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 15N‐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 15N 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.

The noncoding small RNA SsrA is released by Vibrio fischeri and modulates critical host responses

“…Significantly, while the symbionts traverse a long epithelium-lined migration path on their way to the crypts ( Fig 1A ), these host cells show a high degree of localized functional differentiation [ 49 ], with only the epithelium lining the crypt becoming labeled with SsrA ( S4 Fig ). We conclude that the crypt epithelium may be particularly susceptible to bacterial products presented by symbiont-derived OMVs and secreted molecules [ 50 ].…”

“…Given this apparently rapid turnover of SsrA within the crypt epithelium and the absence of evidence of bacterial lysis within the light-organ crypts by either transmission electron microscopy (TEM) [48] or live-dead stain [49], we hypothesized that the major source of the SsrA found within host cells is OMV-delivered. A higher-magnification image (Fig 1E) revealed abundant SsrA within the cytoplasm (but little detected in the nucleus) of crypt [49], with only the epithelium lining the crypt becoming labeled with SsrA (S4 Fig) . We conclude that the crypt epithelium may be particularly susceptible to bacterial products presented by symbiont-derived OMVs and secreted molecules [50].…”

Independent host- and bacterium-based determinants protect a model symbiosis from phage predation

“…While the exact means by which the squid protects V. fischeri remains unclear, we speculate that several processes with broader implications may play a role. For one, the epithelium in the LO is proficient at uptake of small particles, which might allow non-specific consumption or transport of phage before they can infect V. fischeri in the crypts, as has been shown with other eukaryotic cells (Bichet et al, 2021; Cohen et al, 2020; Nguyen et al, 2017). E. scolopes generates flow fields through the beating of cilia on the exterior of the LO, preferentially promoting aggregation of V. fischeri- sized particles around the LO entry points, and it is also possible that these flow patterns may actively exclude phage-sized particles from the LO surface, mechanically protecting V. fischeri as it begins the process of colonizing the squid (Nawroth et al, 2017).…”

Independent host- and bacterium-based determinants protect a model symbiosis from phage predation

“…Although the exact means by which the squid protects V. fischeri remains undefined, we speculate that several processes play a role. First, the LO epithelium is proficient at uptake of small particles, which might allow non-specific consumption or transport of phages before they can infect V. fischeri in the crypts, as has been shown with other eukaryotic cells ( Bichet et al, 2021 ; Cohen et al, 2020 ; Nguyen et al, 2017 ). Second, mucus has been shown in other models to adsorb phages.…”