Concurrent PIEZO1 activation and ATP2B4 blockade
effectively reduce the risk of cerebral malaria and inhibit
in vitro Plasmodium falciparum multiplication in red
blood cells

Mathieu Adjemout, Bruno Pouvelle, Fatou Thiam, Alassane Thiam, Magali

Torres, Samia Nisar, Babacar Mbengue, Alioune Dieye, Pascal Rihet,

Sandrine Marquet

► To cite this version:

Mathieu Adjemout, Bruno Pouvelle, Fatou Thiam, Alassane Thiam, Magali Torres, et al. Concurrent PIEZO1 activation and ATP2B4 blockade effectively reduce the risk of cerebral malaria and inhibit in vitro Plasmodium falciparum multiplication in red blood cells. Genes & Diseases, 2023, 10 (6), pp.2210-2214. 10.1016/j.gendis.2023.02.029. hal-04179608

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# Concurrent PIEZO1 activation and ATP2B4 blockade effectively reduce the risk of cerebral malaria and inhibit *in vitro* Plasmodium falciparum multiplication in red blood cells

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Malaria caused by the *Plasmodium falciparum* parasite is responsible for more than 240 million cases per year and killed 627,000 people in 2020, mostly African children. The malaria parasite is transmitted by mosquitos belonging to the genus *Anopheles*. After an asymptomatic liver stage, the parasite is released into the bloodstream to invade red blood cells (RBCs) and replicate asexually. This erythrocytic phase is associated with a variety of clinical manifestations, including mild and severe malaria. Cerebral malaria (CM) is one of the most severe forms, characterized by the sequestration of parasitized RBCs in the small capillaries of the brain and the local development of cytokine-mediated inflammation. Genetic variants in genes encoding proteins involved in red blood cell physiology are protective factors against severe malaria, as clearly demonstrated for the sickle cell variant of hemoglobin (HbS). Rare gain-of-function mutations in Piezo1, a mechanosensitive calcium channel, are involved in hereditary xerocytosis, a disease characterized by red cell dehydration and mild hemolysis. Interestingly, RBC dehydration is associated with reduced Plasmodium infection *in vitro*¹ suggesting that PIEZO1 polymorphisms may protect against malaria. Recently, a gain-of-function PIEZO1 E756del variant (rs59446030) under positive selection in Africa, was associated with protection against severe malaria in children in Gabon.² However, a large case-control study in Ghana failed to replicate this association.³ In this study, we sought to further characterize the role of this PIEZO1 polymorphism in malaria by confirming its association in a Senegalese population and identifying the genetic interaction with polymorphisms in

the *ATP2B4* gene that encodes PMCA4, the major RBC calcium pump. We prove that activation of Piezo1 modulates intracellular calcium concentration and inhibits *Plasmodium falciparum* multiplication in RBCs. Furthermore, we demonstrated the additive effect of Piezo1 activation and PMCA4 blockade on the inhibition of parasite multiplication in erythrocytes.

The E756del variant of *PIEZOT*, a 3-nucleotide deletion (GGA) in the coding region of the gene, results in the deletion of glutamic acid (Fig. 1A). Genotyping of this mutation was performed in a Senegalese population composed of 82 individuals with CM, 27 with severe malaria, 74 with mild malaria, and 77 healthy controls living in Dakar (Table S1). Genotype frequencies were consistent with Hardy-Weinberg Equilibrium. We found that the E756del variant was strongly associated with protection against CM (P = 0.007, odds ratio = 0.36 [0.17–0.76]) in our Senegalese population using a Fisher’s exact test and logistic regression analysis (Fig. 1B). This association remained significant even when age was included as a covariate in the statistical model (P = 0.009, odds ratio = 0.35 [0.16–0.77]). Our results are consistent with a previous study showing that Piezo1 gain-of-function mice are protected against experimental CM.¹ In addition, we evaluated the effect of this PIEZO1 variant in combination with the *ATP2B4* haplotype of 5 regulatory SNPs (rs11210734-rs1541252-rs1541253-rs1541254-rs1541255) (Table S1) that we previously showed to be associated with severe malaria.⁴ In this study, we showed that individuals having at least one *ATP2B4* minor allele haplotype are protected against CM (P = 0.04, estimated odds ratio = 0.5 [0.26–0.93]) (Fig. 1C). Second, we demonstrated a combined effect between the protective

