Malaria, which is caused by infection with the hemoprotozoan parasite Plasmodium, afflicts approximately 500 million people each year, leading to 2.7 million deaths annually worldwide [1]. Malaria results in diverse clinical outcomes presumably because of dynamic relationships between parasite-expressed virulence factors and individual host responses to these determinants [2]. The last few years have seen a rapid growth in the number of reported genetic associations with susceptibility and resistance to malaria in the host; they are mainly variants in erythrocyte, cytoadherence factor, immune system, and inflammatory genes [3,5]. However, molecular basis of malaria infection remains incompletely defined. Identification of genetic factors in both the host and the parasite involved in malaria susceptibility and resistance would lead not only to a greater understanding of this complex disease, but potentially also to the development of effective medical interventions [2,6].

Interactions between Plasmodium and its host hepatocyte factors are of great research interest [7-9]. On one hand, it is well established that malaria sporozoites exploit hepatocyte growth factor (HGF) and its receptor MET signaling during host invasion [10]. On the other hand, Plasmodium is a life cycle-specific disease that includes liver injury at the erythrocyte stage of the parasite [11]. Indeed, both humans and mice suffer from liver injury after infection with the fatal strains Plasmodium falciparum (Pf) and P. berghei (Pb) [12,13]. Because Pb merozoites do not parasitize liver parenchymal cells in mice, it was proposed that the mechanism of Pb-induced liver injury is likely to be caused by the local production of cytokines that activate lymphocytes residing in the liver [14]. Pathologically, malaria-induced mouse liver injury is characterized by dense infiltration of lymphocytes, and the presence of apoptotic and necrotic hepatocytes [15].

Plasmodium subtilisin-like protease 2 (subtilase, Sub2) is an essential integral membrane serine protease in both Pf and Pb. It functions in the shedding of the ectodomain components of 2 surface proteins, membrane surface protein 1 (MSP1), and apical membrane antigen 1 (AMA1), upon host invasion [16-18]. The importance of the Sub2 gene is evidenced by the demonstration that gene-knock out parasites cannot be recovered [19].

The correlation of HGF and malaria infection is not well understood. In this study, to understand the relationship between the malaria infection-related protein Pf-Sub2 and HGF, the Pf-Sub2 5'untranslated region (UTR) was analyzed and its promoter activity was estimated. In addition, the influence of HGF on its promoter activity was analyzed.

Plasmodium infects liver tissue, so to understand malaria infection it is necessary to understand the relation between hepatocytes and malaria. However, this process is poorly understood. Sporozoites traverse the cytosol of several hepatocytes before invading one by forming a parasitophorous vacuole [20]. The host responds to the traveling parasites by secreting HGF, which renders hepatocytes susceptible to infection [10]. HGF is known to act as an autocrine cytokine in various cancer cells and precancerous cell types [21,22], but it is not normally expressed by hepatocytes or by most other MET-expressing epithelial cells. However, the mechanism of HGF regulation in mammalians is not fully understood.

Moreover, clarifying transcription regulation in Plasmodium genome is onerous because of the genome's abnormal AT-richness [23,24]. Nonetheless, Plasmodium genes are organized similarly to those in eukaryotes and are also monocistronically transcribed [25-27], implying the presence of regulatory sequence elements flanking the coding regions [28,29]. For example, the Pf multidrug resistance gene upstream region can be induced at the level of transcription by antimalarial drugs in chloroquine sensitive parasites [30]. Our results obtained from upstream transcriptional activities of Pf-Sub2 demonstrate that the proximal promoter of Pf-Sub2 is influenced by the HGF. 5'UTR were analyzed for transcriptional elements of which 14 TATA boxes and other elements, e.g., one Oct-1 and c-Myb were observed. Deletion mutants of 5'UTR of Pf-Sub2 were constructed and -1,422/+12 UTR exhibited the strongest promoter activity. To understand the interaction of HGF and 5'UTR of Pf-Sub2, 2 vectors of the molecules are cloned and co-transfected into cells. HGF expression was suppressed by 5'untranslated region of the Pf-Sub2 promoter. In these results, -1,422 to +12 UTR of Pf-Sub2 was applied and contained all transcriptional elements in Table 1. Therefore, further study will be performed what positions of -1,422 to +12 UTR are important to inhibit the HGF expression. Although the effect of HGF on malaria gene expression in cultured cells may be indirect or atypical, the present results indicate the feasibility of regulation of gene(s) important in malaria invasion through host-parasite interaction. In fact, both recombination in subtelomeric regions and chromosome internal rearrangements under host pressure have proven that the parasite can interact with its vertebrate host [31-33]. Specific upstream regions are important in the activity of the protease. Future studies will be aimed at elucidating the details of the role of HGF in malaria sensitivity/resistance and how Sub2 gene expression is influenced by the host HGF signaling cascade.