Abstract
Child undernutrition is a global health issue associated with a high burden of infectious disease. Undernourished children display an overabundance of intestinal pathogens and pathobionts, and these bacteria induce enteric dysfunction in undernourished mice; however, the cause of their overgrowth remains poorly defined. Here, we show that disease-inducing human isolates of Enterobacteriaceae and Bacteroidales spp. are capable of multi-species symbiotic cross-feeding, resulting in synergistic growth of a mixed community in vitro. Growth synergy occurs uniquely under malnourished conditions limited in protein and iron: in this context, Bacteroidales spp. liberate diet- and mucin-derived sugars and Enterobacteriaceae spp. enhance the bioavailability of iron. Analysis of human microbiota datasets reveals that Bacteroidaceae and Enterobacteriaceae are strongly correlated in undernourished children, but not in adequately nourished children, consistent with a diet-dependent growth synergy in the human gut. Together these data suggest that dietary cross-feeding fuels the overgrowth of pathobionts in undernutrition.
Highlights
In the reciprocal analysis, annotated E. coli genes were filtered for genes present in at least 60% of samples and pairwise Spearman’s correlations were again performed against the abundance of Bacteroidaceae and Bacteroides
Statistical analysis was performed in GraphPad Prism (v 9.1.1) or in R Studio (v 1.2.1335) using the packages phyloseq (v 1.32.0), ggplot[2] (v 2.3.3.2), dplyr (v 1.0.2), tidyr (v 1.1.2), vegan (v 2.5.6), RColorBrewer (v 1.1.2), biomformat (v 1.16.0), reshape (v 2.1.4.4), and psych (v 2.0.8)
For in vitro experimental work, parametric tests were chosen based on the normal distribution of bacterial growth outcomes
Summary
Mutants, and plasmids used in this study are listed in Supplementary Table 2. E. coli ΔnanA and ΔfucI mutants were generated using homologous recombination to replace the target gene with a kanamycin resistance cassette (KanR) following a protocol based on lambda red recombination[47]. The KanR locus on pKD13 (Supplementary Table 2) was PCR-amplified using primers with 50 bp 5’ overhangs homologous to the target gene (Supplementary Table 7). Amplicons were purified by phenol chloroform extraction and electroporated into target E. coli carrying pkD46 expressing the lambda red recombinase. Mutants were genotypically confirmed by PCR using primers flanking KanR. Mutant phenotypes were confirmed by aerobic growth in M9 minimal media with fucose or sialic acid as a sole carbon source. Growth was assessed by OD600 using a plate reader (Tecan)
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