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

Nicole B Potchen; Tatiane Macedo Silva; Inderjeet Kalia; Justin B Lack; Sanjay A Desai

doi:10.1371/journal.ppat.1013321

. 2025 Oct 16;21(10):e1013321. doi: 10.1371/journal.ppat.1013321

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

Nicole B Potchen ^1,^¤,^#, Tatiane Macedo Silva ^1,^#, Inderjeet Kalia ^1,^#, Justin B Lack ², Sanjay A Desai ^1,^*

Editor: Tania F de Koning-Ward³

PMCID: PMC12530616 PMID: 41100555

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.

Author summary

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.

Introduction

Malaria parasites evade immune attack by replicating within erythrocytes of their vertebrate hosts. P. falciparum, a virulent and deadly human pathogen, dramatically remodels its host erythrocyte through exported effector proteins that alter the cell’s deformability, cytoadherence and permeability [1–4]. These changes compromise immune evasion because surface display of parasite antigens and altered cellular properties can elicit host immune responses [5–7]. Epigenetic switching of multigene families encoding surface-displayed antigens, such as the PfEMP1 cytoadherence receptor or the RIFIN immunity modulator [8,9], is generally thought to be the parasite’s primary defense against immune recognition.

The clag multigene family is unique because its encoded proteins represent the only surface-displayed antigens conserved throughout the genus Plasmodium. In contrast to other surface exposed antigens, the CLAG proteins do not function in cytoadherence or immune evasion, but instead enable nutrient uptake via formation of the plasmodial surface anion channel (PSAC) [10–14]. While the two copies on chromosome 3 (clag3.1 and clag3.2) are under epigenetic control in P. falciparum [15,16], switching between these paralogs does not alter channel properties but it may finetune parasite replication in human infections [17]. Remarkably, a CLAG3 knockout line exhibits only partially compromised PSAC activity and expands normally in nutrient-rich RPMI 1640-based media, but cannot be propagated in media with more physiological levels of key nutrients [18]. The paralogs encoded by three clag genes on other chromosomes are exported into host cytosol [19], but their roles remain unclear as they have not been linked to PSAC formation [20–23]. Although not subject to mutually exclusive expression like the two clag3 paralogs, clonal variation and a blasticidin S resistant PSAC mutant have established epigenetic regulation of clag2 but not of clag8 or clag9 in P. falciparum [12,15].

We now report copy number variation (CNV) as an alternate parasite strategy for modifying host cell properties. We used in vitro nutrient restriction on CLAG3-null parasites to select a mutant with a nearly 20-fold amplification of the clag2 genomic locus [18]. The resulting increases in CLAG2 expression restored host cell permeability through formation of channels with altered properties. Our studies uncover a capacity for large-scale genome amplification in Plasmodium spp., demonstrate that CNV is a novel strategy for altering host cell properties, reveal evolutionary forces that drive clag gene expansion in Plasmodium spp., and implicate a CLAG2 contribution to nutrient uptake.

Results

Small numbers of CLAG3 knockout parasites persist and expand in nutrient-restricted media

CLAG proteins are translated in late schizont stage parasites and assemble cotranslationally with RhopH2 and RhopH3, two unrelated and conserved parasite proteins, to form the RhopH complex; this complex is targeted to rhoptry organelles in developing merozoites and transferred to the new host cell during erythrocyte invasion [19]. It is then exported into host cytosol, becomes integral at the erythrocyte membrane, and fully induces PSAC activity by the early trophozoite stage [24].

DNA transfection to study clag3 in P. falciparum is complicated by the presence of two copies, clag3.1 and clag3.2, that undergo epigenetic switching and monoallelic expression in most parasite clones [15]. KC5, a laboratory clone that carries a single hybrid clag3 gene termed clag3h [25], was recently used to produce a clag3-null parasite termed C3h-KO [18]. We examined sorbitol and isoleucine permeabilities in synchronous trophozoite-stage cells infected with C3h-KO or the KC5 parent. Both solutes are primarily acquired via PSAC in infected cells [10,26]. Molecular confirmation of these transport studies has been established by knockdown of RhopH2 and RhopH3 [24]. Isoleucine uptake is critical for parasite propagation as it is absent from human hemoglobin and cannot be acquired through hemoglobin digestion [27].

Both solutes exhibited slower osmotic lysis kinetics in C3h-KO (Fig 1A), indicating reduced uptake. Because osmotic lysis kinetics correlate with whole-cell currents in patch-clamp [28,29], these measurements quantitatively estimate permeability reduction (Fig 1B). Sorbitol permeability was reduced by 48 ± 2%; isoleucine exhibited a greater reduction at 68 ± 2% (P < 10^-4, Student’s t test, n = 7–21 trials for each solute and parasite). It is not clear why these permeabilities are differentially affected by the loss of CLAG3, but studies implicating distinct routes through PSAC for these solutes may provide a structural basis [30].

We next examined the effect of reduced host cell permeability on in vitro parasite propagation and found that C3h-KO cultures expanded at rates comparable to KC5 in standard RPMI 1640-based medium (red vs black circles, Fig 1C), a general-purpose formulation with supraphysiological levels of most nutrients. Cultivation in a more physiological medium designed to evaluate PSAC inhibitors under conditions that simulate human plasma (PGIM, PSAC growth inhibition medium) [16], however, revealed a dramatic difference as KC5 cultures continued to expand while the C3h-KO parasitemia failed to increase over 8 days (Fig 1C). KC5 expansion was slower in PGIM than in the standard RPMI 1640-based medium (black triangles vs. black circles, Fig 1C), as has been reported previously for other wild-type parasite lines [16]. This reduced propagation in PGIM presumably reflects growth limitation due to nutrient restriction. Because growth rates in PGIM are comparable to those in human serum without addition of commercial media [16], PGIM may predict in vivo parasite nutrient access and replication more faithfully than RPMI 1640. Thus, stalled growth of the C3h-KO line in PGIM suggests that CLAG3 is essential for parasite expansion in human infections.

Interestingly, microscopic examination revealed occasional apparently healthy C3h-KO infected cells despite PGIM cultivation for more than 10 days (Fig 1D). To examine viability, we cultured C3h-KO in PGIM for various durations and quantified recovery from nutrient restriction upon return to the standard RPMI-based medium (S1A Fig). Each culture quickly resumed normal expansion; increasing the duration of PGIM exposure to 8 days did not increase the lag time (S1B Fig), indicating that a stable fraction of C3h-KO parasites remains viable in PGIM over this duration. One possibility is that compromised nutrient acquisition, imposed here through the combination of reduced host cell permeability and limited nutrient levels in the external medium, triggers a dormant parasite state [31,32].

Growth compromised to a greater extent than expected from permeability reduction

The marked reduction in growth using PGIM is surprising given the incomplete reduction in solute permeabilities in the C3h-KO line. To explore this finding quantitatively, we measured expansion of wild-type parasite cultures in the presence of ISG-21, a potent and specific PSAC inhibitor also known as MBX-2366 [33]. We quantified dose-dependent ISG-21 inhibition for both sorbitol and isoleucine uptake (Fig 1E and 1F). We then estimated the ISG-21 concentration required to reduce each solute’s permeability in wild-type KC5 parasites to levels matching those in C3h-KO without inhibitor addition (right panels, Fig 1E and 1F). The dose-response profiles for sorbitol and isoleucine as well as the ISG-21 concentrations required to yield osmotic lysis halftimes matching values for C3h-KO without inhibitor addition, 1.2 and 3 nM respectively, differed modestly, possibly reflecting these solutes’ distinct transport mechanisms through PSAC [30,34].

We then cultured the KC5 parental line with and without ISG-21 and compared expansion to that of C3h-KO cultivated without inhibitor. Remarkably, although 10 nM ISG-21 reduces permeability to a much greater extent than clag3 knockout, growth of KC5 in PGIM despite addition of this inhibitor is comparatively well-preserved (Fig 1G), indicating that reduced permeability of these solutes does not adequately account for C3h-KO’s failure to expand in PGIM. These findings are consistent with parasite growth as a complex process dependent on PSAC for uptake of many nutrients from an incompletely defined medium and intracellular utilization; there may also be contributions from channel-mediated efflux of soluble metabolic waste products, some of which remain unknown.

