand, attempts to use the erythrocytic stages of the parasite as the source of antigen have met with only limited success with avian3, rodent4 and monkey malaria5,6. Previous attempts to use killed sporozoites of the rodent malarial parasite, Plasmodium berghei, to immunize rodents have been unsuccessful. We therefore sought to determine whether protective immunity to this parasite could be achieved by partial inactivation of the injected sporozoites as opposed to injection of dead parasites. X-irradiation was chosen as the inactivating agent, because of the partial immunity obtained by vaccination with irradiated blood forms of malaria parasites7–9. This communication reports preliminary results on the production of protective immunity in mice by vaccination with X-irradiated sporozoites of P. berghei.

851 citations


"Plasmodium Cysteine Repeat Modular ..." refers background in this paper

  • ...Immunization with sporozoites that are attenuated by irradiation or genetic modification and that infect hepatocytes but arrest development at an early stage can confer protection against subsequent challenge with infectious sporozoites [3,5-15,34]....

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  • ...Author details 1Malaria Unit, Instituto de Medicina Molecular, Faculdade de Medicina de Lisboa, Av. Prof. Egas Moniz, 1649-028 Lisbon, Portugal....

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  • ...Aly AS, Vaughan AM, Kappe SH: Malaria parasite development in the mosquito and infection of the mammalian host....

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  • ...Malaria Journal 2011 10:71....

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  • ...Janse CJ, Haghparast A, Speranca MA, Ramesar J, Kroeze H, del Portillo HA, Waters AP: Malaria parasites lacking eef1a have a normal S/M phase yet grow more slowly due to a longer G1 phase....

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Journal ArticleDOI
08 Aug 1997-Cell
TL;DR: It is demonstrated that by gene targeting in a rodent Plasmodium, TRAP is critical for sporozoite infection of the mosquito salivary glands and the rat liver, and is essential for sporzoite gliding motility in vitro, suggesting that in PlasModium sporozoites, and likely in other Apicomplexa, gliding locomotion and cell invasion have a common molecular basis.

627 citations


"Plasmodium Cysteine Repeat Modular ..." refers background in this paper

  • ...It has previously been thought that oocyst and haemolymph sporozoites are intrinsically less infectious than those that reach the salivary gland, and that infectivity is enhanced by mechanisms that are activated during salivary gland invasion [39,40]....

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Journal ArticleDOI
TL;DR: It is shown that only a proportion of the parasites enter blood capillaries, whereas others are drained by lymphatics, andymph sporozoites stop at the proximal lymph node, where most are degraded inside dendritic leucocytes, but some can partially differentiate into exoerythrocytic stages.
Abstract: Plasmodium, the parasite that causes malaria, is transmitted by a mosquito into the dermis and must reach the liver before infecting erythrocytes and causing disease. We present here a quantitative, real-time analysis of the fate of parasites transmitted in a rodent system. We show that only a proportion of the parasites enter blood capillaries, whereas others are drained by lymphatics. Lymph sporozoites stop at the proximal lymph node, where most are degraded inside dendritic leucocytes, but some can partially differentiate into exoerythrocytic stages. This previously unrecognized step of the parasite life cycle could influence the immune response of the host, and may have implications for vaccination strategies against the preerythrocytic stages of the parasite.

575 citations


"Plasmodium Cysteine Repeat Modular ..." refers background in this paper

  • ...be noted, however, that parasite transmission to a host via mosquito bite is a more complex process requiring sporozoite migration within the skin of the host at the injection site [33,42,43] before entry into a blood vessel and transport to the liver....

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  • ...To assess sporozoite motility, circumsporozoite protein (CSP) gliding trails were visualized by staining with a monoclonal antibody against CSP [24,25]....

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  • ...This phenotype shows some similarity with that of parasites containing mutations in the conserved region II plus of Circumsporozoite Protein (CSP; PBANKA_040320) and in parasite mutants lacking expression of Egress Cysteine Protease 1 (ECP1; PBANKA_030470), PSOP9 (PBANKA_070190) and Sporozoite Invasion-associated Protein 1, (SIAP-1; PBANKA_100620) [35-38]....

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  • ...parasite forward and leaves a trail of CSP [24,32,33]....

