Malaria is still one of the most important tropical diseases in human. In order to understand its complexity and combat against this kind of life, the parasite development, drug sensitivity and protein alteration of parasite under stress such as drug or febrile temperature is challenging. As indicated by in vitro drug sensitivity assay, the standard strain K1 used in this study was found to be chloroquine resistant but sensitive to quinine, mefloquine and artesunate whereas the strain 3D7 was classified as sensitive to chloroquine, quinine, mefloquine and artesunate. It is not surprising to find that one field isolate PF91 presented a moderate resistant to chloroquine because there was evidence showing that the parasite resists to chloroquine in Thailand [13]. There was no evidence indicating ARS resistant. In order to study the effect of stresses toward parasite, the culture of 5% parasite with highly synchronous ring stage P. falciparum was exposed to drug at IC50 concentrations but all of the parasites died. Therefore, chloroquine and artesunate concentration used in all experiments was 40 and 1 nM, respectively.
The human malarial parasite P. falciparum interacted with wide temperature variation during its life cycle, ranging from 25°C or 26°C in the mosquito vector and 37°C in humans, to 41°C during febrile episodes in the patient [2]. This study has examined the influence of two repeated febrile episodes on parasites growth by exposing them to elevated temperature twice, at ring and trophozoite stage (phase A and C) with two recovery phases (phase B and D). It was found that high temperature did not inhibit parasite growth in all stages of standard K1 strain and high temperature has no effect to any stage of K1 in this study. In 1989, Kwiatkowski found that ring stage parasite can resist to high temperature up to 41°C while this temperature can effect to trophozoite stage and developing schizonts were particularly vulnerable [4]. This was represented similarly to the work of Long and his colleagues in 2001 showing that the development of the parasites stopped at late trophozoite and schizonts stage within the first growth cycle at temperature above 40°C [3]. The schizonts appeared pyknotic and hyposegmented and the parasites failed to develop into new cycle. Moreover they found that the effects of hyperthermic conditions parasites growth on wild isolates were similar to those of laboratory-adaptive strain [3]. These are not in agreement with our data and it may be due to the difference in the length of time when the parasites were exposed to high temperature. The parasite cultures were exposed to a long period of high temperature up to 24 h in their experiments while the parasites was exposed twice to the short time of elevated temperature mimicking febrile episode (phase A at 40°C for 2 h and phase C at 40°C for 4 h) in our experiment. The adherence of red blood cells infected with ring stage was also increased considerably by brief heating and fever-induced cytoadherence was associated with enhanced expression of PfEMP-1 on the ring infected red blood cell surface [14]. Moreover, Pavithra and colleagues found that a prior heat shock had a stimulatory effect on parasite development during the subsequent exposure to heat shock. These observations imply that appearance of repeated febrile episodes in malaria patients can hasten intra-erythrocytic development of the parasite [5]. These are in agreement with our results. When the parasites were exposed to Phase A at 40°C for 2 h following Pavithra and colleagues in order to promote prior heat shock effect [5], the laboratory-adaptive strains, K1 and 3D7, under HS developed efficiently to trophozoite and schizonts stage at phase C (p K1 = 0.032, p 3D7 = 0.042 at 95% CI) and the reinfection rate of K1 under HS and 3D7 under HS (p K1 = 0.04, p 3D7 = 0.05 at 95% CI) was also promoted. The previous research found that the effect of hyperthermic conditions on wild isolated parasites growth were similar to those of laboratory-adaptive strain [3]. Our data shows that the survival rate of field isolate PF91 was reduced (p = 0.05 at 95% CI). The effect of temperature on morphology of field isolated parasites was found similarly to that of Oakley and his colleagues [15]. The temperature promoted the presence of pyknotic form or "crisis form" which is the appearance of parasites undergoing death [15]. The appearance of crisis form was significantly evident following 4 h of culture at 41°C [15] whereas the crisis from of field isolate PF91 was found after heating for 2 h following by incubation at 37°C. These results are not found in K1 standard strain but trifling appeared in 3D7.
