Mutant/mutation
The mutant lacks expression of HscB. It also expresses mCherry and luciferase under constitutive promoters. The mutant does not contain a drug-selectable marker.

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
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. 

Additional information
Analysis of a mutant expressing a C-terminal GFP-tagged version of HscB (RMgm-5645) showed the following:
- Evidence for expression in asexual blood stages as shown by flow-cytometric analysis
- Expression in oocysts and in (maturing/mature) liver stages (at 48 and 56 hour after invasion of hepatocytes by sporozoites) where it co-localized with mitochondria (that were stained with the mitochondrial dye MitoView 650).

Previous studies have linked reduced survival of wild type (WT) parasites during liver stage development to host cell autophagy. To investigate whether PbHscB-KO parasites might be impaired in shedding the autophagy marker microtubule-associated protein 1 light chain 3 (LC3) from their parasitophorous vacuole membrane (PVM), WT and PbHscB-KO parasites were fixed at 24 and 48 hpi and stained with LC3 antibodies. At 24 hpi, both WT and PbHscB-KO parasites were heavily decorated with LC3. However, at 48 hpi, approximately half of the WT parasites were able to shed the autophagy marker, while the majority of KO parasites remained LC3-positive. This reduced shedding capacity of the PbHscB-KO parasite correlates with the reduced survival and could explain the lower number of PbHscB-KO parasites present at 48 hpi.

Due to the small size of PbHscB-KO parasites we questioned whether they are still able to initiate late liver stage development.
Therefore, infected cells were fixed at 56 hpi for WT parasites and at 96 hpi for PbHscB-KO parasites. Cells were stained with antibodies against merozoite surface protein 1 (MSP1), which is known to be only expressed at late liver stage (from 48 hpi onwards). MSP1 was readily detected at 56 hpi in WT parasites, while it was weakly detected at 72 hpi (not shown) and more strongly detected at 96 hpi in PbHscB-KO parasites. This observation indicates a significant delay in liver stage development for PbHscB-KO parasites but also suggests that, although delayed, these parasites might be capable of completing their liver stage development.

In the paper a double knockout (dKO) mutant PbHscB-PbMei2 is described (RMgm-5646), that lacks expression of HscB and Mei2 (PF3D7_0623400, PBANKA_1122300).
First, a marker-free PbHscB-KO line was generated using negative selection of blood stages of the HscB-KO line. Subsequently, the PlasmoGEM vector for PbMei2-KO (PbGEM-300555) was transfected into the marker-free PbHscB-KO schizonts. In addition to the PbHscB-PbMei2-double knockout (dKO) parasite line, a PbMei2 single knockout (sKO) line was also included for comparison. 
No significant difference in oocyst size was observed between WT, PbHscB-KO, PbMei2-KO and PbHscB-PbMei2-dKO lines at day 7 and day 14 post-feeding, indicating that both gene deletions are dispensable for oocyst development. This finding was supported when counting the number of sporozoites present in salivary glands at earlier (day 18 and 19 post-feeding) and later (day 24, 26, and 28 post-feeding) time points, which showed no significant difference among the WT and the three KO lines. Automated microscopy analysis revealed that PbHscB-KO parasites exhibited impaired growth at 24, 48 and 56 hpi, while PbHscB-PbMei2-dKO parasites showed an even more pronounced growth defect. PbHscB-KO parasites consistently showed a significant reduction in size at all time points. When both knockouts were combined in the PbHscB-PbMei2-dKO parasites, the phenotype resembled that of PbHscB-KO, displaying a substantial reduction in size and slow growth towards 48- and 56-hours post-infection. Since PbMei2-KO parasites appeared to grow to a normal size, as previously reported for P. yoelii Mei2-KOs, further investigation was conducted on the late liver stage. PbMei2-KO-infected cells were fixed at 56 hpi and stained for MSP1, which is expressed just prior to merozoite formation, and for mitochondria using anti-TgHsp70 antibodies to assess parasite fitness. While WT parasites displayed cytomere stages with the invaginated plasma membrane and a strong MSP1 signal, PbMei2-KO parasites showed neither plasma membrane invaginations nor distinct MSP1 signal. The mitochondria of liver stages of PbMei2-dKO and the double knockout PbHscB-PbMei2-dKO exhibited abnormal accumulations, and the cytoplasmic mCherry signal revealed numerous vacuoles within the parasite. Staining with the PVM marker UIS4 showed that PbMei2-KO parasites at 56 hpi had a corrupted PVM and an apparent leakage into the host cell cytosol was observed. After infection of mice with 100.000 sporozoites blood-stage parasitemia appeared with a 2–3 day delay in PbHscB-KO and a 5–6 day delay in PbMei2-KO sporozoite-injected mice. No breakthrough infections were observed in mice injected with PbHscB-PbMei2-dKO sporozoites for up to 41 days post-injection

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