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  • ...Sporozoite migration through cells was quantified by detection of parasite-wounded hepatocytes using a cell-impermeant fluorescent tracer macromolecule, rhodamin-dextran [24] and staining for CSP. Migration through host cells was quantified as the percentage of dextran-positive non-infected cells, and the number of sporozoites that reach a final hepatocyte for infection and further development, as the percentage of parasites inside dextran-negative cells [24]....

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Journal ArticleDOI
05 Jan 2001-Science
TL;DR: Sporozoite migration through several cells in the mammalian host appears to be essential for the completion of the life cycle of Plasmodium sporozoites.
Abstract: Intracellular bacteria and parasites typically invade host cells through the formation of an internalization vacuole around the invading pathogen. Plasmodium sporozoites, the infective stage of the malaria parasite transmitted by mosquitoes, have an alternative mechanism to enter cells. We observed breaching of the plasma membrane of the host cell followed by rapid repair. This mode of entry did not result in the formation of a vacuole around the sporozoite, and was followed by exit of the parasite from the host cell. Sporozoites traversed the cytosol of several cells before invading a hepatocyte by formation of a parasitophorous vacuole, in which they developed into the next infective stage. Sporozoite migration through several cells in the mammalian host appears to be essential for the completion of the life cycle.

565 citations


"Plasmodium Cysteine Repeat Modular ..." refers background in this paper

  • ...Cells that have been traversed can be visualized using a rhodamine-dextran tracer that enters the cell immediately after membrane disruption and becomes trapped inside after its’ repair [24,32]....

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  • ...Following transmission, sporozoites migrate through several hepatocytes before invading and establishing infection in a final hepatocyte; a feature dependent on sporozoite gliding motility [24,32]....

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  • ...To assess sporozoite motility, circumsporozoite protein (CSP) gliding trails were visualized by staining with a monoclonal antibody against CSP [24,25]....

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  • ...This phenotype shows some similarity with that of parasites containing mutations in the conserved region II plus of Circumsporozoite Protein (CSP; PBANKA_040320) and in parasite mutants lacking expression of Egress Cysteine Protease 1 (ECP1; PBANKA_030470), PSOP9 (PBANKA_070190) and Sporozoite Invasion-associated Protein 1, (SIAP-1; PBANKA_100620) [35-38]....

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  • ...parasite forward and leaves a trail of CSP [24,32,33]....

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Journal ArticleDOI
TL;DR: This protocol for high-efficiency transformation will enable the application of large-scale functional genomics approaches and enables the generation of transformed parasites within 10–15 d.
Abstract: This protocol describes a method of genetic transformation for the rodent malaria parasite Plasmodium berghei with a high transfection efficiency of 10(-3)-10(-4). It provides methods for: (i) in vitro cultivation and purification of the schizont stage;(ii) transfection of DNA constructs containing drug-selectable markers into schizonts using the nonviral Nucleofector technology; and (iii) injection of transfected parasites into mice and subsequent selection of mutants by drug treatment in vivo. Drug selection is described for two (antimalarial) drugs, pyrimethamine and WR92210. The drug-selectable markers currently in use are the pyrimethamine-resistant dihydrofolate reductase (dhfr) gene of Plasmodium or Toxoplasma gondii and the DHFR gene of humans that confer resistance to pyrimethamine and WR92210, respectively. This protocol enables the generation of transformed parasites within 10-15 d. Genetic modification of P. berghei is widely used to investigate gene function in Plasmodium, and this protocol for high-efficiency transformation will enable the application of large-scale functional genomics approaches.

564 citations


"Plasmodium Cysteine Repeat Modular ..." refers methods in this paper

  • ...berghei ANKA strain used was cl15cy1 [16]....

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  • ...Transfection, selection and cloning of mutant parasite lines was performed as described [16] and were carried out in duplicate for each gene, generating Δpcrmp3a and b and Δpcrmp4a and b....

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  • ...Correct integration of the construct into the genome of mutant parasites was analysed by Southern blot analysis of digested genomic DNA and/or of FIGE separated chromosomes [16]....

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  • ...The genes were disrupted using standard genetic modification technologies aimed at disruption of the gene via double crossover integration [16]....

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