Reduction in S. flexneri membrane potential and ATP levels following IgAC5 treatment. (A) A total of 1 × 10⁸ CFU of S. flexneri SJF31 per ml was loaded with the dye JC-1, seeded in 96-well microtiter plates, and then treated with indicated concentrations of IgAC5 (µg/ml) or CCCP (µM). At 2, 15, and 30 min following IgAC5 or CCCP treatment, the relative fluorescence emission signal ratios (530 nm/590 nm) were determined by using a Synergy HT (Bio-Tek, Winooski, VT) fluorescent plate reader with dual excitation (485/20 and 530/25) and dual emission (528/20 and 590/35). The fluorescence emission ratio of control cells was set to 100%. The results are the average values (with standard error) from a representative experiment done in triplicate. The asterisks indicate a significant (P < 0.05) reduction in membrane potential relative to the control samples, as determined by using the Student t test (GraphPad Software, San Diego, CA). SJF31 (ΔtolC::kan), an isogenic derivative of strain M90T, was created using λ red-mediated recombination with pKD4 as the antibiotic resistance marker template (26). It was necessary to use a TolC mutant for these studies to avoid rapid expulsion of JC-1 from the cell cytoplasm (24). (B) A total of 1 × 10⁸ CFU of mid-log-phase S. flexneri strain M90T per ml grown in minimal medium with succinate (0.5%) as the sole carbon source was treated with a phosphate-buffered saline (PBS), IgAC5 (10 µg/ml), or CCCP (100 µM). At the indicated time points, total cellular ATP levels were measured with the BacTiter-Glo (Promega, Madison, WI) luminescence assay. The asterisks indicate significant (P < 0.05) reduction in ATP levels relative to the control samples, as determined by using the Student t test.

IgAC5 suppresses secretion of Ipa proteins by S. flexneri. A total of 1 × 10⁹ CFU per ml of mid-log-phase cultures of S. flexneri strain M90T was treated with IgAC5, control IgA MAbs (e.g., TEPC-15 and Sal4), O-Ag polyclonal rabbit antisera, or CCCP for the indicated times prior to the addition of Congo red (CR; 6 µg/ml) to trigger T3S system-mediated Ipa transport. Ten minutes later, the bacterial cultures were subjected to centrifugation (5 min at 8,000 relative centrifugal force [RCF]). The resulting supernatants were passed through a 0.2-µm filter to remove any contaminating bacterial cells, concentrated with trichloroacetic acid, solubilized in Laemmli sample buffer containing β-mercaptoethanol (β-ME; 5% [vol/vol]), and then analyzed by SDS-PAGE and Western blotting (W.B.) with murine IgG MAbs specific for IpaB or IpaC (as indicated) and chemiluminescent detection (Bio-Rad, Hercules, CA). The amount of IpaB or IpaC was determined by densitometric analysis of the Western blot films. IpaB and IpaC amounts below the level of detection were set to 1. For panel B only, the bacterial pellets were lysed by the addition of 2× Laemmli sample buffer with β-ME and subjected to SDS-PAGE and Western blotting, as indicated above. (A and B) Supernatants (A) or bacterial pellets (B) from cultures were treated as follows: lane 1, no treatment; lane 2, no treatment followed by CR; lane 3, TEPC-15 (5 µg/ml) for 15 min followed by CR; lane 4, IgAC5 (2 µg/ml) for 15 min followed by CR; lane 5, IgAC5 (5 µg/ml) for 15 min followed by CR; lane 6, CCCP (100 µM) for 15 min followed by CR. The relative amounts of IpaB in the cell supernatants are indicated across the top of the panel as “% control.” In this case, the control was the amount of IpaB associated with CR-treated cells (lane 2). (C) Dose-dependent inhibition of IpaB secretion by IgAC5. Shown are IpaB levels in the supernatants of cultures of S. flexneri M90T treated for 15 min with the indicated concentrations of IgAC5 or the S. Typhimurium-specific IgA MAb Sal4 prior to the addition of CR. Cells treated with SalIV and CR (lane 5) served as the positive control for this experiment, and the amounts of IpaB in the supernatants are expressed as a percentage of this control. (D) IpaC secretion is inhibited by O-Ag polyclonal antibodies (PAbs). Cultures of S. flexneri were treated with 2.5 or 5 µg/ml O-Ag PAbs or CCCP (100 µM) for 15 min prior to the addition of CR. Culture supernatants were prepared as indicated above and blotted with an IpaC-specific MAb. The vertical line between lanes 2 and 3 indicates that a lane(s) present in the original Western blot at this location was removed from the final image. The removed lane(s) represents experimental conditions that were not relevant to the figure. (E to F) IgAC5’s effects on Ipa secretion are transient. Cultures of S. flexneri were treated with IgAC5 (5 µg/ml), O-Ag PAbs (5 µg/ml), or CCCP (100 µM) for 45 min (E) or 90 min (F) prior to the addition of CR. Culture supernatants were then blotted with an IpaB-specific MAb. The elevated levels of IpaB in the negative control samples (lanes 1) in these experiments (compared to those described in panels A to D) reflect the baseline accumulation of the proteins in culture supernatants over the prolonged course of the experiments (45 and 90 min in panels E and F, compared to 15 min for experiments described in panels A to D). (G) Dose-dependent inhibition of Ipa secretion by CCCP. Cultures of S. flexneri were treated with indicated concentrations CCCP for 15 min prior to the addition of CR, after which cell supernatants were blotted for IpaC.