In vivo imaging and genetic analysis link bacterial motility and symbiosis in the zebrafish gut

Abstract

Complex microbial communities reside within the intestines of humans and other vertebrates. Remarkably little is known about how these microbial consortia are established in various locations within the gut, how members of these consortia behave within their dynamic ecosystems, or what microbial factors mediate mutually beneficial host-microbial interactions. Using a gnotobiotic zebrafish-Pseudomonas aeruginosa model, we show that the transparency of this vertebrate species, coupled with methods for raising these animals under germ-free conditions can be used to monitor microbial movement and localization within the intestine in vivo and in real time. Germ-free zebrafish colonized with isogenic P. aeruginosa strains containing deletions of genes related to motility and pathogenesis revealed that loss of flagellar function results in attenuation of evolutionarily conserved host innate immune responses but not conserved nutrient responses. These results demonstrate the utility of gnotobiotic zebrafish in defining the behavior and localization of bacteria within the living vertebrate gut, identifying bacterial genes that affect these processes, and assessing the impact of these genes on host-microbial interactions.

Fig. 1.

Gut bacteria display diverse behaviors within the intestines of gnotobiotic zebrafish. (A and D) Whole-mount preparation of a live 3.5-dpf zebrafish colonized since 3 dpf with GFP-expressing P. aeruginosa PAO1 (PAO1 pMF230) demonstrates the transparency of the developing zebrafish intestine. Brightfield microscopy of the anterior intestine (segment 1, A) shows the intestinal lumen (lum) and the adjacent intestinal epithelium (ep). Fluorescence time-lapse microscopy of the same field (D) shows the movements of individual bacteria over the course of 10 frames, or 4 sec (D extracted from SI Movie 3). The locations of individual bacteria in the first (1) and the last (10) frames are numbered accordingly. (B and E) Brightfield (B) and fluorescence time-lapse (E) microscopy of the same field from a live 6-dpf zebrafish, colonized since 3 dpf with PAO1 pMF230, shows increasing bacterial density and behavioral complexity in the midintestine (junction of segments 1 and 2) over the course of 10 frames or 2.6 sec (E extracted from SI Movie 4). Note that the intestines shown in D and E both contain bacteria that are nonmotile in association with the host epithelium or luminal contents (yellow), whereas other bacteria exhibit high rates of motility in both ascending (distal to proximal; red tracks) and descending (green tracks) directions. Note that ascending and descending bacteria were tracked for only the first several frames because they quickly moved out of the focal plane; the first and last frames over which bacteria were tracked are numbered. (C and F) Brightfield (C) and fluorescence time-lapse (F) microscopy of a live 4.5-dpf zebrafish colonized since 3 dpf with DsRed-expressing E. coli MG1655 (MG1655 pRZT3) showing movement of luminal bacteria (green tracks) in the midintestine (segment 1). Over the course of 14 frames or 14 sec (F extracted from SI Movie 5), some bacteria appear adherent to the epithelium or luminal structures (yellow track), whereas most bacterial motion is synchronous and attributed to intestinal motility (green tracks). Anterior is to the left, and dorsal is to the top in all images. (Scale bars: 20 μm.)

Fig. 2.

TEM of gut bacteria in gnotobiotic zebrafish. Transverse sections are shown that include segments 1 and 2 of the intestine of a 6-dpf zebrafish colonized since 3 dpf with P. aeruginosa strain PAO1. (A) Bacteria are clustered together in the luminal space, and some remain close to the host epithelium (arrowhead in A). (B and C) Bacteria (arrowheads) are also observed in association with unidentified electron-dense laminated objects in the lumen (arrows in B) and undergoing fission (C). (Scale bars: A, 3 μm; B, 1 μm; C, 500 nm.)

Fig. 3.

The impact of P. aeruginosa flagellar mutants on host responses in gnotobiotic zebrafish. Expression levels of serum amyloid a (saa), myeloperoxidase (mpo), fasting-induced adipose factor (fiaf), and carnitine palmitoyltransferase 1a (cpt1a) were assessed by qRT-PCR by using RNA extracted from whole 6-dpf larvae colonized since 3 dpf with P. aeruginosa PAK wild-type strain, PAK exsA deletion mutant (PAK ΔexsA), PAK retS deletion mutant (PAK ΔretS), PAK pilA deletion mutant (PAK ΔpilA), PAK fliC deletion mutant (PAK ΔfliC), or PAK motABCD deletion mutant (PAK ΔmotABCD). Data from biological duplicate pools (– animals per pool) were normalized to 18S rRNA levels. Normalized mRNA levels in colonized fish were referenced against age-matched GF controls (ΔΔC_t method), and the results are expressed as mean percent change relative to the PAK wild-type strain ± SEM. ∗∗∗, P < 0.0001; ∗∗, P < 0.001 compared with wild-type, based on a two-tailed Student's t test.