The gut microbiome is associated with susceptibility to febrile malaria in Malian children

Abstract

Malaria is a major public health problem, but many of the factors underlying the pathogenesis of this disease are not well understood, including protection from the development of febrile symptoms, which is observed in individuals residing in areas with moderate-to-high transmission by early adolescence. Here, we demonstrate that susceptibility to febrile malaria following Plasmodium falciparum infection is associated with the composition of the gut microbiome prior to the malaria season in 10-year-old Malian children, but not in younger children. Gnotobiotic mice colonized with the fecal samples of malaria-susceptible children were shown to have a significantly higher parasite burden following Plasmodium infection compared to gnotobiotic mice colonized with the fecal samples of malaria-resistant children. The fecal microbiome of the susceptible children was determined to be enriched for bacteria associated with inflammation, mucin degradation and gut permeability, and to have increased levels of nitric oxide-derived DNA adducts and lower levels of mucus phospholipids compared to the resistant children. Overall, these results indicate that the composition of the gut microbiome is associated with the prospective risk of febrile malaria in Malian children and suggest that modulation of the gut microbiome could decrease malaria morbidity in endemic areas.

Fig. 1. Microbiome composition correlates with susceptibility to febrile malaria in children and high parasite burden in mice.

A Principal coordinate analysis (PCoA) plot of the Bray-Curtis dissimilarity of the human fecal samples using 16S rRNA sequencing. Gavage samples are highlighted by increased point size. B Parasitemia and C AUC of the gavaged gnotobiotic mice by resistant and susceptible outcome groups, and D parasitemia by individual gavage groups. n = 4 mice for each donor used. n = 16 for the overall resistant and susceptible groups. PCoA plots of the Bray-Curtis dissimilarity for the E human and the F murine fecal samples using 16S rRNA sequencing and the updated resistant definition. The P value for the Bray–Curtis distance was determined using a two-sided PERMANOVA, and the P value for the parasitemia was determined using a two-sided Mann–Whitney U test. Blue circles indicate resistant children or low parasitemia mice, and red circles indicate susceptible children or high parasitemia mice. Mean ± standard error of the mean (SEM) indicated for parasitemia and AUC.

Fig. 2. Taxa associated with susceptibility and resistance using 16S rRNA sequencing.

Percentage of the mouse microbiome that is A Eubacterium coprostanoligenes, B Gemmiger formicilis, C Anaerostipes hadrus, D Roseburia faecis, E Clostridum citroniae, F Dorea longicatena, G Coprococcus comes, H Blautia faecis, I Bacteroides intestinalis, J Bacteroides ovatus, K Clostridium sp. FS41, and L Ruminococcus sp. Marseille-P328. Percentage of the human microbiome that is M Streptococcus thermophilus, N Ruminococcus sp. 5_1_39BFAA, O Dorea (metagenome), and P Blautia (unclassified). P values were determined using DESeq2 (∗), corncob (†), MaAsLin2 (‡), and ALDEx2 (§). Data were normalized using the default method for each tool: relative log expression (RLE), no normalization, total sum scaling (TSS), and no normalization, respectively, and the Benjamini–Hochberg method was used to control for multiple comparisons. Blue circles indicate resistant children or low parasitemia mice, and red circles indicate susceptible children or high parasitemia mice. Median indicated on graphs. n = 20 for the resistant mice and n = 12 for the susceptible mice. n = 24 for the resistant children and n = 46 for the susceptible children.

Fig. 3. Network of the top 100 nodes by degree centrality for resistant and susceptible children.

Size of nodes is indicative of degree centrality (number of connected nodes). Hubs (nodes with a degree centrality value above the empirical 95% quantile of all degree centralities in the network) are further highlighted with bold borders and decreased transparency. Nodes are colored by cluster (identified using greedy modularity optimization). Green edges indicate positive interactions, red edges indicate negative interactions, and absolute edge weight is indicated by transparency of the edge.

Fig. 4. Species from the Prevotella/Bacteroides and Streptococcus/Veillonella clusters correlate with the resistant children, and species from the Eubacteriales cluster correlate with the susceptible children.

A Sample plot, B correlation circle plot, and C loadings plot for the sPLS-DA of the metagenomics samples. The top 30 taxa by loading weight are listed, and the colors indicate in which group the taxa have the maximum median count—blue for the resistant group and red for the susceptible group. The ellipses represent 95% confidence intervals in the sample plot, and a correlation coefficient cutoff of 0.35 was used for the correlation circle plot.

Fig. 5. Taxa associated with susceptibility and resistance in the children using shotgun metagenomics.

Percentage of the microbiome that is A Prevotella copri, B Prevotella melaninogenica, C Streptococcus thermophilus, D Veillonella parvula, E Ruminococcus torques, F Ruminococcus gauvreauii, G Dorea formicigenerans, H Dorea longicatena, I Coprococcus comes, J Lachnoclostridium sp. YL32, K Lachnoclostridium phocaeense, L Enterocloster clostridioformis, M Enterocloster bolteae, N Anaerobutyricum hallii, O Blautia producta, and P Sellimonas intestinalis. P-values determined using DESeq2 (∗), corncob (†), MaAsLin2 (‡) and ALDEx2 (§). Data were normalized using the default method for each tool: relative log expression (RLE), no normalization, total sum scaling (TSS), and no normalization, respectively, and the Benjamini–Hochberg method was used to control for multiple comparisons. Blue circles indicate resistant children, and red circles indicate susceptible children. Median indicated on graphs. n = 24 for the resistant children and n = 46 for the susceptible children.

Fig. 6. Metabolite loading weights are evenly distributed.

A Sample plot, B correlation circle plot, and C loadings plot for the sPLS-DA of the metabolomics samples. The top 30 metabolites by loading weight are listed, and the colors indicate in which group the metabolite has the maximum median count—blue for the resistant group and red for the susceptible group. The ellipses represent 95% confidence intervals in the sample plot, and a correlation coefficient cut-off of 0.35 was used for the correlation circle plot.

Fig. 7. Features associated with inflammation and impaired gut barrier function are enriched in children susceptible to febrile malaria symptoms.

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