In vitro selection with PGIM restores host cell permeability via altered channels

As rare parasites were observed in nutrient-restricted cultures (Fig 1D), we used in vitro selection of C3h-KO to identify mutants capable of expansion in PGIM. A gradually increasing parasitemia suggested selection of replicating parasites (Fig 2A). After ~ 2 months, expansion yielded the KO-PGIM parasite pool suitable for molecular and biochemical studies. Cross-contamination with wild-type lines was excluded by PCR, which revealed an unchanged deletion locus in KO-PGIM and no detectable wild-type band (Fig 2B, p1-p2 amplicons); PCR from the unmodified downstream sequence also did not reveal changes that might have resulted from indels or other recombination events (p3-p4 and p3-p5). DNA sequencing of the clag3 promoter and deletion locus excluded mutations that could confer growth advantage to KO-PGIM and further excluded cross-contamination with wild-type parasites.

Remarkably, sorbitol permeability was significantly increased in the selected parasite when compared to C3h-KO (Fig 2C, P < 0.0001, n = 4 trials), reaching levels modestly greater than those of the KC5 wild-type parent (P = 0.07 for comparison to KC5). To explore ongoing changes that may confer additional survival advantages, we continued KO-PGIM propagation in PGIM for over 1 year; transport measurements revealed modest additional increases in sorbitol permeability (Fig 2C, bar graph; 8 trials after 200 days of cultivation in PGIM), suggesting a plateau in uptake kinetics. Isoleucine permeability also increased over this period but was not fully restored to wild-type levels (Fig 2D, P = 0.006 when compared to KC5, n = 8–10 trials each). The correlation between restored growth in nutrient-limited PGIM and increased solute permeabilities suggests that parasite replication depends on sufficient PSAC-mediated uptake of essential solutes under physiological nutrient levels.

To explore whether the increased permeability is reversible and sustained by nutrient restriction, we next returned KO-PGIM to nutrient-rich RPMI 1640-based media to generate a parasite pool we termed KO-rev. Transport studies after continuous cultivation in nutrient-rich standard medium for up to 228 days revealed modest, statistically insignificant decreases in sorbitol and isoleucine permeabilities (Fig 2E and 2F; P ≥ 0.19 for comparisons to KO-PGIM maintained in PGIM). This finding suggests stable increases in permeability not dependent on continued nutrient restriction.

To further evaluate the selected mutant’s stability, we examined parasite expansion rates and found that KO-rev retained its ability to expand in PGIM despite growth in nutrient-rich RPMI 1640-based media for up to 165 days (Fig 2G). Both KO-PGIM and KO-rev could be propagated in PGIM, albeit at rates below that of the wild-type KC5 parent.

Interestingly, our analysis revealed changes in relative solute permeabilities for the selected KO-PGIM mutant that could not be explained by simple upregulation of transport. While cells infected with KC5 parasites have a 2-fold greater permeability to isoleucine than to sorbitol (black bars in Fig 2C and 2D; P < 0.0001), KO-PGIM parasites produce channels with negligible differences between these solutes’ permeabilities (cyan bars in Fig 2F, P = 0.21). This altered preference reflects changes in the channel’s solute selectivity and indicates that the increased permeabilities in KO-PGIM do not result from an increased number of unmodified channels; channels with altered properties are instead required.

We explored channel properties further using protease susceptibility studies as protease treatment of intact infected cells is known to compromise PSAC activity [10,35]. Although proteases cleave many host and parasite proteins on infected cells, genetic mapping and molecular studies have established that CLAG3 cleavage within an exposed variant loop accounts for the transport’s protease susceptibility [36]. Here, we used pronase E, a mixture of nonspecific proteases active against nearly all exposed sites, and found reduced uptake by wild-type cells (Fig 2H, KC5) [36]. In contrast, channels associated with C3h-KO were insensitive to pronase E. Although sorbitol permeability is restored to wild-type levels, the channels on cells infected with KO-PGIM were also unaffected by this treatment (Fig 2H and 2I; P < 0.0001, one-way ANOVA with post-hoc Tukey’s multiple comparisons test; n = 3–8 trials each parasite). Solute uptake into cells infected with C3h-KO and KO-PGIM is resistant to pronase E, further implicating channels with altered properties.

We then used whole-cell patch-clamp to examine channel-mediated Cl^- flux in these clonal lines [28]. While uninfected human erythrocytes have substantial exchange-restricted Cl^- flux through the Cl^-/HC0₃ exchanger [37], they mediate negligible conductive Cl^- flux and have small whole-cell currents at all membrane potentials (V_m) [38]. Induction of PSAC activity on the infected cells permits net Cl^- flux and relatively large currents in whole-cell patch-clamp, which measures net ion flow only at the plasma membrane of a single infected cells (Fig 3A, schematic). These currents have been confidently linked to PSAC through electrophysiological studies of conditional knockdown of either RhopH2 or RhopH3, where net Cl^- transport is abolished [24].

In our studies, KC5 exhibited larger currents at negative membrane potentials (V_m) than at positive values (Fig 3A), consistent with PSAC’s voltage-dependent gating. Remarkably, although organic solute permeabilities are reduced in C3h-KO, we found that Cl^- conductance was not significantly decreased (Fig 3A and 3B; P = 0.99 for post-hoc Tukey’s multiple comparison test; n = 3–15 cells each). Also surprisingly, KO-PGIM produced even larger whole-cell currents (P < 0.0001). The channel’s voltage dependence was not affected by either clag3 knockout or by subsequent PGIM selection (Fig 3C); a modestly greater inward rectification for Ch3-KO and KO-PGIM, apparent in the I-V_m plot as smaller normalized currents at V_m of +100 mV, was not significant (P = 0.05, one-way ANOVA). Voltage-dependent inactivation, a property previously linked to a cleavable cytoplasmic domain on PSAC [39], was also preserved in these lines (Fig 3D). Cell-to-cell variation in inactivation kinetics and magnitude, which appear to reflect variable liberation of cytoplasmic channel components during whole-cell patch-clamp [39], prevented comparison of inactivation time courses. Thus, CLAG3 knockout and in vitro PGIM selections produce complex and unexpected changes in channel properties.

A markedly increased copy number for the clag2 paralog

To examine possible molecular mechanisms for adaptation to nutrient restriction and the observed transport changes, we performed whole-genome long-read sequencing of the KC5 parent, the C3h-KO transfectant, and the KO-PGIM mutant. After quality filtering and alignment to the reference sequence, average read lengths ranged between 10,900 and 11,800 base pairs with median coverages of 128-fold to 160-fold. These long-read lengths revealed a marked increase in clag2 copy number resulting from multiple duplications of a 6.8 kbp region that included the entire ORF and flanking untranslated sequences (Fig 4A and S2 Table).

Fig 4 — A) Integrative Genomics Viewer image of the locus containing *clag2* (PF3D7_0220800), showing sequencing coverage and alignment (cvrg and align) for KC5, *C3h-KO*, and *KO-PGIM*. Coverage is scaled to show the markedly increased coverage of a 6.8 kb region including *clag2* in *KO-PGIM*, suggesting multiple duplication events. Positions matching the reference genome sequence are gray and mismatches are colored depending upon the mutant allele. The amplified region begins 753 bp upstream of the *clag2* transcription start site and ends 15 bp before the mRNA terminus; its chromosomal positions are listed in S2 Table. B) Schematic shows inverted PCR strategy to identify tandem duplication events. Ethidium-stained gel showing that the wild-type KC5 does not produce an amplicon, but that *C3h-KO* produces a single band and PGIM-selected lines produce multiple bands, implicating duplication in *C3h-KO* and many copies in *KO-PGIM* and *KO-rev.* C) Mean ± S.E.M. DNA copy numbers of indicated genes in KC5, *C3h-KO*, *KO-PGIM* and *KO-rev* (left to right bars in each group), estimated by qPCR.

An additional 910 bp deletion in PF3D7_1418600, one member of the multigene 5S rRNA family, was detected in KO-PGIM relative to KC5 and C3h-KO. We also compared genomic sequences to detect mutations relative to the reference and sought differences between the parental and derivative strains. Only two were found in KO-PGIM relative to KC5 and C3h-KO (blue rows, S3 Table). The PF3D7_1418600 deletion and these mutations—a 5’UTR indel in Pf3D7_0608700 and a nonsynonymous mutation in Pf3D7_0821400 encoding a small uncharacterized protein with predicted nuclear localization—were deemed unlikely to contribute to the selected mutant phenotype and were not pursued further.

