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 , 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 . 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 parasitaemia can be easily assessed. 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 6 h exposure (Fig. 1A). The extent of heat-shock-induced death was strain-dependent, with one Kenyan strain (ID9775) being markedly more sensitive than the other two, and 3D7 being of intermediate sensitivity. Interestingly, we observed that the most heat-sensitive field strain (ID9775) was the one that grew most rapidly and robustly in normal culture conditions. These results, measured via the SYBR Green-1 Fluorescence method, which measures levels of parasite DNA, were broadly corroborated by microscopy, conducted on two of the four strains as an independent measure of viable parasite numbers (Fig. 1B). Microscopy tended to yield a lower survival rate, because the SYBR Green-1 Fluorescence method, as used here, also measures background fluorescence from potentially ‘dead’ parasite DNA, whereas microscopy allows live and dead parasites to be distinguished. By contrast to heat shock, lactate exposure did not have a significant adverse effect on parasite survival (Fig. 1A, B) and when both stressors were applied together, results were very similar to those seen after heat shock alone (Fig. 1A, B).
When heat shock was applied to ring-stage parasites, survival after 72 h was more variable between strains than it had been at the trophozoite stage (Fig. 1C, D). Kenyan strain ID3518 was almost completely insensitive, whereas rings of strains ID9775 and ID10668 were significantly affected (27% and 38% death, respectively, i.e. almost as severely affected as their trophozoites). As before, microscopy yielded a more severe assessment of heat-induced death than the SYBR Green-1 Fluorescence assessment (Fig. 1C cf Fig. 1D). Lactate exposure for 6 h at the ring stage again had little effect on parasite survival (Fig. 1C, D).
Heat shock modulates the expression of sirtuins 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 . Hsp70 expression was indeed upregulated, 3–7 fold in most strains, with considerable strain-to-strain variation (p < 0.0001) (Fig. 2A). This response was heat-shock-specific because hsp70 was not upregulated after lactate exposure, as expected.
PfSir2A expression trended upwards in most strains after heat shock, particularly at the 2 h time point and somewhat less so at the 6 h time point. However, upregulation was usually less than 2-fold and did not reach statistical significance (Fig. 2B). PfSir2B expression, meanwhile, also trended upwards, generally by 2-3-fold, and reached statistical significance in several strains (p = 0.0031 at 2 h; p = 0.0495 at 6 h) (Fig. 2C). There was no significant difference in the response to heat shock alone versus heat shock combined with 5 mM lactate, thus suggesting that heat shock was the main factor modulating transcription of the genes measured here.
Since the Sir2A gene appeared only modestly upregulated after heat shock, we designed a second independent approach to measure this response. A luciferase reporter gene was cloned under the presumptive Sir2A promoter (~ 1.7 kb 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 (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.
Heat shock modulates the expression of sirtuins and var genes in P. falciparum ring stages, whereas lactate does not
Figure 2 shows that exposure to heat shock can modulate the expression of both the parasite sirtuins, particularly PfSir2B, at least 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 10 h later, at 18 hpi – around the time of maximum expression for active var gene(s) . 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 . 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 .
Exposure of ring-stage parasites to heat shock induced the upregulation of hsp70 (Fig. 3A), as was previously seen in trophozoites (Fig. 2A). Upregulation was at most 2.5-fold and the magnitude of the response varied between strains, with strain ID9775 appearing almost entirely refractory at the ring stage, despite being clearly responsive at the trophozoite stage. PfSir2A expression trended upwards in both strains immediately after heat shock, similar to the response in trophozoites, but the increase was again less than 2-fold and did not reach statistical significance. Nevertheless, after 10 h of recovery at 37 °C, PfSir2A expression remained elevated, reaching statistical significance in strain ID3518 (Fig. 3B). PfSir2B, meanwhile, showed a response that contrasted with its response in trophozoites: immediately after heat shock, PfSir2B expression was downregulated by ~ 2-fold, but returned to baseline levels following 10 h of recovery (Fig. 3C).
In concert with these changes in sirtuin expression, changes in var gene expression after heat shock were also measured (Fig. 3D, E). Immediately after heat shock (i.e. 8 hpi), both strains showed a general upregulation of var transcription: 1.4-1.6-fold upregulation was measured with the pan-var ‘ATS’ primer (which detects the conserved sequence encoding the ‘Acidic Terminal Segment’ of PfEMP1), although this did not reach statistical significance. Primers specific to the various ups groups A-E suggested that there was particularly significant upregulation of subtelomeric upsA and upsB genes in one of the two strains, ID3518 (Fig. 3D). Interestingly, 10 h later at 18 hpi the overall level of var transcript remained elevated, by ~ 5-fold in the more strongly responding ID3518 strain and ~ 1.6-fold in strain ID9775. However, the most strongly upregulated var gene had changed in both strains. A upsC gene was elevated in strain ID3518, while strain ID9775 had upregulated the upsE gene var2csa (Fig. 3E).
Having established that heat stress could indeed induce changes in both sirtuin and var gene expression when applied to ring-stage parasites, we proceeded to compare this with the transcriptional response to lactate exposure. This caused no consistent change in sirtuin expression (Fig. 4A, B), as had previously been shown in trophozoites, and there was also no clear change in var gene expression at the peak 18 hpi time point. When var expression was measured in young rings at 8 hpi, immediately after lactate exposure, one of the two strains did show a marked upregulation in groups B and C2 var transcripts (Fig. 4C). This, however, was not sustained by 18 hpi (Fig. 4D), casting some doubt upon its potential phenotypic significance.
Finally, we tested the transcriptional effect upon ring-stage parasites of combining heat shock with lactate exposure. In trophozoites, this had yielded similar results – in terms of both parasite survival and hsp70/sirtuin expression – as had heat shock alone. The same was evidently true in ring-stage parasites: hsp70, PfSir2A and PfSir2B expression all responded almost identically regardless of the presence or absence of 5 mM lactate (compare Fig. 5A-C with 3A-C). Var expression responses were likewise broadly similar (Fig. 5D, E) i.e. there was an overall upregulation in transcript levels, stronger in strain ID3518 than ID9775.