An external sensing system in Plasmodium falciparum-infected erythrocytes

Abstract

Background: A number of experiments have previously indicated that Plasmodium falciparum-infected erythrocytes (pRBC) were able to sense host environment. The basis of this ability to detect external cues is not known but in screening signalling molecules from pRBC using commercial antibodies, a 34 kDa phosphorylated molecule that possesses such ability was identified.

Methods: The pRBC were exposed to different culture conditions and proteins were extracted for 1D or 2D gel electrophoresis followed by Western blot. The localization of 34 kDa protein was examined by biochemical fractionation followed by Western blot. High-resolution mass spectrometric analysis of immune precipitants was used to identify this protein and real-time quantitative reverse transcriptase polymerase chain reaction was used for detecting mRNA expression level.

Results: The 34 kDa protein was called PfAB4 has immediate responses (dephosphorylation and rapid turnover) to host environmental stimuli such as serum depletion, osmolality change and cytokine addition. PfAB4 is expressed constitutively throughout the erythrocytic lifecycle with dominant expression in trophozoites 30 h post-infection. Tumour necrosis factor (TNF) treatment induced a transient detectable dephosphorylation of PfAB4 in the ItG strain (2 min after addition) and the level of expression and phosphorylation returned to normal within 1-2 h. PfAB4 localized dominantly in pRBC cytoplasm, with a transient shift to the nucleus under TNF stimulation as shown by biochemical fractionation. High-resolution mass spectrometric analysis of immune precipitants of AB4 antibodies revealed a 34 kDa PfAB4 component as a mixture of proliferating cellular nuclear antigen-1 (PCNA1) and exported protein-2 (EXP2), along with a small number of other inconsistently identified peptides. Different parasite strains have different PfAB4 expression levels, but no significant association between mRNA and PfAB4 levels was seen, indicating that the differences may be at the post-transcriptional, presumably phosphorylation, level. A triple serine phosphorylated PCNA1 peptide was identified from the PfAB4 high expression strain only, providing further evidence that the identity of PfAB4 is PCNA1 in P. falciparum.

Conclusion: A protein element in the human malaria parasite that responds to external cues, including the pro-inflammatory cytokine TNF have been discovered. Treatment results in a transient change in phosphorylation status of the response element, which also migrates from the parasite cytoplasm to the nucleus. The response element has been identified as PfPCNA1. This sensing response could be regulated by a parasite checkpoint system and be analogous to bacterial two-component signal transduction systems.

Fig. 1

Synchronized stage specific ItG parasite lysates at 14, 24, 36 and 46 h after parasite invasion were analysed by Western blot using AB4 antibodies. PfAB4 expressed throughout parasite lifecycle with expression peak between 28 and 32 h. A 90 kDa protein also reacted with AB4 antibodies. The lower part is the Coomassie blue stained gel image used as loading control (a). Panel b shows parasite strains with CIP in vitro treatment, which attenuated the signal indicating the phosphorylation state of PfAB4 proteins

Fig. 2

ItG parasites at mid-trophozoite stage were exposed to TNF (1 ng ml⁻¹) treatment for 2, 5, 15, 30, 60, 120 and 180 min. PBS mock treatment was performed as a control. The expression of PfAB4 from total cell lysates was determined by Western blot using AB4 antibodies (upper panel). The middle panel shows the quantification of PfAB4 level from three independent experiments by scanning autoradiographs. The lower panel shows a Coomassie blue stained image as a loading control

Fig. 3

Different parasite strains have different expression levels of PfAB4. The total protein lysates were extracted from ten well-synchronized parasite strains and analysed for PfAB4 expression level. Dd2, BC12 and PCM7 were repeatedly identified as low expression strains. The lower panel shows the Coomasie blue stained image used as a loading control

Fig. 4

RT-qPCR analysis of selected parasite strains for transcript levels of five candidate genes. Three independent qPCR runs were analysed using the 2^−ΔΔCT method. Data are presented as fold changes against an internal reference gene ASL. The results of Student’s t tests are indicated as *P < 0.05; t tests compared the same gene among different strains

Fig. 5

ItG pRBC were treated with TNF (1 ng ml⁻¹), at the given a time points, followed by cell fractionation using combined methods of saponin lysis and detergent extraction. PfAB4 and other candidates’ expression and localization were revealed by Western blot. PfAB4 is localized in parasite cytoplasm, nuclear and insoluble fractions. In response to TNF treatment there was a transient shift from cytosol and insoluble compartments to the nucleus within 20–30 min. PGK and calcinurin also showed responsiveness to TNF treatment

Fig. 6

Proteins from cytoplasmic and nuclear fractions of ItG pRBC before or after TNF (1 ng ml⁻¹) treatment at the four time points were subjected to 2D gel electrophoresis using narrow pH (3.9–4.9) immobiline Drystrips. PfAB4 expression was revealed by Western blot, showing different patterns of migration in the nucleus and cytoplasm over time

Fig. 7

Treatment with different inhibitors, Calpeptin and Gambogoc Acid. a ItG early trophozoite stage pRBC were treated with/without calpeptin, a calpain inhibitor for 1 h before TNF stimulation, and the expression of PfAB4 and a 90 kDa protein reacting with AB4 antibodies measured. The 100 µM calpeptin treatment caused a block of PfAB4 degradation. b ItG early trophozoite stage pRBC were treated with/without Gambogic acid (Gam), a HSP90 inhibitor overnight before TNF stimulation. The expression of PfAB4 and a 90 kDa protein reacting with AB4 antibodies was measured. Gam 0.5, 1.0 and 1.5 µM inhibited the 90 kDa protein expression and reduced the effect on PfAB4 caused by TNF

Fig. 8

The 32–35 kDa region (covering the PfAB4 proteins) of ItG pRBC from 1D SDS-PAGE were excised and in-gel digested with trypsin. The phosphopeptides were enriched using Pierce Magnetic Titanium Dioxide kit and subjected to Oribitrap mass spectroscopy. This enabled the identification of triple phosphorylation sites in the PfAB4 protein at serine 87, serine 91 and serine 92, which is the PfPCNA1 peptide: LCGANEsVVIssK