Figure 1.

Calcium depletion from the bacterial growth media enhances effector secretion. (a) T3 secretion profiles of the EPEC wild‐type strain (WT) grown in regular DMEM containing 1.8 mmol/L of CaCl₂ (Regular), calcium‐free DMEM (Ca²⁺‐free) or calcium‐free DMEM supplemented with 1.8 mmol/L of CaCl₂ (Ca²⁺‐free + Ca²⁺), and the ∆sepL and ∆escU mutant strains, visualized by SDS‐PAGE stained with Coomassie brilliant blue (CBB) (upper panel). The presence of DnaK, Tir, Map, and EspA in the supernatants (SN) and whole‐cell lysates (WC) was examined by immunoblotting, using anti‐DnaK, anti‐Tir, anti‐Map, and anti‐EspA antibodies (lower panels). (b) Protein secretion profiles of EPEC WT strain overproducing HA‐tagged T3 substrates, grown in the presence or absence of calcium as described in (a). Immunodetection of LEE and non‐LEE‐encoded effectors (NleH2, EspH, and NleC) and the inner rod protein EscI, was performed in the supernatants (SN) and whole‐cell lysates (WC) using specific antibodies against the HA tag. The results shown are representative of three independent experiments. (c) Relative abundance of EspA and Tir proteins in the supernatant of EPEC wild‐type strain grown in the presence or absence of calcium as described in (a). CBB‐stained protein bands were quantified from six independent secretion assays by gel densitometry using the ImageJ software (Schneider, Rasband, & Eliceiri, 2012). The secretion level of EspA and Tir proteins was normalized relative to the secretion level of the EspC autotransporter band. The average and the standard deviation of normalized data are displayed. Significant statistical differences compared with the regular DMEM condition are denoted by asterisks. A p value < .05 was considered statistically significant. *^* p = .002, Wilcoxon‐test