A stealth adhesion factor contributes to Vibrio vulnificus pathogenicity: Flp pili play roles in host invasion, survival in the blood stream and resistance to complement activation
Fig 5
Delayed RtxA1 secretion (A) and cytotoxicity (B) by the Δtad123 mutant cells.
(A) For RtxA1 detection, log-phase V. vulnificus cells were incubated with HeLa cells in 6-well plates at an MOI of 100 bacteria in the presence of 2 μg/ml tetracycline for 35 or 45 min. The cells in each well were lysed with lysis buffer, followed by concentration using the Amicon Ultra-0.5 centrifugal filter apparatus. The RtxA1 toxin was detected by Western blot analysis using an anti-GD domain antibody (RtxA1-C, a band of approximately 130 kDa). (B) Effect of Δtad123 mutations on cytotoxicity against HeLa cells. (C) Restoration of cytotoxicity in tad-complemented strains in the presence of antibiotics. Data shown represent the mean ± SEM of three independent experiments performed with five replicates. WT (pLAFR3), wild type harboring pLAFR3; Δtad123 (pLAFR3), Δtad123 mutant harboring pLAFR3; Δtad123 (pLAFR3::tad1), Δtad123 mutant in trans complemented with pLAFR3::tad1 locus; Δtad123 (pLAFR3::tad2), Δtad123 mutant in trans complemented with pLAFR3::tad2 locus); Δtad123 (pLAFR3::tad3), Δtad123 mutant in trans complemented with pLAFR3::tad3 locus. Statistical analysis was carried out Student’s t test. **, P < 0.01; ***, P < 0.001; ns, not significant.