Tracking the cargo of extracellular symbionts into host tissues with correlated electron microscopy and nanoscale secondary ion mass spectrometry imaging

Abstract

Extracellular bacterial symbionts communicate biochemically with their hosts to establish niches that foster the partnership. Using quantitative ion microprobe isotopic imaging (nanoscale secondary ion mass spectrometry [NanoSIMS]), we surveyed localization of ¹⁵ N-labelled molecules produced by the bacterium Vibrio fischeri within the cells of the symbiotic organ of its host, the Hawaiian bobtail squid, and compared that with either labelled non-specific species or amino acids. In all cases, two areas of the organ's epithelia were significantly more ¹⁵ N enriched: (a) surface ciliated cells, where environmental symbionts are recruited, and (b) the organ's crypts, where the symbiont population resides in the host. Label enrichment in all cases was strongest inside host cell nuclei, preferentially in the euchromatin regions and the nucleoli. This permissiveness demonstrated that uptake of biomolecules is a general mechanism of the epithelia, but the specific responses to V. fischeri cells recruited to the organ's surface are due to some property exclusive to this species. Similarly, in the organ's deeper crypts, the host responds to common bacterial products that only the specific symbiont can present in that location. The application of NanoSIMS allows the discovery of such distinct modes of downstream signalling dependent on location within the host and provides a unique opportunity to study the microbiogeographical patterns of symbiotic dialogue.

Keywords: 15N-labeled bacteria; host-microbe communication; squid-vibrio.

Figure 1

Relevant morphology, anatomy, and ultrastructure of the host, Euprymna scolopes. (A) A diagram of a ventral dissection revealing the mantle cavity with relevant tissues and organs that were examined in this study. br, brain; dg, digestive gland; lo, light organ; mc, mantle cavity; st, statolith; w, white body. (B) A diagram of the bilateral light organ. Relevant surface features (left half) include the juvenile-specific ciliated epithelium with two appendages, anterior (aa) and posterior (pa), and the field of cilia surrounding three pores. Relevant interior features include the three independent regions of symbiont migration and residence. ante, antechamber diverticulum; bs, blood sinus; cr, crypt; du, duct; endo, endothelial cells lining the blood sinus; hc, hemocytes; po, pores. (C) A TEM with symbiont-containing crypt region of the light organ, false colored to correspond to colored regions in (B). cr 1–3, crypts 1–3; ce, crypt epithelium; ct, connective tissue; is, ink sac; ref, reflector.

Figure 2

Uptake of isotope-labeled V. fischeri products into light-organ epithelial cells of a juvenile host. (A) ¹⁵N-enrichment (expressed as δ¹⁵N) in the light organ of a newly-hatched animal at 2 h post-exposure to 10⁶ intact ¹⁵N-labeled V. fischeri cells/ml of seawater; the data were obtained from all nuclei in the mosaic, independent of enrichment level. A composite TEM micrograph and the corresponding mosaic of 65 individual, high-resolution NanoSIMS images. Labeling was abundant and appeared as hotspots in the nucleoli of all light-organ epithelia (e.g., yellow arrows) with which V. fischeri cells interface, i.e., the surface ciliated field, here showing the epithelia of the anterior appendage (ap), the pore (po), the duct/antechamber (du/ante) and crypts (crypt epithelia, ce 1–3); ct, connective tissue; ref, reflector. Inset, upper left, the average level of ¹⁵N-enrichment in cell nuclei in different epithelial regions. Data were derived from the mosaic (Ap, n=56; Po, n=58; Du, n=36; Cr, n=142). Lower case letters indicate statistically significant differences between the light-organ compartments (Kruskal-Wallis test with Nemenyi post hoc, P<0.0001). Error bars represent 1 standard deviation. (B) ¹⁵N-labeled V. fischeri cells migrating through the ducts. (C, C’) TEM (left) and NanoSIMS (right) images of two cells of the crypt epithelium, defining the region scanned (start – end) for ¹⁵N/¹⁴N ratio within the nucleus, showing strong enrichment in the nucleoli (green arrows) and euchromatin (eu); ht, heterochromatin. C’, NanoSIMS at two sensitivities (main, lower and inset higher sensitivity, respectively) to highlight less intense labeling of the chromatin. (D) A representative image of the quantification of ¹⁵N enrichment through the region scanned in (C). Points are averages of NanoSIMS scans (n=10) +/− one standard deviation.

