Fig 1.
Symbiont noncoding RNA, SsrA, localizes within the crypt epithelium.
(A) Diagram of a juvenile squid showing the anatomical location (left) and internal aspects (middle) of the light organ, illustrating one of its two pairs of cea, and three entry pores (“p”) through which the symbionts reach the migration path to internal crypts (“c”). Gray dots inside the sinus of the cea represent symbiosis-induced trafficking of hemocytes. (Right) Illustration of the close contact between the V. fischeri population (green) and the light-organ epithelial cells in a crypt. (B) Relative proportions of types of V. fischeri RNAs present in squid H-lymph or in the RNA cargo of OMVs (S1 Data). (C) Volcano-plot representation of fold-change in gene expression (log2FC) of the RNA cargo in OMVs produced by WT or the ssrA-deletion mutant ΔssrA strain; the only significant difference in RNA content is the presence (in WT) or absence (in ΔssrA) of SsrA. Transcripts with evidence for significant differential expression (FDR < 0.05) are colored in red (S1 Data). (D) Localization of symbiont SsrA transcript by confocal microscopy, 24 h after colonization by WT or ΔssrA bacteria. Left: merged images with orthogonal views; other panels: images of individual labels. (E) Higher magnification of WT V. fischeri cells (green) colonizing the light organ, showing the location of SsrA transcript (magenta) within the cytoplasm of host epithelial cells. cea, ciliated epithelial appendages; FDR, false discovery rate; H-lymph, hemolymph; OMV, outer membrane vesicle; sRNA, small RNA; WT, wild type.
Table 1.
List of abundant small, noncoding RNAs
.
Fig 2.
The ΔssrA mutant is able to initiate colonization normally, but persists poorly.
(A) The number of V. fischeri cfu per light organ at 24 or 48 h post colonization in animals colonized by WT, ΔssrA, or the genetically complemented ΔssrA + ssrA strains. A 1-way Kruskal–Wallis ANOVA, followed by DMC was performed. Ten squid/condition from six different clutches were used in this experiment (n = 60). (B) Levels of hemocyte trafficking into the light organ’s anterior appendages at 16 and 18 h post colonization. Significant differences, as indicated by a 1-way ANOVA with TMC (n = 10) are shown. (C) Levels of hemocyte trafficking 3 h after exposure to 100 μg of OMVs per ml. Significant differences, as indicated by a 1-way ANOVA with TMC (n = 10) are given. (D) The degree of apoptosis in the light organ’s ciliated epithelium, as indicated by the number of acridine orange-staining nuclei, in animals that were uncolonized (APO) or colonized by either WT or the ΔssrA strain (S5 Fig). Statistical significance was determined by a 1-way ANOVA, followed by DMC (n = 10). (E) Specific luminescence (RLU per cfu) of symbionts either within the light organ, or within a homogenate of the light organ, of a 24-h juvenile. Animals were uncolonized (APO) or colonized by either WT or the ΔssrA strain. Significant differences are given, as indicated by a 1-way ANOVA, followed by DMC. The experiment was repeated twice with the same outcome. P value code: ****<0.0001; ***<0.0002; **<0.001; *<0.021 for all graphs. Numerical values for all graphs can be found at S2 Data. APO, aposymbiotic; cfu, colony-forming units; DMC, Dunn’s multiple comparison test; OMV, outer membrane vesicle; RLU, relative light units; TMC, Tukey’s multiple comparison test; WT, wild type.
Fig 3.
Host responses to colonization by WT or ΔssrA differ.
(A) Paraffin-section image of a WT-colonized light organ after 48 h, illustrating how crypt-cell cytoplasmic volume was measured. The nuclear area (black dotted line) was subtracted from the total cell area (yellow line). The areas of 10 epithelial cells in crypt 1, just inside of (i.e., distal to) the bottleneck, were measured per light organ. (A’) Cytoplasmic volume of the crypt epithelium at 24 and 48 h post inoculation with WT, ΔssrA or Δlux strains, or left uncolonized (APO). (n = 5). (B) Heat map depicting fold-change differences in significantly differently expressed genes in light organs colonized by WT, the ΔssrA mutant, or its genetically complemented (ΔssrA + ssrA) strain. Genes that are up-regulated in ΔssrA-colonized animals compared to WT-colonized are indicated in bold. The replicate number for each condition (S3 Data) is indicated beneath the heat map. (C) Localization of the laccase-3 transcript (magenta) on one side of the light organ using hybridization chain-reaction fluorescence in situ hybridization labeling. Light organs were colonized by the indicated strain of GFP-labeled symbionts (green). (C’) Quantification of laccase-3 signal using relative fluorescence intensity of a Z-series image of the light organ (n = 9). P values were calculated using a 1-way ANOVA with TMC. (D) Relative expression of laccase-3 after 24 h post colonization in light organs colonized by WT, ΔssrA, or ΔssrA + ssrA, determined by qRT-PCR. Expression was normalized to ribosomal protein S19 and expressed as 2^ΔΔCT normalized to WT expression. Significant differences are indicated by a 1-way ANOVA with TMC (n = 3). Data are presented as the mean ± SD. P value code: ****<0.0001; ***<0.0002; **<0.001; *<0.021. Numerical data can be found at S3 Data. APO, aposymbiotic; GFP, green fluorescent protein; qRT-PCR, quantitative real-time PCR; TMC, Tukey’s multiple comparison test; WT, wild type.
