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| Peptide Synthesis | For T3SS protein expression and secretion assays, overnight cultures of the E. tarda strains were diluted 1:200 in DMEM, and grown without shaking for 24 h at 25°C under 5% CO2. For pH shift experiments, bacteria cultured overnight in TSB were re-suspended into pre-warmed DMEM (at OD540 = 0.3) at pH 5.5 for 4 hours before being shifted to pH 5.5 or pH 7.2 media for additional 90 min. Secreted (extracellular proteins, ECP) proteins and bacterial lysate proteins (total bacterial proteins, TBP) were then prepared and loaded for immunoblotting according to equivalent OD540 of culture as described (31). ECP and TBP samples were separated on a NuPAGE 12% or 10% gel for electrophoresis in MES or MOPS SDS running buffer (Invitrogen) and transferred onto PVDF membrane (Millipore), before being probed with primary antibodies, including rabbit antibodies against EseB (1:1,000) (32), EseC (1:1,000) (33), EseD (1:1,000) (33), EseG (1:1,000) (13), EvpC (1:5,000) (31), EsaL (1:1000), EsaB (1:2000), and EsaM (1:2000). The anti-EsaL, anti-EsaB, and anti-EsaM antibodies were raised in rabbits against keyhole limpet hemocyanin-conjugated peptides of EsaL [EsaL aa 41 to 54; PTDRQTIVPHAAPG], EsaB [EsaB aa 146 to 159; RDTHPESPFIGRYA], and EsaM [EsaM aa 113 to 126; LLDRVMENPHENGQ], 150 respectively, by Genscript, China, and purified using the specific peptides as the 151 ligand. Horse-radish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Millipore) 152 was used as the secondary antibody. | Get A Quote |
The type III secretion system (T3SS) plays a crucial role in the pathogenesis of many Gram-negative bacteria, including Edwardsiella tarda, an important fish pathogen. Within the E. tarda T3SS, there are three proteins (EsaB/EsaL/EsaM) that are homologous to proteins present in many other bacteria, including SpiC/SsaL/SsaM in Salmonella, SepD/SepL/CesL in EPEC and EHEC, and YscB/YopN/SycN in Yersinia. EsaL was found to interact with both EsaB and EsaM within the bacterial cell, as revealed by a co-immunoprecipitation assay. Moreover, EsaM is required for EsaB stability, and they interact with each other. EsaB, EsaL and EsaM are all indispensible for the secretion of the T3SS translocon pr... More
