Peptide-assisted degradation of the Salmonella MgtC virulence factor

Abstract

MgtC is a virulence factor common to several intracellular pathogens that is required for intramacrophage survival and growth in magnesium-depleted medium. In Salmonella enterica, MgtC is coexpressed with the MgtB magnesium transporter and transcription of the mgtCB operon is induced by magnesium deprivation. Despite the high level of mgtCB transcriptional induction in magnesium-depleted medium, the MgtC protein is hardly detected in a wild-type Salmonella strain. Here, we show that downregulation of MgtC expression is dependent on a hydrophobic peptide, MgtR, which is encoded by the mgtCB operon. Our results suggest that MgtR promotes MgtC degradation by the FtsH protease, providing a negative regulatory feedback. Bacterial two-hybrid assays demonstrate that MgtR interacts with the inner-membrane MgtC protein. We identified mutant derivatives of MgtR and MgtC that prevent both regulation and interaction between the two partners. In macrophages, overexpression of the MgtR peptide led to a decrease of the replication rate of Salmonella. This study highlights the role of peptides in bacterial regulatory mechanisms and provides a natural antagonist of the MgtC virulence factor.

Figure 1

The levels of the MgtC protein are regulated by the 3′ region of the mgtCB operon. (A) Schematic representation of sequences carried by plasmids pEG9091 (mgtC⁺ mgtB⁺), pEG9094 (mgtC⁺) and pEG9091 derivatives. (B) Western blot analysis of E. coli strains transformed with pEG9091, pEG9094 or pEG9091 derivatives. Total extracts were prepared from bacteria grown for 16 h in low-Mg²⁺ medium and were blotted with anti-MgtC antibodies or anti-DnaK antibodies. The band detected above MgtC appears non-specific since it is also found with E. coli strains or S. typhimurium strain NM14. (C) Western blot analysis of E. coli strains transformed with pEG9094 and pBBR1MCS vector or pMgtR. The pMgtR plasmid is a pBBR1MCS derivative that carries a sequence of 150 bp downstream of mgtB (which is highlighted in black in panel A).

Figure 2

The 3′ region of the mgtCB operon encodes a peptide that is involved in MgtC downregulation. (A) The 150-bp regulatory region at the 3′ end of the mgtCB operon includes a 90-bp sequence that could encode a small RNA or a peptide. One of the secondary structures of a putative RNA predicted by Mfold server (http://bioweb.pasteur.fr/seqanal/interfaces/mfold-simple.html) is shown (dG=−15.5 kcal mole⁻¹). Site-directed mutagenesis was performed on pMgtR plasmid at positions 1 (TCGCvCAGA) and 2 (GGTGvAGTC) to destabilize RNA structure without affecting protein sequence. The mutations 3 (TvC) and 4 (ΔC) affected synthesis of a putative ORF, without affecting predicted RNA structure. (B) Western blot analysis of E. coli strains transformed with pEG9094 and mutated pMgtR plasmids. The pBBR1MCS vector and pMgtR plasmid were used as controls. Total extracts were prepared from bacteria grown for 16 h in low-Mg²⁺ medium and were blotted with anti-MgtC antibodies or anti-DnaK antibodies.

Figure 3

Downregulation of MgtC by the induction of a His₆-MgtR peptide in a Salmonella ΔmgtR mutant. Western blot experiments were carried out on Salmonella extracts using anti-MgtC antibodies. (A) Characterization of a Salmonella ΔmgtR mutant and complementation with the pMgtR plasmid. Western blot on S. typhimurium wild-type and ΔmgtR-mutant strains. The pMgtR plasmid or the pBBR1MCS vector was introduced into the ΔmgtR-mutant strain. Extracts were blotted with anti-DnaK antibodies as control. (B) Induction of His₆-MgtR expression by addition of 0.1 mM IPTG decreased the expression of MgtC in a Salmonella ΔmgtR-mutant strain. The lower band is the His₆-MgtR peptide (approximately 5 kDa), which cross-reacts with anti-MgtC antibodies. (C) Kinetic of MgtC degradation by His₆-MgtR. After overnight growth without IPTG in conditions of mgtC transcription (10 μM Mg²⁺), bacteria were shifted to a medium containing 10 mM Mg²⁺ to shut down mgtC transcription and IPTG to induce His₆-MgtR expression. In each case, samples were prepared for Western blot at several time periods after the shift (0, 2 and 4 h). Identical volumes corresponding to a constant number of cells were loaded independently of the time point. (D) Quantification of the levels of MgtC in panel C.

