A host-specific factor is necessary for efficient folding of the autotransporter plasmid-encoded toxin

Abstract

Autotransporters are the most common virulence factors secreted from Gram-negative pathogens. Until recently, autotransporter folding and outer membrane translocation were thought to be self-mediated events that did not require accessory factors. Here, we report that two variants of the autotransporter plasmid-encoded toxin are secreted by a lab strain of Escherichia coli. Biophysical analysis and cell-based toxicity assays demonstrated that only one of the two variants was in a folded, active conformation. The misfolded variant was not produced by a pathogenic strain of enteroaggregative E. coli and did not result from protein overproduction in the lab strain of E. coli. Our data suggest a host-specific factor is required for efficient folding of plasmid-encoded toxin.

Figure 1

Isolation of two functionally distinct variants of Pet from E. coli HB101. (A): Native gel electrophoresis of Pet prior to size exclusion chromatography. The molecular masses (kDa) of protein standards are indicated. Pet was visualized with Coomassie staining. (B): Elution profile of Pet fractions generated with a HiLoad 16/60 Superdex 75 size exclusion column. (C): Either toxin-free media or Pet-containing media at a final protein concentration of 40 μg/ml was added to CHO cells and incubated overnight at 37°C. Phase contrast images were then taken at 10× magnification. Cell rounding is indicative of productive intoxication.

Figure 2

Structural characteristics of the two Pet variants produced by E. coli HB101. (A): Far-UV CD spectra of Pet elution peaks 1 (dashed line) and 2 (solid line) from Fig. 1B. (B): Fluorescence spectra of Pet elution peaks 1 (dashed line) and 2 (solid line) from Fig. 1B.

Figure 3

Stability of Pet after size exclusion chromatography. (A): Pet produced by E. coli HB101 was separated into two fractions by size exclusion chromatography. (B): When the toxin in peak 2 was collected and immediately reapplied to the HiLoad 16/60 Superdex 75 size exclusion column, a single peak corresponding to fraction 2 was obtained. (C) Far-UV CD spectrum of the toxin present in the single peak obtained after a second round of chromatography.

Figure 4

Structural characteristics of Pet produced by E. coli HB101 under suboptimal expression conditions. (A): Pet produced by an E. coli HB101 culture grown at 25°C was subjected to size exclusion chromatography with the HiLoad 16/60 Superdex 75 column. At 25°C, toxin yield was 5% of the amount recovered from HB101 cultures grown at 37°C. (B): Far-UV CD spectrum of Pet peak 1 from panel A, collected between 39.3 and 41 ml of elution volume. (B): Far-UV CD spectrum of Pet peak 2 from panel A, collected between 49.5 and 51.2 ml of elution volume.

Figure 5

Structural state of the Pet variants after denaturation and refolding. The peak 2 and peak 1 toxins produced by E. coli HB101 were denatured in GnHCl and then allowed to refold by removal of the denaturant with dialysis. (A): The elution profile of Pet peak 2 after denaturation and refolding yielded two peaks, fractions 2.1 and 2.2. (B): Far-UV CD spectra of Pet fractions 2.1 (dashed line) and 2.2 (dotted line). (C): The elution profile of Pet peak 1 after denaturation and refolding. (D): Far-UV CD spectra of Pet peak 1 after denaturation and refolding.

Figure 6

Isolation of a single Pet variant from EAEC. Pet was purified from a strain of EAEC that had been genetically modified to eliminate the expression of a second autotransporter. (A): Elution profile of Pet generated with a HiLoad 16/60 Superdex 75 size exclusion column. (B): Far-UV CD spectrum of the toxin present in the major peak shown in panel A.

Figure 7

Homology-modeled structures of SPATE proteins. The structures of Pet and other SPATEs were modeled using hemoglobin protease (PBD_1WXR) as the template. Hydrophobic residues are indicated in yellow. (A) hemoglobin protease; (B) Pet; (C) Sat; (D) EspP; (E) EspC; (F) SepA.