A conserved bacterial genetic basis for commensal-host specificity

Abstract

Animals selectively acquire specific symbiotic gut bacteria from their environments that aid host fitness. To colonize, a symbiont must locate its niche and sustain growth within the gut. Adhesins are bacterial cell surface proteins that facilitate attachment to host tissues and are often virulence factors for opportunistic pathogens. However, the attachments are often transient and nonspecific, and additional mechanisms are required to sustain infection. In this work, we use live imaging of individual symbiotic bacterial cells colonizing the gut of living Drosophila melanogaster to show that Lactiplantibacillus plantarum specifically recognizes the fruit fly foregut as a distinct physical niche. L. plantarum establishes stably within its niche through host-specific adhesins encoded by genes carried on a colonization island. The adhesin binding domains are conserved throughout the Lactobacillales, and the island also encodes a secretion system widely conserved among commensal and pathogenic bacteria.

Fig. 1.. Live imaging of bacterial cells in the Drosophila gut shows that LpWF attaches specifically to a physical niche in the host foregut.

(A and B) Schematics showing the anatomy of the fruit fly gut in the fly (A) and dissected (B), with the symbiotic niche highlighted in red. (C and D) Confocal micrographs of proventriculus (C) and crop (D) colonized by mCherry-labeled LpWF. (E and F) Confocal micrographs of proventriculus (E) and crop (F) colonized by mCherry-labeled LpATCC8014. Scale bars, 20 μm. (G) Schematic and photos showing the Bellymount apparatus for live imaging. (H) X-Z (top), X-Y (middle), and X-Y (bottom) time series images of LpWF by Bellymount. (I) X-Y time series and particle tracking of LpATCC8014. (J and K) Histogram of coefficients of diffusion for individual LpATCC8014 (J) and LpWF (K) cells. n = 500 tracks from five biological replicates. (L) Schematic describing the experimental evolution screen. P, passage. (M) Percentage of flies colonized by each of the 12 experimental replicates at passage 51. n ≥ 72 individual flies from three biological replicates per strain. (N) Percentage of flies colonized by evolved strains diminished with passage number. n ≥ 72 individual flies from three biological replicates per time point. (O) Histogram of coefficients of diffusion for LpWF^R3P51. n = 500 tracks from five biological replicates. Statistics by Kruskal-Wallis test; ****P < 0.0001.

Fig. 2.. A colonization island containing SRRPs drives L. plantarum adhesion to the gut.

(A) Long-read sequencing of LpWF and the experimentally evolved mutants indicates a colonization island. (B and C) PFGE and Southern blotting of plasmid partition genes shows a reduction in length of the linear pKG plasmid that contains the colonization island (B) and loss of the colonization island (C) using probes for the island-specific genes srpA and srpB. (D) Map of the island with functional annotations. See fig. S13 for additional annotations. (E) SRRP protein schematics. (F and G) Negative stain EM of LpWF (F) and LpWF::Δisland (G). Arrowhead indicates fibril structures. Scale bars, 500 nm. (H) Quantification of colonization of flies by LpWF, LpDm13, LpDm48, and mutants. n ≥ 100 flies per strain. (I) Bellymount quantification for LpWF::Δisland. (J and K) Negative stain EM of LpWF carrying CRISPRi with single guide RNA (sgRNA) negative control (J) and sgRNA targeting srpA and srpB (K). (L and M) Bellymount of LpWF carrying CRISPRi with sgRNA negative control (L) and sgRNA targeting srpA and srpB (M). (N) Colonization quantification for CRISPRi knockdowns (KDs) in LpWF. Statistics by Fisher’s exact test; ****P < 0.0001; ns, not significant.

Fig. 3.. The colonization island is widely conserved in host-associated bacteria.

(A) Core genome phylogeny of 36 species and maps of their corresponding colonization islands. (B) Clustering analysis of the binding regions of diverse bacterial colonization islands and representative predicted structures for each of the clusters. See fig. S24 for complete results.