PCR using a reverse primer at the start of the clag2 gene and a forward primer at the gene’s 3’ end excluded detectable tandem duplication in KC5, but revealed a low-level amplification in the C3h-KO line (Fig 4B). This amplification, not reported in previous studies of the C3h-KO knockout [18], may reflect a compensatory change required for growth of successfully transfected cells carrying the engineered CLAG3 knockout. PCR revealed a more dramatic clag2 amplification in KO-PGIM, with several bands and more intense amplicons that parallel the multiple duplication events suggested by whole-genome sequencing. Real-time quantitative PCR (qPCR) was then used to estimate clag2 copy numbers at 2, 18, and 8 in C3h-KO, KO-PGIM, and KO-rev, respectively (Fig 4C; P < 10^-4, n = 4 independent trials).

CLAG2 upregulation accounts for altered channels and growth phenotype

Because clag genes are known to undergo complex epigenetic regulation and silencing, we next used real-time quantitative reverse transcriptase PCR (RT-qPCR) to explore whether genome-level clag2 amplification alters transcription of clag paralogs, rhoph2 and rhoph3. Consistent with the marked copy number increases observed in Fig 4, RT-qPCR using synchronized, trophozoite-infected parasites identified a marked upregulation of clag2 transcription in both KO-PGIM and KO-rev (Fig 5A; P = 2 x 10^-4, one-way ANOVA; n = 3–4 trials each parasite). Significant changes in transcript abundance were not detected for other clag paralogs, rhoph2, or rhoph3; clag3 transcripts remained at or below detection thresholds (Figs 5A and S2). Paralleling an apparent copy number reduction upon return to RPMI 1640-based media (Fig 4C), clag2 expression decreased upon extended propagation in nutrient-rich media (Fig 5A, dark red bars for KO-rev; P = 0.002 in post-hoc multiple comparison test). Although evolving gene copy number, possible promoter and ORF mutations, and cell-level epigenetic marks complicate attempts to correlate copy number and transcript abundance, comparisons of our qPCR and RT-qPCR results (Figs 4C and 5A, respectively) suggest that increased clag2 transcription results primarily from genome-level amplification without controlling effects of epigenetic marks.

Fig 5 — A) Mean ± S.E.M. expression of indicated *clag* and *rhoph* genes in KC5, *C3h-KO*, *KO-PGIM*, and *KO-rev* parasites (black, red, cyan, and dark red bars in each group, respectively; n = 3-4 independent RNA harvests each), normalized to 1.0 for matched measurements using the KC5 wild-type parent. The *msp2* control is included as an unrelated gene with similar stage-specific expression; similar *msp2* transcript levels establish matched harvest of cell cycle stages. B) CRISPR/Cas9 transfection strategy for C-terminal addition of a NanoLuc reporter tag on CLAG2. Indicated primers were used for PCR integration checks (S3A Fig and S1 Table). C) Nano-Glo blot showing the tagged CLAG2 protein in indicated lines. Loading control, anti-aldolase immunoblot at bottom. Notice the increased intensities in *C3h-KO*_C2NLuc and *KO-PGIM*_C2NLuc. Right panel, mean ± S.E.M. CLAG2 band densities, normalized to the loading control and presented in arbitrary units (AU) for each parasite. *n =* 3 independent harvests and blots. D) Osmotic lysis kinetics for *KO-PGIM*_C2NLuc in sorbitol. The lysis halftime t_1/2, 5.6 min, is comparable to untransfected *KO-PGIM*, suggesting that NanoLuc tagging does not alter channel activity. Representative of 2 independent trials.

To examine the effects of increased clag2 transcription, we used CRISPR transfection of KC5, C3h-KO and KO-PGIM to introduce a C-terminal NanoLuc tag on CLAG2 (Fig 5B). Integration PCRs using the resulting limiting dilution clones—KC5_C2NLuc, C3h-KO_C2NLuc and KO-PGIM_C2NLuc—confirmed faithful addition of the tag and replacement of all clag2 genomic copies in each line, without residual wild-type copies of the gene (S3A Fig). Indirect immunofluorescence microscopy revealed that the modified protein was transferred to new erythrocytes after invasion and exported to the host cell cytoplasm with RhopH3 (S3B Fig), as previously reported for each CLAG paralog [19,20].

We then estimated CLAG2 abundance on nitrocellulose blots with the Nano-Glo blotting system, which can quantify NanoLuc expression [40]. ImageJ quantification with loading control normalization revealed marked increases in the tagged protein with KO-PGIM_C2NLuc having greater increases than C3h-KO_C2NLuc (Fig 5C right panel; P = 0.01, n = 3 independent trials each). This C-terminal tag did not compromise the increased solute uptake of the KO-PGIM mutant (Fig 5D). Although the transfection strategy included a glmS element in the 3’ UTR of clag2, our attempts to examine conditional knockdown using this riboswitch were unsuccessful, possibly due the presence of multiple gene copies with separate promoters and glucosamine-associated toxicity on parasite cultures. Cas9-mediated cleavage during transfection, possible changes in channel-mediated nutrient uptake due to CLAG2 tagging, and variable genome remodeling in transfection subclones all may alter clag2 copy number.

We also found that the increased CLAG2 abundance in the untransfected KO-PGIM could be detected in conventional immunoblots due to cross-reactivity with the anti-CLAG3 antibody (S4A Fig), consistent with significant sequence homology over the epitope used for production of this antibody (S4B Fig). We excluded low-level contamination with parasites carrying the wild-type clag3 locus (Fig 2B); ectopic recombination to transfer the distal clag3 sequence that encodes the antibody epitope to another clag paralog [10], as established for other gene families in P. falciparum [41], was also excluded with PCR experiments (S4 C and S4D Fig).

Discussion

Epigenetic switching of variant surface antigens is considered P. falciparum’s primary method of responding to changes in the host environment [42,43]. Switching and expression of single PfEMP1 paralogs, as encoded by members of the var gene family, allows infected cells to escape newly-mounted immune responses [44]. PfEMP1 isoforms bind to distinct endothelial receptors [45], so switching also bestows infected cell tropism for different host tissues and organs. This is particularly advantageous in pregnancy where placental sequestration leads to marked parasite expansion [46]. Epigenetic control of RIFINs, encoded by the rif gene family, is not under the same tight regulation as var gene transcription [47], but upregulation of specific rif genes facilitates evasion through LILRB1 or LAIR1 receptor binding to suppress host immune responses [9]. Frequent recombination between member genes for these variant antigens (50–60 var genes and 150–200 rif genes in P. falciparum clones) also generates distinct paralogs with new properties [48]. Despite the remarkable plasticity of these genes and their transcription profiles, studies have not revealed drastic changes in copy number for these and other variant surface antigens in Plasmodium spp.

We now report copy number variation (CNV) as a distinct parasite mechanism for responding to changes in host plasma. Although CNV generally cannot match the speed and reversibility characteristic of epigenetic regulation, it can provide adaptive advantage under sustained selective pressure. In our study, nutrient restriction appears to have promoted repeated duplication events including the full-length clag2 gene and essential flanking sequences. The gene’s subtelomeric location may have facilitated amplification as such sites are hotspots for chromosome reshuffling [49,50]. Copy number changes can arise in clinical P. falciparum isolates [51], but have been largely limited to intracellular parasite proteins with minimally variant sequences. PfMDR1, a transporter on the parasite’s digestive vacuole, exhibits CNV to acquire resistance to mefloquine, quinine and halofantrine [52,53]; resistant clinical isolates have at most 2–4 copies of the pfmdr1 gene, but they revert to a single copy upon drug removal to reduce the associated fitness cost [54]. GTP cyclohydrolase underwent a similar duplication in some clinical isolates to confer sulfadoxine resistance [55]. Genome-level amplifications are generally considered deleterious, especially when open reading frames are involved [56]. Thus, clag2 amplification to 18 copies in KO-PGIM represents an unprecedented increase. It appears to have been necessary to restore uptake of isoleucine, an essential amino acid, establishing that nutrient restriction provided the selective pressure to drive amplification. KO-rev, as obtained after return of the mutant to nutrient-rich medium, exhibits a lesser copy number increase, consistent with a balance between the benefit of restored nutrient uptake and the fitness cost from high-level amplification.

