EVALUATION OF A SURROGATE IN VITRO MAMMALIAN LOCALIZATION
SYSTEM FOR RECOGNITION OF PLASMODIAL FOOD VACUOLE
TARGETING SIGNAL
Gan C.S. and Sim T. S.
Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore,
Block MD4, 5 Science Drive 2, Singapore 117597
ABSTRACT
The completion of Plasmodium falciparum genomic sequences had revealed
approximately 5300 potential gene products with 60% annotated as hypothetical proteins.
The main task is to define the functional roles of these putative proteins experimentally. The
use of protein localization study could aid in this cause as the strategic location of cellular
proteins underscores their functionality in living systems. This would help in resolving
unknown metabolic pathways which are fundamentally important in understanding and
controlling the parasite pathogenicity. A previous study had successfully employed an in
vitro surrogate mammalian host system to evaluate plasmodial mitochondrial- and nucleartargeted signals. In this study, the surrogate host system’s ability to recognize plasmodial
food vacuole proteins was evaluated. Two protein domains carrying food vacuole signal
peptides were made and subjected to co-localization study with organelle selective probe,
LysoTracker® Red. This fluorescent acidotropic probe is able to specificity stain acidic
lysosomal organelles. The preliminary results had demonstrated that the surrogate host could
recognize plasmodial food vacuole-targeting signal and deliver it to vesicle-like
compartments. After which, co-localization studies identified these compartments as
lysosomal organelles. This demonstrated that the authenticity of putative food vacuole
proteins could be preliminary verified via a surrogate host system.
INTRODUCTION
The genome sequence of P. falciparum, a causative agent of malaria, was uncovered
in year 2002 (Gardner et al. 2002). It revealed that an approximate of 5300 protein encoding
genes was reported with 60% being hypothetical proteins with no similar homologs in other
organisms to provide clues in their functionality (Gardner et al. 2002). These putative
proteins hold the key in resolving remaining unknown metabolic pathways in the parasite.
Characterizing and authenticating them would be fundamentally important for our
understanding and controlling the parasite pathogenicity.
Much research effort had been carried out to study P. falciparum genes. One of effort
made was the design of in vitro mammalian cell as surrogate host to evaluate plasmodial
protein localization (Chan et al, 2006). In that study, mammalian cells were able to
translocate the plasmodial mitochondrial- and nuclear-targeted signals to the respective
organelles, thereby verifying that the cells are able to interpret plasmodial mitochondrial- and
nuclear-targeted signals. This experimental system offers a new and easily accessible
alternative which is able to circumvent the technical glitch in transfecting parasites.
By broadening the scope of using mammalian surrogate system to analyze other
plasmodial organelle targeted putative proteins; it would help in defining the thousands of
proteins that have been identified through genomics efforts. One of the utmost important
organelles to investigate would be the malarial food vacuole. It is known that several
biologically essential processes such as hemoglobin degradation, heme polymerization and
reduction of oxidative stress occur in the food vacuole (Olliaro and Goldberg, 1996).
1 Hence, the aim of this project is to ascertain the use of mammalian surrogate host for
localization study of plasmodial food vacuole proteins. Studies had shown that the targeting
of food vacuole proteins is via three different routes (Tonkin et al. 2006). However, this
project would only be focusing on one of them. This approach seeks to serve as a prelude to
authenticate the putative food vacuole proteins experimentally.
MATERIAL & METHOD
The methods used in this illustrated in the flow chart. (Fig 1)
RESULT
Fluorescence microscopic analysis of transfected CHO cells revealed the expression
of Cat-L, pFP2A, pFP3, pf-MD and pf-GK GFP recombinant constructs in the mammalian
cells. (Not shown). Vesicles-like fluorescence pattern (Fig 2) displayed by Cat-L, pFP2A,
pFP3 was dispersed throughout the cell which was similarly observed in other studies (Linke
et al. 2002 and Taha et al. 2005). This had shown that the mammalian cells could interpret the
signal peptide within the constructs and delivered them to endocytic-like vesicles. Negative
controls cytoplasmic pf-MD and pf-GK which do not possess any organelle-targeting signal,
showed a diffused fluorescence impression throughout the cytosol. In situ staining confirmed
the co-localization of Cat-L, pFP2A and pFP3 with the LysoTracker®Red dye (Fig 2). This
successfully illustrated that mammalian cells could recognize the food vacuole signal
peptides found on pFP2A and pFP3 and transported them into lysosomal compartments.
