Investigation of glycosylation in flagellin biosynthesis and LPS O-antigen biosynthesis in Aeromonas caviae

Aeromonas species are Gram-negative facultative anaerobic bacteria which are
widespread in fresh water and salt water. Aeromonas caviae and Aeromonas
hydrophila are two kinds of mesophilic aeromonads which belong to genus
Aeromonas and are emerging as major pathogens in humans. Flagella are important
pathogenic factors of bacteria and there are two kinds of flagella discovered in A.
caviae and A. hydrophila which are polar flagella and lateral flagella (Rabaan et al.
2001; Tabei et al. 2009; Canals et al. 2006a).
The flagella of Aeromonas are glycosylated and investigation of glycosylation in
flagellin biosynthesis is valuable for the understanding of pathogenicity of Aeromonas
species. The aim of this project is to investigate the glycosylation during flagellin
biosynthesis in A. caviae. It has been discovered that the flagella of A. caviae are
glycosylated 6 to 8 times by Pse5Ac7Ac which is under the control of Maf1 which is
known as flagellin glycosyl-transferase (Parker et al. 2012). Theoretically, Maf1 of A.
caviae Sch3N has the ability to transfer activated pseudaminic acid (CMP-
Pse5Ac7Ac) to the hydroxyl group of serine and threonine residues in the central
immunogenic D2/D3 domain of flagellin of A. caviae Sch3N (Parker et al. 2012; Tabei
et al. 2009). The details of how Maf1 interacts with flagellin will be explored in this
project.
CMP-Pse5Ac7Ac which is the substrate for glycosylation is generated from
UDP-GlcNAc. The biosynthesis of CMP-Pse5Ac7Ac is under the control of flm locus
including flmA, flmB, neuA, neuB, flmD. The pseudaminic acid biosynthetic pathway
has been confirmed in Campylobacter jejuni and related enzymes are homologous
proteins of A. caviae (Schoenhofen et al. 2006). The proteins encoded by the flm
genes will be applied in this project to investigate the biosynthesis of CMP-
Pse5Ac7Ac. In addition, there is evidence that the LPS O-antigen incorporates with
Pse5Ac7Ac. The substrate of O-antigen glycosylation is also CMP-Pse5Ac7Ac
(Tomás 2012). The connections between glycosylation in LPS O-antigen biosynthesis
and flagellin biosynthesis will be investigated in this project.