EL AMBITO DE LA GUERRA EN LA DIMENSION PODER (Acerca de

Previous mass spectrometry study identified UROD, ALAS2 and FECH in human red blood cells (Egan, Jiang et al. 2015). Sigala and colleagues found the haem synthesis pathway in mature red cells can be stimulated by adding exogenous 5-aminolevulinic acid (ALA) that caused the accumulation of the intermediate protoporphyrin IX (PPIX),

indicating the existence and function of enzymes of hALAD, hHMBS, hUROS, hUROD, hCPOX and hPPOX in red blood cells (Sigala, Crowley et al. 2015). However, this study did not prove the existence of ALAS2 and FECH, and it was believed that mature

erythrocytes loss mitochondria and their constituent proteins (Ponka 1997). Clare Smith studied the import evidence of hALAD, hUROD and hFECH in her PhD project reporting these three enzymes were localised inside parasite based on western blot analysis and confocal microscopy (Drakesmith, Schimanski et al. 2005). Results in this chapter

confirmed the presence of hALAD, hHMBS and hUROS with expected size, hALAS2 and hFECH with smaller size based on western blot analysis. The rest enzymes were not detected probably due to a lack of specificity of the antibodies used. The limitation of this work was that repeat experiments were not carried out and different developmental stages were not investigated.Furthermore, immunofluorescent staining and HPLC analysis results confirmed the existence of active hHMBS in RBCs.

This chapter also investigated if the human red blood cell haem enzymes were present in purified parasites. Previous studies demonstrated that hALAD and hFECH was detected in

Plasmodium using western blot and confocal microscopy (Bonday, Taketani et al. 1997, Bonday, Dhanasekaran et al. 2000). However, western blot results in this chapter did not detect the presence of hALAD in parasite possibly due to the antibody specificity. For hFECH, it was not conclusive whether the probed larger band due to non-specific binding or dimer conformation. By contrast, this chapter identified two other enzymes hHMBS and hUROS that were detected inside Plasmodium.

Which haem biosynthetic emzymes are present in red blood cells?

Immunoblotting results in this chapter showed that the human haem enzymes hALAD, hHMBS and hUROS were detected in the mature red blood cells. Two rate-limiting haem enzymes hALAS2 and hFECH were detected with a smaller molecular weight, suggesting these two enzymes might exist in RBCs as forms of truncated protein. Studies have shown

degradation during the process of maturation (Griffiths, Kupzig et al. 2012, Chu, Sinha et al. 2018). If the truncated protein is a product of the autophagosome destruction, co- localisation experiments with mitochondria and autophagosome markers can be used to investigate this hypothesis. hUROD was not identified in mature red blood cells with western blot analysis, which was inconsistent with previous functional study (Sigala, Crowley et al. 2015). But the western blot data is preliminary due to experiment samples insufficiency and antibody specificity. If these enzymes were not detected in mature RBCs with Western blot analysis, they certainly would be impossible to be probed in Plasmodium

fraction with the same method. Results of the western blotting analysis with antibodies specifically against hHMBS and hUROS showed that these two enzymes existed in uninfected red blood cell lysate, and these antibodies also detected an identical band in purified parasite samples. One concern arising from the Western blotting analysis is whether the anti-human antibody is also cross-reacting with the Plasmodium counterparts. In the case of hUROS, it is unlikely to have the cross-reaction problem because there is no gene coding for UROS enzyme homologous in the Plasmodium genome

(http://plasmodb.org/plasmo/app/record/gene/PF3D7_1209600). In the case of hHMBS, this is also unlikely because the amino acid sequence identity between hHMBS and

PfHmbs is only 16% and the molecular weight of PfHmbs is around 45 kD, which is larger than hHMBS size (37-39 kD).

Is hHMBS newly identified to be imported by intraerythrocytic Plasmodium?

Western blotting was employed using antibodies specifically against hHMBS protein in this chapter to investigate the parasite uptake of hHMBS at red blood cell stage. The anti- hHMBS antibody detected a single unique band with expected size of hHMBS in

uninfected red blood cell lysate, uRBC membrane fraction and purified parasite fraction. These results newly showed evidence that hHMBS was present in the mature red blood cells and seemed to be imported by Plasmodium. However, this cannot be certain due to the red blood cell contamination. Western blot analysis showed that a limited amount of human red blood cell membrane proteins Ankyrin1 and Band3 were detected in parasite fraction, indicating it is relatively free from RBC contamination. Thus, other investigations were conducted using immunofluorescent imaging and measurement of HMBS activity in

ΔPfHmbs parasite line.

Immunofluorescent imaging analysis was conducted by fixing thin smears of iRBCs with the use of anti-hHMBS antibody and immunofluorescent microscopy, and it detected

erythrocyte HMBS within intraerythrocytic P. falciparum. Due to the resolution limitation of immunofluorescent imaging, future work using specific organelle molecular markers combined with confocal microscopy and immuno Electronic Microscopy may provide a deeper insight, such as the parasite subcellular localisation of hHMBS. The HPLC

measurement of the HMBS activity from whole 3D7-iRBC was significantly higher than the

ΔPfHmbs-iRBC, but the HMBS activity of saponin-lysed 3D7 and PfHmbs-KO parasite lines both showed no activity. Perhaps the saponin treatment inhibited all HMBS activity, and the Percol collected whole iRBC results were actually more indicative. Theoretically, 3D7-iRBC could contain both human and parasite HMBS enzyme while only hHMBS contributed to the activity in ΔPfHmbs-iRBC. Other purification methods such as magnetic beads can be used to collect iRBC with less uRBC contamination. Together, it might dispute the hypothesis of importing active hHMBS, suggesting the hHMBS could function in the red blood cell fraction. However, further experiments are required such as