Composition of the gut microbiota modulates the severity of malaria

Abstract

Plasmodium infections result in clinical presentations that range from asymptomatic to severe malaria, resulting in ∼1 million deaths annually. Despite this toll on humanity, the factors that determine disease severity remain poorly understood. Here, we show that the gut microbiota of mice influences the pathogenesis of malaria. Genetically similar mice from different commercial vendors, which exhibited differences in their gut bacterial community, had significant differences in parasite burden and mortality after infection with multiple Plasmodium species. Germfree mice that received cecal content transplants from "resistant" or "susceptible" mice had low and high parasite burdens, respectively, demonstrating the gut microbiota shaped the severity of malaria. Among differences in the gut flora were increased abundances of Lactobacillus and Bifidobacterium in resistant mice. Susceptible mice treated with antibiotics followed by yogurt made from these bacterial genera displayed a decreased parasite burden. Consistent with differences in parasite burden, resistant mice exhibited an elevated humoral immune response compared with susceptible mice. Collectively, these results identify the composition of the gut microbiota as a previously unidentified risk factor for severe malaria and modulation of the gut microbiota (e.g., probiotics) as a potential treatment to decrease parasite burden.

Keywords: Plasmodium; gut microbiome; severe malaria.

Fig. 1.

Plasmodium parasite burden, morbidity, and mortality vary by mouse vendor and diet. C57BL/6 mice were infected with P. yoelii parasitized RBCs. (A) Fraction of RBCs infected with P. yoelii (percentage of parasitemia). (B) AUC analysis. Data were analyzed by one-way ANOVA and Tukey’s multiple comparison posttest. (C) Percentage of weight loss following infection. Data were analyzed by one-way ANOVA. (D) Survival of mice following infection. Survival curves were analyzed by log-rank (Mantel–Cox) test. (E–H) Mice were fed either NIH-31 or Teklad 22/5 diet before and after P. yoelii infection. (E) Percentage of parasitemia following P. yoelii infection. (F) AUC analysis. Data were analyzed by one-way ANOVA and Tukey’s multiple comparison posttest. (G) Percentage of weight loss following infection. Data were analyzed by one-way ANOVA. (H) Survival of mice following infection. Survival curves were analyzed by log-rank (Mantel–Cox) test. Data (mean ± SE) in A–F and H are cumulative results (n = 8–10 mice per group) from two experiments. Data (mean ± SD) in G are from four to five mice per group from one experiment. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. n.s., not significant; p.i., postinfection.

Fig. 2.

Susceptibility to malaria correlates with differences in cecal bacteria populations. (A) Bacterial families that were identified as being significantly enriched in Jax or Tac mice. (B) Bacterial families identified as being significantly enriched in NCI or Har mice. Data (mean ± SE) in A and B are from six mice per group and extracted from analysis in SI Appendix, Fig. 6C. Data were analyzed by the Kruskal–Wallis test.

Fig. 3.

Gut microbiome shapes susceptibility to severe malaria. GF mice were colonized with cecal contents from Jax or NCI mice. (A) Bacterial population analysis was performed using nonmetric multidimensional scaling, as described in SI Appendix, Fig. 6. (B–D) Colonized GF mice and control Jax and NCI mice were infected with P. yoelii. (B) Percentage of parasitemia following P. yoelii infection. (C) AUC analysis. Data (mean ± SE) in B and C from four to five mice per group are representative of two experiments. Data were analyzed by one-way ANOVA and Tukey’s multiple comparison posttest. (D) Survival of mice following infection. Data are cumulative results (n = 8–10 mice per group) from two experiments. Survival curves were analyzed by log-rank (Mantel–Cox) test. ****P < 0.0001. Env, environment.

Fig. 4.

Susceptible mice treated with yogurt have decreased parasitemia and morbidity. Jax and NCI mice were left untreated (control), treated with antibiotics for 3 wk and then left untreated for an additional 3 wk (Abx), left untreated for 3 wk followed by treatment with yogurt five times per week for 3 wk (Yogurt), or treated with antibiotics for 3 wk followed by treatment with yogurt five times per week for 3 wk (Abx+Yogurt). Mice were then infected with P. yoelii. Yogurt-treated mice continued to receive yogurt five times per week following infection. (A) Percentage of parasitemia following P. yoelii infection. (B) AUC analysis. Data were analyzed by one-way ANOVA and Tukey’s multiple comparison posttest. (C) Percentage of weight loss following infection. Data were analyzed by one-way ANOVA. Data (mean ± SE) in A–C are cumulative results (n = 3–10 mice per group) from two experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 5.

Resistant Jax mice have an elevated cellular and humoral immune response to Plasmodium. Jax and NCI mice were infected with P. yoelii. Total number of CD4⁺CD11a^hiCD49d^hi cells (A), Tfh cells (B), and germinal center (GC) B cells (C) per spleen on the indicated day. Data (mean ± SE) are cumulative results (n = 5–10 mice per data point) from three experiments. (D) Serum MSP1₁₉-specific Ab end-point titers. Data (mean ± SE) are cumulative results (n = 3–7 mice per data point) from two experiments. Numbers in the panels represent the fold difference between the means of the Jax and NCI mice. Data were analyzed by an unpaired two-tailed t test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.