<footnote>Peer review under responsibility of Chongqing Medical University.</footnote>
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<table><thead><tr><th rowspan="2">Variant</th><th rowspan="2">Minor allele</th><th rowspan="2">Protective genotype</th><th rowspan="2">Ctrl (n=77)</th><th rowspan="2">MM (n=74)</th><th rowspan="2">SM (n=27)</th><th rowspan="2">CM (n=82)</th><th colspan="3">Ctrl vs MM</th></tr><tr><th>Odd ratio (95% CI)</th><th>Odd ratio (95% CI)</th><th>Odd ratio (95% CI)</th></tr></thead><tbody><tr><td>rs59446030<br>(GGA>-)</td><td>E756Del</td><td>E756/E756Del</td><td>28 (36%)</td><td>18 (24%)</td><td>9 (33%)</td><td>14 (17%)</td><td>0.56 (0.28 - 1.14)</td><td>0.88 (0.35 - 2.21)</td><td><strong>0.36** (0.17 - 0.76)</strong></td></tr></tbody></table>

<table><thead><tr><th rowspan="2">Variant present</th><th rowspan="2">Ctrl</th><th rowspan="2">CM</th><th rowspan="2">Odd ratio (95% CI)</th><th rowspan="2">P-value (two-tailed)</th></tr></thead><tbody><tr><td><strong>PIEZ01</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Risk genotypes (DEL/DEL + WT/WT)</td><td>49</td><td>68</td><td>1</td><td>-</td></tr><tr><td>Protective genotype (WT/DEL)</td><td>28</td><td>14</td><td>0.36 (0.17-0.76)</td><td>0.007</td></tr><tr><td><strong>ATP2B4</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Risk haplotype (Major/Major)</td><td>26</td><td>45</td><td>1</td><td>-</td></tr><tr><td>Protective haplotypes (Major/Minor + Minor/Minor)</td><td>49</td><td>42</td><td>0.50 (0.26-0.93)</td><td>0.04</td></tr><tr><td><strong>Combination</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Risk genotypes PIEZO1/haplotype ATP2B4</td><td>17</td><td>33</td><td>1</td><td>-</td></tr><tr><td>Protective genotypes PIEZO1/haplotype ATP2B4</td><td>17</td><td>5</td><td>0.15 (0.05-0.48)</td><td>0.0009</td></tr><tr><td>Interaction PIEZO1 × ATP2B4</td><td></td><td></td><td>0.28 (0.13-0.61)</td><td>0.001</td></tr></tbody></table>

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<figure><img src="image_9.png" /><figcaption>**Figure 1** The combination of genetic variants in the *ATP2B4* and *PIEZ01* genes protects against cerebral malaria by altering calcium homeostasis that inhibits *P. falciparum* reinvasion. (A) Description of the E756Del mutation of *PIEZ01*. The E756Del mutation (rs59446030) is a 3-base pair DNA deletion in a GGA repeat and results in a deletion of a glutamate (E) amino acid. The ancestral genotype consists of 8 repeats of the GGA motif, compared to 7 repeats in E756Del mutation. Genotyping was performed</figcaption></figure>
genotype of *PIEZ01* and the protective haplotype of *ATP2B4*
(P = 9.10–4, odds ratio = 0.15 [0.05–0.48]) (Fig. 1C). We
also identified a significant genetic interaction between
*ATP2B4* and *PIEZ01* variants using logistic regression anal-
ysis (P = 1.10–3 (odds ratio = 0.28 [0.13–0.61]) (Fig. 1C).
For individuals heterozygous and homozygous for the
haplotype with minor *ATP2B4* alleles, the odds ratio was
0.5. In contrast, for individuals with both the protective
heterozygous genotype for *PIEZ01* and the protective
*ATP2B4* haplotype, the odds ratio was 0.15, indicating
stronger protection against CM. *ATP2B4* encodes PMCA4, a
calcium pump, and E756Del is a gain-of-function mutation
that increases the activity of the mechanosensitive cationic
channel Piezo1, both of which are expressed on the surface
of RBCs. To assess *in vitro* the effect of Piezo1 activation on
intracellular calcium, we incubated human RBCs from a
healthy blood group O donor provided by the local blood
bank (Etablissement Français du Sang) with Yoda 1, a spe-
cific activator of Piezo1. As shown in Figure 1D, in each of
the two experiments performed, cells treated with Yoda1
had a 10-fold increased calcium concentration (P < 0.0001)
compared with cells without Yoda1. Similarly, a meta-
analysis considering MFI, SD, and sample size showed
increased calcium concentration in Yoda1-treated cells
(P < 0.001). Yoda1 (in the micromolar range) causes
increased opening of Piezo1 in response to mechanical
stimulation, thus allowing massive calcium entry. In the