The effect of temperature on drug susceptibility of parasites was tested to the parasites exposed to heat at ring stage for 2 h followed by drug sensitivity tests immediately. The result indicated that IC50 value to chloroquine, mefloquine and artesunate of strain K1 under HS was higher than that of K1 under non-HS significantly (p = 0.04 at 95% CI). The IC50 value of K1 under non-HS with quinine was higher than of K1 under HS. The effect of temperature on K1 drug susceptibility was similar to that of 3D7 only toward artesunate. It was found that IC50 value to artesunate of 3D7 under heat shock was higher than that of 3D7 under non- HS condition (p = 0.04 at 95% CI). The IC50 value to chloroquine, quinine and mefloquine) of 3D7 under non-HS was higher than that of 3D7 under HS (p = 0.04 at 95% CI). The IC50 value of field isolate under HS could not be calculated because heat shock causing death in parasites.
The IC50 value of K1 under HS was higher than that of K1 under non-HS whereas the IC50 of 3D7 under non-HS was higher than that of 3D7 under HS This might be explained by the effect of high temperature toward drug transporter reported by Cecilia and her colleagues in 1997 [16]. The chloroquine uptake by P. falciparum infected erythrocyte is temperature-dependent and saturable and the existence of a specific parasite-encoded protein that facilitates chloroquine uptake. For strain with chloroquine resistant, the temperature does not effect to chloroquine uptake whereas in sensitive strain, temperature effects to chloroquine uptake. The increasing in temperature increases chloroquine uptake [16]. Although, there was no report in the effect of high temperature toward mefloquine, quinine and artesunate, high temperature might have some effects to drug transport or drug mechanism by direct or indirect way as shown in our study. When the parasites were heated at 40°C for 2 h and cultured back at 37°C until new ring infected (NRF), the IC50 value of strain K1 and 3D7 was similar to that of heating at ring stage for 2 h followed by drug susceptibility testing immediately. The IC50 of field isolate PF91 under HS and non-HS was not significantly different (p = 0.5 at 95% CI). Therefore, high temperature shows some effects only toward the drug susceptibility of standard or laboratory strain K1 or 3D7 but not to field isolate.
In order to observe the effect of temperature and drug by mimicking the situation of treatment failure in malaria patients coped with fever, it was found that high temperature had no effect to the development of parasite with chloroquine resistant (K1 strain). When K1 strain was cultured with 40 nM drug under heat at ring stage for 2 h (phase A), parasite developed to phase B similarly to the other groups grown under HS or non-HS with or without drug when compared by the total number of parasite between these groups. These might be due to the concentration of chloroquine used which is lower than IC50 of K1 strain therefore the drug can not harm the parasite. Additionally, K1 is chloroquine resistant strain and it might have some proposed mechanisms such as increasing in vacuolar pH, enhancing in drug efflux, reducing in drug binding, losing in chloroquine transporter and changing in glutathione-S-dehydrogenese (GSH) protein. After culture parasites at phase B (37°C) for 18 h followed by heating again at 40°C for 4 h, the resistant strain was tolerant to high temperature by comparing the total parasite number between these groups. However, almost parasites developed to schizont and trophozoite. After culture parasites at phase D, the data indicated that temperature did not effect to the reinfection rate of the parasite as compared by the number of parasite at phase D.
The total parasite number of strain 3D7 between non-HS and non-HS with chloroquine after phase B was significantly different (p = 0.05 at 95%CI). This data illustrates the effect of drug toward 3D7 development. The total number of parasite between 3D7 under HS and under HS with chloroquine was also significantly different (p = 0.05 at 95%CI). This might be due to the increase in chloroquine uptake [16]. Moreover, the total number of parasite between non-HS with chloroquine and HS with chloroquine was significantly different (p = 0.05 at 95%CI) indicating that temperature might also increase chloroquine drug uptake of the parasite [16]. The total number of parasite at phase C and D between these groups are significantly different. Therefore, high temperature increases drug uptake and also coped with second heat shock might induce higher drug uptake in the parasite [16].
