An external sensing system in Plasmodium falciparum-infected erythrocytes.

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.

Wolbachia lipoproteins: abundance, localisation and serology of Wolbachia peptidoglycan associated lipoprotein and the Type IV Secretion System component, VirB6 from Brugia malayi and Aedes albopictus.

BACKGROUND: Lipoproteins are the major agonists of Wolbachia-dependent inflammatory pathogenesis in filariasis and a validated target for drug discovery. Here we characterise the abundance, localisation and serology of the Wolbachia lipoproteins: Wolbachia peptidoglycan associated lipoprotein and the Type IV Secretion System component, VirB6. METHODS: We used proteomics to confirm lipoprotein presence and relative abundance; fractionation, immunoblotting and confocal and electron immuno-microscopy for localisation and ELISA for serological analysis. RESULTS: Proteomic analysis of Brugia malayi adult female protein extracts confirmed the presence of two lipoproteins, previously predicted through bioinformatics: Wolbachia peptidoglycan associated lipoprotein (wBmPAL) and the Type IV Secretion System component, VirB6 (wBmVirB6). wBmPAL was among the most abundant Wolbachia proteins present in an extract of adult female worms with wBmVirB6 only detected at a much lower abundance. This differential abundance was reflected in the immunogold-labelling, which showed wBmPAL localised at numerous sites within the bacterial membranes, whereas wBmVirB6 was present as a single cluster on each bacterial cell and also located within the bacterial membranes. Immunoblotting of fractionated extracts confirmed the localisation of wBmPAL to membranes and its absence from cytosolic fractions of C6/36 mosquito cells infected with wAlbB. In whole worm mounts, antibody labelling of both lipoproteins were associated with Wolbachia. Serological analysis showed that both proteins were immunogenic and raised antibody responses in the majority of individuals infected with Wuchereria bancrofti. CONCLUSIONS: Two Wolbachia lipoproteins, wBmPAL and wBmVirB6, are present in extracts of Brugia malayi with wBmPAL among the most abundant of Wolbachia proteins. Both lipoproteins localised to bacterial membranes with wBmVirB6 present as a single cluster suggesting a single Type IV Secretory System on each Wolbachia cell.