Role of Aeromonas hydrophila flagella glycosylation in adhesion to Hep-2 cells, biofilm formation and immune stimulation.

Polar flagellin proteins from Aeromonas hydrophila strain AH-3 (serotype O34) were found to be O-glycosylated with a heterogeneous heptasaccharide glycan. Two mutants with altered (light and strong) polar flagella glycosylation still able to produce flagella were previously obtained, as well as mutants lacking the O34-antigen lipopolysaccharide (LPS) but with unaltered polar flagella glycosylation. We compared these mutants, altogether with the wild type strain, in different studies to conclude that polar flagella glycosylation is extremely important for A. hydrophila adhesion to Hep-2 cells and biofilm formation. Furthermore, the polar flagella glycosylation is an important factor for the immune stimulation of IL-8 production via toll receptor 5 (TLR5).

Aeromonas hydrophila lateral flagellar gene transcriptional hierarchy.

Aeromonas hydrophila AH-3 lateral flagella are not assembled when bacteria grow in liquid media; however, lateral flagellar genes are transcribed. Our results indicate that A. hydrophila lateral flagellar genes are transcribed at three levels (class I to III genes) and share some similarities with, but have many important differences from, genes of Vibrio parahaemolyticus. A. hydrophila lateral flagellum class I gene transcription is σ(70) dependent, which is consistent with the fact that lateral flagellum is constitutively transcribed, in contrast to the characteristics of V. parahaemolyticus. The fact that multiple genes are included in class I highlights that lateral flagellar genes are less hierarchically transcribed than polar flagellum genes. The A. hydrophila lafK-fliEJL gene cluster (where the subscript L distinguishes genes for lateral flagella from those for polar flagella) is exclusively from class I and is in V. parahaemolyticus class I and II. Furthermore, the A. hydrophila flgAMNL cluster is not transcribed from the σ(54)/LafK-dependent promoter and does not contain class II genes. Here, we propose a gene transcriptional hierarchy for the A. hydrophila lateral flagella.

Differential glycosylation of polar and lateral flagellins in Aeromonas hydrophila AH-3.

Polar and lateral flagellin proteins from Aeromonas hydrophila strain AH-3 (serotype O34) were found to be glycosylated with different carbohydrate moieties. The lateral flagellin was modified at three sites in O-linkage, with a single monosaccharide of 376 Da, which we show to be a pseudaminic acid derivative. The polar flagellin was modified with a heterogeneous glycan, comprised of a heptasaccharide, linked through the same 376-Da sugar to the protein backbone, also in O-linkage. In-frame deletion mutants of pseudaminic acid biosynthetic genes pseB and pseF homologues resulted in abolition of polar and lateral flagellar formation by posttranscriptional regulation of the flagellins, which was restored by complementation with wild type pseB or F homologues or Campylobacter pseB and F.

Transcriptional hierarchy of Aeromonas hydrophila polar-flagellum genes.

Aeromonas hydrophila polar-flagellum class I gene transcription is σ70 dependent, which is consistent with the fact that the A. hydrophila polar flagellum is constitutively expressed. In contrast to other bacteria with dual flagellar systems such as Vibrio parahaemolyticus, the A. hydrophila LafK protein does not compensate for the lack of the polar-flagellum regulator FlrA (V. parahaemolyticus FlaK homologue). This is consistent with the fact that the A. hydrophila FlrA mutation abolishes polar-flagellum formation in liquid and on solid surfaces but does not affect inducible lateral-flagellum formation. The results highlight that the polar- and lateral-flagellum interconnections and control networks are specific and that there are differences between the dual flagellar systems in A. hydrophila and V. parahaemolyticus. Furthermore, our results indicate that the A. hydrophila polar-flagellum transcriptional hierarchy (also in class II, III, and IV genes) shares some similarities with but has many important differences from the transcriptional hierarchies of Vibrio cholerae and Pseudomonas aeruginosa. The A. hydrophila flhF and flhG genes are essential for the assembly of a functional polar flagellum because in-frame mutants fail to swim in liquid medium and lack the polar flagellum. In Vibrio and Pseudomonas flhG disruption increases the number of polar flagella per cell, and Pseudomonas flhF disruption gives an aberrant placement of flagellum. Here, we propose the gene transcriptional hierarchy for the A. hydrophila polar flagellum.

