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. 2019 Nov 13;10:2551. doi: 10.3389/fmicb.2019.02551

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Copyright © 2019 Tseytin, Mitrovic, David, Langenfeld, Zarivach, Diepold and Sal-Man.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Replacement of EscS TMDs by an alternative hydrophobic sequence abolishes T3SS activity. (A) 3D structure of EscS_wt monomeric subunit within the EscR₅S₄T complex (green). The hydrophobic regions, TMD1_ex and TMD2_ex, that were replaced to 7L9A are marked in cyan and red, respectively. (B) Protein secretion profiles of EPEC WT, ΔescN, ΔescS, and ΔescS or WT strains carrying the pEscS_wt-HA, pEscS-TMD1_ex-HA or pEscS-TMD2_ex-HA plasmids grown under T3SS-inducing conditions. The secreted fractions were concentrated and analyzed by SDS-PAGE and Coomassie staining. The secreted translocator EspA, EspB, and EspD are marked on the right of the gel. Also, indicated is the location of EspC, which is not secreted via the T3SS. The ΔescS strain carrying the plasmid encoding EscS_wt-HA showed proper T3SS activity while replacement of either TMD1 or TMD2 to an alternative hydrophobic sequence resulted in non-functional T3SS. Expression of pEscS-TMD1_ex-HA or pEscS-TMD2_ex-HA within the WT EPEC strain had no dominant-negative effect on the T3SS activity. (C) EscS_wt-HA, EscS-TMD1_ex-HA and EscS-TMD2_ex-HA expression was confirmed in the bacterial pellets obtained in (B) by SDS-PAGE and western blot analysis with an anti-HA antibody. EscS-TMD1_ex-HA migrated slightly slower than EscS_wt-HA and EscS-TMD2_ex-HA. (D) EPEC ΔescS carrying either pEscS_wt-HA, pEscS-TMD1_ex-HA, or pEscS-TMD2_ex-HA, were grown under T3SS-inducing conditions, fractionated into periplasmic (Peri), cytoplasmic (Cyto), and membrane (Mem) fractions and analyzed by western blot analysis with an anti-HA antibody. To confirm correct bacterial fractionation, the western blots were probed with anti-DnaK (cytoplasmic marker), anti-MBP (periplasmic marker), and anti-intimin (membrane marker) antibodies. (E) Proteins extracted from of HeLa cells infected with WT, ΔescN, ΔescS, or ΔescS carrying the pEscS_wt-HA, pEscS-TMD1_ex-HA or pEscS-TMD2_ex-HA, were subjected to western blot analysis using anti-JNK antibody and anti-actin (loading control). JNK and its degradation fragments are indicated. WT EPEC showed massive degradation of JNK relative to the uninfected sample and the samples infected with ΔescN or ΔescS mutant strains. EPEC ΔescS complemented with pEscS_wt-HA showed a JNK degradation profile similar to that of WT EPEC, indicating a functional complementation, while the ΔescS transformed with EscS exchanged TMDs (ΔescS+ pEscS-TMD1_ex-HA or ΔescS+ pEscS-TMD2_ex-HA) vectors showed a JNK profile similar to that of the uninfected sample.