Inhibition of Salmonella enterica serovar typhimurium motility and entry into epithelial cells by a protective antilipopolysaccharide monoclonal immunoglobulin A antibody

Abstract

Secretory immunoglobulin A (SIgA) antibodies directed against the O antigen of lipopolysaccharide (LPS) are the primary determinants of mucosal immunity to gram-negative enteric pathogens. However, the underlying mechanisms by which these antibodies interfere with bacterial colonization and invasion of intestinal epithelial cells are not well understood. In this study, we report that Sal4, a protective, anti-O5-specific monoclonal IgA, is a potent inhibitor of Salmonella enterica serovar Typhimurium flagellum-based motility. Using video light microscopy, we observed that Sal4 completely and virtually instantaneously "paralyzed" laboratory and clinical strains of serovar Typhimurium. Sal4-mediated motility arrest preceded and occurred independently of agglutination. Polyclonal anti-LPS IgG antibodies and F(ab)(2) fragments were as potent as was Sal4 at impeding bacterial motility, whereas monovalent Fab fragments were 5- to 10-fold less effective. To determine whether motility arrest can fully account for Sal4's protective capacity in vitro, we performed epithelial cell infection assays in which the requirement for flagellar motility in adherence and invasion was bypassed by centrifugation. Under these conditions, Sal4-treated serovar Typhimurium cells remained noninvasive, revealing that the monoclonal IgA, in addition to interfering with motility, has an effect on bacterial uptake into epithelial cells. Sal4 did not, however, inhibit bacterial uptake into mouse macrophages, indicating that the antibody interferes specifically with Salmonella pathogenicity island 1 (SPI-1)-dependent, but not SPI-1-independent, entry into host cells. These results reveal a previously unrecognized capacity of SIgA to "disarm" microbial pathogens on mucosal surfaces and prevent colonization and invasion of the intestinal epithelium.

FIG. 1.

Measurement of bacterial migration in agar motility assays. Strain JS107 was stab inoculated into 0.3% LB agar containing TEPC-15 (5 μg/ml) or Sal4 (5 μg/ml) and incubated at 37°C for 8 h. The diameter (cm) of the migration was measured at hourly intervals. The results shown are the averages of the results for two plates.

FIG. 2.

Sal4 arrests motility of serovar Typhimurium cells in a dose- and time-dependent manner. Wild-type serovar Typhimurium cells (strain JS107) or an oafA mutant (strain JS93) were mixed with Sal4 at indicated concentrations, spotted on glass microscope slides, and imaged by video light microscopy at the indicated time points. Motility was expressed as percentage of control motility: (% motile bacteria in experimental sample/% motile bacteria in control samples) × 100. Control samples were treated with TEPC-15 (10 μg/ml) for 15 min, as described in Materials and Methods. Each value represents the average (with standard error) of the results from four to six independent video recordings. An asterisk indicates a statistically significant (P ≤ 0.01) reduction in motility in comparison to that of TEPC-15-treated bacteria.

FIG. 3.

Sal4 blocks serovar Typhimurium invasion of epithelial cells, even when the requirement for motility is bypassed through centrifugation. (A) Wild-type (strain JS107) and oafA mutant (strain JS93) serovar Typhimurium cells were mixed 1:1 and incubated for 15 min with 5 μg/ml TEPC-15 or Sal4 before being applied to MDCK II cell monolayers in 96-well microtiter plates and subjected (or not) to brief centrifugation, as described in Materials and Methods, so as to promote (or not promote) bacterium-epithelial cell contact. The results shown are the average values (with standard errors) from three independent experiments, each done in triplicate. An asterisk indicates a statistically significant (P ≤ 0.05) reduction in invasion in the presence of Sal4 in comparison to that in the TEPC-15 control. (B) Centrifugation enhances uptake of a nonmotile mutant of serovar Typhimurium. Cells of the wild type and a nonmotile motB serovar Typhimurium strain were mixed 1:1 before being applied to MDCK II cell monolayers in 96-well microtiter plates and subjected (or not) to brief centrifugation. The results shown are the average values (with standard errors) from three independent experiments, each done in triplicate. An asterisk indicates that bacterial invasion in the presence of centrifugation (+) was significantly (P ≤ 0.05) greater than in the absence of centrifugation (−).

FIG. 4.

Sal4 does not impede serovar Typhimurium attachment to epithelial cells. The wild-type or ΔSPI-1 strain of serovar Typhimurium was mixed with the oafA mutant at a 1:1 ratio, incubated without antibody (white bars) or with Sal4 (5 μg/ml; black bars) for 15 min, applied to HeLa cells seeded in 96-well microtiter plates, and then subjected to centrifugation (10 min at 4°C) to promote bacterium-epithelial cell adherence. Immediately thereafter, the HeLa cells were washed to remove unbound bacteria and then lysed with 1% Triton X-100. The number of bacteria in the lysates (CFU) was determined by serial dilution onto LB. The CI reflects the attachment of wild-type or ΔSPI-1 strain cells to HeLa cells relative to the attachment of oafA mutant cells. Sal4 marginally enhanced attachment of both the wild-type (P = 0.06) and the ΔSPI1 (P = 0.02) serovar Typhimurium strain to HeLa cells, possibly due to antibody-mediated agglutination that may have occurred during centrifugation.

FIG. 5.

Anti-LPS antibodies inhibit invasion by serovar Typhimurium cells of epithelial cells independent of bacterial agglutination. Serovar Typhimurium strain JS107 was incubated with TEPC-15 (5 μg/ml), Sal4 (5 μg/ml), polyclonal anti-LPS F(ab)₂ (5 μg/ml), or Fab fragments (14 μg/ml) for 15 min and then applied to HeLa cells seeded in 96-well microtiter plates. The noninvasive ΔSPI-1 strain was used in parallel as a control. Due to the reactivity of polyclonal anti-O antibodies with the oafA strain of serovar Typhimurium, we were unable to perform competitive invasion assays in this experiment. Therefore, invasion is expressed as percentage of initial inoculum. The data shown are the average values (with standard errors of the means) from a single representative experiment done in triplicate. An asterisk indicates a statistically significant (P ≤ 0.05) reduction in invasion in the presence of Sal4 in comparison to that of the TEPC-15 control. +, present.

FIG. 6.

Serovar Typhimurium uptake by murine macrophages in the presence of Sal4. A 1:1 mixture of wild-type and oafA strain serovar Typhimurium cells was incubated with Sal4 (5 μg/ml), TEPC-15 (5 μg/ml), or PBS and then applied to J774 cells seeded in 96-well microtiter plates. The CI, which reflects the ratio of wild-type serovar Typhimurium cells to oafA strain cells that were taken up into J774 cells, was determined as described in Materials and Methods. The results shown are from a single representative experiment done in quadruplicate. The asterisk indicates that Sal4 enhanced (P ≤ 0.05) the uptake of serovar Typhimurium cells into macrophages in comparison to that of TEPC-15-treated bacteria, possibly due to the fact that Sal4 was used under agglutinating conditions in this assay. +, present.