Mutant/mutation
The mutant expression a C-terminal GFP-tagged version of HscB. It also expresses mCherry and luciferase under constitutive promoters. 

Protein (function)
HscB is a putative mitochondrial iron-sulfur (Fe-S) protein assembly chaperone HscB (heat shock cognate B). HscB belongs to the Hsp40 protein family, known as heat shock cognate B chaperones, and it operates in conjunction with an Hsp70 family member, HscA, for the assembly of Fe-S proteins. Fe-S proteins possess an iron sulfur cluster (ISC) as a cofactor, enabling efficient electron transport and involvement in various cellular redox reactions. Examples of Fe-S proteins include aconitase of the tricarboxylic acid (TCA) cycle and succinate dehydrogenase of the respiratory chain [40, 41]. In most eukaryotic cells, the biogenesis of ISCs and Fe-S proteins occurs within the mitochondria via the ISC pathway. Plasmodium parasites, however, possess an additional ISC biogenesis pathway in the apicoplast, a vestigial plastid resembling the chloroplast [43, 44]. This pathway is known as the sulfur mobilization (SUF) pathway and has been shown to be essential for asexual blood stage development. Conditional knockout experiments targeting SufS, the first enzyme in the
SUF pathway during oocyst development, resulted in impaired sporozoite formation, underscoring the significance of the SUF pathway in sporozoite development. It has been reported previously that the deletion of hscB in P. berghei parasites did not exhibit significant impairment during the mosquito stage development.

Phenotype
Analysis of the mutant expressing a C-terminal GFP-tagged version of HscB showed the following:
- Evidence for expression in asexual blood stages as shown by flow-cytometric analysis
- Expression in oocysts. On day 12 post-feeding, we extracted midguts from the mosquitoes and stained them with the mitochondrial dye MitoView 650, while DNA was stained with Hoechst 33342. Live confocal microscopy imaging revealed overlapping signals between PbHscB-GFP and the mitochondrial dye, indicating that PbHscB-GFP localized to the mitochondria at this stage
- Expression in (maturing/mature) liver stages. The cells were fixed at 48 and 56 hours post-infection (hpi) and stained with antibodies against GFP and Toxoplasma gondii Hsp70 (TgHsp70), which has been shown to recognize P. berghei mitochondrial Hsp70. Although PbHscB-GFP expression was weak, a clear overlap between the PbHscB-GFP signal and the mitochondrial marker TgHsp70 was observed at both time points

Additional information
Analysis of a mutant lacking expression of HscB (RMgm-5640) showed the following:
Normal numbers of oocysts were produced (with a similar size as wild type oocysts). Normal numbers of (salivary gland) sporozoites were produced. Infection of mice with PbHscB-KO sporozoites resulted in blood stage infections with a prolonged prepatent period. All mice infected with wild type (WT) sporozoites were blood stage parasites positive on day 3 post-injection, while mice infected with PbHscB-KO sporozoites were positive on days 5 to 7 post-injection. PbHscB-KO liver stages appeared smaller compared to WT at 24, 48 and 56 hpi. Comparison of the parasite numbers at 6 and 48 hpi indicated reduced survival in the PbHscB-KO parasites. Merozoite surface protein 1 (MSP1) expression in liver stages at 56, 72 and 96 hpi. 

Other mutants