Analysis of factors that affect FlgM-dependent type III secretion for protein purification with Salmonella enterica serovar Typhimurium

The FlgM protein is secreted in response to flagellar hook-basal body secretion and can be used as a secretion signal to direct selected protein secretion via the flagellar type III secretion (T3S) system [H. M. Singer, M. Erhardt, A. M. Steiner, M. M. Zhang, D. Yoshikami, G. Bulaj, B. M. Olivera, and K. T. Hughes, mBio 3(3):e00115-12, 2012, http://dx.doi.org/10.1128/mBio.00115-12]. Conditions known to affect flagellar gene expression, FlgM stability, and flagellar T3S were tested either alone or in combination to determine their effects on levels of secreted FlgM. These conditions included mutations that affect activity of the flagellar FlhD4C2 master regulatory protein complex or the FlgM T3S chaperone σ(28), the removal of Salmonella pathogenicity island 1 (Spi1), the removal of flagellar late secretion substrates that could compete with FlgM for secretion, and changes in the ionic strength of the growth medium. Conditions that enhanced FlgM secretion were combined in order to maximize levels of secreted FlgM. An optimized FlgM secretion strain was used to secrete and isolate otherwise difficult-to-produce proteins and peptides fused to the C terminus of FlgM. These include cysteine-rich, hydrophobic peptides (conotoxins δ-SVIE and MrVIA), nodule-specific, cysteine-rich antimicrobial peptides (NCR), and a malaria surface antigen domain of apical membrane antigen AMA-1.