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

Abstract

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.

FIG 1

Increased levels of secreted FlgM under P_araBAD overexpression conditions. Western blot analysis of supernatant fractions of strains overexpressing FlgM from the chromosomal P_araBAD promoter. Overnight cultures were diluted 100-fold in LB medium and incubated at 37°C for 2 h, followed by addition of arabinose to 0.2% to induce excess FlgM expression. After 5 h of further incubation at 37°C, cells were separated from the supernatant by centrifugation and TCA was added to the cell supernatant to precipitate secreted proteins (see Materials and Methods). For the secreted proteins, 100 OD units sample was loaded; for the whole-cell protein, 50 OD units sample was loaded. Anti-FlgM antibody was used to determine levels of secreted and whole-cell FlgM (Sec, secreted FlgM; WC, whole-cell FlgM; Ara, 0.2% arabinose). P*, parent strain, TH18500 (ΔaraBAD1156::flgM⁺).

FIG 2

Effect of flhDC operon expression on levels of secreted FlgM and cell motility. (A) Secreted levels of FlgM in strains affected in flhDC operon expression were determined by quantitative Western blot analysis using anti-FlgM antibody to detect FlgM in the supernatant of the spent growth medium. (Sec FlgM, secreted FlgM; Sol FlgM, cellular soluble FlgM; Insol FlgM, cellular insoluble FlgM; WC DnaK, whole cellular DnaK). P*, parent strain, TH18500 (ΔaraBAD1156::flgM⁺). (B) Relative FlgM secreted levels. (C) Swim phenotype on soft agar plates of strain TH18649 (ΔPflhDC8089::tetR-PtetA ΔaraBAD1156::flgM⁺) with no inducer added, 1 μg/ml anyhdrotetracycline (ATc) added, and both ATc and 0.2% arabinose (Ara) added. (D) Motility assay for various mutant backgrounds depicted with ΔaraBAD1156::flgM⁺ in the absence and presence of added 0.2% arabinose. WT, wild type.

FIG 3

Effect of fliA (σ²⁸) alleles on levels of secreted FlgM and cell motility. (A) Levels of secreted FlgM were determined for strains carrying different alleles of the σ²⁸ structural gene fliA by quantitative Western blot analysis using anti-FlgM antibody to detect FlgM in the supernatant of the spent growth medium (Sec FlgM, secreted FlgM; WC FlgM, whole cellular FlgM; WC FliA, whole cellular FliA; WC DnaK, whole cellular DnaK). P*, parent strain, TH18500 (ΔaraBAD1156::flgM⁺). (B) Relative levels of secreted FlgM. (C) Motility assay with various mutant backgrounds, depicted with the ΔaraBAD1156::flgM⁺ strain (WT), in the absence and presence of added 0.2% arabinose.

FIG 4

Effect of flagellar late substrate deletion, growth phase, and fljB^enx vh2 mutations on levels of secreted FlgM. (A) Secreted levels of FlgM were determined for strains carrying different flagellum late substrate gene (flgK, flgL, fliC, fliD, and fljB) deletion mutants, flagellum-specific phase (Δhin-5717 and Δhin-5718) mutants, and fljB^enx vh2 mutants by quantitative Western blot analysis using anti-FlgM antibody to detected FlgM in the supernatant of the spent growth medium. Sec FlgM, secreted FlgM; Sol FlgM, cellular soluble FlgM; Insol FlgM, cellular insoluble FlgM; WC DnaK, whole cellular DnaK. P*, parent strain, TH18500 (ΔaraBAD1156::flgM⁺). (B) Relative secreted FlgM levels.

FIG 5

Effects of late flagellum T3S chaperone deletion mutations on levels of secreted FlgM. (A) Secreted levels of FlgM were determined for strains carrying different deletion mutant alleles of the flagellar T3S chaperone genes flgN, fliS, and fliT by quantitative Western blot analysis using anti-FlgM antibody to detect FlgM in the supernatant of the spent growth medium. Sec FlgM, secreted FlgM; WC FlgM, whole cellular FlgM; WC DnaK, whole cellular DnaK. P*, parent strain, TH18500 (ΔaraBAD1156::flgM⁺). (B) Relative levels of secreted FlgM.

FIG 6

Effects of Spi-1 and Spi-2 deletions on levels of secreted FlgM. (A) Secreted levels of FlgM were determined for strains carrying deletion mutant alleles of the either the Spi1 or Spi2 gene by quantitative Western blot analysis using anti-FlgM antibody to detect FlgM in the supernatant of the spent growth medium. Sec FlgM, secreted FlgM; WC FlgM, whole cellular FlgM; WC DnaK, whole cellular DnaK. P*, parent strain, TH18500 (ΔaraBAD1156::flgM⁺). (B) Relative levels of secreted FlgM.

