Role of flm Locus in Mesophilic Aeromonas Species Adherence

Abstract

The adherence mechanism of Aeromonas caviae Sch3N to HEp-2 cells was initially investigated through four mini-Tn5 mutants that showed a 10-fold decrease in adherence. These mutants lost motility, flagella, and their lipopolysaccharide (LPS) O antigen (O-Ag). Three genes, flmB-neuA-flmD, were found to be interrupted by the transposon insertions; additionally, two other genes, one lying upstream (flmA) and one downstream (neuB), were found to be clustered in the same operon. While the flmA and flmB genes were present in all mesophilic Aeromonas spp. (A. hydrophila, A. caviae, A. veronii bv. veronii, and A. veronii bv. sobria) tested, this was not the case for the neuA-flmD-neuB genes. Construction and characterization of flmB insertion mutants in five other mesophilic Aeromonas strains revealed the loss of motility, flagella, and adherence but did not alter the LPS composition of these strains. Taking Mesophilic Aeromonas has been associated with gastrointestinal and wound infections of healthy humans, and less commonly with septicemias of immunocompromised patients (12). A. caviae, in particular, is reported as the most prevalent paediatric enteropathogenic species of the genus (26, 39).A number of putative pathogenicity determinants have been reported for aeromonads; these include toxins, adhesins, and invasins (34). There is still little known about their adherence process, although long-wavy pili have been implicated as important colonization factors of A. hydrophila and A. veronii bv. sobria (14). However, many clinical isolates are poorly piliated or nonpiliated (15) In this study, we describe five genes of A. caviae that belong to a putative operon involved in the biosynthesis of LPS O-Ag and flagellum assembly. We have also investigated the distribution of these genes among the mesophilic Aeromonas species, which allowed us to draw conclusions about the roles of these two surface structures in the adherence of aeromonads to human epithelial cells. MATERIALS AND METHODSBacterial strains, plasmids, and growth conditions. Bacterial strains and plasmids used in this study are listed in Table 1. E. coli strains were grown on Luria-Bertani (LB) Miller broth and LB Miller agar, while Aeromonas strains were grown either on tryptic soy broth or agar or in brain heart infusion broth (BHIB) (Oxoid). Ampicillin (50 g/ml), nalidixic acid (50 g/ml), kanamycin (50 g/ml), chloramphenicol (25 g/ml), rifampin (100 g/ml), and tetracycline (20 g/ml) were added to the different media when needed.HEp-2 cell culture and adherence assay. Tissue culture was maintained as described by Thornley et al. (34). The adherence assay was conducted as a slight modification of that described by Carrello et al. (4). Bacteria were grown statically in BHIB at 37°C, harvested by gentle centrifugation (1,600 ϫ g for 5 min), and resuspended in phosphate-buffered saline (PBS), pH 7.2, at approximately 10 6 to 10 7 CFU/ml (A 600 Ϸ 0.07). The monolayer was infected with 1 ml of the bacterial suspension for 90 min at 37°C in 5% CO 2 . Following infec...

Polar Flagellum Biogenesis inAeromonas hydrophila

“…4). This fact together with the knowledge that A. hydrophila AH-3 polar flagellins are glycosylated (unpublished observation), similarly to the A. caviae polar flagellins (25,53), may suggest that the encoded protein (Maf-1) is involved in posttranslational polar flagellum glycosylation, but their exact role in flagellar biosynthesis remains unknown. The A. hydrophila polar flagellum region 3 (Table 3) showed an organization similar to that of the genes downstream of flaM in V. parahaemolyticus polar flagellum region 2, with the absence of the motor genes (34,41) (Fig.…”

“…In humans, Aeromonas hydrophila belonging to hybridization group 1 (HG1) and HG3, A. veronii biovar sobria (HG8/HG10), and A. caviae (HG4) have been associated with gastrointestinal and extraintestinal diseases, such as wound infections of healthy humans, and less commonly with septicemia of immunocompromised patients (30). The swimming motility of all mesophilic aeromonads has been linked to a single polar unsheathed flagellum, expressed constitutively, which is required for adherence to and invasion of human and fish cell lines (25,43,53,63). Moreover, 50% to 60% of mesophilic aeromonads are able to produce many unsheathed peritrichous lateral flagella when grown in viscous environments or over surfaces (58), which increase bacterial adherence and are required for swarming motility and biofilm formation (23).…”

Campylobacter Protein Glycosylation Affects Host Cell Interactions

“…The protein also shows homology to proteins involved in glycosylation of pilin in Neisseria spp. (20, 31) and flagellin in Caulobacter crescentus (23) and Aeromonas caviae (13,32).Growth comparisons. Cell morphology, as determined by transmission electron microscopy, was similar for 81-176 and pglB and pglE mutants (results not shown).…”

“…The protein also shows homology to proteins involved in glycosylation of pilin in Neisseria spp. (20, 31) and flagellin in Caulobacter crescentus (23) and Aeromonas caviae (13,32).…”

A Colonization Factor (Production of Lateral Flagella) of Mesophilic Aeromonas spp. Is Inactive in Aeromonas salmonicida Strains

“…Aeromonas strains seem to be involved in bacterial adhesion to eukaryotic cells and the ability to form biofilms (3,5,19). In order to see if the A. salmonicida laf genes were fully functional, we tested them for the ability to complement the mesophilic Aeromonas laf mutants for adhesion to HEp-2 cells and biofilm formation in vitro by using previously described assays (18,23).…”