1. Aeromonas salmonicida proliferation and quorum sensing in response to mucins isolated from Atlantic salmon skin and intestine
Here, we cultured A. salmonicida in the presence of mucins purified from skin, pyloric caeca, proximal and distal intestine from five healthy Atlantic salmons, and analyzed growth rate and bacterial communication through quorum sensing molecules. Intestinal mucins enhanced A. salmonicida growth, whereas skin mucins had no effect. The increase in growth was positively affected by longer glycan chains of mucins and higher ratio of sialic acid. Enzymatic desialylation of mucins enhanced proliferation further. Mucins from all sites decreased the production of autoinducer-II (AI-II) signal molecules. Desialylation organ specifically altered the level of AI-II molecules produced by A. salmonicida. Thus, it appears that although mucins of the intestinal tract stimulated A. salmonicida growth, presumably reflecting that the pathogen senses the right target niche, the sialylated mucin glycans seem to act as a defense mechanism and limit the growth response. Mucin inhibition of QS may be an additional host defense mechanism influenced by the level of sialylation on salmon mucins.
János Tamás Padra, PhD
University of Gothenburg, Sweden
Mucins in Infection and Cancer Team

2. Furunculosis disease
Causative agent of furunculosis disease: Aeromonas salmonicida
Gram-negative, facultative anaerobic, non-motile rod
Furuncle-like swelling, ulcerative lesions, bowel inflammation – often fatal
Infects mainly salmonids but described in other fish species in contact with salmonids
A. salmonicida has a devastating impact on cultivated fish, particularly the Salmonids

3. Role of Mucins Form mucus layer with other secretions Barrier function
Dense carbohydrate coating can hold water
Protects against proteolysis
Lubrication
Other

4. Sites of interest
Skin

5. Mucin structure 2-11 residues in glycan chains
Skin O-glycans are short (2-5)
Intestinal mucin O-glycans are longer (up till 11)
Point at the border between mammalian and fish and point out that the structures there are from our samples

6. Previous results

7. The salmon O-glycans are highly sialylated (78-90%)
Jin et al., 2015

8. AB-PAS staining Skin Pyloric ceca Distal intestine Proximal intestine
Jin et al., 2015

9. Intestinal mucins promote A. salmonicida growth
Defined medium (DM)+Glucose
DM only
Time difference on the slide, glucose 5x growth speeed but mucins can be utilized, click in the next
30 min
160 min

10. Adhesion – dual role? DM+Glucose DM Legends on the figure
refer to Saras talk

11. ”Large” mucins nurture better
DM+Glucose DM

12. Do sialic acids promote growth?
DM+Glucose DM

13. Aeromonas salmonicida – tactics of colonization
A. salmonicida is provided with tools to degrade mucins and use as C-source
β-galactosidase
β-N-acetylhexosaminidase
GlcNAc-6-phosphate deacetylase
Glucosamine-6-phosphate deaminase
Sialidase?

14. Sialidase present?

15. Mucin building blocks on proliferation

16. Desialylated mucins further increase growth

17. Quorum sensing A form of communication with small molecules
Responsible for the group behaviour of bacteria
Alters metabolism, luminescence, virulence etc.
Three forms are known at present:
Intra-species communication
Inter-species communication
Vibrio intra genera communication

18. Aeromonas salmonicida ”language”
Autoinducer-1 – Acylhomoserine lactons
AsaI-autoinducer synthase
AsaR-autoinducer receptor
Autoinducer-2 system – A. hydrophila – 4,5-Dihydroxy-2,3-pentanedione
LuxS-autoinducer synthase
LuxP-autoinducer receptor
LuxU-autoinducer phosphorelay protein
LuxO-autoinducer regulator
Other system(s)???

19. Effect of autoinduction on growth
N-Butyryl-homoserine lactone
(BOC Sciences)
(S)-4,5-Dihydroxy-2,3-pentandione (DPD, AI-2) Autoinducer-2

20. BHL (C4 HSL) growth response

21. DPD growth response

22. Ai-2 timecourse

23. Mucins Ai-2 communication
Conclusion in title for all the slides possible move dc to the left

24. Reduction in sialic acids differentially affects QS in the organ groups

25. Conclusion
Intestinal mucins feed the bacteria but reduce the level of communication. Balancing between commensals and pathogens through AI-1 system?
Skin mucins seem to be inhibitory for A. salmonicida colonization and infection.
Sialic acids in Proximal intestine and Skin has an especially high importance in QS
AI-1 and AI-2 timecourse with mucin effect

26. Acknowledgements
Prof. Sara Lindén , Dr. Chunsheng Jin and the rest of the Mucins in Infection and Cancer Team Prof. Kristina Sundell Dr. Henrik Sundh