Biochemical characterization of the SPATE members EspPα and EspI

Abstract

The activity of serine proteases is influenced by their substrate specificity as well as by the physicochemical conditions. Here, we present the characterization of key biochemical features of the two SPATE members EspPα and EspI from Shiga-toxin producing Escherichia coli (STEC) and enterohemorrhagic E. coli (EHEC). Both proteases show high activity at conditions mimicking the human blood stream. Optimal activities were observed at slightly alkaline pH and low millimolar concentrations of the divalent cations Ca2+ and Mg2+ at physiological temperatures indicating a function in the human host. Furthermore, we provide the first cleavage profile for EspI demonstrating pronounced specificity of this protease.

Figure 1

Purification of EspPα and EspI (left) SDS-PAGE of purified EspPα. *, EspPα autoproteolysis product; (right) SDS-PAGE of purified EspI. *, EspI autoproteolysis product. M = Molecular weight marker. Purity (including autoproteolysis products) of both samples was >95% as determined by densitometrical analysis of SDS-PAGE gels.

Figure 2

Temperature optimum and heat denaturation of EspPα (a) Relative activity of EspPα at varying incubation temperatures. Relative activity is normalized to T_opt at ~40 °C, n = 8; (b) Effect of 30 min pre-incubation at elevated temperatures on the activity of EspPα at 37 °C. Pre-incubation temperatures are given in the x-axis and the relative activity was subsequently determined at 37 °C. The negative control was incubated for 30 min at 20 °C. Relative activity is normalized to the negative control, n = 8.

Figure 3

Temperature optimum and heat denaturation of EspI (a) Relative activity of EspI at varying incubation temperatures. Relative activity is normalized to T_opt at ~38 °C, n = 3; (b) Effect of 120 min pre-incubation at elevated temperatures on the activity of EspI at 37 °C. Pre-incubation temperatures are given in the x-axis and the relative activity was subsequently determined at 37 °C. The negative control was incubated for 120 min at 20 °C. Relative activity is normalized to the negative control, n = 3.

Figure 4

Determination of the pH optimum of EspPα. Activity of EspPα was determined in the pH range from 5.5 to 9.1. The proteolytic activity is expressed relative to pH_opt at ~7.4, n = 5.

Figure 5

Determination of the pH optimum of EspI. Activity of EspI was determined in the pH range from 5.9 to 9.4. The proteolytic activity is expressed relative to pH_opt at ~7.5. Note that EspI activity in TRIS buffer is higher than in tetraborate buffer. n = 4.

Figure 6

Influence of buffer composition on the activity of EspPα. Relative activity is normalized to the maximal activity observed for 82.5 mM Mg²⁺. n = 8.

Figure 7

Influence of buffer composition on the activity of EspI. (a) Relative activity is normalized to the maximal activity observed for 186 mM Mg²⁺, n = 4; (b) Detailed view of cation concentrations in the range from 0 to 9.6 (Na⁺) or 9.3 mM (Ca²⁺, Mg²⁺).

Figure 8

Cleavage profile of EspI. Chromogenic substrates were incubated with EspI. Activity is normalized to the maximal activity observed after incubation of Suc-Ala-Ala-Pro-Leu-pNA, n = 3.