Quantification and Surface Localization of the Hemolysin A Type I Secretion System at the Endogenous Level and under Conditions of Overexpression

Abstract

Secretion systems are essential for Gram-negative bacteria, as these nanomachineries allow communication with the outside world by exporting proteins into the extracellular space or directly into the cytosol of a host cell. For example, type I secretion systems (T1SS) secrete a broad range of substrates across both membranes into the extracellular space. One well-known example is the hemolysin A (HlyA) T1SS from Escherichia coli, which consists of an ABC transporter (HlyB), a membrane fusion protein (HlyD), the outer membrane protein TolC, and the substrate HlyA, a member of the family of repeats in toxins (RTX) toxins. Here, we determined the amount of TolC at the endogenous level (parental strain, UTI89) and under conditions of overexpression [T7 expression system, BL21(DE3)-BD]. The overall amount of TolC was not influenced by the overexpression of the HlyBD complex. Moving one step further, we determined the localization of the HlyA T1SS by superresolution microscopy. In contrast to other bacterial secretion systems, no polarization was observed with respect to endogenous or overexpression levels. Additionally, the cell growth and division cycle did not influence polarization. Most importantly, the size of the observed T1SS clusters did not correlate with the recently proposed outer membrane islands. These data indicate that T1SS clusters at the outer membrane, generating domains of so-far-undescribed identity. IMPORTANCE Uropathogenic Escherichia coli (UPEC) strains cause about 110 million urinary tract infections each year worldwide, representing a global burden to the health care system. UPEC strains secrete many virulence factors, among these, the TX toxin hemolysin A via a cognate T1SS into the extracellular space. In this study, we determined the endogenous copy number of the HlyA T1SS in UTI89 and analyzed the surface localization in BL21(DE3)-BD and UTI89, respectively. With approximately 800 copies of the T1SS in UTI89, this is one of the highest expressed bacterial secretion systems. Furthermore, and in clear contrast to other secretion systems, no polarized surface localization was detected. Finally, quantitative analysis of the superresolution data revealed that clusters of the HlyA T1SS are not related to the recently identified outer membrane protein islands. These data provide insights into the quantitative molecular architecture of the HlyA T1SS.

Keywords: T1SS; TolC; membrane cluster; membrane localization; secretion systems; type I secretion.

FIG 1

Quantification of TolC trimers. Bar diagram of the quantification of TolC by quantitative Western blot analysis. Blue, BL21(DE3)-BD cells; red, UTI89 cells; +, induction of eGFP-HlyA and eGFP-HlyAc, respectively; −, no induction during the experiment; ns, not significant. In the case of UTI89, HlyB and HlyD were not overexpressed, and only the endogenous protein levels were present. In BL21(DE3)-BD, HlyB and HlyD were also overexpressed. One-way ANOVA (P < 0.05) and Tukey’s multiple-comparison test indicated no significance differences. Bars represent the mean value of at least five independent quantifications, and the error bars represent the standard error of the mean.

FIG 2

Quantification of active T1SS in E. coli BL21(DE3)-BD and UTI89. Error bars represent the standard error of the mean of three independent experiments. **, P < 0.01.

FIG 3

Growth analysis of E. coli UTI89. (A) Western blotting against HlyB (left) and HlyD (right) of wild-type UTI89. Time points after inoculation (in hours) at which the samples were taken are indicated. Overnight cultures (o/n) corresponded to approximately 14 h. Polyclonal antibodies were used as stated. Arrows indicate the size of the corresponding full-length proteins. (B) Growth curve of E. coli UTI89 in a 96-well plate (for further details, see Materials and Methods). The curve represents the average of three biological replicates, each of which was performed in quadruplicate.

FIG 4

Surface staining of stalled eGFP-HlyA T1SS does not indicate a specific accumulation of the complex at the tip. (A) eGFP-HlyA expression was induced in BL21(DE3)-BD or UTI89, and cells were fixed after 2 h of expression. DNA was stained by DAPI, and cell surface-exposed HlyA was stained by immunofluorescence labeling. Cells were imaged by SIM. Maximum-intensity projections (MIPs) of raw data before processing show the DAPI channel in blue (DAPI), the eGFP channel in green (eGFP-HlyA), and the HlyA channel in red (anti-HlyA). Contrast was identical to the conditions applied to nonexpressing BL21(DE3)-BD and wild-type UTI89 cells (Fig. S4 in the supplemental material). Processed SIM MIPs are shown on the right (SIM). White square indicates the area displayed in panel B. Scale bar, 5 μm. (B, Left) Inset of SIM-processed MIP. (B, Middle) The first analysis step highlights the area of the bacterial cell (blue) and the detected maxima (white). (B, Right) The final output of the analysis included a color-coded classification for each bacterium highlighting both tips and the cell body areas. The selected bacterium displayed no specific accumulation at any tip, indicated by blue outline. Details on color coding can be found in the supplementary information under “Classification and quality management.” Scale bar, 1 μm. (C) Quantification of nonaccumulating and one tip- and both tip-accumulated HlyA signals. Staining and analysis were performed in duplicate using 3 to 5 different clones and at least 15 images for each replicate. In total, 1,411 UTI89 bacteria were analyzed with respect to a potential accumulation of the stalled HlyA T1SS at the tips, and 771 bacteria were analyzed in the case of BL21(DE3)-BD.

FIG 5

Quantification of anti-HlyA antibody cluster size in SIM data. Signals identified and used to estimate the polarized localization analysis (Fig. 4) were analyzed for full width half maximum (FWHM) as size parameter. Values were summed in bins of 5-nm sizes and plotted using the calculated FWHM in micrometer on the x axis and the absolute amount of occurring cluster sizes per bin on the y axis. A calculated normal distribution of the data was overlaid in blue. On top of the graph, a box-and-whisker plot of the distribution is displayed indicating the median (vertical line), average value (square), interquartile range (box), and 5%/95% (whiskers).

FIG 6

Anti-HlyA antibody clusters distribution in median bacteria shape and dependence on cell length. (A) Relative density plot of anti-HlyA antibody signals of BL21(DE3)-BD cells (left) and UTI89 (right) to a normalized median bacterial shape. Longitudinal and transversal size is indicated on the axes. (B) Anti-HlyA antibody signals of BL21(DE3)-BD (left) and UTI89 (right) cells are plotted on vertical lines sorted by cell length (x axis). Relative anti-HlyA signal densities are displayed for both figures as color-coded heatmap ranging from 1 (white) to 0 (black) as indicated on the right side panel.