Immunoglobulin-mediated agglutination of and biofilm formation by Escherichia coli K-12 require the type 1 pilus fiber

Abstract

The binding of human secretory immunoglobulin A (SIgA), the primary immunoglobulin in the gut, to Escherichia coli is thought to be dependent on type 1 pili. Type 1 pili are filamentous bacterial surface attachment organelles comprised principally of a single protein, the product of the fimA gene. A minor component of the pilus fiber (the product of the fimH gene, termed the adhesin) mediates attachment to a variety of host cell molecules in a mannose inhibitable interaction that has been extensively described. We found that the aggregation of E. coli K-12 by human secretory IgA (SIgA) was dependent on the presence of the pilus fiber, even in the absence of the mannose specific adhesin or in the presence of 25 mM alpha-CH(3)Man. The presence of pilus without adhesin also facilitated SIgA-mediated biofilm formation on polystyrene, although biofilm formation was stronger in the presence of the adhesin. IgM also mediated aggregation and biofilm formation in a manner dependent on pili with or without adhesin. These findings indicate that the pilus fiber, even in the absence of the adhesin, may play a role in biologically important processes. Under conditions in which E. coli was agglutinated by SIgA, the binding of SIgA to E. coli was not increased by the presence of the pili, with or without adhesin. This observation suggests that the pili, with or without adhesin, affect factors such as cell surface rigidity or electrostatic repulsion, which can affect agglutination but which do not necessarily determine the level of bound immunoglobulin.

FIG. 1.

Agglutination of various strains of E. coli in the presence of SIgA. The numbers and sizes of particles after the addition of 0.5 mg of SIgA/ml to various strains of E. coli (ORN225, ORN226, and ORN227; see Table 1) were measured as a function of time with a Coulter counter. For presentation purposes, smaller aggregates (3 to 6 μm) (A) were plotted separately than larger aggregates (>6 μm) (B), which formed slower than the smaller aggregates. Experiments were performed in duplicate, and the standard errors are shown.

FIG. 2.

SIgA-mediated biofilm formation of E. coli as a function of pilus and adhesin expression (A) Experiments with chromosomal mutants. Tubes A contain no bacteria, tubes B contain E. coli (chromosomal mutants) without pilus or adhesin (ORN225; Table 1), tubes C contain E. coli with pilus but no adhesin (ORN226; Table 1), and tubes D contain E. coli with pilus and adhesin (ORN227; Table 1). (B) Quantification of the SIgA-mediated biofilm growth by various E. coli expressing pili with adhesin, pili without adhesin, or neither pili nor adhesin. Chromosomal mutants were the same used in the experiment described in Fig. 2A. Plasmid constructs expressing pili with adhesin (ORN201 containing pSH2; Table 1), pili without adhesin (ORN201 containing pORN307; Table 1), or neither pili nor adhesin (ORN201 containing pACY184; Table 1) were also used. Biofilm growth did not occur in the absence of immunoglobulin (see Fig. 3), indicating that the biofilm growth was mediated by SIgA. The growth is shown on a log scale. Growth was quantified by measuring the amount of ¹⁴C-glucose incorporated into the biofilm as described in Materials and Methods. Experiments were performed in duplicate, and the standard errors are shown.

FIG. 3.

IgM-mediated aggregation and biofilm formation of E. coli. (A) Agglutination of various E. coli (chromosomal mutants ORN225, ORN226, and ORN227; Table 1) in the presence or absence of human IgM. In contrast to results obtained with SIgA, aggregates greater than 6 μm in diameter were not formed by any of the E. coli in the presence of the IgM. (B) IgM-mediated biofilm formation by various E. coli (chromosomal mutants). All experiments were performed in duplicate, and the standard errors are shown.

FIG. 4.

SIgA binding to E. coli as a function of pilus and adhesin expression. (A) Immunoprecipitation of SIgA (1.0 mg/ml) was carried out for 2 h at 22.5°C with 7.2 × 10⁷ E. coli/ml. The unbound SIgA was washed away, bacteria and bound SIgA were solubilized in SDS, and the amount of SIgA was quantified by immunoblotting and densitometric evaluation of the blots. Chromosomal mutants (ORN225, ORN226, and ORN227; Table 1) were utilized in the study. The experiment was conducted in duplicate, and the standard errors are shown. (B) Binding of human SIgA to immobilized E. coli (ORN225, ORN226, and ORN227; Table 1) expressing pilus with or without adhesin and to E. coli expressing no pilus. Binding of SIgA to E. coli was determined by ELISA with 0.5 mg of SIgA/ml to probe the E. coli. The number of E. coli bound to the plate is shown in the inset (note log scale). The number of E. coli bound was determined at various positions in the well by inserting a grid in each well (94-μm cell screen filter) and counting the number of E. coli in a given section manually. The ELISA was run in quadruplicate, and the standard errors are shown.