Distinct Expression of Immunoglobulin-Binding Proteins in Shiga Toxin-Producing Escherichia coli Implicates High Protein Stability and a Characteristic Phenotype

Abstract

Several immunoglobulin-binding proteins of Escherichia coli (Eib) have been isolated from both non-pathogenic and pathogenic E. coli strains. Shiga toxin (Stx)-producing E. coli (STEC) contain eibG either as a single gene or in combination with eibC, while other E. coli strains harbour single or multiple eib genes. The Eib proteins bind human immunoglobulins in a non-immune manner and contribute to bacterial chain-like adherence to human epithelial cells. In this study, the EibG expression in several STEC strains was analysed under different environmental conditions. STEC produced high levels of EibG in complex media and lower levels in low-grade and minimal media under static growth conditions. This characteristic was independent on the Eib subtypes. Microscopically, EibG-expressing STEC exhibited chain formation and aggregation in all employed media, while aggregates were only visible after growth in complex medium. Once expressed, EibG proteins demonstrate high stability during prolonged incubation. Our findings indicate that the regulation of the expression of Eib proteins is highly complex, although the protein levels vary among STEC strains. However, positive upregulation conditions generally result in distinct phenotypes of the isolates.

Keywords: Shiga toxin-producing Escherichia coli; expression; immunoglobulin-binding protein G; regulation.

Figure 1

Differential EibG protein levels detected in various STEC by immunoblotting. Proteins derived from EibG-positive STEC strains were visualised on immunoblots using HRP-conjugated IgG Fc fragments and EibG signal intensities were quantified by densitometry. (A) After cultivation without agitation, proteins from the STEC strains (5 µg each) were separated, immunoblotted (left graphic) and EibG specific signal intensities were calculated (right graphic). To control for variations in the methodology, at least three independent immunoblots were included into each calculation. For comparison, EibG signal intensities of STEC representing the highest signals on an immunoblot were defined as 1.0. Variations of repeated SDS-PAGE runs were expressed as standard deviations (±SD of the means). (B) EibG positive STEC were cultivated with (+) and without (−) agitation. Proteins were finally separated in dilutions as indicated by SDS-PAGE, immunoblotted, and specific proteins were visualised immunologically (left blot). EibG signals were quantified using the imager technique (right graphic). Black and white circles represent EibG signal intensities after static and shaking conditions, respectively. (C) To demonstrate reproducibility of the high and low expression of various EibG subtypes, colonies from the wild-type strains carrying the α-type and the γ-type were cultivated in three independent probes with agitation at 180 rpm (a) and under static growth conditions (s) at 37 °C. For precise identification different protein amounts were loaded onto gels as 4 µg and 2 µg for strain 2875/96 (α-type) and 7.5 µg and 3.8 µg for strain 0520/99 (γ-type), separated electrophoretically and immunoblotted. Sizes of the marker proteins are indicated (M).

Figure 2

The composition of the growth medium has an impact on EibG protein expression in STEC and on other Eib proteins in E. coli. EibG-positive STEC, as well as ECOR2 and ECOR9, were cultivated with agitation at 180 rpm and under static growth conditions (0 rpm) at 37 °C in the following media: LB broth as a complex medium, yeast extract medium (HM) and minimal medium (M9). For the distinctive differentiation of EibG expression levels, various protein amounts (in the graph interrupted by a line) of 2 µg and 7 µg for strain 2875/96 (α-type), 4 µg and 7 µg for strain 0520/99 (γ-type), and 1 µg and 7 µg for strain 3671/97 (harbouring genes eibC and eibG) were separated and immunoblotted, as indicated earlier. For ECOR2 and ECOR9 strains, 3 µg and 5 µg of proteins were loaded, respectively. Sizes of the marker proteins are indicated (M).

Figure 3

Diverse phenotypes are displayed by EibG-producing strain 2875/96 in different nutrient media. Strain 2875/96 was incubated without shaking at 37 °C for 20 h. (A) Bacteria aggregated and formed deposits in the complex media (LB). In reduced media, such as the yeast extract medium (HM) and the minimal medium (M9), cells grew homogeneously. (B) Microscopically, however, cells exhibited aggregates and clumps in all media. Figures show images after crystal violet staining.

Figure 4

EibG is highly expressed in the stationary phase under static growth conditions. Strain 2875/96 was incubated under static growth conditions (− agitation) and with shaking (+ agitation). Bacterial growth and protein content were determined at different time intervals as indicated. Protein aliquots of 3 µg (A) and 2 µg (B) per lane were immunoblotted and EibG signals were visualized using HRP-conjugated human IgG Fc fragment. Molecular masses of the standard proteins are indicated. Growth of bacteria was measured photometrically and followed by values of the optical density (closed circles and black lines) of these exemplary setups for short (A) and long incubation (B). Grey bars represent the signal intensities of EibG given as computer internal units. The results represent one out of three independent curves.

Figure 5

Once expressed, EibG proteins demonstrate high stability during prolonged incubation. For the adequate synthesis of EibG, strains 2875/96, 0520/99, and 3671/97 were incubated statically in LB broth without agitation (−) for 24 h. (A) These cultures were re-inoculated into a fresh medium at a 1:100 dilution followed by incubation with (+) and without (−) shaking. Aliquots were harvested after indicated time periods. Proteins (4 µg per lane) were immunoblotted and EibG were detected using human IgG Fc fragment antibody. (B) After static growth for 24 h and expression of EibG, the culture of STEC strain 2875/96 was aliquoted and incubation was extended with (+) and without (−) shaking. To inhibit protein and EibG synthesis, one aliquot was laced with tetracycline (20 µg/mL) (+). Cells were harvested at the indicated periods of time and proteins from the aliquots were immunoblotted. Molecular masses of standard proteins are indicated.