Genome-wide screen identifies host colonization determinants in a bacterial gut symbiont

Abstract

Animal guts are often colonized by host-specialized bacterial species to the exclusion of other transient microorganisms, but the genetic basis of colonization ability is largely unknown. The bacterium Snodgrassella alvi is a dominant gut symbiont in honey bees, specialized in colonizing the hindgut epithelium. We developed methods for transposon-based mutagenesis in S. alvi and, using high-throughput DNA sequencing, screened genome-wide transposon insertion (Tn-seq) and transcriptome (RNA-seq) libraries to characterize both the essential genome and the genes facilitating host colonization. Comparison of Tn-seq results from laboratory cultures and from monoinoculated worker bees reveal that 519 of 2,226 protein-coding genes in S. alvi are essential in culture, whereas 399 are not essential but are beneficial for gut colonization. Genes facilitating colonization fall into three broad functional categories: extracellular interactions, metabolism, and stress responses. Extracellular components with strong fitness benefits in vivo include trimeric autotransporter adhesins, O antigens, and type IV pili (T4P). Experiments with T4P mutants establish that T4P in S. alvi likely function in attachment and biofilm formation, with knockouts experiencing a competitive disadvantage in vivo. Metabolic processes promoting colonization include essential amino acid biosynthesis and iron acquisition pathways, implying nutrient scarcity within the hindgut environment. Mechanisms to deal with various stressors, such as for the repair of double-stranded DNA breaks and protein quality control, are also critical in vivo. This genome-wide study identifies numerous genetic networks underlying colonization by a gut commensal in its native host environment, including some known from more targeted studies in other host-microbe symbioses.

Keywords: Neisseriaceae; microbiota; symbiosis; transposon mutagenesis; type IV pilus.

Fig. 1.

Cross-sections of the honey bee ileum, showing colonization by S. alvi in 4-d-old workers inoculated (A) experimentally with laboratory-cultured S. alvi wkB2 and (B) naturally within the hive. S. alvi localization (purple) is derived from an overlay of a DNA stain (red) and an rRNA probe specific to S. alvi (blue). Methods and B are adapted from ref. .

Fig. 2.

Determining the S. alvi gene set essential for growth and beneficial for gut colonization. (A) Genome of S. alvi strain wkB2 with locations of transposon insertions in bee gut samples (inside track) and on culture media (outside track). Callouts illustrate three categories of genes identified from this approach. (B) The essential genome of S. alvi wkB2 on laboratory culture plates. (C) Tn-seq and RNA-seq results for S. alvi genes in the bee gut compared with culture plate growth. For Tn-seq, only genes with significant fitness benefit are highlighted.

Fig. 3.

Genes required or beneficial for S. alvi colonization of the honey bee ileum. Pathways and regulation inferred from characterized homologs in other bacterial species (e.g., E. coli and Neisseria spp.). Gene products and pathways that confer fitness benefits in the gut are colored yellow or blue (two colors are used to facilitate visualization of different pathways and gene products within complexes). Genes that may be beneficial, but do not reach significance, are denoted by an asterisk. Dataset S2 and SI Appendix, Figs. S2 and S3 provide details.

Fig. 4.

In vivo competitive ability of mutants implicated in Tn-seq screen as having roles in gut colonization. (A–D) Roles of pilF and pilG in biofilm formation and host colonization. (A) Crystal violet-stained surface biofilms in polypropylene wells. (B) Mean solubilized crystal violet-stained biofilm, as absorbance at OD₆₀₀ (n = 16 wells per strain, P = 1.91 × 10⁻¹³, Kruskal–Wallis multiple comparisons). (C) Scanning electron micrographs of surface biofilms at the air/liquid interface of strains grown in vitro. (D and E) Ratio of viable transposon mutants to wild-type following coinoculation of bee guts. *P < 0.029, **P < 0.005. (D) n = 7 bees per condition (Kruskal–Wallis test). (E) n = 4 bees per condition (Mann–Whitney test).