Scientists claim the malaria parasite can use one of two members of the same but unexpected family of its own genes to generate a channel protein that is inserted into the host blood cell membrane to increase the influx of nutrients needed to support its survival.
The new work has helped to answer a long-standing question about the plasmodial surface anion channel (PSAC) that is widely understood to act as this nutrient gateway: Is the channel produced by the parasite itself and inserted into the blood cell membrane, or is PSAC a native human channel that was present in the red blood cell membrane all the time but in a quiescent state until the parasite took up residence ?
The combination of approaches and experimental techniques used by scientists at the NIAID's Laboratory of Malaria and Vector Research has provided persuasive evidence for the former. It also suggests that the parasite can change which of the two channel protein genes it switches on according to selective pressures.
Surprisingly, both belong to the parasite’s own clag3 gene family, which has previously been thought to play a role in sticking infected cells to blood vessel linings. These results are published in Cell in a paper titled “Malaria Parasite clag3 Genes Determine Channel-Mediated Nutrient Uptake by Infected Red Blood Cells.”
P. falciparum and all studied malaria parasites increase their host erythrocyte cells’ permeability to a range of solutes including nutrients required for intracellular parasite growth, explain corresponding author Sanjay A. Desai, M.D., and colleagues. Dr. Desai is one of the co-discoverers of the PSAC. While it hasn’t, to date, been clear whether the channel is of human or parasitic origin, PSAC is nevertheless considered a potential antimalarial drug target, especially as studies suggest that the parasite can alter the permeability of the cell membrane to acquire resistance to existing antimalarials.
Dr. Desai’s team has been working to thrown more light on the genetic origins and workings of PSAC. The newly published series of studies combine high-throughput screens for novel channel inhibitors, electrophysiology to reveal direct inhibitor action on PSAC, linkage analysis in a P. falciparum genetic cross, DNA transfection experiments, in vitro selections, and mutant analysis to demonstrate the role of two paralogous genes in PSAC activity.
The researchers first screened a compound library to identify a candidate that specifically inhibited the uptake of nutrients by erythrocytes infected with either Plasmodium falciparum line HB3 or Dd2 but not both. The rationale behind this was that if the PSAC protein is indeed made by the parasite itself, then genetic variability could play a key role in determining whether an inhibitory compound is effective against one but not another parasitic line.
They homed in on a compound, subsequently given the name ISPA-28 (for isolate-specific PSAC antagonist), that blocked nutrient uptake by Dd2- but not HB3-infected cells. Further tests showed the compound was also ineffective against a large number of different parasite lines and that it acted directly by blocking PSAC in the Dd2-infected cells.
Evaluating the effects of ISPA-28 on PSAC activity in red blood cells infected with recombinant progeny clones generated from a Dd2 x HB3 genetic cross showed that compound only blocked nutrient uptake by those cells that had inherited a certain collection of genes originating from the Dd2 cell line.
To try and identify which of these gene products ISPA-28 was acting on, the researchers further generated Dd2 parasites transfected with alleles of the same candidate genes carried by the HB3 strain. Erythrocytes infected with D2d transfectants that expressed HB3 alleles for PFC0110w and PFC0120w were less susceptible to the blocking effects of ISPA-28 on nutrient uptake than the parent D2d parasite.
PFC0120w and PFC0110w are members of the clag multigene family that is conserved in P. falciparum and all plasmodia evaluated to date. They are believed to play a role in the adherence of infected cells to blood vessel linings. This makes their apparent role in protein transport particularly unexpected, Dr. Desai and colleagues admit.
The paralogs of PFC0120w and PFC0110w are clag3.1 and clag3.2, located on chromosome 3. P. falciparum carries three additional paralogs, on chromosomes 2, 8, and 9. Dd2 expresses the clag3.1 version of the clag3 gene, whereas HB3 expresses clag3.2.
This differential expression was exploited to examine what the researchers called “the unexpected possibility” that clag3 is, in fact, the originator of PSAC. To do this they used an allelic exchange strategy to integrate part of Dd2’s clag3.1 gene into the clag3.2 gene expressed by HB3.
Surprisingly, erythrocytes infected with these modified HB3 parasites did indeed show inhibition of PSAC on administration of the ISPA-28 compound, albeit at level between that of the unaffected native HB3-infected cells and the inhibited Dd2-infected cells.
Interestingly, some of the progeny from the original Dd2 x HB3 cross that had inherited the complete Dd2 clag3.1 gene were less adversely affected by ISPA-28 than their parent. Further genetic analysis suggested that the some of the progeny were able to switch on clag3.2 expression, indicating epigenetic mechanisms had been activated.
To test whether selective pressures can trigger the parasites to switch on different clag genes, the researchers placed erythrocytes infected with Dd2 x HB3 progeny parasites in solutions containing ISPA-28 and high enough concentrations of sorbitol to cause the host cells to lyse. Under these conditions parasites that can selectively switch on the clag3.1 gene would be at an advantage, as the ISPA-28 would shut down the nutrient channel and prevent too much sorbitol entering the cell. Real-time qPCR using primers specific for each of the 5 clag genes confirmed that selection with sorbitol and ISPA-28 did indeed reproducibly increase clag3.1 expression while decreasing that of clag3.2. Subsequent studies confirmed that the clag3 gene product is associated with the host cell membrane, providing yet more evidence that it is, indeed, PSAC.
Nevertheless, there are still structural questions that will need answering, the authors admit. “We envision two models for how the protein may contribute to channel activity. In one, the clag3 product alone forms a novel microbial nutrient and ion channel.
"In the other, the clag3 product interacts with one or more other proteins to form functional PSAC.” If the second scenario is correct, then additional subunits with which the clag3 product interacts could also either be parasite-encoded or resident host proteins.
“Our findings open several new directions for future research,” the team adds. “Most importantly, they permit molecular studies into the mechanisms of PSAC-inhibitor interaction and the process of solute recognition and permeation through this unusual channel. A second direction will be to explore the trafficking of parasite-encoded channel components to the host membrane.”
Additional studies suggested that the clag products are packaged up and stored in the parasite and then secreted into the host cell and transported across the cytosol to the erythrocyte cell membrane after infection.
The existence of paralogous genes and variable inhibitor affinities may seem to represent hurdles to future drug or vaccine discovery efforts, the authors admit. However, they add, “While polymorphic sites on the channel have permitted linkage analysis and gene identification, most of the coding region is highly conserved in P. falciparum and other malaria parasites, consistent with conserved selectivity and transport biophysics in all infected cells.”