Identification of a putative glycosyltransferase responsible for the transfer of pseudaminic acid onto the polar flagellin of Aeromonas caviae Sch3N

Abstract

Motility in Aeromonas caviae, in a liquid environment (in broth culture), is mediated by a single polar flagellum encoded by the fla genes. The polar flagellum filament of A. caviae is composed of two flagellin subunits, FlaA and FlaB, which undergo O-linked glycosylation with six to eight pseudaminic acid glycans linked to serine and threonine residues in their central region. The flm genetic locus in A. caviae is required for flagellin glycosylation and the addition of pseudaminic acid (Pse) onto the lipopolysaccharide (LPS) O-antigen. However, none of the flm genes appear to encode a candidate glycotransferase that might add the Pse moiety to FlaA/B. The motility-associated factors (Maf proteins) are considered as candidate transferase enzymes, largely due to their conserved proximity to flagellar biosynthesis loci in a number of pathogens. Bioinformatic analysis performed in this study indicated that the genome of A. caviae encodes a single maf gene homologue (maf1). A maf mutant was generated and phenotypic analysis showed it is both nonmotile and lacks polar flagella. In contrast to flm mutants, it had no effect on the LPS O-antigen pattern and has the ability to swarm. Analysis of flaA transcription by reverse transcriptase PCR (RT-PCR) showed that its transcription was unaltered in the maf mutant while a His-tagged version of the FlaA flagellin protein produced from a plasmid was detected in an unglycosylated intracellular form in the maf strain. Complementation of the maf strain in trans partially restored motility, but increased levels of glycosylated flagellin to above wild-type levels. Overexpression of maf inhibited motility, indicating a dominant negative effect, possibly caused by high amounts of glycosylated flagellin inhibiting assembly of the flagellum. These data provide evidence that maf1, a pseudaminyl transferase, is responsible for glycosylation of flagellin and suggest that this event occurs prior to secretion through the flagellar Type III secretion system.

Aeromonas Flagella and Colonisation Mechanisms

“…The genes encoding Maf proteins are present in the flagellar glycosylation loci of bacteria that glycosylate their flagellins with nonulosonic acids (Parker, Day-Williams, Tomás, Stafford, & Shaw, 2012). Putative maf homologues have been identified in a number of Aeromonas species, including the clinically important A. caviae (Parker et al, 2012), A. hydrophila and A. veronii ( J. Shaw unpublished), as well as A. diversa and A. dhakensis (Shaw unpublished). In addition, maf homologues have also been identified in the psychrophilic fish pathogens A. salmonicida and A. molluscorum (Reith et al, 2008;Shaw unpublished).…”

“…However, glycosylation occurs on the flagellin D2/D3 domain and not the flagellin D0 domain recognised by TLR-5. Evidence from A. caviae Sch3 suggests that the glycosylated flagellin is recognised better by antisera than the unglycosylated form (Parker et al, 2012).…”

“…The paralogous family of Maf proteins were first identified in the flagellar glycosylation islands of C. jejuni (Karlyshev, Linton, Gregson, & Wren, 2002). The genes encoding Maf proteins are present in the flagellar glycosylation loci of bacteria that glycosylate their flagellins with nonulosonic acids (Parker, Day-Williams, Tomás, Stafford, & Shaw, 2012). Putative maf homologues have been identified in a number of Aeromonas species, including the clinically important A. caviae (Parker et al, 2012), A. hydrophila and A. veronii ( J. Shaw unpublished), as well as A. diversa and A. dhakensis (Shaw unpublished).…”

“…2). The genome of A. caviae encodes a single maf homologue (Parker et al, 2012) and its flagellin has been shown to be homogenously glycosylated (Tabei et al, 2009), making it an ideal model organism to use for the study of flagellin glycosylation.…”

Pen and Pal Are Nucleotide-Sugar Dehydratases That Convert UDP-GlcNAc to UDP-6-Deoxy-d-GlcNAc-5,6-ene and Then to UDP-4-Keto-6-deoxy-l-AltNAc for CMP-Pseudaminic Acid Synthesis in Bacillus thuringiensis*

“…and Helicobacter pylori, the central domain of flagellin is post-translationally modified by the addition of pseudaminic acid (5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-␣-L-manno-2-nonulopyranosonic acid, Pse 3 ) (23, 24) that is O-linked to serine or threonine residues (25). Mutants lacking glycosylated flagellin are affected in both their motility and their ability to infect their host (26).…”