There are many potential explanations for why an 18-fold increase in clag2 copy number was required for KO-PGIM expansion in PGIM. These include lower transcription and translation rates for clag2 than clag3 (S2 Fig), differing affinities of CLAG paralogs for interaction and transport in the RhopH complex, differing efficiencies of channel subunit delivery and insertion at the host membrane (a highly regulated process for other ion channels [57]), variable subunit stoichiometries in functional channels, and differences in relative permeabilities of key nutrients in the channels formed by combinations of expressed paralogs. These possibilities may be explored when the PSAC structure is solved.

As with mutations and indels, CNV appears to rise regularly in parasite populations only to disappear quickly in the absence of associated survival advantage. CLAG3 knockout to produce C3h-KO provided adequate survival advantage to yield a detectable subpopulation with 2 or more copies of clag2 despite cultivation in nutrient-rich RPMI 1640-based medium (Fig 4B); greater selective pressure upon transfer to PGIM presumably enabled growth of individual parasites carrying larger scale clag2 amplifications. Thus, our findings help establish the pathogen’s remarkable capacity for CNV under appropriate and sustained selective pressure.

When compared to other multigene copies, clag genes exhibit an unparalleled range of species-specific family sizes in Plasmodium spp. with some species such as P. berghei having only two copies and others exhibiting marked expansion, with the gorilla parasite P. blacklocki having over 35 copies [58,59]. Even within a single species, P. falciparum isolates are also known to have clag copy numbers that range between 3 and 7 even without engineered clag3 knockout [60]. The reasons for this variable expansion are unclear, but our findings suggest nutrient restriction in specific hosts may be a key driving force. Copy number increases appear to be followed by ongoing evolution of members through mutations [60]. Interestingly, despite variable expansion, each examined Plasmodium species has a single clag closely related to the P. falciparum clag9 gene while all other members group with P. falciparum clag3. While a distinct role for clag9 has been proposed and debated [21,23], the encoded CLAG9 protein shares key phenotypes with the CLAG3 group: translation in schizonts, trafficking as a ternary complex with RhopH2 and RhopH3 via merozoite rhoptries into new erythrocytes upon invasion, and similar membrane topologies at the host cell surface [59].

In combination with copy number, epigenetic regulation of clag member genes serves to regulate total production of CLAG protein but the reasons for this complex regulation are unknown [61]. One possibility, switching between members to allow fine-tuning of nutrient uptake in response to changing host nutritional status [17], is appealing and consistent with some of our findings, but is unfortunately not associated with differences in nutrient permeability, as might be predicted [16]. Another possibility, switching between clag3 genes and potential up- and down-regulation of other paralogs to permit immune evasion, is also unsatisfying, given that other multigene families require a larger number of paralogs to achieve satisfactory escape from mounted immune responses [62]. In addition to changes in PSAC-associated subunits, nutrient stress may also produce adaptive changes in the intracellular pathogen including altered expression of sugar and amino acid transporters; one example is PfAAT1, an amino acid transporter and drug susceptibility modulator localized at the parasite digestive vacuole [63].

We propose that this complex multi-layered regulation may be driven by a need to achieve adequate nutrient uptake while maintaining a sufficiently low Na⁺ permeability to avoid premature osmotic lysis of infected cells. While PSAC has an unparalleled ability to exclude this cation [64], there is a nonzero Na⁺ leak through the channel [65]. Upregulated nutrient permeability, as required when the host may be malnourished, must be achieved without increasing Na⁺ uptake to levels that would lead to infected cell lysis before lifecycle completion. Fine-tuning of each paralog’s expression may enable balanced permeabilities for multiple solutes in response to host nutritional status [15,20]. Consistent with this model, in vitro selections of mutant channel phenotypes, in the present study and in production of a mutant with reduced blasticidin S uptake [11,12,66], invariably leads to reversion to wild-type phenotypes when selection is removed.

Methods

Parasite culture and in vitro selection

Asexual P. falciparum parasites including KC5 and its derivative C3h-KO, KO-PGIM, and KO-rev lines were cultivated at 5% hematocrit in O⁺ human erythrocytes (Interstate Blood Bank, Inc.). All cultures were maintained with regular media changes under 5% O₂, 5% CO₂, 90% N₂ at 37 °C. Standard, nutrient-rich propagation used RPMI 1640 medium supplemented with 25 mM HEPES, 31 mM NaHCO₃, 0.37 mM hypoxanthine, 10 µg/mL gentamicin, and 0.5% w/v NZ Microbio BSA (MP Biomedicals).

In vitro selection for C3h-KO parasites capable of expansion in physiological, nutrient-restricted medium was performed using PGIM medium with modifications [16]. PGIM was prepared using glutamine and isoleucine deficient RPMI 1640 (US Biological) supplemented with 25 mM HEPES, 31 mM NaHCO₃, 3.01 µM hypoxanthine, 11.4 µM L-isoleucine, 10 µg/mL gentamicin, and 0.5% w/v NZ Microbio BSA. Erythrocytes were washed in PGIM before addition to cultures to minimize carryover of supraphysiological nutrient levels. Propagation was evaluated with microscopic examination of Giemsa-stained smears. Expansion rate analysis used sorbitol-synchronized cultures seeded at 0.5% initial ring-stage parasitemias. Some comparisons of parasite growth rates used blinded examination of smears to exclude observer bias. Two independent trials of in vitro PGIM selection produced similar results.

DNA sequencing and PCR were performed with selected lines to exclude contamination by other parasite strains and to evaluate possible genomic recombination. DNA was isolated from parasite cultures using Quick-DNA Miniprep kit (Zymo Research) according to the manufacturer’s protocol. Primers used are listed in S1 Table.

Solute uptake measurements

PSAC-mediated uptake of solutes was measured using transmittance-based tracking of infected cell osmotic lysis. Synchronous cultures containing trophozoite- and schizont-stage infected cells were harvested and enriched to > 95% parasitemia using percoll-sorbitol density gradient separation. Prior studies have found that trophozoite- and schizont-infected cells have maximally induced PSAC activity on their host cells. Enriched cells were washed in saline (150 mM NaCl, 20 mM HEPES, pH 7.4) before resuspension at 0.125% hematocrit in lysis solution (280 mM sorbitol or 280 mM isoleucine with 20 mM HEPES, 0.1 mg/mL BSA, pH 7.4) at 37 °C. Solute uptake and the resulting osmotic lysis were tracked at 15–20 s intervals by recording transmittance of 700 nm light through the cell suspension (DU800 spectrophotometer with Peltier temperature control, Beckman Coulter). Where used, ISG-21, a potent PSAC inhibitor [16], was freshly prepared in lysis solution before each experiment. Solute permeabilities and inhibitor efficacies were determined using locally developed code (SigmaPlot, Systat), based on a two-compartment model of infected cell osmotic lysis [29].

Protein blots

CLAG protein expression was evaluated with immunoblotting of cell membranes. Sorbitol-synchronized cultures were evaluated for maturity by examination of serial Giemsa-stained smears and harvested at matched levels of trophozoite- and schizont-infected cells. Infected cells were enriched by percoll-sorbitol separation and lysed in hypotonic buffer (7.5 mM Na₂HPO₄, 1 mM EDTA, 1 mM PMSF at pH 7.5) at 4 °C. Membrane-associated proteins were enriched by centrifugation (14,000 x g, 30 min at 4 °C) and solubilized in modified Laemmli buffer with a final 6% SDS concentration. Proteins were separated by electrophoresis in 4–15% Mini-PROTEAN TGX gels (Bio-RAD) and transferred to nitrocellulose membrane. This membrane was blocked for 1 h in 3% milk solution in TBST (150 mM NaCl, 20 mM TrisHCl, pH 7.4 with 0.1% Tween20) at room temperature. Primary anti-CLAG3 and secondary HRP-conjugated anti-mouse antibodies were applied in blocking buffer as described previously [24]. Bound antibodies were visualized on Hyblot X-ray film after addition of Clarity Western ECL chemiluminescent substrate (Bio-Rad).

