A PorX/PorY and σP Feedforward Regulatory Loop Controls Gene Expression Essential for Porphyromonas gingivalis Virulence

Abstract

The PorX/PorY two-component system in the periodontal pathogen Porphyromonas gingivalis controls the expression of the por genes, encoding a type IX secretion system, and the sigP gene, encoding sigma factor σ^P. Previous results implied that PorX/PorY and σ^P formed a regulatory cascade because the PorX/PorY-activated σ^P enhanced the por genes, including porT, via binding to their promoters. We recently showed that PorX also binds to the por promoters, thus suggesting that an alternative mechanism is required for the PorX/PorY- and σ^P-governed expression. Here, our in vitro assays show the PorX response regulator binds to the sigP promoter at a sequence shared with the porT promoter and enhances its transcription, mediated by a reconstituted P. gingivalis RNA polymerase holoenzyme. Merely producing σ^P in trans fails to reverse the porT transcription in a porX mutant, which further argues against the action of the proposed regulatory cascade. An in vitro transcription assay using a reconstituted RNA polymerase-σ^P holoenzyme verifies the direct role of PorX in porT transcription, since transcription is enhanced by a pure PorX protein. Accordingly, we propose that the PorX/PorY system coordinates with σ^P to construct a coherent regulatory mechanism, known as the feedforward loop. Specifically, PorX will not only bind to the sigP promoter to stimulate the expression of σ^P, but also bind to the porT promoter to facilitate the RNA polymerase-σ^P-dependent transcription. Importantly, mutations at the porX and sigP genes attenuate bacterial virulence in a mouse model, demonstrating that this regulatory mechanism is essential for P. gingivalis pathogenesis. IMPORTANCE The anaerobic bacterium Porphyromonas gingivalis is not only the major etiologic agent for chronic periodontitis, but also prevalent in some common noncommunicable diseases such as cardiovascular disease, Alzheimer's disease, and rheumatoid arthritis. We present genetic, biochemical, and biological results to demonstrate that the PorX/PorY two-component system and sigma factor σ^P build a specific regulatory network to coordinately control transcription of the genes encoding the type IX secretion system, and perhaps also other virulence factors. Results in this study verify that the response regulator PorX stimulates the expression of the genes encoding both σ^P and the type IX secretion system by binding to their promoters. This study also provides evidence that σ^P, like the PorX/PorY system, contributes to P. gingivalis virulence in a mouse model.

Keywords: PorX/PorY; Porphyromonas gingivalis; extracytoplasmic function sigma factor P; feedforward loop; in vitro transcription; transcription regulation; two-component system; type IX secretion system; virulence factors.

FIG 1

The PorX/PorY system and σ^P coordinate transcription in P. gingivalis. (A to C) mRNA levels of the porT gene (A), the PGN_0341 gene (B), and the PGN_1639 gene (C) in the 33277 wild-type strain, the ΔporX mutant (YS19181), and the ΔsigP mutant (YS17717) carrying pT-COW, p-porX (pYS18679, pT-COW-P_{PGN_1016}-porX), or p-sigP (pYS19107, pT-COW-P_sigP-sigP). The mRNA level in the wild-type strain was set to 1 for calculation. Results are representative of three independent experiments. *, P < 0.05; **, P < 0.01; versus wild type by t test. (D) The growth of wild-type 33277 strain with pT-COW (vector), ΔporX mutant (YS19181), and ΔsigP mutant (YS17717) carrying pT-COW, p-porX, or p-sigP, respectively, on a blood BHI plate containing tetracycline (0.5 μg/ml). Results are representative of four independent experiments.

FIG 2

The PorX response regulator binds to the sigP promoter region. (A) The mRNA levels of the sigP gene in the 33277 wild-type strain and the ΔporX mutant (YS19181) carrying pT-COW, p-porX, or p-sigP. Results are representative of three independent experiments. (B) EMSA analysis for binding of PorX to the sigP promoter. ³²P-labeled sigP DNA fragment (40 fmol) was incubated with PorX-_C-His₆ protein at the indicated amount. Lane 5 is the same as lane 4 but supplemented with nonlabeled (cold) sigP DNA fragment (1 pmol). The PorX/DNA mixtures were subjected to 5% PAGE. The location of DNA migration was detected by autoradiography. Arrows indicate the shifted bands after DNA fragments were associated with the PorX-_C-His₆ protein. The experiment was repeated twice. (C) DNase footprinting analysis of the sigP promoter fragment amplified with primers ³²P-3043 and 3044 for the coding strand and increasing amounts of PorX-c-His₆ protein. Products were separated in polyacrylamide DNA sequencing electrophoresis and the bands were detected by autoradiography. The bracket indicates the region protected by the PorX-_C-His₆ protein. Underlined DNA sequence (right of gel) indicates the PorX-protected nucleotides in the sigP promoter. The ladder M corresponds to the same ³²P-labeled sigP promoter fragment and degraded by the Maxam and Gilbert reaction. Results were repeated multiple times. (D) The DNA sequence of the sigP promoter region. Underlining corresponds to the PorX-protected region characterized in (C). Capital letters represent the sigP start codon. Numbering begins from the adenine nucleotide of the start codon. Highlighted sequences are shared by the PorX-binding site in the porT promoter (also shown in panel E). (E) The homologous sequences of the PorX-binding sites in the sigP and porT promoters. Vertical lines represent the identical nucleotides in the two sequences. Arrows represent the complementary nucleotides exhibited in the two sequences. Highlighted sequences are shared in these two promoters.

