# Nutrient stress dramatically increases malaria parasite clag2 copy number to increase host cell permeability and enable pathogen survival

## TL;DR

Malaria parasites adapt to low-nutrient conditions by increasing CLAG2 gene copies to maintain nutrient uptake when CLAG3 is missing.

## Contribution

Discovery that CLAG2 gene amplification compensates for CLAG3 deletion under nutrient stress in malaria parasites.

## Key findings

- CLAG3-null parasites cannot grow in nutrient-limited media but can adapt through CLAG2 amplification.
- CLAG2 gene amplification restores nutrient uptake via modified channels in the absence of CLAG3.
- Malaria parasites show genome plasticity by altering gene copy numbers in response to environmental stress.

## Abstract

To grow and replicate in erythrocytes, malaria parasites must increase the host cell’s permeability to a broad range of nutrients. The plasmodial surface anion channel (PSAC) mediates this increased permeability and has been linked to CLAG3, a protein encoded by a multigene family conserved in Plasmodium spp. Surprisingly, an CLAG3 knockout parasite produced in P. falciparum exhibits incomplete reductions in PSAC activity, propagates normally in standard nutrient-rich media, but is unable to expand in modified media with more physiological levels of key nutrients. To explore these unexpected findings, we used in vitro selections on a CLAG3-null parasite and obtained a mutant capable of expansion under nutrient-limiting conditions. This growth was associated with restored solute uptake despite absence of CLAG3 protein. The mutant parasite expressed channels with characteristics of PSAC though with altered solute selectivity and lack of protease susceptibility, suggesting a modified channel and genome-level changes in the pathogen. Whole-genome sequencing revealed a dramatically increased clag2 copy number without other relevant changes. Quantitative PCR and DNA transfection confirmed increased production of the clag2 gene product. These findings implicate CLAG2 in direct formation of nutrient channels, suggest a new model that accounts for variable expansion of clag genes in Plasmodium spp., and uncover a dramatic genome plasticity available to malaria parasites.

Malaria parasites grow within circulating red blood cells and acquire nutrients from human and animal plasma via a pore they insert in the host membrane. This pore is linked to CLAG3, a protein conserved in all examined malaria parasites. Surprisingly, deletion of CLAG3 only partially reduces formation of the nutrient pores, allowing parasites to grow normally under standard culture conditions that provide high levels of nutrients. This CLAG3-null parasite could not grow in modified media with two nutrients reduced to levels resembling those in human plasma. Here, we used prolonged culture of the CLAG3-null parasite in nutrient-limited medium to produce a mutant that can grow at near-normal rates. Despite its inability to express CLAG3, this mutant increased its nutrient uptake using pores with altered properties. Molecular studies revealed DNA-level amplification of the gene encoding CLAG2, a closely related protein from another parasite chromosome. Our findings suggest that malaria parasites can change their DNA in response to sustained changes in nutrient availability as may occur with host migration, climate change, or introduction into new host species.