RBC, increased intracellular calcium activates the GARDOS
channel, resulting in potassium efflux, water loss, and
cellular dehydration.⁵ Piezo1 is an important channel for
maintaining calcium homeostasis and RBC volume. Then,
using synchronized mature stages parasites (*Plasmodium
falciparum* 3D7 strain) incubated in the presence of 10 µM
of Yoda1 for three days, we showed that after two in-
vasions, parasitemia increased from 0.2% to 5% in the
control culture without Yoda1 while it dropped to 0.32% in
the culture with Yoda1 (P = 0.002) (Fig. 1E). As shown in
Figure 1F, the percentage of inhibition reached 94% after
two invasions on day 3, confirming that the increase in
intracellular calcium in erythrocytes prevents parasite
multiplication. We thus demonstrated *in vitro* that parasite
multiplication is significantly inhibited by the chemical
activation of Piezo1 in agreement with a previous study. We
have previously shown that the *ATP2B4* minor allele
haplotype is associated with a decrease in enhancer ac-
tivity and, consequently a decrease in the long PMCA4a and
PMCA4b transcripts, the two isoforms expressed mainly in
the brain and red blood cells respectively.⁴ This decrease in
*ATP2B4* expression leads to increased intracellular calcium⁴
probably due to a functional impairment of active calcium
extrusion. To evaluate the effect of PMCA4 inhibition on
parasite growth, experiments were performed in the
presence of ATA (aurintricarboxylic), a specific inhibitor of
PMCA4. At 100 µM of ATA, we observed approximately 44%