Aeromonas hydrophila motY is essential for polar flagellum function, and requires coordinate expression of motX and Pom proteins.

By the analysis of the Aeromonas hydrophila ATCC7966(T) genome we identified A. hydrophila AH-3 MotY. A. hydrophila MotY, like MotX, is essential for the polar flagellum function energized by an electrochemical potential of Na(+) as coupling ion, but is not involved in lateral flagella function energized by the proton motive force. Thus, the A. hydrophila polar flagellum stator is a complex integrated by two essential proteins, MotX and MotY, which interact with one of two redundant pairs of proteins, PomAB and PomA(2)B(2). In an A. hydrophila motX mutant, polar flagellum motility is restored by motX complementation, but the ability of the A. hydrophila motY mutant to swim is not restored by introduction of the wild-type motY alone. However, its polar flagellum motility is restored when motX and -Y are expressed together from the same plasmid promoter. Finally, even though both the redundant A. hydrophila polar flagellum stators, PomAB and PomA(2)B(2), are energized by the Na(+) ion, they cannot be exchanged. Furthermore, Vibrio parahaemolyticus PomAB and Pseudomonas aeruginosa MotAB or MotCD are unable to restore swimming motility in A. hydrophila polar flagellum stator mutants.

Two redundant sodium-driven stator motor proteins are involved in Aeromonas hydrophila polar flagellum rotation.

Motility is an essential characteristic for mesophilic Aeromonas strains. We identified a new polar flagellum region (region 6) in the A. hydrophila AH-3 (serotype O34) chromosome that contained two additional polar stator genes, named pomA2 and pomB2. A. hydrophila PomA2 and PomB2 are highly homologous to other sodium-conducting polar flagellum stator motors as well as to the previously described A. hydrophila AH-3 PomA and PomB. pomAB and pomA2B2 were present in all the mesophilic Aeromonas strains tested and were independent of the strains' ability to produce lateral flagella. Unlike MotX, which is a stator protein that is essential for polar flagellum rotation, here we demonstrate that PomAB and PomA2B2 are redundant sets of proteins, as neither set on its own is essential for polar flagellum motility in either aqueous or high-viscosity environments. Both PomAB and PomA2B2 are sodium-coupled stator complexes, although PomA2B2 is more sensitive to low concentrations of sodium than PomAB. Furthermore, the level of transcription in aqueous and high-viscosity environments of pomA2B2 is reduced compared to that of pomAB. The A. hydrophila AH-3 polar flagellum is the first case described in which two redundant sodium-driven stator motor proteins (PomAB and PomA2B2) are found.

An Aeromonas caviae genomic island is required for both O-antigen lipopolysaccharide biosynthesis and flagellin glycosylation.

Aeromonas caviae Sch3N possesses a small genomic island that is involved in both flagellin glycosylation and lipopolysaccharide (LPS) O-antigen biosynthesis. This island appears to have been laterally acquired as it is flanked by insertion element-like sequences and has a much lower G+C content than the average aeromonad G+C content. Most of the gene products encoded by the island are orthologues of proteins that have been shown to be involved in pseudaminic acid biosynthesis and flagellin glycosylation in both Campylobacter jejuni and Helicobacter pylori. Two of the genes, lst and lsg, are LPS specific as mutation of them results in the loss of only a band for the LPS O-antigen. Lsg encodes a putative Wzx flippase, and mutation of Lsg affects only LPS; this finding supports the notion that flagellin glycosylation occurs within the cell before the flagellins are exported and assembled and not at the surface once the sugar has been exported. The proteins encoded by flmA, flmB, neuA, flmD, and neuB are thought to make up a pseudaminic acid biosynthetic pathway, and mutation of any of these genes resulted in the loss of motility, flagellar expression, and a band for the LPS O-antigen. Furthermore, pseudaminic acid was shown to be present on both flagellin subunits that make up the polar flagellum filament, to be present in the LPS O-antigen of the A. caviae wild-type strain, and to be absent from the A. caviae flmD mutant strain.

Aeromonas hydrophila flagella glycosylation: involvement of a lipid carrier.

