Localisation of laminin within Plasmodium berghei oocysts and the midgut epithelial cells of Anopheles stephensi

<p>Abstract</p> <p>Background</p> <p>Oocysts of the malaria parasite form and develop in close proximity to the mosquito midgut basal lamina and it has been proposed that components of this structure play a crucial role in the development and maturation of oocysts that produce infective sporozoites. It is further suggested that oocysts incorporate basal lamina proteins into their capsule and that this provides them with a means to evade recognition by the mosquito's immune system. The site of production of basal lamina proteins in insects is controversial and it is still unclear whether haemocytes or midgut epithelial cells are the main source of components of the mosquito midgut basal lamina. Of the multiple molecules that compose the basal lamina, laminin is known to interact with a number of <it>Plasmodium </it>proteins. In this study, the localisation of mosquito laminin within the capsule and cytoplasm of <it>Plasmodium berghei </it>oocysts and in the midgut epithelial cells of <it>Anopheles stephensi </it>was investigated.</p> <p>Results</p> <p>An ultrastructural examination of midgut sections from infected and uninfected <it>An. stephensi </it>was performed. Post-embedded immunogold labelling demonstrated the presence of laminin within the mosquito basal lamina. Laminin was also detected on the outer surface of the oocyst capsule, incorporated within the capsule and associated with sporozoites forming within the oocysts. Laminin was also found within cells of the midgut epithelium, providing support for the hypothesis that these cells contribute towards the formation of the midgut basal lamina.</p> <p>Conclusion</p> <p>We suggest that ookinetes may become coated in laminin as they pass through the midgut epithelium. Thereafter, laminin secreted by midgut epithelial cells and/or haemocytes, binds to the outer surface of the oocyst capsule and that some passes through and is incorporated into the developing oocysts. The localisation of laminin on sporozoites was unexpected and the importance of this observation is less clear.</p

The microneme proteins CTRP and SOAP are not essential for Plasmodium berghei ookinete to oocyst transformation in vitro in a cell free system

<p>Abstract</p> <p>Background</p> <p>Two <it>Plasmodium berghei </it>ookinete micronemal proteins, circumsporozoite and TRAP related protein (CTRP) and secreted ookinete adhesive protein (SOAP) both interact with the basal lamina component laminin. Following gene disruption studies it has been proposed that, apart from their role in motility, these proteins may be required for interactions leading to ookinete-to-oocyst transformation.</p> <p>Methods</p> <p>CTRP and SOAP null mutant <it>P. berghei </it>ookinetes were compared to <it>P. berghei </it>ANKA wild-type for their ability to transform and grow <it>in vitro</it>. To confirm <it>in vitro </it>findings for <it>P</it>. <it>berghei </it>CTRP-KO ookinetes were injected into the haemocoel of <it>An</it>opheles <it>gambiae </it>female mosquitoes.</p> <p>Results</p> <p>Transformation, growth, and viability were comparable for the gene disrupted and wild-type parasites. <it>P</it>. <it>berghei </it>CTRP-KO ookinetes were able to transform into oocysts in the haemocoel of <it>An</it>. <it>gambiae </it>mosquitoes.</p> <p>Conclusion</p> <p>Neither CTRP nor SOAP is required for parasite transformation <it>in vitro</it>. By-passing the midgut lumen allows for the transformation of <it>P</it>. <it>berghei </it>CTRP-KO ookinetes suggesting that it is not required for transformation <it>in vivo</it>.</p

clag9 Is Not Essential for PfEMP1 Surface Expression in Non-Cytoadherent Plasmodium falciparum Parasites with a Chromosome 9 Deletion

BACKGROUND: The expression of the clonally variant virulence factor PfEMP1 mediates the sequestration of Plasmodium falciparum infected erythrocytes in the host vasculature and contributes to chronic infection. Non-cytoadherent parasites with a chromosome 9 deletion lack clag9, a gene linked to cytoadhesion in previous studies. Here we present new clag9 data that challenge this view and show that surface the non-cytoadherence phenotype is linked to the expression of a non-functional PfEMP1. METHODOLOGY/PRINCIPAL FINDINGS: Loss of adhesion in P. falciparum D10, a parasite line with a large chromosome 9 deletion, was investigated. Surface iodination analysis of non-cytoadherent D10 parasites and COS-7 surface expression of the CD36-binding PfEMP1 CIDR1α domain were performed and showed that these parasites express an unusual trypsin-resistant, non-functional PfEMP1 at the erythrocyte surface. However, the CIDR1α domain of this var gene expressed in COS-7 cells showed strong binding to CD36. Atomic Force Microscopy showed a slightly modified D10 knob morphology compared to adherent parasites. Trafficking of PfEMP1 and KAHRP remained functional in D10. We link the non-cytoadherence phenotype to a chromosome 9 breakage and healing event resulting in the loss of 25 subtelomeric genes including clag9. In contrast to previous studies, knockout of the clag9 gene from 3D7 did not interfere with parasite adhesion to CD36. CONCLUSIONS/SIGNIFICANCE: Our data show the surface expression of non-functional PfEMP1 in D10 strongly indicating that genes other than clag9 deleted from chromosome 9 are involved in this virulence process possibly via post-translational modifications

The microneme proteins CTRP and SOAP are not essential for ookinete to oocyst transformation in a cell free system-0

Rect immunofluoresence assay. A) mature ookinete; (B, C) ; (D) oocyst.<p><b>Copyright information:</b></p><p>Taken from "The microneme proteins CTRP and SOAP are not essential for ookinete to oocyst transformation in a cell free system"</p><p>http://www.malariajournal.com/content/7/1/82</p><p>Malaria Journal 2008;7():82-82.</p><p>Published online 19 May 2008</p><p>PMCID:PMC2427035.</p><p></p

The microneme proteins CTRP and SOAP are not essential for ookinete to oocyst transformation in a cell free system-4

Rect immunofluoresence assay. A) mature ookinete; (B, C) ; (D) oocyst.<p><b>Copyright information:</b></p><p>Taken from "The microneme proteins CTRP and SOAP are not essential for ookinete to oocyst transformation in a cell free system"</p><p>http://www.malariajournal.com/content/7/1/82</p><p>Malaria Journal 2008;7():82-82.</p><p>Published online 19 May 2008</p><p>PMCID:PMC2427035.</p><p></p

The microneme proteins CTRP and SOAP are not essential for ookinete to oocyst transformation in a cell free system-5

) and Malpighian tubules (MT) are also shown. CTRP-KO ookinetes were injected 18 h post-culture into the haemocoel of 4-day old KIL females and allowed to develop for 7 days prior to mosquito dissection.<p><b>Copyright information:</b></p><p>Taken from "The microneme proteins CTRP and SOAP are not essential for ookinete to oocyst transformation in a cell free system"</p><p>http://www.malariajournal.com/content/7/1/82</p><p>Malaria Journal 2008;7():82-82.</p><p>Published online 19 May 2008</p><p>PMCID:PMC2427035.</p><p></p