Nano-Glo blots (HiBiT blotting system, Promega) were used to quantify CLAG2 abundance in engineered lines expressing NanoLuc-tagged protein using antibody-free blotting to measure luminescence on membranes [40]. After SDS-PAGE and transfer to nitrocellulose membranes, blots were washed in 150 mM NaCl, 20 mM Tris-HCl (pH 7.4) with 0.1% Tween 20. Furimazine was then added in blotting buffer at a 1:500 dilution before imaging and band intensity quantification using ImageJ software (NIH) and normalization with an aldolase loading control (HRP anti-Plasmodium aldolase antibody, Abcam).

Protease susceptibility

Enriched late-stage infected cells were washed and resuspended in PBS-2 (140 mM NaCl, 2.7 mM KCl, 9.6 mM Na₂HPO₄, 1.5 mM KH₂PO₄, 0.6 mM CaCl₂, 1 mM MgCl₂, pH 7.4) with 0.5 mg/mL pronase E (Sigma Aldrich) at 2.5% hematocrit. Cells were incubated with protease for 1 h at 37 °C. The cells were then washed with ice-cold PBS-2 supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1 mM EDTA before resuspension in saline for solute uptake measurements or hypotonic buffer for harvest of membranes and immunoblotting as described above.

Whole-genome sequencing and analysis

The parental KC5, C3h-KO transfectant, and the selected KO-PGIM P. falciparum clones were sequenced using the PacBio SMRT Sequel II Cell with standard PacBio protocol. The samples yielded 259,683–390,238 ccs reads each, with mean read lengths of 10.9-11.8 kb and mean read qualities of QV29.7 to QV30.3. CCS reads were mapped to the PF3D7 reference genome (GCA_000002765) using pbmm2 v1.16.0 (https://github.com/PacificBiosciences/pbmm2), and resulting BAM files underwent INDEL realignment using GATK v3.8-1 [67]. Variant calling was then performed using the HaplotypeCaller from GATK v4.1.9.0 followed by joint genotyping. Resulting raw variants were then filtered with SNPs and INDELs less than 2 base pairs in length filtered at QD < 2.0 and INDELs ≥ 2 base pairs filtered at QD < 5.0, as described [68]. Variants with < 0.75 frequency in each sample were removed to minimize mapping artifacts and exclude low-quality regions or variant positions with high clonal diversity. Highly clustered variants that presumably represent mismapped, problematic regions were visually confirmed as artifacts before exclusion from downstream analysis. Structural variant calling was performed using pbsv v2.9.0 (https://github.com/PacificBiosciences/pbsv); structural variants were annotated using the PF3D7 annotation (GCA_000002765) and VEP release 111 [69]. The raw sequencing data generated for this study have been deposited in the NCBI Sequence Read Archive (SRA) under BioProject accession PRJNA1327946.

Quantitative PCR for gene copy number

Genomic DNA was isolated from cultured parasites and used for clag gene copy number determination after assessing quality and concentration by NanoDrop (Thermo Fisher Scientific). Gene-specific primers (S1 Table) were used in dye-based qPCR (Luna Universal qPCR Master Mix, New England Biolabs) with amplification tracked in a real-time thermal cycler (Biorad CFX96). Ct values from three replicates were averaged and normalized to zero for alpha tubulin 1 (PF3D7_0903700). Copy number was estimated using the 2^-ΔΔC_T method from four independent DNA harvests.

Quantitative reverse transcriptase PCR

Gene expression was measured with real-time PCR using total RNA harvested from tightly synchronized cultures. Cultures were synchronized with two sorbitol treatments separated by 6 h. Serial smears were then used to obtain matched, schizont-stage infected cells, which were then further synchronized with percoll-sorbitol enrichment before freezing in TRIzol reagent. Total RNA was isolated using Direct-zol RNA MiniPrep kit (Zymo Research) according to the manufacturer’s protocol. Isolated total RNA (800 ng) was annealed with 250 ng random hexamer primer, heated to 65 °C for five minutes and slowly cooled. First strand cDNA was synthesized with SuperScript IV Reverse Transcriptase (ThermoFisher) according to the manufacture’s protocol. The resulting cDNA was diluted 10-fold and used for qRT-PCR (QuantiTect SYBR Green, Qiagen), as described previously [18]. Primers were designed to yield specific ~120 bp amplicons for indicated genes and are listed in S1 Table. qRT-PCR was carried out using the iCycler IQ multicolor real-time PCR system (Bio-Rad) programmed for denaturation at 95˚C for 15 min followed by 40 cycles of annealing at 52 °C and extension at 62˚C for 30 s each.

Gene expression was quantified using the 2^-ΔΔC_T method [70], with α-tubulin 1 (PF3D7_0903700) as a constitutively expressed loading control; msp2 was included as a stage-specific control as its transcription matches the preferential expression of clag and rhoph genes at the mature schizont stage. Normalized expression is also reported by comparison to matched trials using RNA from wild-type KC5. All reactions were performed in triplicate and are presented as the mean ± S.E.M. of results from 3-4 independent RNA harvests for each transfectant or selected mutant.

DNA transfection

CRISPR/Cas9 transfection was used to produce engineered lines that express CLAG2 with a C-terminal NanoLuc tag. A previously generated pL7 plasmid for epitope tagging CLAG2 with a high-scoring sgRNA, 5’- ACATACTAGTAATATCCAAA-3’, was modified using InFusion cloning (Clontech) to introduce a full-length NanoLuc tag after an unmodified CLAG2 protein with a two-residue linker (Bgl11 restriction site). A bsd selection marker cassette was also cloned into the plasmid and verified by sequencing. Cas9 was expressed from a separate pUF1-Cas9 plasmid. After transfection of KC5, C3h-KO, and KO-PGIM, cultures were selected with 2.5 µg/mL blasticidin S and 1.5 µM DSM-1 (5-methyl[1,2, 4]triazolo[1,5-a]pyrimidin-7-yl)naphthalen-2-ylamine). KO-PGIM was cultivated in PGIM throughout the transfection process. After parasite outgrowth and PCR confirmation of integration, limiting dilution cloning was performed for all transfectant lines without further exposure to blasticidin S. All experiments were performed with sequence-verified clones.

Indirect Immunofluorescence microscopy

Indirect immunofluorescence assays (IFA) were performed using freshly prepared thin blood smears after fixation with 1:1 acetone: methanol at -20 °C. After air drying, slides were blocked with 3% BSA in PBS for 1 h at RT and incubated with primary antibody in blocking buffer (rabbit anti-RhopH3 at 1:1000; mouse anti-NanoLuc antibody at 1:100, Promega) for 3 h at RT under a coverslip. After two washes, secondary antibodies (Alexa Fluor 488-conjugated goat anti-rabbit at 1:500, Invitrogen; Alexa Fluor 594-conjugated goat anti-mouse at 1:500, Invitrogen) with 1 μg/mL 4’,6-diamidino-2-phenylindole (DAPI; Genetex) was added in blocking buffer and incubated for 1 h at RT. After washes, slides were dried and mounted using ProLong Diamond Antifade Mountant (Molecular Probes). Images were collected on a Leica SP8 microscope using a 63x oil immersion objective. DAPI, Alexa Fluor 488, and Alexa Fluor 594 fluorescence were captured using excitation/emission wavelengths of 377/447 nm, 499/520 nm, and 590/618 nm, respectively. Images were processed using Leica LAS X and Huygens Essential software.

Statistical analysis

Numerical data are shown as mean ± S.E.M of independent trials, as reported. Statistical significance was calculated using unpaired Student’s t-test or one-way ANOVA with post-hoc testing as appropriate. Significance was accepted at P < 0.05.

Supporting information

S1 Fig. Rare C3h-KO parasites seen during PGIM cultivation resume expansion upon transfer to nutrient-rich medium.