Polar flagellin proteins from Aeromonas hydrophila strain AH-3 (serotype O34) were found to be O-glycosylated with a heterogeneous glycan. Mutants unable to produce WecP or Gne enzymes showed altered motility, and the study of their polar flagellin glycosylation showed that the patterns of glycosylation differed from that observed with wild type polar flagellin. This suggested the involvement of a lipid carrier in glycosylation. A gene coding for an enzyme linking sugar to a lipid carrier was identified in strain AH-3 (WecX) and subsequent mutation abolished completely motility, flagella production by EM, and flagellin glycosylation. This is the first report of a lipid carrier involved in flagella O-glycosylation. A molecular model has been proposed. The results obtained suggested that the N-acetylhexosamines are N-acetylgalactosamines and that the heptasaccharide is completely independent of the O34-antigen lipopolysaccharide. Furthermore, by comparing the mutants with differing degrees of polar flagellin glycosylation, we established their importance in A. hydrophila flagella formation and motility.

Aeromonas hydrophila AH-3 AexT is an ADP-ribosylating toxin secreted through the type III secretion system.

We cloned and sequenced an ADP-ribosylating toxin (AexT) from a mesophilic Aeromonas hydrophila strain AH-3 with a type III secretion system (T3SS). This toxin only showed homology, in genes and proteins, with the first half of A. salmonicida AexT. The A. hydrophila AexT showed ADP-ribosyltransferase activity, translocation through the T3SS system, and this A. hydrophila T3SS system is inducible under calcium-depleted conditions. The A. hydrophila aexT mutant showed a slight reduction in their virulence assayed by several methods when compared to the wild-type strain, while an A. hydrophila T3SS mutant is highly reduced in virulence on the same assays. The A. hydrophila AexT is the first described and the smallest T3SS effector toxin found in mesophilic Aeromonas with a functional T3SS.

Non-structural flagella genes affecting both polar and lateral flagella-mediated motility in Aeromonas hydrophila.

An Aeromonas hydrophila AH-3 miniTn5 mutant unable to produce polar and lateral flagella was isolated, in which the transposon was inserted into a gene whose encoded protein was an orthologue of the Campylobacter jejuni motility accessory factor (Maf) protein. In addition to this gene, several other related genes were found in this cluster that was adjacent to the region 2 genes of the polar flagellum. Mutation of the A. hydrophila AH-3 maf-2, neuB-like, flmD or neuA-like genes resulted in non-motile cells that were unable to swim or swarm due to the absence of both polar and lateral flagella. However, both polar and lateral flagellins were present but were unglycosylated. Although the A. hydrophila AH-3 or Aeromonas caviae Sch3N genes did not hybridize with each other at the nucleotide level, the gene products were able to fully complement the mutations in either bacterium. Furthermore, well-characterized C. jejuni genes involved in flagella glycosylation (Cj1293, -1294 and -1317) were fully able to complement A. hydrophila mutants in the corresponding genes (flmA, flmB and neuB-like). It was concluded that the maf-2, neuB-like, flmD and neuA-like genes are involved in the glycosylation of both the polar and the lateral flagella in Aeromonas strains.

Mesophilic Aeromonas UDP-glucose pyrophosphorylase (GalU) mutants show two types of lipopolysaccharide structures and reduced virulence.

A mutation in galU that causes the lack of O34-antigen lipopolysaccharide (LPS) in Aeromonas hydrophila strain AH-3 was identified. It was proved that A. hydrophila GalU is a UDP-glucose pyrophosphorylase responsible for synthesis of UDP-glucose from glucose 1-phosphate and UTP. The galU mutant from this strain showed two types of LPS structures, represented by two bands on LPS gels. The first one (slow-migrating band in gels) corresponds to a rough strain having the complete core, with two significant differences: it lacks the terminal galactose residue from the LPS-core and 4-amino-4-deoxyarabinose residues from phosphate groups in lipid A. The second one (fast-migrating band in gels) corresponds to a deeply truncated structure with the LPS-core restricted to one 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) and three l-glycero-d-manno-heptose residues. galU mutants in several motile mesophilic Aeromonas strains from serotypes O1, O2, O11, O18, O21 and O44 were also devoid of the O-antigen LPS. The galU mutation reduced to less than 1 % the survival of these Aeromonas strains in serum, decreased the ability of these strains to adhere and reduced by 1.5 or 2 log units the virulence of Aeromonas serotype O34 strains in a septicaemia model in either fish or mice. All the changes observed in the galU mutants were rescued by the introduction of the corresponding single wild-type gene.