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Parasite Biology

Source: TropIKA

Title of the session: Parasite Biology

Date: 5 November 2009

Agenda item: Scientific session 24

Session theme: Parasite Genetics

Meeting room: Aberdares

Chair(s):

• Louis Miller (Rockville)
• Fousseyni Toure (Franceville)

Presenters:

• Alicia Couto, Buenos Aires University
• Shailja Singh, International Centre for Genetic Engineering and Biotechnology
• Beth Mutai, Walter Reed Project/ Kenya Medical Research Institute
• Lígia Gonçalves, Instituto Gulbenkian de Ciência
• Abdirahman Abdi, University of Glasgow
• Silvia Portugal, Universidade de Lisboa
• Luc Reininger, Institut national de la santé et de la recherche médicale (INSERM)

Official rapporteur:

TropIKA rapporteur: Daniel Kiboi

Major topics:

• THE GLYCOSPHINGOLIPID PATHWAY AS TARGET FOR NEW ANTIMALARIAL DRUGS [16694906]
• SIGNALS FOR SEQUENTIAL RELEASE OF APICAL ORGANELLES DURING ERYTHROCYTE INVASION BY MALARIA PARASITES [16196093]
• APOPTOTIC MACHINERY IN PLASMODIUM FALCIPARUM GROWING IN A CONTINUOUS CULTURE [16574236]
• FAS-PATHWAY MODULATION BY P.BERGHEI LIVER STAGE [16699842]
• ASSESSMENT OF PLASMODIUM FALCIPARUM TYROSINE KINASE-LIKE (TKL) PROTEIN KINASES AS POTENTIAL ANTI-MALARIAL DRUG TARGETS. [16228941]
• MALARIA LIVER INFECTION IS DECREASED DURING BLOOD-STAGE INFECTION [16299455]
• REVERSE GENETICS OF THE PLASMODIUM FALCIPARUM AURORA-RELATED PROTEIN KINASE PFARK-1 [16696871]

Keywords:

• Antimalarial drugs
• Novel drug targets

Scope: Potential novel antimalarial drug targets were covered in the session, the selectivity and sensitivity of the novel target in relation to humans was reported.

REPORT ON ORIGINAL SESSION

Overview

Exploration of novel drug targets in Plasmodium falciparum is a germane aspect in the fight against malaria to provide platform for screening of novel antimalarial and to replenish the drugs already lost to resistance. Two identified drug targets have been validated for the development of new anti-malarials: glycosyceramide synthase, an enzyme controlling synthesis of essential sphingomyelin in Plasmodium falciparum, and Plasmodium falciparum Aurora-related protein kinase-1 (Pfark-1), an essential gene in P. falciparum parasite development. A study on existence of in vitro apoptotic machinery in Plasmodium falciparum operating in tandem with parasite density was presented. The apoptotic network provides an opportunity for exploration of potential drug targets. In a different study, a model ascertaining that an ongoing Plasmodium blood stage infection significantly decreases the ability of sporozoites to establish a secondary liver infection was presented. Emphasis on the better understanding of blood-liver stage interaction would give insights into why in endemic populations, high transmission areas associate with partial protection from severe malaria.

CONTEXT AND ISSUE

• Glycosphingolipids pathway in Plasmodium falciparum is a potential drug target,

• Is Plasmodium falciparum Aurora-related protein kinase Pfark-1 a potential drug target?

• Apoptotic machinery in Plasmodium falciparum is a potential drug targets pathway

Key facts and figures

• The need to identify Plasmodium falciparum survival tactics in the apoptotic machinery as potential drug target pathway

• glycosyceramide synthase is an essential enzyme controlling synthesis of essential sphingomyelin in Plasmodium falciparum

• Aurora-related protein kinase Pfark-1is involved in spindle pole body maturation mitotic spindle assembly and coordination or early mitotic events

Initiatives on the ground; experience/s derived

• In screening for apoptotic machinery parasite growth, DNA fragmentation and Metacaspase gene expression, were monitored by Giemsa-stained thin smears, flow cytometry and quantitative real time reverse transcriptase polymerase chain reaction (qRT-PCR) using metacaspase gene specific primers

• In exploring the blood and liver stages parasites in Plasmodium berghei, Silvia Portugal, Universidade de Lisboa, gene expression levels and histological inspection revealed lower hepatocyte infection and decreased parasite development with rise in blood stage parasite.

• In exploring the Aurora-related protein kinases for drug targets P. falciparum 3D7 parasites were transfected with Pfark-1 KO and GFP constructs and selected for resistance to blasticidin. Integration of plasmid constructs by homologous recombination was monitored by PCR

Research Findings

• Plasmodium berghei blood stage infections decreases establishment of secondary liver stages. Interaction of these parasites lifecycle stages could potentially explain the partial protection from severe malaria evidenced in high transmission areas

• Plasmodium falciparum Aurora-related protein kinase-1 (Pfark-1) identified and validated as potential target for antimalarial drug development

• Results on existence of in vitro apoptotic machinery in Plasmodium falciparum and the potential of the apoptotic network for exploration of potential drug targets

Lessons learned

• Iron availability limit parasites exo-erythrocytic development of liver stages and in screening for blood liver stage interaction, iron should be monitored

• Decrease of parasite density in an in vitro system is not always associated with apoptosis

Issues raised, obstacles, difficulties

In assessing the apoptotic studies several phenomenon occur in the in vitro system, for instance decrease in parasites density could result from elevated gametocytogenesis; deficiency of nutrient in an in vitro system results to death of cells and which could be wrongly recorded are apoptosis.

Future plans

A better understanding on how Plasmodium blood and liver-stages interact will give insights into why endemic populations in high transmission associate with partial protection from severe disease.

Development and screening of chemical compounds targeting Plasmodium falciparum Aurora-related protein kinase-1 (Pfark-1)

FINDINGS AND CONCLUSIONS

From formal presentations

Translational Research Impact

• Screening of chemical compounds on the identified Glycosyceramide synthase and Aurora-related protein kinase-1 (Pfark-1) proteins

Knowledge gap created

• Exploring the in vitro Plasmodium falciparum apoptotic pathway to screen for potential drug target

• Exploring the in vivo Plasmodium falciparum apoptotic pathway using field isolates

• Explore how Plasmodium blood and liver-stages interact in decreasing the establishment of a secondary liver-stage

• Validation of Glycosyceramide synthase as an essential protein in Plasmodium falciparum by confirmation absence of bypass sources of sphingolipids in the parasite

From open discussions/debates

Identification of glycosyceramide synthase as potential drug target raises the question of prospects of parasite bypassing the sphingolipids pathway and obtaining the sphingolipids from different sources.

Identified conclusions

Main points of agreement

Plasmodium falciparum Aurora-related protein kinase-1 (Pfark-1) is a potential drug target and be can be used for the screening of chemical libraries for activity

Main points of divergence

The Plasmodium falciparum apoptotic studies in vitro system could be confounded by several phenomenons such as decreased nutrient levels and gametocytogenesis

Recommendations

• The Plasmodium falciparum apoptotic phenomenon evidenced in vitro should be explored in an in vivo system using field isolates. Validation of this network in field isolates

• To explore the interaction of Plasmodium blood and liver-stages in decreasing the establishment of a secondary liver-stage

• The validation of Glycosyceramide synthase as an essential protein in Plasmodium falciparum by confirming absence of bypass sources of sphingolipids in the parasite

Personal observations from rapporteur

The challenge in identification of unique drug target proteins in Plasmodium parasites has been the alternative bypass pathways utilized by the parasite

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