Characterization of the novel Maurer's clefts protein MAHRP1 in "Plasmodium falciparum"

Plasmodium falciparum causes the worst form of human malaria and is responsible for 1-2
million deaths annually. A vaccine is not available and resistance to drugs is widespread. One
approach to tackle these problems is to identify new intervention targets. However, this is
hampered by a limited understanding of many aspects of P. falciparum biology.
The morbidity and mortality associated with malaria is due to the asexual erythrocyte stages
of P. falciparum. While most intracellular pathogens interact with an active host cell, the
malaria parasite develops within the red blood cell (RBC) that is devoid of all organelles and
any protein trafficking machinery. The parasite resides in a parasitophorous vacuole (PV),
which is encircled by a parasitophorous vacuolar membrane (PVM). The parasite modifies its
host cell by establishing membranous structures in the RBC cytoplasm. These comprise discshaped
structures at the RBC periphery called Maurer’s clefts (MC) with an elusive function.
In addition the parasite modifies the surface of the infected RBC by exporting own proteins
that contribute to the virulence of P. falciparum. A key protein in this process is the variant
surface antigen, P. falciparum erythrocyte membrane protein 1 (PfEMP1), which mediates
adherence of infected RBCs to host endothelial cells. This is thought to prevent the infected
RBC of being cleared by the spleen. Furthermore the parasite evades the immune system by a
process called antigenic variation, the switching of the expression between different members
of PfEMP1.
As mature RBCs lack a cellular trafficking machinery, the parasite establishes its own
secretory system for exporting proteins beyond the parasite membrane (PM) through the PVM
and across the host cell cytoplasm to the MCs and the RBC membrane. The mechanisms and
molecular apparatus involved in this process are not completely understood. A classical signal
sequence is sufficient to direct proteins into the endoplasmatic reticulum (ER) with default
release into the PV. Onward transport across the PVM requires an additional signature termed
Plasmodium export element (PEXEL) or vacuolar targeting signal (VTS).
The aim of this thesis was to characterize a novel transmembrane (TM) protein termed
membrane-associated histidine-rich protein 1 (MAHRP1). It is transcribed exclusively in
early stages, has a C-terminus with approximately 30% histidines present as DHGH repeats
and localizes to the Maurer’s clefts. MAHRP1 has no classical signal sequence and no
PEXEL/VTS motif and it is thus unclear how it is directed to the MCs.
Histidine-rich proteins have been shown to be effective cation binders. The histidine-rich
region of MAHRP1 was recombinantly expressed and used for interaction studies with the
toxic waste product ferriprotoporphyrine (FP), which accumulates upon degradation of
hemoglobin -a main nutrient source of the parasite. MAHRP1 specifically interacts with FP
and binding stoichiometry correlates with the amount of DHGH repeats. The bound FP has
increased peroxidase-like activity and is 10-fold more susceptible to H2O2-induced
degradation compared with unbound FP. These properties of MAHRP1 suggest it may play a
protective role against oxidative stress at the MCs.
To investigate the amino acids responsible for correct trafficking of MAHRP1 to the MCs,
plasmids were generated encoding different green fluorescent protein (GFP)-tagged domains
of MAHRP1 and parasites were subsequently transfected with these plasmids. Analysis of
transfectants showed that the full length MAHRP1- GFP is successfully trafficked to the MCs,
whereas the domains TM or TM-C-terminus were retained at the ER. Dissection of the Nterminus
revealed a segment of 18 amino acids containing a motif with limited similarity to
the PEXEL/VTS motif, and which is needed for export of MAHRP1 to the MCs.
Fluorescence photobleaching and time-lapse imaging techniques indicate that MAHRP1-GFP
is initially trafficked to isolated subdomains in the PV/PVM that appear to represent nascent
MCs. The data suggest that the MCs bud from the PVM and diffuse within the RBC
cytoplasm before taking up residence at the cell periphery.
To understand the function of MAHRP1, a mutant with a mahrp1 disruption and MAHRP1
ablation was generated. MCs are still formed in the absence of MAHRP1 but the export of
PfEMP1 is interrupted at the PM/PVM interface. As a consequence, no PfEMP1 is detected
on the surface of infected RBCs. By contrast, export of other selected proteins appears to be
uninhibited. This indicates that MAHRP1 plays an essential role in the export of major
virulence factor PfEMP1 and thus represents an interesting intervention target in the battle
against malaria.