Figure 3

The ¹⁵N-enrichments in tissues not directly associating with the symbiont. Nuclei and nucleoli (hotspots) in light organ non-epithelial tissues (A-C) and in epithelial tissues in other squid organs (D-F) after 3.5 h inoculation with ¹⁵N-labelled V. fischeri cells. Left panels show tissue structure (NanoSIMS ¹²C¹⁴N⁻ image). Right panels show corresponding, quantified NanoSIMS ¹⁵N/¹⁴N ratio image. (G) Representative ¹⁵N-enrichments in nuclei of a single animal (excluding the nucleolus when visible) in different tissues across the body. Non-epithelial tissue inside the light organ (l.o.; n=72): connective tissue (n=59), and tissues outside of the light organ: gill epithelia (n=34), blood vessel endothelial cells (n=48), gut epithelia (n=100), digestive gland cells (n=31), brain (n=16); retina (r; n=11), tentacles (n=29), white body (n=27), and epithelium supporting statolith (n=27). Only enriched tissues were considered in the statistical analysis. Lower case letters indicate statistically significant differences between the different tissues (Kruskal-Wallis test with Nemenyi post hoc, P<0.0001). Points are averages +/− one standard deviation. Dashed line, limit of detection, or no difference between the control value for the natural distribution of ¹⁵N over ¹⁴N in the sample.

Figure 4

¹⁵N-enrichment (δ¹⁵N) in juvenile tissues with ¹⁵N-labeled non-symbiotic Vibrionaceae cells. Animals were analyzed at 3.5 h post inoculation. (A/B) Left panels, tissue structure (NanoSIMS ¹²C¹⁴N⁻ image); right panels, corresponding quantified image of NanoSIMS ¹⁵N/¹⁴N ratio; nucleoli, green arrows. Ap, anterior appendage of the light organ. (C) Average ¹⁵N enrichment in the nucleus (n = 236, V. campbellii; n=196, P. leiognathi) and nucleolus (n = 73, V. campbellii; n=57, P. leiognathi) of the light-organ surface epithelium from 5–7 individual animals. Points are average +/− one standard deviation. Lower case letters indicate statistically significant differences between the light-organ compartments (Kruskal-Wallis test with Nemenyi post hoc, P<0.0001).

Figure 5

¹⁵N enrichment of light organ regions upon exposure to outer membrane vesicles (OMVs). (A) Relative Labeling Index (RLI) for ¹⁵N-enrichment in light organs exposed for different time periods. Left, to intact V. fischeri cells for 2 h (n=3) or 3.5 h (n=6); 6–10 nucleoli/region for each animal; right, to purified OMVs for 1 h (n=5) or 3 h (n=4). Ap, anterior appendage (Cells: n=37, 2h; n=90, 3.5 h; OMVs: n=44, 1h; n=48, 3 h); Po, pore (Cells: n=44, 2 h; n=54, 3.5h; OMVS: n=55, 1h; n=42, 3 h); Cr, crypt (cells: n=80, 2 h; n=73, 3.5 h; OMVs: n=65, 1h; n=44, 3 h). Points are averages +/− one standard deviation. Lower case letters indicate statistically significant differences between the light-organ compartments (Two-way ANOVA with Tukey post hoc, P<0.0001). (B) Quantification of the distribution of ¹⁵N-enrichment in subcellular regions of the anterior appendage and crypt in animals exposed to the OMVs of V. fischeri (n=3), V. campbellii (n=5), and E. coli (n=3). For each animal, cytoplasm n =31–64; nucleus n= 101–373; nucleolus n=829–868. Points are averages +/− one standard deviation. Lower case letters indicate significant differences between the cell organelles (Kruskal-Wallis test with Nemenyi post hoc, P<0.0001); asterisks indicate significant differences between bacteria species (Kruskal-Wallis test, P<0.0001). (C) A comparison of ¹⁵N enrichment in epithelium of the superficial anterior appendage of the light organ between animals exposed to labeled OMVs from V. fischeri or V. campbellii. Images of NanoSIMS ¹⁵N/¹⁴N ratios; inset, corresponding mosaic TEM micrographs; nucleoli, green arrows.