Aeromonas salmonicida proliferation and quorum sensing in response to mucins isolated from Atlantic salmon skin and intestine

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1 Aeromonas salmonicida proliferation and quorum sensing in response to mucins isolated from Atlantic salmon skin and intestine 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)

6 Previous results

7 The salmon O-glycans are highly sialylated (78-9%) Jin et al., 215

8 AB-PAS staining Skin Pyloric ceca Proximal intestine Distal intestine Jin et al., 215

9 Intestinal mucins promote A. salmonicida growth Defined medium (DM)+Glucose DM only AB reduction (%) *** *** AB reduction% * ** *** 1 Skin Pyloric Proximal Distal Control 1 Skin Pyloric Proximal Distal Control 3 min 16 min

10 Adhesion dual role? DM+Glucose Binding (OD45) Normalized proliferation (Alamar b. reduction) Normalized proliferation (Alamar b. reduction) Binding (OD45) DM

11 Large mucins nurture better DM+Glucose DM Normalized proliferation (Alamar b. reduction) 3 r 18 =.64, p< AVG weight of glycan structures (g/mol) Normalized proliferation (Alamar b. reduction) 6 r 18 =,57, p< AVG weight of glycan structures (g/mol)

12 Do sialic acids promote growth? DM+Glucose DM Normalized proliferation (Alamar b. reduction) r 18 =.5, p< Sialic acid residue/ chain Skin Pyloric Proximal Distal Normalized proliferation (Alamar b. reduction) r 18 =.63, p< Sialic acid residue/chain Skin Pyloric Proximal Distal

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 p<.5 Skin Proliferation difference from MOCK treated (%) Mock treated Sial A treated p<.1 Pyloric caeca Proliferation difference from MOCK treated (%) Proximal intestine Mock treated Sial A treated Mock treated Sial A treated Distal intestine Proliferation difference from MOCK treated (%) Proliferation difference from MOCK treated (%) -2 p<.1 Mock treated Sial A treated

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 Relative AB reduction ** ** *** *** *** *** BHL concentration (µm)

21 DPD growth response Relative AB reduction(%) DPD(µM)

22 AI-2 production (normalized for positive c.) Ai-2 timecourse OD56 AI T(h) Growth (OD56)

23 Mucins Ai-2 communication *** ** * Luminescence count Control Skin Pyloric Proximal Distal

24 Reduction in sialic acids differentially affects QS in the organ groups Skin Pyloric Difference from MOCK treated (%) -5 p<.1 Mock Sialidase A Difference from MOCK treated (%) Proximal -5 p<.1 Mock Sialidase A Distal 15 p<.1 15 p< Difference from MOCK treated (%) Difference from MOCK treated (%) -5-5 Mock Sialidase A Mock Sialidase A

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