by PCR amplification using the forward primer 5'-CAGGCAGGATGCAGTGAGTG-3' and the reverse primer 5'-GGACATGGCACAGCA-
GACTG-3', followed by Sanger sequencing. The chromatogram shows the result of three observed genotypes, homozygous E756,
heterozygous E756/E756Del, and homozygous E756Del. (B) Association of the *PIEZ01* mutation with cerebral malaria in the Sen-
egalese population-based study. A total of 260 individuals were genotyped, including 77 healthy individuals (Control, Ctrl), 74
subjects with mild malaria (MM) characterized by symptoms such as headache, fever, nausea, 27 cases of severe malaria (SM)
without neurological symptoms, and 82 cases of cerebral malaria (CM) characterized by deep coma (GCS <9). We performed the
statistical analysis with IBM SPSS Statistics. Fisher's exact test was used to calculate *P*-values without any covariate (P = 0.006). (C)
Association of *PIEZ01* mutation and *ATP2B4* regulatory variants with cerebral malaria. *P*-values and odds ratios were calculated
using logistic regression to assess the association of *PIEZ01* and *ATP2B4* genetic variants separately or in combination. Considering
the combination of the protective genotype of *PIEZ01* with the protective haplotypes of *ATP2B4*, the association was highly sig-
nificant by both Fisher's exact test and logistic regression. (D) Visualization of calcium concentration in red blood cells without
(blue) and with (pink) Yoda 1 treatment. Culture of red blood cells from healthy donor blood was performed for 24 h in the presence
of the agonist Yoda1 at a final concentration of 10 µM. Intracellular calcium was measured by flow cytometry with Fluo-4. Fluo-
rescence intensity was estimated on 20,000 labeled red blood cells. Analyzing each of the two experiments performed with Yoda1-
treated cells showed a significant increase in calcium concentration compared with cells without Yoda1 (P < 0.0001). (E) Estimation
of *in vitro* parasitemia without (blue) and with (pink) Yoda1. *P. falciparum* (3D7 strain) culture was synchronized by sorbitol
treatment. 24 h later, the percentage of parasitemia was brought to 0.2% and the parasites were cultured in the presence of 10 µM
of Yoda1 for 2 reinvasions. Parasitemia was determined by flow cytometry, using SYBR Green to label the parasites, after 24 h and
72 h of culture in the presence of Yoda1. A paired sample *t*-test was performed to assess the impact of Yoda1 on the parasite.
During the first and second reinvasion, the percentage of parasitemia was lower in the presence of Yoda1 (P = 0.0007 and
P = 0.002, respectively). (F) Inhibition of parasite invasion. Percentage inhibition of parasite invasion was estimated on days 1 and
3 of infected red blood cells culture in the presence of 10 µM Yoda1. (G) *In vitro* inhibition of parasite invasion using ATA
(aurintricarboxylic acid), a specific inhibitor of PMCA4 (the plasma membrane calcium ATPase 4) encoded by *ATP2B4* and/or Yoda1
an activator of *PIEZ01*. A culture of *P. falciparum* (3D7 strain) was synchronized by sorbitol treatment and incubated 24 h later in
the presence of either Yoda1 at 1.8 µM (green), ATA at 100 µM (brown), or both ATA at 100 µM and Yoda1 at 1.8 µM (orange). After
24 h of treatment, the parasitemia of each culture was determined by flow cytometry, using SYBR Green to label the parasites.
Student's *t*-test was used to calculate *P*-values. Stronger inhibition was observed by combining Yoda1 and ATA (P = 0.001 and
P < 0.001 compared to Yoda1 and ATA, respectively). No statistical difference was observed between Yoda1 and ATA (*P*-
value = 0.254). (H) Conformation of the human *PIEZ01* protein. Representation of the monomer of human *PIEZ01* constructed by
sequence homology with mice (Uniprot KB - E2JF22) using the SWISS-MODEL. The result was visualized on PyMOL. Position E756 is
shown in the red frame. The region containing this mutation in humans is not conserved in mice, which does not allow us to
establish a 3D structure of this region by sequence homology. Data are represented ± standard deviation. **P** < 0.01, ***P** < 0.001.
ns, not significant.
parasite inhibition, and similarly, we observed 45% inhibi-
tion in the presence of Yoda1 at 1.8 µM (Fig. 1G). Inter-
estingly, when ATA and Yoda1 were added simultaneously,
we achieved significantly stronger inhibition of 59%
(Fig. 1G) compared with Yoda1 (P = 0.001) or ATA
(P < 0.001), supporting the additive effect of PIEZO1 and
ATP2B4 variants. To date, no human structure of Piezo1 has
been established, and the region containing the E756Del
mutation is not conserved in the mouse model, making it
impossible to establish the impact of this mutation on the
3D conformation of the protein (Fig. 1H). However, we can
assume that the E756Del mutation alters the secondary
structure of Piezo1, resulting in a gain of function.
Furthermore, the PIEZO1 mutation and ATP2B4 regulatory
variants may play a key role in the protection against CM
through the biological function of these genes in other cell
types such as T cells, B cells, and astrocytes. Indeed, the
increase of intracellular calcium is one of the activation
signals of the T cells promoting the elimination of the
parasite. In addition, the increase in intracellular calcium
in astrocytes leads to a reduction in the production of pro-
inflammatory cytokines and chemokines such as IL-1β,
TNFα, and CXCL1 and thus may protect against CM. More-
over, PIEZO1 appears to play an important role in B cell
responses to membrane-associated antigens.

In conclusion, we confirmed that the Piezo1 gain-of-
function mutation is associated with protection against CM
in a Senegalese population. Our results also indicate that
the combination of the PIEZO1 mutation and the ATP2B4
SNPs further protects individuals against CM probably
because they disrupt cell calcium homeostasis. Indeed, the
activation of Piezo1 and the alteration of PMCA4 simulta-
neously affect parasite multiplication in vitro because of
increased intracellular calcium.

Ethics declaration

The Comité d'Ethique de la Recherche de l'Université Cheikh Anta Diop de Dakar has approved the protocols. Each participant, their parents, or legal guardians for any minors before inclusion has received written or verbal information in their native language and given written consent.