The field isolate is moderate resistant to chloroquine and also the temperature has extremely effect toward parasite development. This might explain the reason why almost parasite after phase A can not develop to phase B. Moreover, this might be due to the drug concentration which is close to IC50 value coping with another factor that is high temperature. These might promote more drug uptake into the parasite and more parasites died showing the direct effect of temperature.
When culture under high temperature with artesunate, the total parasite number of all strains including K1, 3D7 and PF91 was extremely decreased at phase B and the parasite was not found at phase C and D. The decrease in the number of parasite in phase B, K1 (p = 0.01 at 95% CI), 3D7 (p = 0.005 at 95% CI), field isolate PF91 (p = 0.01 at 95% CI) might be from the effect of artesunate coped with temperature. This might indicate that all parasites died at phase C or the sensitivity of this method is not enough to detect. There was no parasite reinfection during phase D. This might be from two reasons, first is the level of the parasite after phase C was too low to detect by the conventional method and the second is parasite died since phase B.
It was found that anti-pf HSP70 binds to the proteins extracted from all conditions. Not surprisingly, two chaperones, HSP70 and 90 orthologs, which have been implicated in the heat shock response across the phylogenetic spectrum of life [15] meaning that pf Hsp70s are stress response that will be expressed when the parasite exposed to the stress such as high temperature or drug. The proteome extracted from K1 grown under heat shock with chloroquine, anti-pf Hsp70 interacted with four bands whereas anti-pf Hsp70 interacted with only one band in another strain under different conditions. After protein analysis by MALDI-TOF peptide mass fingerprint, the 120 kDa band from all conditions is identified as heat shock protein 70 or pfHSP70s. Higher molecular weight might be caused by the complex formation of heat shock protein as molecular chaperone. The pf Hsp70 are chaperone complex consisting of pf Hsp 90, PfPP5, tubulin and an additional protein that is unidentified. The complex was similar to that of higher eukaryotes both in term of size as well as composition [5]. Normally, pf Hsp 70 have six homolog which are pf Hsp70-1 with molecular mass of 74 kDa [6],pf Hsp70-2 with molecular mass of 78 kDa [7], pf Hsp70-x with molecular mass of 76 kDa [17], pf Hsp70-3 with molecular mass of 73 kDa [6], pf Hsp70-z with molecular mass of 100 kDa and pf Hsp70-y with molecular mass of 108 kDa [17]. The 83 kDa hybridized band was identified as elongation factor 1-α (EF-1α). From the previous research studied in rat brain, it was found that heat shock factor 1 (HSF-1) correlated with Hsp70, Hsp 27 and Hsp 90 was found after heating at 41°C. HSF-1 probably binds to Hsp70 and then EF-1α binds to this complex to undergo phosphorylation [18]. The parasite might release EF-1α with direct availability for complex formation with HSF-1 and heat shock receptor (HSR) in the nuclease. Temperature and drug stress possibly changes the capacity of EF-1α to form a complex with other components of the initiation complex. These are major possibilities that will be explored in the future. The 60 kDa hybridized band was identified as pf Hsp 86. pf Hsp 86 is the one of HSP 90 homolog which is normally binding with Hsp70 to form a functional complex [19]. The 40 kDa hybridized band was identified as phosphoethanolamine N-methyltransferase. This enzyme catalyzes the transfer of a methyl group from S-adenosyl-L-methionine to the amino group of the tetrahydrobenzylisoquinoline alkaloid coclaurine [20, 21]. This is a unique N-methyltransferase in the biosynthesis of benzylisoquinoline alkaloids. The previous studies did not present any correlation between heat shock protein and this enzyme so far. The reason why pf Hsp70 could bind to this protein will be explored in the future.