Directionality of substrate translocation of the hemolysin A Type I secretion system

Abstract

Type 1 secretion systems (T1SS) of Gram-negative bacteria are responsible for the secretion of various proteases, lipases, S-layer proteins or toxins into the extracellular space. The paradigm of these systems is the hemolysin A (HlyA) T1SS of Escherichia coli. This multiple membrane protein complex is able to secrete the toxin HlyA in one step across both E. coli membranes. Common to all secreted T1SS substrates is a C-terminal secretion sequence being necessary as well as sufficient for secretion. However, it is not known whether transport occurs directionally, i.e. the N- or the C-terminus of T1SS substrates is secreted first. We have addressed this question by constructing HlyA fusions with the rapidly folding eGFP resulting in a stalled T1SS. Differential labeling and subsequent fluorescence microscopic detection of C- and N-terminal parts of the fusions allowed us to demonstrate vectorial transport of HlyA through the T1SS with the C-terminus appearing first outside the bacterial cells.

Figure 1. The eGFP-HlyAc fusion protein stalls the translocator and prevents HlyAc secretion.

(a) SDS-PAGE analysis of the HlyAc secretion level (with Coomassie blue staining) in the culture supernatant over time without and with induction of eGFP-HlyAc expression. (b) Relative intensity of the SDS-PAGE bands illustrates HlyAc secretion levels without and with induction of eGFP-HlyAc. (c) Western blot analysis of the cells (comparison with the supernatants in (a)) show that eGFP-HlyAc is only present in the induced cells and that the levels of HlyB and HlyD remain constant over time.

Figure 2. The eGFP-HlyAc-Δss fusion protein is unable to block the translocator.

(a) SDS-PAGE analysis of the HlyAc secretion level in the culture supernatant over time without and with induction of eGFP-HlyAc-Δss expression. (b) Relative intensity of the SDS-PAGE bands illustrates the HlyAc secretion levels without and with induction of eGFP-HlyAc-Δss. (c) Western blot analysis of the cells (correlating to the supernatants in (a)) show that eGFP-HlyAc-Δss is only present in the induced cells and that the level of HlyB and HlyD remain constant over time.

Figure 3. Detection of the surface exposed HlyAc fragment of eGFP-HlyAc by confocal laser scanning microscopy.

E. coli cells expressed HlyB and HlyD, as well as additional eGFP-HlyAc and eGFP-HlyAc-Δss. Shown is the eGFP fluorescence (left panel) of the fusion proteins, the HlyA mediated Cy3 fluorescence at the cell surface (second left panel), merged images of eGFP and Cy3 fluorescence (second right panel) and differential interference contrast (DIC) images of the cells (right panel). The different combinations of proteins employed are indicated to the left.

Figure 4

(a) Relative cell fluorescence of eGFP. All values were normalized to the eGFP fluorescence of the eGFP-HlyAc fusion protein (error bars represent the standard error of the mean) after subtraction of autofluorescence. (b) Relative fluorescence of Cy3. All values are normalized to Cy3 fluorescence of the eGFP-HlyAc fusion protein (error bars represent the standard error of the mean) after subtraction of autofluorescence. The different combinations of proteins employed are indicated below the bars.

Figure 5. Detection of the surface exposed HlyA fragment of eGFP-HlyA by confocal laser scanning microscopy.

E. coli cells expressed HlyB and HlyD, as well as additional eGFP-HlyAc and eGFP-HlyAc-Δss, respectively. Shown is the eGFP fluorescence (left panel) of the fusion proteins, the HlyA mediated Cy3 fluorescence at the cell surface (second left panel), merged images of eGFP and Cy3 fluorescence (second right panel) and differential interference contrast (DIC) images of the cells (right panel). The different combinations of proteins tested are indicated to the left.

Figure 6

(a) Relative cell fluorescence of eGFP. All values were normalized to the eGFP fluorescence of the eGFP-HlyAc fusion protein (error bars represent the standard error of the mean) after subtraction of autofluorescence. (b) Relative fluorescence of Cy3. All values are normalized to Cy3 fluorescence of the eGFP-HlyAc fusion protein (error bars represent the standard error of the mean) after subtraction of autofluorescence. The different combinations of proteins employed are indicated below the bars.

Figure 7. Detection of the surface exposed HlyA fragment of eGFP-HlyAc and eGFP-HlyA by structured illumination microscopy.

Shown are maximum intensity projections of z-stacks of representative E. coli cells expressing eGFP-HlyAc or eGFP-HlyA together with HlyB and HlyD. The eGFP fluorescence (left panel in green) is displayed in wide field mode, the HlyA signal (medium panel in red) is displayed in high resolution mode after SIM processing. The right panel shows merged images derived from eGFP and Cy3 fluorescence recordings.

Figure 8. Model for secretion by the T1SS.

The T1SS is indicated by an oval box and the substrate, here HlyA, is depicted as a red line, while eGFP is shown in green (pdb file 2Y0G). HlyAc is secreted (indicated by the grey arrow) and folds in the extracellular space due to the higher calcium concentration. eGFP-HlyAc can only enter the T1SS but is not able to complete the secretion process (indicated by the dotted grey arrow) due to the fast folding of eGFP and stalling inside the translocator. The model also assumes that cell surface exposed HlyAc fragment of the fusion protein folds due to the higher extracellular calcium concentration. Calcium ions are indicted by small black boxes. For further details see text.