Discussion
Malaria is a parasitic disease that prevails in the world's poorest regions severely hampering socio-economic development. In humans, it is caused by five protozoan species belonging to the genus
Plasmodium of which
P. falciparum accounts for
99% of all malaria-associated deaths. Strong correlations have been established between high concentrations of Histidine-rich protein II (HRPII), produced by
P. falciparum, and higher mortality and severity of malaria outcomes, including cerebral malaria.
The neuropathogenesis of cerebral malaria remains unclear and it presents as a multifactorial pathogenesis depending from the patient status and multiple parasitic virulence mechanisms. Recent data identify HRPII as one of the critical virulence factors for the onset of cerebral malaria as its ability to disrupt the Blood-Brain Barrier by itself. The suggested pathogenic mechanism includes activating the NF-kB pathways and the inflammasome in brain endothelial cells (even in the absence of other parasite factors, or immune cells, or astrocytes) with the release of hIL1β. These events then trigger the loss of integrity of the BBB, both in
in vitro model systems and
in vivo. It remains largely unknown how these cellular responses are mediated by HRPII binding to the cell and which HRPII-binding molecules are required to trigger this response. In fact, HRPII has shown high affinity towards some glycosaminoglycans, divalent cations, heme, and others, all of which could play binding, structural or cofactorial role. This study aims to investigate the early brain endothelial response of HRPII through the analysis of gene activation/repression using RNAseq data and provide a better understanding of the metabolite patterns related to HRPII-mediated effects.
The disruption of the BBB due to HRPII at concentrations comparable to its serum levels during infection has been previously shown using directly HRPII-producing parasites or purified HRPII either from parasites and parasite-conditioned media or from
E. coli recombinant expression. Using RNA-seq and
in-vitro BBB model system, we have investigated the HRPII-triggered transcriptional changes in endothelial cells. We used HRPII protein either produced and purified from standard
E. coli BL21(DE3) or a BL21(DE3)-derived mutant lacking lipopolysaccharide endotoxin, called Clear-coli® (Lucigen). This comparison aimed to rule out any contribution to the cellular responses of traces of endotoxin eventually carried over during purification from
E. coli and bound to HRPII, possibly via the repetitive glycan polymer, commonly referred to as O antigen. An extensive LPS-removal step using Triton-X114 was performed in both purifications, and the purified protein was negative at the LAL-test. We have then analysed the cellular transcriptional responses over three time points, 0, 3h, and 12h. By including gene responses to LPS only, we have also analysed the differences between endotoxin and HRPII activation of the endothelial cells.
The RNA was extracted from eight conditions in quadruplicate, and the libraries were prepared, assessed for high quality, and then sequenced using an Illumina HiSeq. On average, a sequence depth of 26.9 ± 0.8 million reads per sample was obtained and about 99% mapped to the human genome. The average length-mapped of ~255bp was higher than expected (~200bp), and the sequencing depth per library achieved in this experiment was sufficient for downstream differential expression analyses. Our analysis of the RNAseq data revealed that HRPII purified from Clear coli® did not trigger the activation of the innate immune response, supporting the hypothesis that endothelial response to HRPII requires costimulatory molecul
