The emergence of new animal models to better understand infectious processes in humans is a challenge for human health. The development of marine models is also important to better understand the impact of climate change on marine ecology. From a "One Health" perspective, exploring the interaction between pathogenic bacteria such as Vibrio parahaemolyticus and a marine animal model such as the sea anemone Exaiptasia pallida is promising.
Here, we use GFP-expressing V. parahaemolyticus to investigate different infection methods (balneation and injection) to gain a better understanding of the dissemination of V. parahaemolyticus in the whole organism. The data presented in this study showed that the animal responds physiologically similarly to both infection methods, resulting in tentacles retraction and tissue dissociation after 30 hours of infection (Figure 1). These morphological alterations were similar to those observed in E. pallida exposed to equivalent quantities of Vibrio spp. and during equivalent time of infection (Zaragoza et al., 2014).
E. pallida consists of 2 layers: the ectoderm, which is covered by a mucus layer, and the gastroderm, which contains Symbiodiniaceae (Figure 6). The two layers are separated by mesoglea, consisting mainly of myonemes (muscle cells) and few phagocytic cells (Mydlarz et al., 2008). The ectoderm and gastroderm contain different types of cells: epithelial cells, mucocytes, cnidocytes, and cells containing granules. All these cell types may be involved in tolerance responses to symbionts and bacteria from the microbiota and in the different stages of innate immune responses to pathogens.
Confocal microscopy localized Vp-GFP close to the ectoderm during the early period of infection in both methods. However, the quantity of adherent bacteria was higher in animals infected by injection than in animals infected by balneation (Figure 2). This could be explained by the stress-induced during injection causing animal retraction and "mucus-like" expulsion containing bacteria (Figure 1). Expulsed Vp-GFP could be trapped on the mucus covering the ectoderm. This hypothesis is supported by a significant presence of mucocytes in the ectoderm of E. pallida (Supplementary Figure 2).
Mucus and antimicrobial peptides are the most important components in the innate immune defense against pathogens in humans and invertebrates (Geitani et al., 2020). Mucus, which covers the whole surface of the epithelial layer, forms a barrier preventing the access of pathogenic bacteria to the cells (Fransolet et al., 2013). As shown in this study, V. parahaemolyticus colocalized with mucus found on the surface of the ectoderm (Figure 3). In the latter time of infection, the bacteria adhering to the ectoderm disappeared. It could be explained by different hypotheses. On one hand, the mutualistic and commensal microorganisms living in, which is known to improve fitness of the holobiont and compete with potential pathogens (Rohwer et al., 2002; Thompson et al., 2014). And on another hand, by the synthesis of antimicrobial peptides that recognized the pathogenic bacteria trapped in the mucus and destroyed them. Sea anemones are a rich source of biologically active compounds with hemolytic, cytotoxic, and antimicrobial activities (Kvetkina et al., 2021; Shnit-Orland & Kushmaro, 2009). A transcriptomic analysis performed in our laboratory identified an up-regulation of the expression of antimicrobial peptides in E. pallida infected by V. parahaemolyticus (Seneca et al., 2020). Further identification of these antimicrobial peptides is of interest as biologically active compounds with potential application to human health.
V. parahaemolyticus were detected in the gastroderm as early as 6 hours after injection, whereas they were not found in the gastroderm in animals infected by balneation until 24 hours. These data suggest that bacteria, introduced directly into the coelenteron bypassing the natural defense mechanisms of anemones provided by the mucus, have been directly recognized as pathogenic bacteria by immune cells present in the gastroderm (Fransolet et al., 2013). Moreover, the number of Vp-GFP decreased significantly after 24 hours in anemones infected by injection (Figure 4). This could be explained by a faster recruitment of immune cells caused by the syringe injury.
In Cnidaria, the gastroderm plays an important role in immune recognition of commensals and pathogens. This is supported by the localization of commensal algal symbionts found mainly along the gastroderm (Figure 6). Phagocytosis was identified as the primary mechanism required to the establishment of symbiosis with the dinoflagellates (Snyder et al., 2021). Phagocytosis of bacteria by endodermal epithelial cells from the gastric cavity has already been reported in Hydra, demonstrating the implication of this tissue in the immune response to pathogens and non-pathogenic organisms (Augustin & Bosch, 2010). Interestingly, in E. pallida infected by both methods, we observed that in the gastroderm Vp-GFP were localized in clusters reaching approximately 10 µm of diameter, suggesting a process of phagocytosis. Phagocytic immune cells named in cnidarians ‘’Amoebocytes’’ are involved in antimicrobial defenses of anthozoans (C Hutton & Smith, 1996; Mydlarz et al., 2008; Rosental et al., 2017). This cell population includes multiple cell types that, in response to wound healing, bleaching, and disease, migrate to the site of the lesion (Palmer et al., 2011).
Recently, fluorescence-activated cell sorting (FACS) and microscopy have been used to identify distinct cell populations engulfing bacteria or carboxylated fluorescent beads in the coral Pocillopora damicornis and the sea anemone Nematostella vectensis (Snyder et al., 2021). Gastrodermal cells, exhibiting amoebocytes behavior, have been identified upon exposure of Aiptasia sp. to microplastics (Okubo et al., 2018). Little is known about their implication in the anti-infection process of E. pallida. Our recent research on E. pallida amebocyte-like cells has shown their implication in the engulfment of Vp-GFP into vesicles thanks spectral flow cytometry cell sorting (Billaud et al., 2023). This result allowed us for the first time to reveal immune cells in E. pallida which present spectral similarities with “human monocyte” and are localized in mesenterial filaments. This tissue is involved in many processes including reproduction, locomotion, nutrition and immunity (Hartenstein & Martinez, 2019; Steinmetz, 2019; Van-Praët, 1985). In the present study, by using pHrodo™ Red Zymosan showing colocalization with the Vp-GFP in the mesenteries, we confirmed the conservation process of phagocytosis. Process that we already proved to be separated from the nutritive zone (Billaud et al., 2023).
In our experimental model of infection, the immune function in response to Vp-GFP is privileged, but at this stage of investigation we cannot exclude the managment of bacteria as well. In many invertebrate phyla, enterocytes of the digestive system operate as effective phagocytes, named as “enteric phagocytes” (Hartenstein & Martinez, 2019). The global changes occurring in situ which induce Vibrio spp. seasonal increases concentration urge to better understand Aiptasia infectious process (Baker-Austin et al., 2013; DePaola et al., 2003; Martinez-Urtaza et al., 2010).
In conclusion, by using GFP-expressing Vp, we have shown that we can infect the anemone E. pallida and localize the bacteria in the different tissues of the whole animal and quantify them. Here, we introduced and validated the two methods of infection: balneation and injection. The use of both techniques of infection may provide useful information about the physiological process of infection. Both methods showed colocalization of the bacteria with the mucus present on the ectoderm that disappeared at late time of infection. Both methods showed that Vp forms a cluster in the gastrodermis suggesting that this tissue is the main entry route of bacteria. The use of both techniques allows to localize the phagocytic cells found activated in the mesenterial filaments. This study opens new areas of research for the identification of antimicrobial peptides from Exaiptasia pallida and the characterization of the amebocyte-like cells involved in the anti-infectious process.