FIG 7

Effects of cellular protease mutant alleles on levels of secreted FlgM and cell motility. (A) Secreted levels of FlgM were determined for strains carrying deletion mutant alleles of the ompT, degP, clpA, clpX, or clpP gene with and without a functional flhDC operon by quantitative Western blot analysis using anti-FlgM antibody to detect FlgM in the supernatant of the spent growth medium. Sec FlgM, secreted FlgM; WC FlgM, whole cellular FlgM; WC DnaK, whole cellular DnaK. P*, parent strain, TH18500 (ΔaraBAD1156::flgM⁺). (B) Relative levels of secreted FlgM in the flhD⁺C⁺ and flhDC null backgrounds and relative whole-cell FlgM accumulation level in flhDC null background. (C) Motility assay in various mutant backgrounds, depicted with the ΔaraBAD1156::flgM⁺ strain (WT) in the absence and presence of added 0.2% arabinose.

FIG 8

Effects of NaCl and KCl ionic strengths on levels of secreted FlgM and cell motility. (A) Secreted levels of FlgM were determined with strain P*, the parent strain, TH18500 (ΔaraBAD1156::flgM⁺), in LB-0.2% arabinose medium with NaCl and KCl added at concentrations depicted by quantitative Western blot analysis, using anti-FlgM antibody to detect FlgM in the supernatant of the spent growth medium. Sec FlgM, secreted FlgM; Sol FlgM, cellular soluble FlgM; Insol FlgM, cellular insoluble FlgM; WC DnaK, whole cellular DnaK. (B) Relative levels of secreted FlgM. (C) Motility assay using various concentrations of NaCl and KCl, depicted with strain TH18500, in the absence and presence of added 0.2% arabinose (Ara).

FIG 9

Effects of different combined mutations on levels of secreted FlgM. (A) Secreted levels of FlgM were determined for strains carrying different combined mutations, depicted by quantitative Western blot analysis using anti-FlgM antibody to detect FlgM in the supernatant of the spent growth medium (Sec FlgM, secreted FlgM; WC FlgM, whole cellular FlgM; WC DnaK, whole cellular DnaK). P*, parent strain, TH18500 (ΔaraBAD1156::flgM⁺). (B) Relative levels of secreted FlgM.

FIG 10

Effects of different combined mutations and added salt concentrations on levels of secreted FlgM-6His-TEV-δ-SVIE and FlgM-6His-ETK-δ-SVIE. (A, B, and C) Levels of secreted FlgM-6His-TEV-δ-SVIE and FlgM-6His-ETK-δ-SVIE were determined for strains carrying different combined mutations, depicted by quantitative Western blot analysis using anti-FlgM antibody to detected FlgM-6His-TEV-δ-SVIE and FlgM-6His-ETK-δ-SVIE in the supernatant of the spent growth medium (Sec FlgM-6His-TEV-SVIE, secreted FlgM-6His-TEV-δ-SVIE; Sec FlgM-6H-ETK-δ-SVIE, secreted FlgM-6His-ETK-δ-SVIE; Sec FlgM, secreted FlgM; WC DnaK, whole cellular DnaK). Relative levels of secreted FlgM-6His-TEV-δ-SVIE and FlgM-6His-ETK-δ-SVIE are presented at the right of the gels. P*, parent strain, TH17831 (ΔaraBAD1124::flgM-6His-TEV-δ-SVIE).

FIG 11

Secretion of AMA1, NCR, and MrVIA peptides fused to a His-tagged FlgM secretion signal. (A) Schematic diagram of the constructs tested. (B) Coomassie-stained SDS-PAGE gel of the supernatant from a culture expressing FlgM-His6-ETK-AMA1 in an optimized secretion strain (TH20685). The supernatant was concentrated 20-fold by TCA precipitation. (C) Western blot against 20 μl of nonconcentrated supernatant from panel B with anti-His antibody. (D) Coomassie-stained SDS-PAGE gel of the supernatant from cultures expressing three different NCR peptides fused to a His-tagged FlgM secretion signal in an optimized secretion strain background (NCR247, TH20221; NCR057, TH20222; NCR224, TH20223). The supernatants were concentrated 20-fold by TCA precipitation. (E) Coomassie-stained SDS-PAGE gel of the supernatant from a culture expressing conopeptide MrVIA fused to a His-tagged FlgM secretion signal in an optimized secretion strain background (TH20492). The supernatants were concentrated 20-fold by TCA precipitation. (F) Western blot against 20 μl of nonconcentrated supernatant from strain TH20492 (FlgM-His-ETK-MrVIA) with anti-His antibody grown under different arabinose induction concentrations.