Evaluation of the immunomodulatory activity of Plasmodium falciparum hsp70-1
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Date
2014
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University of Zululand
Abstract
Heat shock proteins (Hsps) are conserved molecules that constitute a major part of the cell’s molecular chaperone system (protein folding machinery). Plasmodium falciparum Hsps play an important cytoprotective role ensuring that the malaria parasite survives under the harsh conditions that prevail in the host environment. P. falciparum Hsp70-1 (PfHsp70-1) is a ubiquitous, cytosol-localised Hsp70 that is essential for parasite survival. Apart from their role as molecular chaperones, it is believed that some Hsps of parasitic origin are capable of modulating host immunity through signal transduction (chaperokine role). Most investigations focusing on the chaperokine functions of Hsps use recombinant forms of the proteins produced in E. coli. The main drawback is that the recombinant proteins co-purify with lipopolysaccharides (LPS). Although LPS removal techniques have been developed, they do not completely remove these contaminants, leading to confounding data as LPS are active immune modulants. The current study sought to investigate the immunomodulatory role of PfHsp70-1. A recombinant form of
the protein was produced in three bacterial expression hosts (E. coli XL1 Blue, E. coli ClearColi BL21 and Brevibacillus choshinensis). The protein was expressed attached to an N-terminal polyhistidine tag to facilitate purification by nickel affinity chromatography. PfHsp70-1 produced using the E. coli ClearColi BL21 and Brevibacillus expression systems was associated with no detectable traces of LPS. The protein exhibited no immunomodulatory function when it was exposed to macrophage cells cultured in vitro. However, PfHsp70-1 expressed in E. coli XL1 Blue was tainted with LPS contaminants and exhibited apparent immunomodulatory function suggesting that the LPS background was responsible for the signal. Findings, from this study, suggest that endotoxin-free PfHsp70-1 does not possess immunomdulatory function. Furthermore, this study confirms that E. coli ClearColi BL21 and Brevibacillus expression are Evaluation of the immunomodulatory activity of Plasmodium falciparum Hsp70-1 reliable expression hosts for the production of recombinant protein for use in immunomodulatory studies. Furthermore, cytokine production was induced on PMN cells that were exposed to a
protein preparation consisting of the N-terminal ATPase subdomain of PfHsp70-1. However, the ATPase subdomain is known to be aggregation prone. thus, this may explain its apparent immune modulatory function. Polymyxin-B has routinely been used to neutralise the adverse effects of LPS from recombinant Hsps produced using traditional E. coli. Polymyxin-B is a cationic cyclic antibiotic that binds and aggregates LPS. However, the effects of polymyxin-B treatment on the integrity of the chaperones during LPS removal is still unclear. This study, therefore, sought to investigate the effect of polymyxin-B on PfHsp70-1’s chaperone role, by investigating its effect on the thermal stability and structural conformation of PfHsp70-1. Results from this study clearly indicate that polymyxin-B interacts and interferes with the chaperone function of PfHsp70-1. Thus polymixin-B could potentially inhibit PfHsp70-1 function, thus, interfering with parasite growth. In, Conclusion this report demonstrates that recombinant forms of PfHsp70-1 can easily be produced without LPS contamination using E. coli ClearColi BL21 and Brevibacillus expression systems. Furthermore this study demonstrates that PfHsp70-1
proteins do not have immunomodulatory activity and that previously reported activity could have been due to the presence of co-purified LPS. Lastly this report discusses the prospects of using polymyxin-B’s as a potential antimalarial therapeutic drug.
Description
A thesis submitted to the Faculty of Science and Agriculture in fulfilment of the requirements for the Degree of Doctor of Philosophy in the Department of Biochemistry and Microbiology at the University of Zululand, South Africa, 2014
Keywords
Plasmodium falciparum HSP70-1 --immunomodulatory activity