2 DISCUSSION
In this study, GFP was deployed as a visualizing tool to study the geography of
plasmodial food vacuole proteins within the mammalian cells. Ever since the discovery of
GFP, it had upheaval the approach in conducting protein localization studies. The ease of
GFP application had helped to facilitate the proceeding of extensive experiments in numerous
studies (Kumar et al. 2002, Huh et al. 2003). Generally, GFP is highly stable against harsh
condition such as heat or pH. It does not require the utilization of expensive specific antibody
or any external cofactors. Furthermore, the ability to employ GFP in living cells offers a new
dimension in viewing the dynamic translocation of cellular proteins.
In the co-localization study, LysoTracker® Red was employed as a visualizing probes
to stains the lysosomal organelle in the cells. LysoTracker® Red is basically a fluorescent
acidotropic probes which consist of a fluorophore linked to a weak base that is only partially
protonated at neutral pH. In cellular compartments with low pH, it is believed that the probe
will get protonated and start to accumulate and stain the compartment. This probe had been
widely used to visualize lysosomal organelle. However, LysoTracker® Red could also stain
other acidic compartments such as trans-Golgi vesicles. Hence, to improve on this method,
the GFP expression vectors should be co-transfected with recombinant vectors containing
known lysosomal signals tagged with different fluorescent markers such as red fluorescent
protein (DsRed)
The hypothesis of this study is that mammalian cells are able to recognize malarial
food vacuole targeting signal as a lysosomal signal and deliver it to lysosomal organelles.
This hypothesis was based on the similar characteristics shared between lysosome and
plasmodial food vacuole (Bainton, 1981). They are both organelles bounded by a single
membrane, playing primary role in the intracellular digestive system, and have a similar
battery of degradative enzymes. Both organelles also have acidic physiological environment
[Biagini et al, 2003, Ohkuma and Poole, 1978). Furthermore, sub-cellular localization
predication programme, LOCtree, had predicted the localization of falcipain 2A and falcipain
3 to the lysosomes.
Indeed, the results clearly indicated that the CHO cells had recognized the food
vacuole signal peptides and sorted them into cellular compartments. From the co-localization
study suggested that those cellular compartments were lysosomal-related organelles. Hence,
the authenticity of putative food vacuole proteins could be preliminary verified by its subcellular localization in the mammalian cells. This would help to define some of the
hypothetical proteins that were unveiled in the P. falciparum genome sequences. Besides, as
suggested by Chan et al. (2006), this experimental system could provide new avenue to study
parasites which have no reliable in vitro culturing technique.
To further evaluate the robustness of this system, other food vacuole signal peptides
such as dipeptide aminopeptidase 1 (DPAP1) and FYVE domain-containing protein (FCP)
should be included. They were known to have different trafficking pathway as compared with
falcipains. Besides, targeting signal does not reside exclusively within the prodomain of the
protein. For example, falcilysin does not contain any prodomain and the targeting signal was
suggested to be within the mature domain. All these information implied that the use of
targeting signals within pFP2A and pFP3 is not comprehensive enough to reflect the food
vacuole proteins population. As a result, more experiment would have to be carried out to
fully establish this system.
3 CONCLUSION
In conclusion, the results had successfully demonstrated that the authenticity of
putative food vacuole proteins could be preliminary verified via the surrogate host system.
This system could serve as a useful preliminary approach to study the functionality of novel
food vacuole proteins.
ACKNOWLEDGEMENTS
I would like to thank my supervisor, A/P Sim Tiow-Suan for giving me such a
wonderful opportunity to work on an UROPS project and experience a challenging journey of
scientific research. Her sound guidance and advice would be deeply remembered in the
coming years of my life. I would also like to extend my gratitude to all my laboratory seniors
which included Doreen, Mdm Seah, Wenjie, Jasmine, Jason, Huiyu, Chun Song, Jun Ji and
Jane for their time and effort in assisting and advising me. Their encouragement, support, and
humour have made my stay in the laboratory truly a memorable experience. Finally, I would
like to thank my family for their tender loving care.
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