Mutant/mutation
The mutant lacks expression of HMGB1

Protein (function)
High mobility group box (HMGB) proteins are non-histone chromosome-binding nuclear proteins that contain at least one HMG-box domain and are expressed in almost all the eukaryotes. Many eukaryotes contain a large number of HMGB proteins and most of them possess one or two HMG-boxes, although transcription factor like UBF1 contains up to six HMG-boxes. In addition to nuclear functions such as chromatin organization, transcriptional regulation, and DNA repair, HMGB proteins have evolved to perform cytosolic and extracellular functions. A typical example for the cytosolic function is the regulation of autophagy and mitophagy by binding of human/mouse HMGB1 to key
autophagy proteins like Beclin1 and Atg5 through its active shuttling between nucleus and cytosol. When present extracellular either through active secretion or passive release, HMGB1 serves as a cytokine or danger-associated molecular pattern (DAMP) by mediating the inflammation. Depending on the overall redox state and interaction with various receptors such as TLR4, RAGE etc., HMGB1 can induce the proinflammatory responses. The genome of malaria parasite encodes four HMGB proteins - HMGB1, HMGB2, HMGB3 and HMGB4 that are conserved across the Plasmodium species. While HMGB1 and HMGB2 are of around 100 amino acids in length with one HMG-box, HMGB3 consists of over 2000 amino acids with two HMG-boxes and HMGB4 has around 250 amino acids with one HMG-box. Of the four HMGBs, HMGB1 and HMGB2 have been studied so far. In vivo studies carried out in mice infected with P. berghei (Pb; rodent parasite) HMGB2 (PBANKA_0712900; PF3D7_0817900;  knockout (KO) parasites have shown significant protection from experimental cerebral malaria (ECM). Another study carried out in the rodent parasite, P. yoelii (Py), has shown that the deletion of HMGB2 (PY17X_0713100; PF3D7_0817900; PY17X_0713100) leads to a prominent reduction of oocyst formation in the mosquitoes. In case of HMGB1, a ChIP-seq study carried out with PfHMGB1 (PF3D7_0817900) wildtype (WT) and KO lines has shown that HMGB1 deletion disrupts centromere-/telomere-dependent nuclear architecture leading to a complete silencing of var expression

Phenotype
Growth analysis performed for the asexual stages in Balb/c mice infected with 105 PbHMGB1KO parasitized RBCs indicated a delay of ~3 days in comparison with PbWT parasite growth. Mice infected with PbHMGB1KO parasites displayed a subsequent clearance when the blood parasitemia reached around 15-30%. While all the mice infected with PbWT parasites succumbed to anemia within day 19 post-infection, PbHMGB1KO-infected mice were completely protected against disease and complete clearance of blood parasitemia and the absence of recrudescence infections.
Eevidence is presented for splenic clearance of PbHMGB1KO parasites and that PbHMGB1 regulates pir expression.
 
Additional information
The clearance of PbHMGB1KO parasites and the protective phenotype of PbHMGB1KO-infected mice were also reproducible with the inoculum of 10, 102 or 106 parasitized RBCs through intravenous route. To rule out the subsequent clearance of PbHMGB1KO asexual stage infections occurring due to any growth defect in the parasite per se, PbHMGB1KO parasites were collected during the protective phase when the blood parasitemia fell to 0.1%. When such parasites were injected into naïve Balb/c mice, they could successfully establish the blood stage infections leading to an increase in the blood parasitemia followed by subsequent clearance. The protective phenotype of PbHMGB1KO parasites was also examined in CBA/CaJ mice - an in vivo mouse model for ECM, by injecting 105 PbHMGB1KO-parasitized RBCs through intraperitoneal route. As observed for Balb/c mice, PbHMGB1KO parasites displayed a growth delay in CBA/CaJ mice. While all the PbWT-infected CBA/CaJ mice died within 9 days post-infection with ~80% of them showing the typical symptoms of ECM, none of the PbHMGB1KO-infected mice showed ECM. There was also a complete protection from mortality due to ECM and/or anemia with subsequent clearance of blood parasitemia.
Eevidence is presented for splenic clearance of PbHMGB1KO parasites and that PbHMGB1 regulates pir expression.
 
Analysis of a mutant expressing a C-terminal GFP-tagged version of HMGB1 (RMgm-5612, PbWT(HMGB1-GFP)) showed the following:
- Western analysis carried out with the lysates of PbWTHMGB1-GFP blood stage parasites confirmed the expression of a 41 kDa PbHMGB1-GFP fusion protein
- Live imaging studies performed with PbWTHMGB1-GFP transgenic parasites showed the colocalization of GFP fluorescence with the DNA staining of 4′,6-diamidino-2-phenylindole (DAPI), indicating the nuclear localization of PbHMGB1
- PbHMGB1 is undetectable in plasma of infected mice
To examine the extracellular presence of PbHMGB1, plasma samples were collected from mice infected with PbWTHMGB1-GFP parasites showing ~10% blood parasitemia and ELISA was carried out with GFP antibodies

Evidence is presented for the following:
- PbHMGB1 lacks TNF-a stimulatory activity
Plasmodium HMGB1 lack the A box and C-terminal acidic tail that are present in mammalian HMGB1. The A box contains two critical cysteine residues (Cys23 and Cys45) whose redox status determines the chemoattractant and proinflammatory functions of mammalian HMGB1. Further, the third critical cysteine residue (Cys106) present in the TNF-a stimulatory domain of B box in mammalian HMGB1 is also absent in Plasmodium HMGB1
- PbHMGB1 is undetectable in plasma

Other mutants