A) C3h-KO expansion after cultivation in PGIM for 0, 2, 4, 6, or 8 days prior to return to RPMI 1640-based medium (left to right curves; symbols represent mean of 3 trials). Increasing the duration of PGIM exposure does not extend the lag before resumed growth. B) Time to 2% parasitemia after return to RPMI 1640-based medium for C3h-KO cultures seeded in PGIM for indicated number of days. Symbols represent mean ± S.E.M. estimated by linear interpolation of daily parasitemia estimates from microscopic observation; n = 3 trials each. Recovery time does not increase with extended PGIM exposure, indicating that these parasites remain viable.

(TIF)

ppat.1013321.s001.TIF^{(121.1KB, TIF)}

S2 Fig. Transcript abundances for clag and rhoph genes.

Mean ± S.E.M. transcription of indicated clag and rhoph genes in KC5, C3h-KO, KO-PGIM, and KO-rev parasites (black, red, cyan, and dark red bars in each group, respectively), calculated as 2^(-ΔCt) using ΔCt relative to the α-tubulin loading control. msp2, unrelated gene with similar stage-specific expression. Values reflect the RT-qPCR experiments shown in Fig 5A without normalization to the wild-type control.

(TIF)

ppat.1013321.s002.TIF^{(123.3KB, TIF)}

S3 Fig. Engineered parasites carrying a NanoLuc reporter at CLAG2 C-terminus.

A) Ethidium-stained gels using indicated primers and DNA from transfectant clones and the wild-type KC5 parent. Primer pair p8-p9 is specific for the wild-type clag2 locus while other pairs yield amplicons upon integration. Loss of the p8-p9 amplicon in KC5_C2NLuc, C3h-KO_C2NLuc, and KO-PGIM_C2NLuc indicates complete replacement of all genomic clag2 copies with the integration cassette, which adds a C-terminal NanoLuc reporter. Expected sizes (in bp): p8-p9, 605; p8-p10, 619; p8-p11, 1027; p8-p12, 1465; p8-p13, 1975 in transfectant lines and 1066 in wild-type KC5. Primer sequences are in S1 Table. B) Indirect immunofluorescence confocal microscopy images of a trophozoite-stage KO-PGIM_C2NLuc parasite probed with anti-RhopH3 and anti-NanoLuc antibodies (green and red, respectively). Scale bar, 5 µm. The NanoLuc-tagged CLAG2 protein is delivered to infected cells after invasion and is exported into host cytosol [24].

(TIF)

ppat.1013321.s003.tif^{(2MB, tif)}

S4 Fig. Increased CLAG2 production in KO-PGIM is detected by a weakly cross-reactive anti-CLAG3 antibody.

A) Anti-CLAG3 immunoblot using total membranes from indicated parasites. Samples used percoll-sorbitol enriched infected cells and were match-loaded. A low-intensity band is detected in KO-PGIM lysates but absent in the unselected C3h-KO parent. B) Sequence alignment of KC5 CLAG2 sequence with the recombinant epitope used to produce anti-CLAG3 antibody. Identical residues are highlighted in red; the ruler is numbered according to CLAG2. As the band detected in KO-PGIM reflects cross-reaction with the CLAG3 C-terminal epitope, we did not attempt to quantify band intensities. C) Ethidium-stained gels showing unaltered detection of clag genes in KO-PGIM when compared to the wild-type KC5 line. PCR of clag8 was performed in two fragments to facilitate specific detection. Ribbon schematic at top shows the sites recognized by each primer; primer sequences are in S1 Table. D) Gels showing absence of ectopic recombination between clag3 and other paralogs in either KC5 or KO-PGIM.

(TIF)

ppat.1013321.s004.TIF^{(2.2MB, TIF)}

S1 Table. Primers used in this study.

(XLSX)

ppat.1013321.s005.xlsx^{(14.4KB, xlsx)}

S2 Table. Structural variants in KO-PGIM.

Structural variants from whole-genome sequencing filtered for variants that differ between C3h-KO and KO-PGIM. The two filtered variants are tabulated with the impacted genes, chromosomal location, structural length, mutant allele, genotypes, and flanking sequencing depth.

(XLSX)

ppat.1013321.s006.xlsx^{(10KB, xlsx)}

S3 Table. Mutations and indels in C3h-KO and KO-PGIM.

Variants identified from whole-genome sequencing of parental and mutant strains and mapped to the 3D7 reference strain. Following initial quality filtering outlined in the Methods, variants were filtered that did not differ between the 3 strains. The KC5, C3h-KO, and KO-PGIM genotypes are reported as 0 when identical to the reference and 1 when a mutant allele was called. The VAR column reflects the number of parasites that differed from 3D7 at the indicated position. Columns with.DP and.GT suffixes indicate read depth and the primary allele for each parasite, respectively. Blue highlighted rows indicate high-confidence mutations in KO-PGIM relative to C3h-KO; green highlights reflect those in C3h-KO relative to wild-type KC5.

(XLSX)

ppat.1013321.s007.xlsx^{(23.4KB, xlsx)}

Acknowledgments

We thank BEI Resources for DSM-1 and Anna Crater for intellectual and technical input during early stages of these studies.

Data Availability

All relevant data are within the manuscript and its Supporting Information files. The raw sequencing data generated for this study have been deposited in the NCBI Sequence Read Archive (SRA) under BioProject accession PRJNA1327946.

Funding Statement

This work was funded by the Division of Intramural Research, NIAID/NIH. N.P. was a recipient of an Intramural NIAID Research Opportunities (INRO) Training Award. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS Pathog. doi: 10.1371/journal.ppat.1013321.r001

Decision Letter 0

Tania de Koning-Ward

7 Aug 2025

PPATHOGENS-D-25-01488

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

PLOS Pathogens

Dear Dr. Desai,

Thank you for submitting your manuscript to PLOS Pathogens. After careful consideration, we feel that it has merit but does not fully meet PLOS Pathogens's publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript within 30 days Oct 06 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plospathogens@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/ppathogens/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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If you would like to make changes to your financial disclosure, competing interests statement, or data availability statement, please make these updates within the submission form at the time of resubmission. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

We look forward to receiving your revised manuscript.

Kind regards,

Tania F. de Koning-Ward

Academic Editor

PLOS Pathogens

Dominique Soldati-Favre

Section Editor

PLOS Pathogens

Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

Additional Editor Comments:

Your manuscript has been reviewed by three assessors and the consensus was that the work was well executed and provided compelling evidence that amplification of the clag2 gene in P. falciparum parasites lacking CLAG3 permit growth of the parasite under nutrient-limiting conditions via the restoration of solute uptake. Whilst no new experiments are requested by the reviewers, some conclusions require a more cautious interpretation. In addition, some further clarifications and discussion points are also required. These are outlined in the reviewers comments below. Finally, the sequencing data should be deposited in a public repository.

Journal Requirements:

1) Please ensure that the CRediT author contributions listed for every co-author are completed accurately and in full.

At this stage, the following Authors/Authors require contributions: Sanjay A. Desai. Please ensure that the full contributions of each author are acknowledged in the "Add/Edit/Remove Authors" section of our submission form.

The list of CRediT author contributions may be found here: https://journals.plos.org/plospathogens/s/authorship#loc-author-contributions

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https://journals.plos.org/plospathogens/s/figures

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- State the initials, alongside each funding source, of each author to receive each grant. For example: "This work was supported by the National Institutes of Health (####### to AM; ###### to CJ) and the National Science Foundation (###### to AM)."

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If you did not receive any funding for this study, please simply state: u201cThe authors received no specific funding for this work.u201d

Reviewers' Comments:

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: This manuscript presents compelling and well-executed work that challenges the prevailing view of PSAC composition by demonstrating that P. falciparum parasites lacking CLAG3 can restore solute uptake and growth under nutrient-limited conditions via amplification of the clag2 gene. The authors provide strong genetic and phenotypic evidence suggesting that CLAG2 can compensate for the loss of CLAG3, leading to a revised model of channel formation and nutrient acquisition in the parasite. This finding highlights the genomic plasticity of P. falciparum and extends our understanding of how the parasite adapts to environmental stress.

However, some conclusions—specifically those regarding isoleucine uptake via PSAC—require a more cautious interpretation. Isoleucine is a bulky, hydrophobic amino acid not generally considered a primary PSAC substrate. The observed reduction in isoleucine uptake in CLAG3 knockout lines may reflect indirect effects on parasite physiology or other transport systems (e.g., amino acid transporters such as PfAAT1 (PF3D7_0629500)) rather than direct passage through PSAC. Acknowledging this limitation and refining some interpretations would strengthen the manuscript. Additionally, clarification of the parasite stage at which permeability and expression assays were performed is important, particularly in the context of possible altered IDC duration under nutrient-limited conditions.