Fig 4.
The absence of SsrA in the epithelium, but not SsrA activity in the symbiont, weakens the host.
(A) Visualization by HCR of SsrA transcript (magenta) in a whole-mount light organ, 24 h after colonization with a GFP-labeled ΔsmpB strain of Vf (green). A representative confocal image indicates that symbiont SsrA transcript is within the crypt epithelial cells. Scale bar, left panel = 30 μm; see S6A Fig. (B) Kaplan-Meier survival plot of juvenile squid colonized by WT, ΔssrA, the complement (ΔssrA + ssrA), or ΔsmpB strains. A calculation based on three separate experiments (S6B Fig) is shown, consisting of WT (n = 59), ΔssrA (n = 59), ΔssrA + ssrA (n = 57), or ΔsmpB (n = 56) colonized animals. Survival-curve analysis by a log-rank Mantel-Cox test, with Bonferroni multiple-testing adjustment for pairwise comparisons. P value = 0.016. (C) Dry-weight measurements of juvenile squid immediately after hatching (“Hatch”) or at 4 d post hatching when kept APO or colonized with WT, ΔssrA, or a dark-mutant (Δlux) strain. Analysis by a 1-way ANOVA with TMC indicated that hatchlings had a significantly greater dry weight compared to all other conditions (P < 0.0001). Data are represented as the median, with 95% confidence intervals. (D) Left: dorsal view of a juvenile squid, illustrating the location of the internal yolk sac (dotted box). Right: representative confocal Z-stack image of a hatchling yolk sac stained with the lipophilic lipidspot-488 (green) and depicting how the area (dotted region) was measured; scale bar = 100 μm. (D’) Quantification of the internal yolk-sac area was determined from confocal Z-stack images. Data are represented as means ± SD, analyzed by a 1-way ANOVA with TMC. (E) Representative SEM images of the yolk sac of a hatchling squid and animals colonized for 2 d by the WT or the ΔssrA-mutant strain. Scale bar = 100 μm; P value code: ****<0.0001; ***<0.0002; **<0.001; *<0.021 for all figures. Numerical values found at S4 Data. APO, aposymbiotic; GFP, green fluorescent protein; HCR, hybridization chain reaction; ns, not significant; SEM, scanning electron microscopy; TMC, Tukey’s multiple comparison test; Vf, V. fischeri; WT, wild type.
Fig 5.
SsrA taken up by hemocytes may be detected through host cytosolic RNA sensors.
(A) HCR visualization of SsrA transcript (magenta) in a whole-mount light organ, 24 h after colonization with a GFP-labeled WT strain (green). A representative confocal image indicates that symbiont SsrA transcript is within the crypt epithelial cells (nuclei, TO-PRO-3; blue). White arrow indicates symbiont SsrA transcript in a host hemocyte (“h”) within the crypt space. (B) Changes in gene expression 30 min after challenging isolated juvenile hemocytes with OMVs purified from exponential cultures of either WT or ΔssrA cells, or after addition of DPBS (“Mock”). Relative expression levels were determined by qPCR for C3 and RIG-I. Error bars = SD, (n = 3); P value code: *<0.021 (S5 Data). (C) A hypothetical model for SsrA modulation of host immune response. During WT colonization, OMVs containing SsrA enter the host cell. The OMV cargo is released into the cytoplasm, where SsrA associates with RIG-I, triggering a signaling cascade that induces RIG-I’s own up-regulation as well as the activation of an IFN-like response for symbiont modulation. During ΔssrA colonization, there is no SsrA to associate with RIG-I. As a consequence, there is no modulation of IFN response, leading to inflammation. This result leads to a continued production of antibacterial laccase and cell swelling and an overall diminished robustness of the host due to the rapid depletion of its yolk sac, resulting from the demands of the increased immune response. C3, complement protein 3; DPBS, Dulbecco’s phosphate-buffered saline; GFP, green fluorescent protein; HCR, hybridization chain reaction; IFN, interferon; OMV, outer membrane vesicle; qPCR, quantitative PCR; RIG-I, retinoic-acid inducible gene-I; WT, wild type.