Abstract

Polar and but not lateral flagellin proteins from Aeromonas hydrophila strain AH-1 (serotype O11) were found to be glycosylated. Top-down mass spectrometry studies of purified polar flagellins suggested the presence of a 403 Da glycan of mass. Bottom-up mass spectrometry studies showed the polar flagellin peptides to be modified with 403 Da glycans in O-linkage. The MS fragmentation pattern of this putative glycan was similar to that of pseudaminic acid derivative. Mutants lacking the biosynthesis of pseudaminic acid (pseB and pseI homologues) were unable to produce polar flagella but no changes were observed in lateral flagella by post-transcriptional regulation of the flagellin. Complementation was achieved by reintroduction of the wild-type pseB and pseI. We compared two pathogenic features (adhesion to eukaryotic cells and biofilm production) between the wild-type strain and two kinds of mutants: mutants lacking polar flagella glycosylation and lacking the O11-antigen lipopolysaccharide (LPS) but with unaltered polar flagella glycosylation. Results suggest that polar flagella glycosylation is extremely important for A. hydrophila AH-1 adhesion to Hep-2 cells and biofilm formation. In addition, we show the importance of the polar flagella glycosylation for immune stimulation of IL-8 production via toll-“like” receptor 5 (TLR5).

Highlights

  • Mesophilic Aeromonas spp. strains are important pathogens of humans and lower vertebrates, including amphibians, reptiles, and fish [1]

  • In the current study we show that polar but not lateral flagellins of A. hydrophila strain AH-1 from serotype O11 [16] are modified at multiple sites with putative glycans, which we propose to be pseudaminic acid-like moieties

  • The partial DNA sequence of the AH-1 lateral flagella cluster allowed us to identify two lateral flagellins (LafA1 and A2) in this strain. This compares with a single lateral flagellin observed in strain AH-3 [22]

Read more

Summary

Introduction

Mesophilic Aeromonas spp. strains are important pathogens of humans and lower vertebrates, including amphibians, reptiles, and fish [1]. Strains from Aeromonas hydrophila, A. veronii biovar veronii, or sobria are described as virulent for humans [3] and fish [4]; these strains are serologically related by their O-antigen lipopolysaccharide (LPS) (serotype O11) This has a known chemical structure containing O-polysaccharide chains of homogeneous chain length [5]. Several studies have shown that both the polar and lateral flagella systems of the mesophilic Aeromonas spp. are involved in adherence to both biotic and abiotic surfaces, as well as in the biofilm formation [10]. We recently characterized the O11 antigen LPS from the same strain A. hydrophila AH-1, and obtained mutants lacking this LPS structure [18] This allowed us to evaluate the importance of both flagellin and LPS in adhesion to Hep-2 cells and biofilm formation. The importance of polar flagella glycosylation for immune stimulation of IL-8 production via toll-“like” receptor 5 (TLR5) was evaluated

Results
Adhesion to HEp-2 Cells and Biofilm Formation
IL-8 Immune Stimulation
DDisisccuusssioionn
Experimental Section
DNA Techniques
Construction of Defined Mutants
Plasmid Constructions
Flagella Purification
4.11. Biofilm Formation
Full Text
Paper version not known

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call

Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.