The Gut Microbiota Protects Bees from Invasion by a Bacterial Pathogen

Abstract

Commensal microbes in animal guts often help to exclude bacterial pathogens. In honey bees, perturbing or depleting the gut microbiota increases host mortality rates upon challenge with the opportunistic pathogen Serratia marcescens, suggesting antagonism between S. marcescens and one or more members of the bee gut microbiota. In laboratory culture, S. marcescens uses a type VI secretion system (T6SS) to kill bacterial competitors, but the role of this T6SS within hosts is unknown. Using infection assays, we determined how the microbiota impacts the abundance and persistence of S. marcescens in the gut and visualized colocalization of S. marcescens with specific community members in situ. Using T6SS-deficient S. marcescens strains, we measured T6SS-dependent killing of gut isolates in vitro and compared the persistence of mutant and wild-type strains in the gut. We found that S. marcescens is rapidly eliminated in the presence of the microbiota but persists in microbiota-free guts. Protection is reduced in monocolonized and antibiotic-treated bees, possibly because different symbionts occupy distinct niches. Serratia marcescens uses a T6SS to antagonize Escherichia coli and other S. marcescens strains but shows limited ability to kill bee symbionts. Furthermore, wild-type and T6SS-deficient S. marcescens strains achieved similar abundance and persistence in bee guts. Thus, an intact gut microbiota offers robust protection against this common pathogen, whose T6SSs do not confer the ability to compete with commensal species. IMPORTANCE Bacteria living within guts of animals can provide protection against infection by pathogens. Some pathogens have been shown to use a molecular weapon known as a T6SS to kill beneficial bacteria during invasion of the mouse gut. In this study, we examined how bacteria native to the honey bee gut work together to exclude the opportunistic pathogen Serratia marcescens. Although S. marcescens has a T6SS that can kill bacteria, bee gut bacteria seem resistant to its effects. This limitation may partially explain why ingestion of S. marcescens is rarely lethal to insects with healthy gut communities.

Keywords: Apis mellifera; Serratia marcescens; T6SS; colonization resistance; microbiota.

FIG 1

Serratia marcescens is eliminated from the guts of bees colonized by commensal species. (A) Total S. marcescens kz11 abundance in the midgut and hindgut of bees with a conventional, perturbed, or absent gut microbiota. Newly emerged bees from a single hive were inoculated with a conventional gut community (CV), inoculated with a conventional community and later treated with Tet (Tet), or kept microbiota free (MF). *, P < 0.05; ***, P < 0.005, Kruskal-Wallis test with Dunn’s multiple-comparison test. (B) S. marcescens abundance in bees colonized by individual gut taxa. MF bees were inoculated with representative strains of core gut taxa (Lactobacillus sp. Firm-4 DSM 26254 and DSM 26255, Lactobacillus sp. Firm-5 wkB8 and wkB10, S. alvi wkB2, G. apicola wkB1 and PEB0154, and G. apis PEB0162 and PEB0183), all isolates in combination, or homogenized gut of a bee collected from the hive. Bees were exposed for 1 day to 4 × 10⁸ S. marcescens cells/ml in sugar syrup and dissected 1 day after the end of exposure. S. marcescens abundance was quantified by counting CFU. Box and whisker plots show the minimum, first quartile, median, third quartile, and maximum. ***, P < 0.005, one-way ANOVA with Tukey’s multiple-comparison test.

FIG 2

Gut commensals compete with Serratia marcescens for space. (A) Diagram of the honey bee gut, showing a cross-section of the ileum. (B) Representative cross-sections of ilea from bees colonized by S. alvi (white) and S. marcescens (magenta). MF bees were inoculated with S. alvi wkB2 and exposed to S. marcescens after 5 days. S. alvi and S. marcescens were visualized using fluorescent probes that hybridize to 16S rRNA, while Sytox green stain indicates the presence of host nuclei and bacterial cells (green). (C) Cross-section of the ileum of a bee inoculated with gut homogenate (CV) and then exposed to S. marcescens (magenta). Sytox blue stain was used to label host nuclei and bacterial cells (green). Dashed white lines on the composite images outline the lumen of the gut.

FIG 3

Serratia marcescens kz11 uses a T6SS to antagonize other S. marcescens strains and one bee commensal. (A) Open reading frame (ORF) map of the T6SS loci in S. marcescens kz11, with structural genes in dark pink, auxiliary genes (such as adapters and effectors) in medium pink, genes for posttranscriptional regulation in light pink, and hypothetical genes encoding proteins of unknown function in white. (B) Recovery of E. coli K-12 Tn7-Gm^R after 4 h of coculture with the indicated S. marcescens strains. Competitions began with approximately 10⁷ E. coli cells and a 1:4 ratio of E. coli to S. marcescens. (C) Recovery of E. coli K-12 and S. marcescens strain Db11, kz19, and kz11 CFU after 4 h of coculture with WT S. marcescens kz11 (black), SmE1E2 (gray), or buffer (white). (D) Recovery of E. coli K-12 and gut commensal G. apicola wkB1, wkB7, and PEB0154, G. apis PEB0162 and PEB0183, and S. alvi wkB2 CFU after coculture with S. marcescens. Target CFU were measured through plate counts on selective media. Letters indicate significant differences between treatment groups (one-way ANOVA with Tukey’s multiple-comparison test, P < 0.05). ns, not significant.

FIG 4

T6SS-deficient Serratia marcescens strains do not differ from the WT strain in fitness within the bee gut. Percentages of sampled bees infected with S. marcescens (top) and abundance of S. marcescens in the midguts and hindguts of individuals (bottom) 1 to 4 days after exposure are shown. MF bees were exposed to WT S. marcescens (A) or to SmE1E2 (B). Conventionalized (CV) bees were exposed to WT S. marcescens (C) or to SmE1E2 (D). Bees were exposed to 4 × 10⁸ S. marcescens cells/ml in sugar syrup for 1 day. Abundance was quantified by counting CFU. Box and whisker plots show range, first and third quartiles, and median. *, P < 0.05; **, P < 0.005, Kruskal-Wallis test with Dunn’s multiple-comparison test.