Heat shock adversely affects parasite survival whereas lactate exposure does not
In order to choose appropriate parameters for exposing parasites to either heat shock or elevated lactate, we first measured parasite survival following such exposures, both at the ring stage and at the trophozoite stage. Trophozoites are known to be more sensitive to heat shock than rings [39], and although comparable stage-specific data are not published for lactate sensitivity, the trophozoite is also the most metabolically active stage, at which lactate production is highest. Cultures were treated at either 2 ± 2 hpi, or at 28 ± 4 hpi (primarily young rings and young trophozoites respectively). Exposure to stress was for 2 h or 6 h and consisted of heat shock at 40°C or 5 mM added lactate. These were chosen to mimic, respectively, common levels of fever in the human host and the WHO threshold for hyperlactataemia that defines severe malaria [2]. Survival was measured 48 h after treatment of the trophozoites or 72 h after treatment of the rings, thus allowing reinvaded rings to develop into trophozoites, when the DNA content can parallel parasite numbers. Four parasite strains were used: the reference strain 3D7 and three recently laboratory-adapted strains derived from Kenyan malaria patients.
In trophozoites, heat shock resulted in 0-20% death following a 2 h exposure and 20-40% death following a 6h exposure (Fig. 1A). The extent of heat-shock-induced death appeared strain-dependent, with one strain (ID9775) appearing more sensitive. This field strain (ID9775) grew most rapidly and robustly in normal culture conditions. By contrast to heat shock, lactate exposure did not appear to have an adverse effect on parasite survival (Fig.1A) and when both stressors were applied together, results were very similar to those seen after heat shock alone (Fig. 1A).
When heat shock was applied to ring-stage parasites, survival after 72h was more variable between strains than it had been at the trophozoite stage (Fig. 1 B). Kenyan strain ID3518 was almost completely insensitive, whereas rings of strains ID9775 and ID10668 were affected (27% and 38% death, respectively, i.e. almost as severely affected as their trophozoites). Lactate exposure for 6h at the ring stage again had little effect on parasite survival (Fig. 1 B).
Heat shock modulates the expression of PfSir2B in P. falciparum trophozoites
Having established heat shock conditions that caused only a moderate amount of parasite death in both trophozoites and rings, we measured the expression of sirtuin genes immediately after heat shock, as well as the expression of the gene encoding heat shock protein 70 (hsp70) as a positive control. These experiments were conducted first on trophozoites: the stage that was previously reported to upregulate PfSir2A expression after heat shock [34]. Across the strains, hsp70 expression was indeed upregulated (Fig. 2A) by an average of 3 – 5 folds (Table 1) in response to heat shock but not to lactate exposure, as expected.
PfSir2A appeared to be unaffected by heat shock or lactate treatment (Fig. 2B, Table 1). PfSir2B expression was increased after 2 h of heat shock but not after 6 h (Fig. 2C) with mean fold changes ranging from 1.5 – 2 folds (Table 1). This response was again heat-shock-specific because lactate versus no lactate was not significantly different, thus suggesting that heat shock was the main factor modulating the transcription of PfSir2B.
Table 1: Mean (95% CI) fold changes in the expression of hsp70, Sir2A and Sir2B in trophozoites after a 2 h or 6 h exposure to 5mM Lactate or 40°C heat shock, alone or in combination.
Gene/Time of exposure
|
Lactate 5mM
|
40°C
|
40°C + Lactate 5mM
|
Hsp70 2h
|
1.39 (0.49 to 3.89)
|
4.92 (1.73 to 14.00)
|
3.69 (1.06 to 12.88)
|
Hsp70 6h
|
1.06 (0.57 to 1.99)
|
4.70 (2.06 to 10.72)
|
3.42 (1.20 to 9.76)
|
Sir2A 2h
|
1.11 (0.43 to 2.87)
|
1.37 (0.57 to 3.26)
|
1.90 (0.64 to 5.64)
|
Sir2A 6h
|
1.06 (0.42 to 2.73)
|
1.21 (0.52 to 2.87)
|
1.23 (0.53 to 2.88)
|
Sir2B 2h
|
1.28 (0.59 to 2.76)
|
1.88 (1.11 to 3.20)
|
1.45 (0.76 to 2.77)
|
Sir2B 6h
|
1.07 (0.46 to 2.49)
|
1.98 (0.86 to 4.54)
|
1.48 (0.49 to 4.57)
|
Since there was no change in Sir2A expression after heat shock, we designed a second independent approach to measure this response. A luciferase reporter gene was cloned under the presumptive Sir2A promoter (~1.7kb of the gene’s upstream sequence) and the reporter gene was transfected into 3D7 parasites, where it was shown to follow an expression profile similar to that of the endogenous Sir2a gene across the intraerythrocytic cycle [40](Additional file 2, Figure S1B). Unfortunately, although this system may be useful in other applications, it proved unsuitable for heat shock experiments because the luciferase was severely destabilised at 40°C: activity dropped by ~80% after a 2 h heat shock, irrespective of the promoter driving luciferase expression (Additional file 2, Figure S1C), thus obscuring any promoter-mediated regulation at the transcriptional level.
Pilot study on the alteration of sirtuins and var genes in P. falciparum ring stages after exposure to heat shock and lactate
Figure 2 shows that exposure to heat shock modulates the expression of PfSir2B, in trophozoite-stage parasites. We therefore proceeded to investigate the same responses in ring-stage parasites, i.e. the stage at which var genes are also expressed. Sirtuin and var gene expression were measured immediately after the rings had been exposed to heat shock, elevated lactate, or both stressors combined for 6 h, i.e. expression was measured at 8 hpi. The same genes were also measured at 16 h (i.e. 10 h after the stressors were removed), at 18 hpi – around the time of maximum expression for active var gene(s) [41]. Kenyan field strains (ID9775 and ID3518) were selected for these experiments and, since var gene families are hyper-diverse, var expression was measured using general primer sets that were previously developed to detect conserved regions within each ups-group of var genes [10]. These same primers were used in the study of Gambian field strains which originally reported the association between var gene expression, sirtuin expression, patient fever and hyperlactataemia [11].
Preliminary findings show that on exposure of ring-stage parasites to heat shock, hsp70 was upregulated (Fig. 3A) and Sir2A (Fig. 3B) trended upwards both after stress and 10 h of recovery at 37°C (16 h after the onset of heat shock), while Sir2B (Fig. 3C) trended downwards only after stress. These changes in Sir2A and Sir2B are not statistically significant.
The pattern of expression of var genes was nondescript (Fig. 4A-I) but may be clearer if more samples are studied. However, the var genes belonging to all the groups are upregulated after the 10 h of no stress (p values are not shown for the time categorical factor). This most likely means that var genes are increasingly expressed with time.