Sickle Cell Hemoglobin Genotypes Affect Malaria Parasite Growth and Correlate with Exosomal miR-451a and let-7i-5p Levels

Abstract

Malaria affects a significant portion of the global population, with 247 million cases in 2021, primarily in Africa. However, certain hemoglobinopathies, such as sickle cell trait (SCT), have been linked to lower mortality rates in malaria patients. Hemoglobin (Hb) mutations, including HbS and HbC, can cause sickle cell disease (SCD) when both alleles are inherited (HbSS and HbSC). In SCT, one allele is inherited and paired with a normal allele (HbAS, HbAC). The high prevalence of these alleles in Africa may be attributed to their protective effect against malaria. Biomarkers are crucial for SCD and malaria diagnosis and prognosis. Studies indicate that miRNAs, specifically miR-451a and let-7i-5p, are differentially expressed in HbSS and HbAS compared to controls. Our research examined the levels of exosomal miR-451a and let-7i-5p in red blood cells (RBCs) and infected red blood cells (iRBCs) from multiple sickle Hb genotypes and their impact on parasite growth. We assessed exosomal miR-451a and let-7i-5p levels in vitro in RBC and iRBC supernatants. Exosomal miRNAs exhibited distinct expression patterns in iRBCs from individuals with different sickle Hb genotypes. Additionally, we discovered a correlation between let-7i-5p levels and trophozoite count. Exosomal miR-451a and let-7i-5p could modulate SCD and malaria severity and serve as potential biomarkers for malaria vaccines and therapies.

Keywords: exosomes; extracellular microvesicles; malaria; microRNA; parasitemia; red blood cells; sickle cell disease.

Figure 1

Parasite count percentage over 16 days culture of (A) all sickle Hb genotypes; (B) HbAA (red); (C) HbAC (green); (D) HbAS (orange); (E) HbSS (blue); (F) HbSC (purple); (G) HbCC (pink); and (H) area under the curve (AUC) for each genotype. AUC was used to estimate the overall parasite counts over 16 days. HbAA had significantly (p = 0.03) more parasites compared to the HbCC group. (I) A one-way ANOVA and Tukey’s multiple comparison test on all the genotypes. The median parasitemia was significantly lower in HbSS (p = 0.0015) and HbCC (p < 0.0001) groups compared to HbAA and in both groups compared to HbAC (p = 0.0135 and p < 0.0001, respectively). Additionally, the HbCC genotype had a significantly lower median parasitemia compared to HbAS and HbSC, with p = 0.0004 and p = 0.0003, respectively.

Figure 2

Ring, trophozoite, and schizont count for each sickle Hb genotype over 16 days. Red is for HbAA, orange for HbAS, green for HbAC, blue for HbSS, purple for HbSC, and pink for HbCC. Area under the curve (AUC) was used to estimate parasite counts over 16 days. A one-way ANOVA and a Tukey’s multiple comparison test were used. (A) Ring counts for each sickle Hb genotype. (B) AUC of overall ring count for each genotype. (C) Trophozoite counts for each sickle Hb genotype. (D) AUC for overall trophozoite count for each sickle Hb genotype. (E) Schizont counts for each sickle Hb genotype. (F) AUC for overall schizont count for each genotype.

Figure 3

Gametocyte phase 1–5 counts for each sickle Hb genotype over 16 days. Red is for HbAA, orange for HbAS, green for HbAC, blue for HbSS, purple for HbSC, and pink for HbCC. Area under the curve (AUC) was used to estimate parasite counts over 16 days. A one-way ANOVA and a Tukey’s multiple comparison test were used. (A,B) illustrate phase-1 gametocyte counts for each genotype. (C,D) illustrate phase-2 gametocyte counts. (E,F) illustrate phase-3 gametocyte counts. (G,H) illustrate phase-4 gametocyte counts. (I,J) illustrate phase-5 gametocyte counts.

Figure 4

Exosomal miR-451a and let-7i-5p levels in Plasmodium-infected RBC and non-infected RBC of different sickle Hb genotypes. Area under the curve (AUC) was used to estimate exosomal miRNA levels over 16 days. A Pearson correlation was used for all correlations, and a linear regression was run. (A) shows the exosomal miR-451a levels of malaria-negative RBC with different hemoglobin (Hb) genotypes over 16 days. (B) presents the AUC for exosomal miR-451a levels over 16 days for each genotype without malaria. (C) displays the exosomal miR-451a levels of malaria-positive RBC with different sickle Hb genotypes over 16 days. (D) presents the AUC for exosomal miR-451a levels over 16 days for each sickle Hb genotype’s infected RBCs. (E–H) show the correlation between exosomal miR-451a levels and parasite count percentage on days 3, 8, 9, and 16, respectively. A significant positive correlation was observed on days 3 and 8 (R² = 0.29 and p = 0.02). (F) includes the equation of the regression line (Y = −0.2316*X + 1.422). (I,J) display the exosomal let-7i-5p levels of malaria-negative RBC with different sickle Hb genotypes over 16 days and the AUC for exosomal let-7i-5p levels over 16 days for each genotype without malaria, respectively. (K) shows the exosomal let-7i-5p levels of malaria-positive RBC with different sickle Hb genotypes over 16 days. (L) presents the AUC for exosomal let-7i-5p levels over 16 days for each sickle Hb genotype’s infected RBCs. (M–P) show the correlation between exosomal let-7i-5p levels and parasite count percentage on days 3, 8, 9, and 16, respectively. The trend changes from a positive correlation on days 3 and 8 to a negative correlation on day 9, where let-7i-5p decreases with an increase in parasite count. (O) includes the equation of the regression line (Y = −0.2302*X − 0.08344).

Figure 5

Exosomal miR-451 and let-7i-5p correlation and comparison between sickle Hb genotypes for days 3, 8, 9, and 16. Statistical analyses were performed using ANOVA and Tukey’s multiple comparison or Pearson correlation tests as appropriate, with significance determined for malaria-positive or negative sickle Hb genotypes unless stated otherwise. (A) displays miRNA gene expression on day 3, where no significant differences were found between malaria-positive or negative sickle Hb genotypes. In (B), miR-451a expression was significantly lower in HbSS- compared to HbSC- on day 8 (p = 0.02), while miR-451a was significantly higher in HbSS+ compared to HbSS (p = 0.03) and let-7i-5p was significantly higher in HbAC+ compared to HbAC- (p = 0.04) using an unpaired t-test. (C,D) demonstrate no significant differences in miRNA expression levels on days 9 and 16, respectively, for malaria-positive or negative sickle Hb genotypes. In (E–H), there were significant correlations found between exosomal miR-451a and let-7i-5p levels for malaria-negative sickle Hb genotypes on days 3, 8, 9, and 16, with respective equations of Y = 0.6803*X + 1.901, Y = 0.8521*X + 2.365, Y = 0.4235*X + 0.9760, and Y = 0.3904*X + 0.8477. The correlations had R² values of 0.48, 0.61, 0.24, and 0.45 and were all significant with p-values of 0.0003, <0.0001, 0.02, and 0.009, respectively. (I–L) display no significant correlations between exosomal miR-451a and let-7i-5p levels for malaria-positive sickle Hb genotypes on days 3, 8, 9, or 16, respectively, with respective equations of Y = −0.1365*X – 0.1045, Y = −0.07589*X – 0.07005, Y = −0.1509*X – 0.2939, and Y = 0.2378*X + 0.1944. The p-values for all correlations were greater than 0.05. Additionally, the figures show the log-fold change in miRNA expression levels for days 3, 8, 9, and 16.