Rapid effects of a protective O-polysaccharide-specific monoclonal IgA on Vibrio cholerae agglutination, motility, and surface morphology

Abstract

2D6 is a dimeric monoclonal immunoglobulin A (IgA) specific for the nonreducing terminal residue of Ogawa O-polysaccharide (OPS) of Vibrio cholerae. It was previously demonstrated that 2D6 IgA is sufficient to passively protect suckling mice from oral challenge with virulent V. cholerae O395. In this study, we sought to define the mechanism by which 2D6 IgA antibody protects the intestinal epithelium from V. cholerae infection. In a mouse ligated-ileal-loop assay, 2D6 IgA promoted V. cholerae agglutination in the intestinal lumen and limited the ability of the bacteria to associate with the epithelium, particularly within the crypt regions. In vitro fluorescence digital video microscopy analysis of antibody-treated V. cholerae in liquid medium revealed that 2D6 IgA not only induced the rapid (5- to 10-min) onset of agglutination but was an equally potent inhibitor of bacterial motility. Scanning electron microscopy showed that 2D6 IgA promoted flagellum-flagellum cross-linking, as well as flagellar entanglement with bacterial bodies, suggesting that motility arrest may be a consequence of flagellar tethering. However, monovalent 2D6 Fab fragments also inhibited V. cholerae motility, demonstrating that antibody-mediated agglutination and motility arrest are separate phenomena. While 2D6 IgA is neither bactericidal nor bacteriostatic, exposure of V. cholerae to 2D6 IgA (or Fab fragments) resulted in a 5-fold increase in surface-associated blebs, as well an onset of a wrinkled surface morphotype. We propose that the protective immunity conferred by 2D6 IgA is the result of multifactorial effects on V. cholerae, including agglutination, motility arrest, and possibly outer membrane stress.

FIG 1

2D6 IgA reduces V. cholerae attachment to epithelial surfaces in vivo. V. cholerae O395 was either untreated (A) or treated with 2D6 IgA (B) or 2D6 Fab fragments (C) and injected into 1-cm ligated ileal loops of BALB/c mice, as described in Materials and Methods. After 30 min, the loops were excised, frozen, cryosectioned, and stained with fluorescently labeled V. cholerae antiserum (red) or CD11c (blue) to detect mucosal dendritic cells (DCs). Stained cryosections were then visualized by confocal microscopy. In each case, the panels in the right-hand column are zoomed-in images of the panels in the left-hand columns. In panels A, V. cholerae was observed in close association with the epithelium (arrows) and penetrating the intestinal crypts (arrowheads). In panels B, 2D6 IgA-treated bacteria were largely aggregated in the lumen (arrows) and rarely in the crypts (arrowheads). In panels C, 2D6 Fab fragment-treated V. cholerae cells were observed in close association with villus epithelium and in the crypts (arrowhead). Scale bars, left column, 300 μm; right column, 50 μm. Abbreviations: PP, Peyer's patches; V, villi; L, lumen.

FIG 2

2D6 IgA rapidly inhibits V. cholerae motility in liquid media. A mid-log-phase culture of V. cholerae strain RT4273 was treated with 2D6 IgA or 2D6 Fab fragments, spotted onto a microscope slide, mounted on Nikon TI inverted microscope equipped with a CoolSnap HQ2 digital camera, and imaged for 20 min, as described in Materials and Methods. (A) Quantitation (percentage) of the number of motile and nonmotile cells at each time point following antibody treatment. There was a significant reduction (P < 0.01, Student's t test) in bacterial motility following treatment of V. cholerae with 2D6 IgA or 2D6 Fab fragments at all time points examined compared to control (untreated) cells. (B) Assessment of bacterial agglutination by measurement of mean fluorescent intensity (MFI) following 2D6 IgA treatment. In the absence of 2D6 IgA, there was no measurable agglutination. Student's t test was used to determine significance (*, P < 0.05; ***, P = 0.0003).

FIG 3

SEM analysis reveals that 2D6 IgA treatment induces membrane wrinkling, blebbing, and flagellar tethering. Mid-log-phase cultures of V. cholerae O395 were treated with Sal4 IgA (A and B) or 2D6 IgA (C to E). Cells were collected at 20 min (A and C), 60 min (B and D), or 120 min (E) following antibody treatment and processed for SEM, as described in Materials and Methods. (A and B) Control-treated V. cholerae cells were evenly distributed across the porous filter membrane, and there was no evidence of bacterial agglutination or clumping. (C to E) Compared to control cells, 2D6 IgA-treated V. cholerae cells were aggregated (compare arrows in panels A and C) and were notably more wrinkled (compare panels B and D). In addition, following 60 min (D) and 120 min (E) of treatment with 2D6 IgA, there was notable flagellum-flagellum cross-linking, flagellar entanglement with neighboring bacterial bodies, and even flagellar knotting (arrows). (D) V. cholerae cells treated with 2D6 IgA were also associated with an increased number of surface blebs (arrow). Note that TCP was not observed in these images, because the bacteria were cultured under non-toxin-inducing conditions. Scale bars: panels A and C, 2 μm; panels B and D, 200 nm; panel E, 1 μm.

FIG 4

Increased blebs on the surface of V. cholerae following 2D6 IgA treatment. Mid-log-phase cultures of V. cholerae O395 were treated with 2D6 IgA for 60 min and then processed for SEM, as described in the legend to Fig. 3. (A) Surface blebs (arrows) on cells following 2D6 IgA treatment. Scale bar, 1 μm. (B) Quantitation of blebs per bacterium in control or 2D6 IgA-treated V. cholerae. In general, blebs were heterogeneous in size (30 to 160 μm) following 2D6 IgA treatment compared to those in control cells, where they were less numerous and more homogenous (40 to 60 μm). Student's t test was used to determine significance (P < 0.0001).

FIG 5

Treatment of V. cholerae with 2D6 Fab fragments results in membrane wrinkles and blebs. Mid-log-phase cultures of V. cholerae O395 were treated with 2D6 Fab fragments for 60 min and then processed for SEM, as described in the legend to Fig. 3. (A) Control cell with relatively smooth surface pattern and single polar flagellum. (B and C) Cells treated with 2D6 Fab fragments were wrinkled and had numerous blebs (arrows), similar to those observed following 2D6 IgA treatment. Moreover, there was notable debris on the filter surface (arrowheads) that was absent from the control cells. Scale bars: panels A and B, 1 μm; panel C, 200 nm.