Broad specificity of monoclonal IgA (TEPC15-IgA) for enteric bacteria via phosphorylcholine-mediated interaction

Abstract

Immunoglobulin A (IgA) is notable for its broad specificity toward multiple bacteria. Phosphorylcholine (PC) plays a role in the infection of pathogenic bacteria carrying PC and in the induction of IgA responses in the host immune system. The commercially available mouse monoclonal IgA, TEPC15-IgA, is a distinctive antibody with specificity for PC, warranting further exploration of its response to PC-bearing enteric bacteria. In this study, using 17 different enteric bacteria, including 3 aerobic and 14 anerobic bacteria that could be cultured in vitro, we confirmed that TEPC15-IgA recognizes 4 bacterial species: Lactobacillus taiwanensis, Limosilactobacillus frumenti, Streptococcus infantis, and Escherichia coli, although reactivity varied. Interestingly, TEPC15-IgA did not react with four of six Lactobacillus species used. Moreover, distinct target molecules associated with PC in L. taiwanensis and L. frumenti were evident, differing in molecular weight. These findings suggest that the natural generation of PC-specific IgA could prevent PC-mediated infections and potentially facilitate the formation of a microflora rich in indigenous bacteria with PC, particularly in the gastrointestinal tract.

Keywords: enteric bacteria; immune system; immunoglobulin A; phosphorylcholine.

Fig. 1.

The dimeric structure of TEPC-IgA was confirmed by Sodium Dodecyl Sulfate−Poly-Acrylamide Gel Electrophoresis (SDS-PAGE) and western blot analyses. (A) Dimeric IgA was detected under nonreducing condition on SDS-PAGE gels stained with SimplyBlue and on western blot membranes treated with either anti-IgA or anti-J chain. (B) Heavy chain and J-chain, both of which are IgA components, were separately detected under reducing condition.

Fig. 2.

The reaction of TEPC15-IgA to enteric bacteria was investigated by enzyme-linked immunosorbent assay (ELISA). Two Lactobacillus spp. (i.e., Limosilactobacillus frumenti and Lactobacillus taiwanensis) were highly recognized by TEPC15-IgA. In addition, two other bacteria (i.e., Eschericia coli and Streptococcus infantis) were slightly recognized by TEPC15-immunoglobulin A. The reactions of TEPC15-IgA to each bacterium were examined in quadruplicate and are shown as the mean ± SD. Positive reactions were defined as optical density (OD)₄₅₀ values obtained from serial dilutions of TEPC15-IgA (1,000–2 ng/μL) that were >0.1 compared with the OD₄₅₀ value obtained from the most dilute TEPC15-IgA (1 ng/μL used as a control). For ELISA analyses that showed positive reactions, the Kruskal–Wallis test followed by the Dunn test were performed to determine the difference from the control reaction (1 ng/μL of TEPC15-IgA). *P<0.05, **P<0.01, ***P<0.001 vs. OD₄₅₀ obtained using 1 ng/μL of TEPC15-IgA.

Fig. 3.

Presence of phosphorylcholine (PC) in two enteric bacteria recognized by TEPC15-IgA as confirmed by additional enzyme-linked immunosorbent assay (ELISA) analyses. Two Lactobacillus spp. (i.e., Limosilactobacillus frumenti and Lactobacillus taiwanensis) harbored PC, as shown by detection using anti-PC antibodies (BH8-IgM). The reactions of BH8-immungoglobulin (Ig) M to each bacterium were examined in quadruplicate and are shown as the mean ± SD. Positive reactions were defined as OD₄₅₀ values obtained from serial dilutions of BH8-IgM (1,000–2 ng/μL) that were >0.1 compared with the OD₄₅₀ value obtained from the most dilute BH8-IgM (1 ng/μL used as a control). For ELISA analyses that showed positive reactions, a Kruskal–Wallis test followed by the Dunn test were performed to determine the difference from the control reaction (1 ng/μl of BH8-IgM). *P<0.05, **P<0.01, ***P<0.001 vs. OD₄₅₀ obtained using 1 ng/μL of BH8-IgM.

Fig. 4.

The reaction of TEPC15-IgA to phosphorylcholine (PC) itself, which is carried by Limosilactobacillus frumenti, was verified by a neutralization study. (A) The reactivity of TEPC15-IgA to PC itself was confirmed in a concentration-dependent manner by enzyme-linked immunosorbent assay (ELISA) using PC-conjugated keyhole limpet hemocyanin (PC-KLH). The reactions of TEPC15-IgA to PC-KLH were examined in quadruplicate and are shown as the mean ± SD. (B) In addition, the specificity of TEPC15-IgA to PC itself harbored by Limosilactobacillus frumenti was examined in quadruplicate by use of TEPC15-IgA pretreated with (or without) PC-KLH. Statistical analyses were performed by the Kruskal–Wallis test followed by the Dunn test. (A) *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs. the OD₄₅₀ obtained by use of 1 ng/μL of TEPC15-IgA. (B) **P<0.01 vs. control [TEPC15-IgA without PC-conjugated keyhole limpet hemocyanin (PC-KLH) treatment].

Fig. 5.

The molecular weight of phosphorylcholine (PC) -containing molecules recognized by TEPC15-IgA was examined by Sodium Dodecyl Sulfate−Poly-Acrylamide Gel Electrophoresis (SDS-PAGE) and western blot analysis. Cell lysates prepared from 17 bacteria used in this study were loaded onto SDS-PAGE and transferred to western blot membranes. TEPC15-IgA reacted with molecules of different molecular weights present in the cell lysates of two strains (Lactobacillus taiwanensis and Limosilactobacillus frumenti). PC-conjugated keyhole limpet hemocyanin (PC-KLH) was also used as a positive control to see the reaction of TEPC15-IgA in western blot analysis.