A novel gnotobiotic experimental system for Atlantic salmon (Salmo salar L.) reveals a microbial influence on mucosal barrier function and adipose tissue accumulation during the yolk sac stage.

Gnotobiotic models have had a crucial role in studying the effect that commensal microbiota has on the health of their animal hosts. Despite their physiological and ecological diversity, teleost fishes are still underrepresented in gnotobiotic research. Moreover, a better understanding of host-microbe interactions in farmed fish has the potential to contribute to sustainable global food supply. We have developed a novel gnotobiotic experimental system that includes the derivation of fertilized eggs of farmed and wild Atlantic salmon, and gnotobiotic husbandry of fry during the yolk sac stage. We used a microscopy-based approach to estimate the barrier function of the skin mucus layer and used this measurement to select the derivation procedure that minimized adverse effects on the skin mucosa. We also used this method to demonstrate that the mucus barrier was reduced in germ-free fry when compared to fry colonized with two different bacterial communities. This alteration in the mucus barrier was preceded by an increase in the number of cells containing neutral mucosubstances in the anterior segment of the body, but without changes in the number of cells containing acidic substances in any of the other segments studied along the body axis. In addition, we showed how the microbial status of the fry temporarily affected body size and the utilization of internal yolk stores during the yolk sac stage. Finally, we showed that the presence of bacterial communities associated with the fry, as well as their composition, affected the size of adipose tissue. Fry colonized with water from a lake had a larger visceral adipose tissue depot than both conventionally raised and germ-free fry. Together, our results show that this novel gnotobiotic experimental system is a useful tool for the study of host-microbe interactions in this species of aquacultural importance.

Elucidating bacterial adhesion to mucosal surface by an original AFM approach.

BACKGROUND: Fish skin represents an ancient vertebrate mucosal surface, sharing characteristics with other mucosal surfaces including those of the intestine. The skin mucosa is continuously exposed to microbes in the surrounding water and is therefore important in the first line defense against environmental pathogens by preventing bacteria from accessing the underlying surfaces. Understanding the microbe-host interactions at the fish skin mucosa is highly relevant in order to understand and control infection, commensalism, colonization, persistence, infection, and disease. Here we investigate the interactions between the pathogenic bacteria Aeromonas salmonicida (A. salmonicida) and Yersinia ruckeri (Y. ruckeri), respectively, and the skin mucosal surface of Atlantic salmon fry using AFM force spectroscopy. RESULTS: The results obtained revealed that when retracting probes functionalized with bacteria from surfaces coated with immobilized mucins, isolated from salmon mucosal surfaces, rupture events reflecting the disruption of adhesive interactions were observed, with rupture strengths centered around 200 pN. However, when retracting probes functionalized with bacteria from the intact mucosal surface of salmon fish fry no adhesive interactions could be detected. Furthermore, rheological measurements revealed a near fluid-like behavior for the fish fry skin mucus. Taken together, the experimental data indicate that the adhesion between the mucin molecules within the mucous layer may be significantly weaker than the interaction between the bacteria and the mucin molecules. The bacteria, immobilized on the AFM probe, do bind to individual mucins in the mucosal layer, but are released from the near fluid mucus with little resistance upon retraction of the AFM probe, to which they are immobilized. CONCLUSION: The data provided in the current paper reveal that A. salmonicida and Y. ruckeri do bind to the immobilized mucins. However, when retracting the bacteria from intact mucosal surfaces, no adhesive interactions are detected. These observations suggest a mechanism underlying the protective function of the mucosal surface based on the clearing of potential threats by adhering them to loosely attached mucus that is subsequently released from the fish skin.

Co association of mucus modulating agents and nanoparticles for mucosal drug delivery.

Nanoparticulate drug delivery systems (nDDS) offer a variety of options when it comes to routes of administration. One possible path is crossing mucosal barriers, such as in the airways and in the GI tract, for systemic distribution or local treatment. The main challenge with this administration route is that the size and surface properties of the nanoparticles, as opposed to small molecular drugs, very often results in mucosal capture, immobilization and removal, which in turn results in a very low bioavailability. Strategies to overcome this challenge do exist, like surface 'stealth' modification with PEG. Here we review an alternative or supplemental strategy, co-association of mucus modulating agents with the nDDS to improve bioavailability, where the nDDS may be surface modified or unmodified. This contribution presents some examples on how possible co-association systems may be achieved, using currently marketed mucolytic drugs, alternative formulations or novel agents.

Alterations in mucus barrier function and matrix structure induced by guluronate oligomers.

The effect of guluronate oligomers on the barrier properties of mucous matrices was investigated in terms of the mobility of nanoparticles in mucous matrices by fluorescence recovery after photobleaching (FRAP), cellular uptake of nanoparticles in mucus secreting cells (HT29-MTX), and mucin matrix architecture by scanning electron microscopy (SEM). Guluronate oligomers improved nanoparticle mobility in both native and highly purified mucus matrices and improved cellular uptake of nanoparticles through a mucus layer. Addition of guluronate oligomers to mucin matrices resulted in a decrease in the density of network cross-links and an increase in matrix pore size. Based on these data, we conclude that guluronate oligomers are able to improve nanoparticle mobility in several mucus matrices and alter network architecture in mucin matrices in a manner that suggests a reduction in barrier function. As such, there may be a potential application for guluronate oligomers in mucosal delivery of nanomedicines.

Oligosaccharides as modulators of rheology in complex mucous systems.

Mucus rheology is integral to physiological function with the exact secretion rheology resulting from the macromolecular components, both mucin and nonmucin, and the interactions between these macromolecules. Here we present data demonstrating that low-molecular-weight guluronic acid oligomers extracted from alginate are able to disrupt intermolecular interactions in both purified and native mucous systems, resulting in rheological changes that are compatible with a lower cross-link density and thus reduced resistance to deformation. Additionally, these changes are associated with altered physiological function, raising the possibility of the use of such oligomers in biomedical applications.