FIG 3

PorX promotes sigP transcription in vitro, mediated by a reconstituted P. gingivalis RNA polymerase-σ^D. (A) In vitro transcription of a 275-bp template (T₁) from the sigP promoter containing the first 29 coding nucleotides was conducted as described in the Materials and Methods. Left braces indicate the P₁ and P₂ transcripts synthesized by 50 nM of RNA polymerase-σ^D (RNAP-σ^D) from reactions supplemented with different amounts of the PorX-_C-His₆ protein. The ladder M corresponds to a PCR product generated with primers 3044 and ³²P-labeled primer 3043 and degraded by the Maxam and Gilbert reaction. (B) The DNA sequence of the sigP promoter region. Underlining corresponds to the PorX-protected region. Blue dashed frames correspond to the regions labeled as p₁ and p₂, respectively, where transcription was initiated. The highlighted sequence corresponds to the wild-type sequence which was substituted by the sequence (Sub) in red capital letters. Numbering begins from the adenine nucleotide of the start codon (underlined capital letters). (C) In vitro transcription of the sigP templates (T₁ and T_1-sub) with the wild-type sequence and a substituted sequence, respectively. Blue left braces indicate the transcripts, P₁ and P₂, produced from the reaction with template T₁. (D) In vitro transcription of the sigP templates (T₁ and T₂) containing the first 29 and 45 coding nucleotides, respectively. Blue right braces indicate the transcripts, P₁ and P₂, produced from the reaction with template T₁, and red right braces indicate the transcripts P₁’ and P₂’, produced from the reaction with template T₂. Double arrows indicate that P₁ and P₂ are 16 nucleotides shorter than P₁’ and P₂’, respectively. Results in A, C, and D were repeated two times.

FIG 4

PorX and σ^P promote porT transcription in vitro. (A) In vitro transcription of a porT template containing its promoter and the first 48 coding nucleotides was conducted as described in the Materials and Methods. The left panel represents the transcripts, labeled as S₁ and S₂, respectively, synthesized in the reactions with different amounts of RNAP-σ^P with and 100 nM PorX-_C-His₆ protein. The right panel represents the products synthesized in the reactions with different amounts of RNAP-σ^D and 100 nM PorX-c-His₆ protein. The ladder M corresponds to a PCR product generated with primers 4026 and ³²P-labeled primer 4025 and degraded by the Maxam and Gilbert reaction. (B) The DNA sequence of the porT promoter region. Underlined sequences correspond to the PorX-protected regions and are also labeled as I and II, respectively. Bold letters, labeled as s₁ and s₂, correspond to the transcription initiation sites detected from the in vitro transcription. Underlined capital letters present the porT start codon. (C) In vitro transcription of porT in the reactions supplemented with 50 nM RNAP-σ^P and different amounts of PorX-_C-His₆ protein. The ladder M is the same as in A. Results in A and C were repeated two times.

FIG 5

The PorX/PorY-determined virulence of P. gingivalis W83 strains. (A) Virulence test using groups of BALB/c mice (n = 5) that were subcutaneously injected with P. gingivalis W83 wild-type, ΔsigP (YS18145), and ΔporX (YS19145) strains, respectively. (B) Survival curves of the results from A (n = 5; P < 0.0001). Three sets of experiments were carried out.

FIG 6

Feedforward loop model illustrating the PorX/PorY- and σ^P-dependent regulatory mechanism. In P. gingivalis, PorX/PorY and σ^P build a feedforward loop. The PorY sensor kinase phosphorylates its cognate PorX response regulator. The phosphorylated PorX protein binds to the sigP promoter at the PorX-binding site and upregulates transcription of the sigP gene. The PorX/PorY-stimulated σ^P protein and RNA polymerase core enzyme build a holoenzyme. Then, phosphorylated PorX protein and RNAP σ^P holoenzyme coordinately activate transcription of their target genes by simultaneously binding to their promoters at the PorX-binding sites and the σ^P recognition site, respectively. The inset illustrates the PorX/PorY σ^P feedforward loop that modulates por expression.