Reviewer #2: The manuscript by Potchen et al. is a continuation of previous work from the group, in which they showed that CLAG3 proteins are an essential component of the parasite-encoded PSAC channel, involved in nutrient transport, and later demonstrated that CLAG3 is not essential for parasite growth in medium with supraphysiological nutrient concentrations (i.e., RPMI) but is essential for growth in medium with limited concentration of specific nutrients (PGIM). They also previously suggested (but not conclusively demonstrated) that other CLAG paralogs are also involved in PSAC formation.

Here the authors show that adaptation of a CLAG3-defficient P. falciparum line to grow in PGIM was associated with a compensatory amplification of the clag2 locus. This implies that CLAG2 can compensate for absence of CLAG3, providing evidence for the involvement of CLAG2 in PSAC formation. The results presented also demonstrate that CLAG2 and CLAG3 form channels with different characteristics (e.g., in terms of solute specificity).

The main conclusions are convincing and well demonstrated by the data. The manuscript is technically sound and clearly written, and the figures are also clearly presented. I only have minor suggestions to improve the manuscript, which do not require new experiments.

Reviewer #3: PSAC is a parasite-induced nutrient channel in the RBC membrane important for parasite growth under physiological conditions. CLAG3 is key component of PSAC and this study shows that loss of CLAG3 expression arrests parasite growth in physiological media.

Loss of CLAG3 reduced Ile transport 3-4 fold but did not abolish it, indicating that transport is not fully dependent on CLAG3.

Long-term maintenance of clag3-deficient parasites in physiological media can select for revertant mutants that recover the ability to grow in physiological media and the transport rate of Sorbitol/Ile is restored to parental levels.

Changes in the relative transport of sorbitol vs Ile make it clear that the channel composition must be changing.

This reversion phenotype is associated with an 18-fold increase in the copy numper of clag2.

Subsequent growth of the revertants in RPMI leads to a reduction from 18 to 9 clag2 copies.

Change in copy number correlates well with expression change at mRNA level.

Tagging of the clag2 copies with NLuc showed ~3x increased protein levels in revertant relative to the C3h-KO parent but also shows ~4x increase in C3h-KO relative to KC5 parents with the intact C3h gene.

This is a well designed and executed study that sheds new light on how parasites can acquire nutrients and the relative functions of clag2 and clag3.

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: No new experiments are strictly required for acceptance.

However, the following points should be addressed in the text to clarify interpretations and add context to the results:

1. Refine the interpretation of isoleucine as a PSAC substrate:

The manuscript currently suggests that isoleucine is acquired via PSAC (e.g., Page 4, lines 14–16). This statement should be revised or qualified, as current knowledge does not support isoleucine as a primary PSAC substrate due to its size and hydrophobicity. The authors should consider alternative explanations for the observed decrease in isoleucine uptake in CLAG3-deficient lines.

2. Clarify parasite stage and IDC timing under PGIM conditions:

According to Subudhi et al transcriptomic data (DOI: 10.1038/s41467-020-16593-y) deposited in PlasmoDB, clag3.1 and clag3.2 are expressed primarily between 34–48 hours post-invasion. Thus, since CLAG3 expression is stage-specific, it is essential to state whether parasite stages were matched across conditions. However, the experimental timepoints (in hours post-invasion) used for permeability assays and RNA/protein analyses are not clearly stated. Also, the PGIM medium could affect the parasite’s IDC, potentially prolonging the cycle, this could influence gene expression and protein levels. The authors should specify the time post-invasion at which experimental assays were performed, and whether parasite staging was carefully matched between conditions to ensure valid comparisons.

Reviewer #2: -

Reviewer #3: 1. While the correlation between increased clag2 and growth is strong, the authors would ideally directly effect the role of clag2 expression level on growth/transport modulating expression via the glmS element introduced during the Nluc tagging (as long as glucosamine transport does not depend on PSAC).

The standard approach using Blasticidin-selectable plasmids may be complicated if there is a major change in blasticidin transport, which would need to be tested.

2. The mutational signature of the copy numbers suggest that amplification might have occurred in two steps, where the parental copy was amplified 8-10 fold, followed the acquisition of additional mutations one one of these copies, which was them amplified again. This is most apparent in the 3'UTR. This could be a sign that the additional mutations in clag2 are adaptive to growth in PGIM and leading to differential transport activities of the two sets of clag2 copies. Do any of these mutations result in coding changes? If not, this can be disregarded.

3. Figure 5C:re-check genomic copy number after tagging, as C3h-KO had 1 copy earlier and similar mRNA but the C3h-KO-Nluc has higher protein expression. CAS9 cleavage & repair at the locus could have led to changes in copy number.

4. Is CLAG2-dependent transport in C3h-KO-PGIM susceptible to inhibition by ISG-21 or residual transport inhibitors PRT1-4?

5. Can the fact that CLAG3-loss has a major effect on growth in PGIM but lesser effects on transport and lysis be explained be differences in the intracellular concentrations of Ile required for growth vs lysis?

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: 1.Wording revisions:

Revise sentence on Page 4, lines 14–16 to something more cautious, e.g.:

"We examined sorbitol and isoleucine permeabilities in C3h-KO and KC5; while sorbitol is a well-established PSAC substrate, isoleucine uptake may involve additional or alternative transport pathways."

2.Suggestion for future experiments (optional, in discussion) to better understand the link between isoleucine uptake and direct PSAC-mediated transport, the authors could consider:

- Performing competition assays using sorbitol or other known PSAC substrates to determine if isoleucine uptake is competitively inhibited.

- Conducting transcriptomic analysis comparing WT and CLAG3-KO lines, with a focus on amino acid transporter expression.

- Generating and testing a PfAAT1 KO or knockdown strain in the clag3-KO background to assess compensatory or synergistic effects on isoleucine uptake.

3. Address clag2 pre-existence (Figure 4B):

The observed clag2 gene amplification under amino acid-limited conditions is a striking example of adaptive genome remodeling. As the authors note, this supports a broader model where nutrient stress, like drug pressure, can drive copy number variations to enhance parasite survival. This aligns with known mechanisms for resistance involving pfmdr1 or pfpm2/3. However, in Figure 4B, clag2 amplification may appear detectable in the C3h-KO parental line, (although not confirmed by WGS) suggesting that some degree of clag2 amplification may have pre-existed in the population, raising the possibility of selection on a pre-existing subpopulation rather than de novo amplification. The authors should discuss whether low-level pre-existing amplification may have been selected under PGIM pressure, or whether this reflects background noise. This clarification will help support the model of clag2-driven adaptation.

4. Figure S2A clarification:

In Figure S2A, the agarose gel analysis supports successful genome editing. However, assuming the primer pairs match those illustrated in Figure 5B, the p8 + p13 combination should, in principle, yield a smaller band in the KC5 line (wild-type for that locus). If this band is absent, please clarify whether this was expected or provide an explanation for the observed result.

5. Minor formatting consistency:

Ensure that acronyms (PGIM) are defined at first use.

Reviewer #2: -It is surprising that the copy number of clag2 increased up to 18-fold. Why are 18 copies of clag2 needed to compensate deletion of one copy of clag3? This massive increase may suggest that the CLAG2-based channel is less efficient than the CLAG3-based channel for the acquisition of the nutrients that are limiting in PGIM. An alternative possibility is that clag2 has a weaker promoter than clag3, which would imply that more copies of clag2 are needed to achieve the same transcript or protein levels. The latter possibility is supported by RNA-seq datasets available in PlasmoDB and several published RT-qPCR analyses. I recommend that the authors discuss these possible reasons for the unexpectedly high level of genetic amplification of clag2.

-In fig. 5A, data is presented in a way that does not enable comparison of clag3 transcript levels in the parental line with clag2 transcript levels in the KO, KO-PGIM and KO-rev lines (only values for each transcript relative to the parental line are shown). Please provide a figure (a new panel in Fig. 5 or a new supplementary figure) that enables this comparison, showing the transcript levels (relative to tubuline) for each clag gene in each parasite line. It would be interesting to see if total clag transcript levels increase in the KO-PGIM line or, in spite of massive clag2 amplification, total clag transcripts remain more or less constant, because of the weaker promoter of clag2.

