Aeromonas salmonicida Growth in Response to Atlantic Salmon Mucins Differs between Epithelial Sites, Is Governed by Sialylated and N -Acetylhexosamine-Containing O -Glycans, and Is Affected by Ca ²⁺

Abstract

Aeromonas salmonicida causes furunculosis in salmonids and is a threat to Atlantic salmon aquaculture. The epithelial surfaces that the pathogen colonizes are covered by a mucus layer predominantly comprised of secreted mucins. By using mass spectrometry to identify mucin glycan structures with and without enzymatic removal of glycan residues, coupled to measurements of bacterial growth, we show here that the complex Atlantic salmon intestinal mucin glycans enhance A. salmonicida growth, whereas the more simple skin mucin glycans do not. Of the glycan residues present terminally on the salmon mucins, only N-acetylglucosamine (GlcNAc) enhances growth. Sialic acids, which have an abundance of 75% among terminal glycans from skin and of Ͻ50% among intestinal glycans, cannot be removed or used by A. salmonicida for growth-enhancing purposes, and they shield internal GlcNAc from utilization. A Ca 2ϩ concentration above 0.1 mM is needed for A. salmonicida to be able to utilize mucins for growth-promoting purposes, and 10 mM further enhances both A. salmonicida growth in response to mucins and binding of the bacterium to mucins. In conclusion, GlcNAc and sialic acids are important determinants of the A. salmonicida interaction with its host at the mucosal surface. Furthermore, since the mucin glycan repertoire affects pathogen growth, the glycan repertoire may be a factor to take into account during breeding and selection of strains for aquaculture.KEYWORDS Atlantic salmon, GlcNAc, HexNAc, NeuAc, O-glycan, calcium, furunculosis, mucin, proliferation T he Atlantic salmon is an anadromous salmonid with a life cycle that includes both freshwater (FW) and seawater (SW) stages. This is reflected in current husbandry protocols, where juveniles (parr) are grown in FW until they have undergone a developmental stage, i.e., the parr-smolt transformation or smoltification, preparing the fish for a life in the sea. After successful smoltification, wild fish, called smolts, normally migrate from their native FW streams into the sea. Farmed smolts are instead usually transferred to large net pens in the sea for growth until slaughter (this growth phase is termed "on-growth"). The global yearly production rate of Atlantic salmon, Salmo salar L., exceeded 2.3 million tons in the year 2014, and the yearly production is expected to continue to grow even more (2).Fish primary barriers, the skin, gills, and gut, are in direct contact with the environment and capable of nurturing both bacteria and viruses (3). Diseases are easily spread

Helicobacter suis infection alters glycosylation and decreases the pathogen growth inhibiting effect and binding avidity of gastric mucins

“…Instead, we observed a positive correlation between growth and the abundance of acidic and fucosylated structures and a negative correlation with the abundance of galactose terminating structures on the mucins, and culture with fucose and galactose monosaccharides supported these effects. We have previously cultured another bacteria (Aeromonas salmonicida) with twice as high concentration of galactose without inhibition of growth, 21 suggesting that the inhibition of growth seen in the current study is not affecting all bacteria. Each mucin carried 35-75 glycans, and the growth response to the mucins is thus likely to be the sum of multiple glycans.…”

“…18 The high diversity of mucin O-glycans provides an extensive repertoire of bacterial attachment sites and O-glycans take part in host-pathogen interactions in the gastrointestinal tract. [19][20][21][22] Mucus can be beneficial for bacteria by providing nutrients for growth 23,24 and protecting bacteria from the harmful environment. 25 Mucins can also prevent close contact of the pathogen with the gastric epithelial cells by binding, which may contribute to removing the bacteria from their niche with the gastric emptying.…”

Role of Sialic Acid in Brachyspira hyodysenteriae Adhesion to Pig Colonic Mucins

“…Twenty-five microliters of colonic mucins from five B. hyodysenteriae-infected pigs and six healthy pigs (2) and the carbohydrates galactose, fucose, NeuAc, NeuGc, GlcNAc, and GalNAc (Sigma-Aldrich, St. Louis, MO, USA) diluted in PBS (100 g/ml) were added to a sterile Falcon 96-well plate (Corning Life Sciences, Corning, NY, USA). B. hyodysenteriae was harvested from the agar plates, washed in PBS, and resuspended in carbohydrate-free medium (40) to an optical density at 600 nm (OD 600 ) of 0.1. RealTime-Glo cell viability assay reagent (Promega, Madison, WI, USA) was diluted 1/1,000 in the bacterial suspension, and 225 l of the bacterial suspension with the reagent was added per well.…”

Biological and Ecological Roles of External Fish Mucus: A Review

“…Although specific molecules responsible for this activity have not yet been identified, O'toole et al [126] suggested that free amino acids and carbohydrates could act as chemoattractant molecules in trout mucus (skin and intestinal) while Klesius et al [127] suggested that a lectin-like substance might be responsible for bacterial chemotaxis in catfish (Ictalurus punctatus). In recent studies, Padra and colleagues found that fish mucosal sialic acids and specifically N-acetylglucosamine (GlcNac) play an important role in the pathogenic bacteria Aeromonas salmonicida growth and binding to its host [152,153].…”