Author contributions

SM and PR designed and supervised the project. MA gen-
otyped the E756Del variant, and SN and MT participated in
ATP2B4 genotyping. AT extracted the DNA samples and
performed whole genome amplification. AT, FT, and BM
were involved in the recruitment, the follow-up of Sene-
galese individuals, and the collection and interpretation of

biological data, under AD supervision. BP performed
cytometry experiments for intracellular calcium levels and
parasite growth rate estimation. MA, PR, and SM per-
formed statistical analyses and interpreted the results. MA
and SM wrote the paper. SM and PR supervised the whole
research and provided guidance. All authors read the
manuscript and approved the final version before sub-
mitting the article.

Conflict of interests

The authors declare no conflict of interests.

Funding

This work was supported by the African Higher Education Centers of Excellence Project (CEA-SAMEF) at UCAD, the Pasteur Institute in Dakar, the Pasteur Institute in Paris, the French Embassy in Senegal, INSERM, and Aix-Marseille University. MA and SN were supported by a Ph.D. fellowship from the French Ministry of Research and the Higher Education Commission (HEC) in Pakistan, respectively. This work received support from the French Government under the France 2030 Investment Plan, as part of the Initiative d'Excellence d'Aix-Marseille Université - A*MIDEX - Institute MarMaRa (No. AMX-19-IET-007).

Acknowledgements

We thank all the individuals who participated in the study.

**Appendix A. Supplementary data**

Supplementary data to this article can be found online at
https://doi.org/10.1016/j.gendis.2023.02.029.

References

1. Ma S, Cahalan S, LaMonte G, et al. Common PIEZO1 allele in African populations causes RBC dehydration and attenuates Plasmodium infection. *Cell*. 2018;173(2):443–455.

2. Nguetse CN, Purington N, Ebel ER, et al. A common polymorphism in the mechanosensitive ion channel PIEZO1 is associated with protection from severe malaria in humans. *Proc Natl Acad Sci U S A*. 2020;117(16):9074–9081.

3. Thye T, Evans JA, Ruge G, et al. Human genetic variant E756del in the ion channel PIEZO1 not associated with protection from severe malaria in a large Ghanaian study. *J Hum Genet*. 2022; 67(1):65–76.

4. Nisar S, Torres M, Thiam A, et al. Identification of ATP2B4 regulatory element containing functional genetic variants associated with severe malaria. *Int J Mol Sci*. 2022;23(9):4849.
5. Maher AD, Kuchel PW. The Gárdos channel: a review of the Ca²⁺-activated K⁺ channel in human erythrocytes. *Int J Biochem Cell Biol*. 2003;35(8):1182–1197.

Mathieu Adjemout<sup>a</sup>, Bruno Pouvelle<sup>a</sup>, Fatou Thiam<sup>b</sup>, Alassane Thiam<sup>c</sup>, Magali Torres<sup>a</sup>, Samia Nisar<sup>a</sup>, Babacar Mbengue<sup>d</sup>, Alioune Dieye<sup>d</sup>, Pascal Rihet<sup>a,1,*</sup>, Sandrine Marquet<sup>a,e,1,*</sup>

<sup>a</sup> Institut National de la Santé Et de la Recherche Médicale (INSERM), Unité Mixte de Recherche (UMR) U1090, Aix Marseille Université, TAGC Theories and Approaches of Genomic Complexity, Institut MarMaRa, Marseille, France

<sup>b</sup> Groupe de Recherche Biotechnologie Appliquée et Bioprocédés Environnementaux (GRBA-BE), Ecole

Supérieure Polytechnique, Université Cheikh Anta Diop de Dakar, Dakar BP5005, Senegal

<sup>c</sup> Pole D'Immunophysiopathologie & Maladies Infectieuses (IMI), Institut Pasteur de Dakar, Dakar BP 220, Senegal

<sup>d</sup> Service D'Immunologie, Université Cheikh Anta Diop de Dakar, Dakar BP5005, Senegal

<sup>e</sup> Aix Marseille University, CNRS, Marseille 13009, France

*Corresponding author. Aix Marseille University, INSERM, TAGC, Marseille, 13288, France.

E-mail addresses: pascal.rihet@univ-amu.fr (P. Rihet), sandrine.marquet@univ-amu.fr (S. Marquet)

1 October 2022
Available online 27 March 2023

<footnote n="1"><sup>1</sup> These authors contributed equally to this work.</footnote>