-For a paper focused on increased clag2 expression as an adaptive mechanism, it would be relevant to discuss that clag2 was previously described as a clonally variant gene regulated by heterochromatin (at the epigenetic level), similar to clag3.1 and clag3.2 but unlike clag8 and clag9. This was demonstrated by transcriptional analysis of subclones that do or do not express the gene, and by genome-wide analysis of heterochromatin (a trait associated with clonally variant gene expression) showing that this epigenetic mark is present in clag2, clag3.1 and clag3.2 but not in clag8 or clag9. Clonally variant expression implies that the expression of this gene is dynamic and switching off the gene can provide a selective advantage under specific conditions, which fits with the observation that copy number decreased in the KO-rev line.

-Some statements in the manuscript may suggest that copy number variation is a usual clag genes regulation mechanism that the parasite can use in natural infections to adapt to changes in the host environment (e.g., a malnourished host). This is missleading. Copy number variation was observed in an artificial parasite line with no functional clag3 gene, a situation that does not occur in the field (no natural isolates without functional clag3 genes have so far been reported, to the best of my knowledge). Furthermore, large copy number variation has not been described in field isolates. It is also important to note that copy number variation cannot provide the rapid and dynamic plasticity needed for adaptation to the changing conditions of the human host. Therefore, in natural infections, regulation of clag3 genes most likely relies on epigenetic mechanisms, which are much faster and dynamic than genetic changes. Studying the adaptive alterations in the CLAG3-defficient line to grow in PGIM was instrumental to establish the role of CLAG2 in the formation of PSAC, but statements that misleadingly suggest that clags copy number variation may be a common mechanism of parasite adaptation should be modified (mainly end of Abstract and Author Summary, and second paragraph of the Discussion: p.13 line 10 and elsewhere).

-The results described in page 6 (Fig. 1E-G) indicate that the moderate decrease in permeability for the solutes tested in the KO line cannot account for the large growth defect of this parasite line in PGIM. The results presented afterwards do not provide an explanation for this intriguing observation, as increased CLAG2 levels restore permeability and largely revert the severe growth defect. Do the authors have an explanation, even if it is speculative, for why a small reduction in permeability in the KO line results in a severe growth defect? (larger than the effect on growth of a drug with a comparable impact on permeability). This should be discussed (in the Discussion).

-End of page 9 and elsewhere. Why do the authors call this observation “unexpected”? If the gene encoding CLAG3 has been disrupted, it is expected that a channel with the properties of a CLAG3-based PSAC cannot be present in the cells.

-Please indicated the position of the predicted TSSs for clag2 relative to the genetic amplification in Fig. 4A, using a zoomed-in view of the locus. The TSSs for most genes are described in several genome-wide datasets and available in PlasmoDB. This would give an idea of which is the functional unit amplified, presumably containing the full clag2 promoter.

-End of page 14. The data presented strongly suggest that different CLAGs confer different specificity and transport properties to the PSAC channel. Ref. 16 does not disprove this possibility, because it is possible that differences in nutrient permeability associated with different clag paralogs occur only for specific nutrients, which was not tested in ref 16. This should be made clear.

Reviewer #3: Does synchrony and stage of C3h-KO alter the fraction of "dormant" parasites that drive recrudescence when switched back to RPMI?

Figures would benefit from within-panel labels and have plenty of white space to fit them.

FigS2A: repeating the primer diagram would be helpful for parsing the figure.

FigS2B: Is this the expected localization pattern for clag2? Is there any localization to the RBC membrane ?

The discussion would be better off starting with the second paragraph as clag2 CNV is not an example of antigenic switching but rather a compensatory mechanism in response to the engineered loss of clag3 and selection with reduced nutrient serum, similar to other CNV that occur in response to drug selections.

Fig5C: Is the Nluc still active on the membrane even after being run on a denaturing gel? Or was this probed with anti-Nluc antibody? Non-experts in patch-clamping (like me) might benefit for a more context and elaboration of the results in Figure 3.

**********

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Reviewer #2: No

Reviewer #3: Yes: Björn Kafsack

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PLoS Pathog. 2025 Oct 16;21(10):e1013321. doi: 10.1371/journal.ppat.1013321.r002

Author response to Decision Letter 1

30 Sep 2025

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PLoS Pathog. doi: 10.1371/journal.ppat.1013321.r003

Decision Letter 1

Tania de Koning-Ward

3 Oct 2025

Dear Dr. Desai,

We are pleased to inform you that your manuscript 'Nutrient stress dramatically increases malaria parasite clag2 copy number to increase host cell permeability and enable pathogen survival' has been provisionally accepted for publication in PLOS Pathogens.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Tania F. de Koning-Ward

Academic Editor

PLOS Pathogens

Dominique Soldati-Favre

Section Editor

PLOS Pathogens

Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

Michael Malim

Editor-in-Chief

PLOS Pathogens

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***********************************************************

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PLoS Pathog. doi: 10.1371/journal.ppat.1013321.r004

Acceptance letter

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Dear Dr. Desai,

We are delighted to inform you that your manuscript, "Nutrient stress dramatically increases malaria parasite clag2 copy number to increase host cell permeability and enable pathogen survival," has been formally accepted for publication in PLOS Pathogens.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Rare C3h-KO parasites seen during PGIM cultivation resume expansion upon transfer to nutrient-rich medium.

(TIF)

ppat.1013321.s001.TIF^{(121.1KB, TIF)}

S2 Fig. Transcript abundances for clag and rhoph genes.

(TIF)

ppat.1013321.s002.TIF^{(123.3KB, TIF)}

S3 Fig. Engineered parasites carrying a NanoLuc reporter at CLAG2 C-terminus.

(TIF)

ppat.1013321.s003.tif^{(2MB, tif)}

S4 Fig. Increased CLAG2 production in KO-PGIM is detected by a weakly cross-reactive anti-CLAG3 antibody.

(TIF)

ppat.1013321.s004.TIF^{(2.2MB, TIF)}

S1 Table. Primers used in this study.

(XLSX)

ppat.1013321.s005.xlsx^{(14.4KB, xlsx)}

S2 Table. Structural variants in KO-PGIM.

(XLSX)

ppat.1013321.s006.xlsx^{(10KB, xlsx)}

S3 Table. Mutations and indels in C3h-KO and KO-PGIM.

(XLSX)

ppat.1013321.s007.xlsx^{(23.4KB, xlsx)}

Attachment

Submitted filename: Itemized Responses to Reviewers 092925.docx

ppat.1013321.s008.docx^{(41.6KB, docx)}

Data Availability Statement

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PERMALINK

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

Nicole B Potchen

Tatiane Macedo Silva

Inderjeet Kalia

Justin B Lack

Sanjay A Desai

Roles

Abstract

Author summary

Introduction

Results

Small numbers of CLAG3 knockout parasites persist and expand in nutrient-restricted media

Fig 1. Compromised growth of a CLAG3 knockout in nutrient-limited medium with rare persister parasites.

Growth compromised to a greater extent than expected from permeability reduction

In vitro selection with PGIM restores host cell permeability via altered channels

Fig 2. Growth in PGIM after in vitro selection is linked to altered channel properties.

Fig 3. Whole-cell patch-clamp reveals altered solute selectivities.

A markedly increased copy number for the clag2 paralog

Fig 4. Dramatic clag2 amplification in KO-PGIM.

CLAG2 upregulation accounts for altered channels and growth phenotype

Fig 5. Increased clag2 transcription and translation.

Discussion

Methods

Parasite culture and in vitro selection

Solute uptake measurements

Protein blots

Protease susceptibility

Whole-genome sequencing and analysis

Quantitative PCR for gene copy number

Quantitative reverse transcriptase PCR

DNA transfection

Indirect Immunofluorescence microscopy

Statistical analysis

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Tania de Koning-Ward

Roles

Author response to Decision Letter 1

Decision Letter 1

Tania de Koning-Ward

Roles

Acceptance letter

Tania de Koning-Ward

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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