Figure 4

The mgtR sequence belongs to mgtCB operon. RT–PCR experiments were performed on total RNA of wild-type S. typhimurium extracted after 30-min growth with 10 μM Mg²⁺ (L) or with 10 mM Mg²⁺ (H). (A) Schematic representation of the fragments amplified by RT–PCR. (B) Analysis of DNA fragments amplified by RT–PCR on agarose gel. RT–PCR of gapA is used as control. No band was amplified in the absence of reverse transcriptase (not shown).

Figure 5

The FtsH protease is involved in MgtC negative regulation mediated by MgtR. The thermosensitive E. coli ftsH mutant and the isogenic ftsH⁺ strain were transformed by plasmids pEG9094 (mgtC⁺) or pEG9091 del3 (mgtC⁺ mgtR⁺) (Figure 1A). Strains were cultured in low-Mg²⁺ medium for 36 h at 30°C or for 7 h at 30°C before an 18-h shift at 42°C. Total extracts were blotted with anti-MgtC antibodies or anti-DnaK antibodies.

Figure 6

Amino-acid residues of MgtR important for MgtC degradation. (A) Position of the mutations on MgtR sequence. The predicted transmembrane domain is shaded. Changes that lead to loss of function are highlighted with circles. (B) Western blot analysis of crude extracts prepared from strains with mutant MgtR encoded by pMgtR derivatives. Extracts were prepared from E. coli strains carrying the pEG9094 plasmid (mgtC⁺) and the mutated pMgtR plasmids. The pBBR1MCS vector and pMgtR plasmid were used as control. Total extracts were blotted with anti-MgtC antibodies or anti-DnaK antibodies. (C) Western blot analysis of crude extracts prepared from strains with mutant MgtR encoded by pQEMgtR derivatives. Extracts were prepared from S. typhimurium ΔmgtR strain carrying the mutated pQEMgtR plasmids. The pQE30 vector and pQEMgtR plasmid were used as control. Total extracts were blotted with anti-MgtC antibodies that recognize both MgtC and His₆-MgtR.

Figure 7

Identification of MgtC mutants that are insensitive to downregulation by MgtR. (A) Schematic representation of MgtC protein topology. Mutations that promoted MgtC expression on Western blot are clustered in the second cytoplasmic loop. (B) Western blot analysis of MgtC mutants. MgtC mutations were carried out on the pNM12 plasmid. Extracts were prepared from E. coli strains carrying pMgtR plasmid or the pBBR1MCS vector, and the pNM12 plasmid (mgtC⁺) or the mutated pNM12 derivatives.

Figure 8

Analysis of in vivo interaction between MgtC and MgtR using the BACHT system. The E. coli BTH101 strain was co-transformed with plasmids encoding MgtC-T18 and T25-MgtR fusions. Assays were carried out at 30°C. Four mutations, L15R, L15P, A24R and A24P, were introduced in the T25-MgtR fusion. Three mutations, E84A, G85A and N92T were introduced in the MgtC-T18 fusion. The basal level of β-galactosidase activity measured with vector is approximately 60 Miller units.

Figure 9

Role of Ala-coil motifs in MgtC–MgtR interaction. (A) Ala-coil motifs and mutagenesis on MgtR and MgtC TM4 sequences. (B) Effect of Ala-coil mutations on the interaction between MgtC and MgtR in vivo using the BACHT system. Assays were carried out at 30°C. (C) Effect of Ala-coil mutations in MgtR on MgtC expression. Extracts were prepared from S. typhimurium ΔmgtR strain carrying the mutated pQEMgtR plasmids and blotted with anti-MgtC antibodies. The pQE30 vector and pQEMgtR plasmid were used as control.

Figure 10

Role of MgtR in S. typhimurium intramacrophage growth. The replication of Salmonella strains in J774 macrophages was evaluated 18 h after infection. Data represent the mean values plus standard errors from at least three independent experiments. (A) Analysis of a ΔmgtR-mutant strain in comparison with a wild-type strain and a ΔmgtC mutant. Values presented are the percentage relative to that of the wild-type S. typhimurium 14028s. (B) Analysis of a wild-type Salmonella strain that carries the pMgtR plasmid. Values presented are the percentage relative to that of the wild-type S. typhimurium 14028s with the pBBR1MCS vector.

Figure 11

Model for the role of MgtR in MgtC expression: the MgtR peptide binds directly to MgtC at the cytoplasmic membrane. The interaction between MgtC and MgtR would unfold MgtC and make it a target for degradation by the FtsH protease.