A two-component system regulates gene expression of the type IX secretion component proteins via an ECF sigma factor

Abstract

The periodontopathogen Porphyromonas gingivalis secretes potent pathogenic proteases, gingipains, via the type IX secretion system (T9SS). This system comprises at least 11 components; however, the regulatory mechanism of their expression has not yet been elucidated. Here, we found that the PorY (PGN_2001)-PorX (PGN_1019)-SigP (PGN_0274) cascade is involved in the regulation of T9SS. Surface plasmon resonance (SPR) analysis revealed a direct interaction between a recombinant PorY (rPorY) and a recombinant PorX (rPorX). rPorY autophosphorylated and transferred a phosphoryl group to rPorX in the presence of Mn(2+). These results demonstrate that PorX and PorY act as a response regulator and a histidine kinase, respectively, of a two component system (TCS), although they are separately encoded on the chromosome. T9SS component-encoding genes were down-regulated in a mutant deficient in a putative extracytoplasmic function (ECF) sigma factor, PGN_0274 (SigP), similar to the porX mutant. Electrophoretic gel shift assays showed that rSigP bound to the putative promoter regions of T9SS component-encoding genes. The SigP protein was lacking in the porX mutant. Co-immunoprecipitation and SPR analysis revealed the direct interaction between SigP and PorX. Together, these results indicate that the PorXY TCS regulates T9SS-mediated protein secretion via the SigP ECF sigma factor.

Figure 1. Structure and localization of PorX and PorY.

(A) Schematic representation of the functional domains of PorX and PorY. The symbols denote the following, RR, response regulatory domain; PglZ, PglZ motif; ALP, alkaline phosphatase-like core domain; SS, signal sequence; TM, transmembrane domain; HK1, histidine kinase phosphoacceptor domain; HK2, histidine kinase ATPase domain. Arrows below the schema indicate the recombinant proteins generated in this study. The domains were predicted by the Kyoto Encyclopedia of Genes and Genomes (KEGG) Sequence Similarity DataBase (KEGG SSDB). (B) Subcellular localization of PorX and PorY in P. gingivalis. Fractionated cell lysates of the wild-type strain ATCC 33277 were subjected to immunodetection with antisera against PorX and PorY. WC, whole cell lysate; C/P, cytoplasm/periplasm; CE, cell envelope; IM, inner membrane; OM, outer membrane. Arrowheads indicate the immunoreacting PorX and PorY bands. (C) SDS-PAGE profile of the purified recombinant proteins.

Figure 2. Interaction between PorX and PorY.

(A) Affinity assays between rPorY and rPorX by surface plasmon resonance (SPR) analysis. rPorX (0.31–4.92 μM) were injected into the sensor chip immobilized by rPorY. BSA exhibited a negative interaction with rPorY. (B) In vitro phosphorylation of rPorY and rPorX. For autophosphorylation assay, rPorY (1 μg) was incubated with [³²P-γ]ATP for 1, 5, 10, and 20 min at 0 °C. For transphosphorylation assay, rPorX (5 μg) and excess cold ATP (1 mM) were added to aliquots of rPorY 5 min after start of autophosphorylation and further incubated at 37 °C for indicated periods. The relative intensities of the radiolabelled protein bands were indicated in the graph after normalization. (C) The effects of compounds on the autophosphorylation of rPorY and the transfer of a phosphoryl group from rPorY to rPorX were examined. Phosphorylation assays were performed in the presence or absence of 10 mM MgCl₂ and 1 mM MnCl₂.

Figure 3. Construction of sigP deletion mutants and the complement strain and their colonial pigmentation.

(A) Gene and protein structure of P. gingivalis sigP. The deletion mutants were generated by double recombination of the 500-bp targeted genes and the introduction of ermF. (B) Colony pigmentation in P. gingivalis sigP deletion mutants and the complement strain. (C) Purification of the recombinant SigP containing N-terminal thioredoxin- and C-terminal His₆-tags. E. coli cells expressing the rSigP protein were lysed by sonication followed by centrifugation to separate a soluble fraction (Soluble) and an insoluble fraction (Insoluble). The soluble cell extracts were applied to nickel-nitrilotriacetic acid agarose to separate a free passed fraction (Ni-FP) and an adsorbed fraction (Ni-ad.).

Figure 4. Involvement of SigP in the transcription of T9SS components.

(A) Correlation in gene expression between porX mutant (KDP363) and sigP mutant (KDP314). Gene expression was measured by the custom tiling microarrays spanning the whole genome of P. gingivalis ATCC 33277. The expression level for each coding sequence was normalized with the constant from the 16S rRNA gene and represented as a ratio to that from wild type. Experiments were performed three times with independently prepared labelled cDNAs. The genes encoding T9SS components are represented in red. (B) EMSA assay of the promoter regions of T9SS component genes by rSigP. Probes corresponding to the possible promoter regions were generated by PCR and labelled with digoxigenin. Binding specificity was tested by competition with 100-fold excess of the appropriate unlabelled probe. PGN numbers for genes are indicated in parenthesis.

Figure 5. Expression of SigP in the porX mutant.

(A) Western blot analysis of SigP in ATCC 33277 (WT), a porX-deficient mutant (KDP363), and the porX/porX⁺ complement strain (KDP372). Whole cell lysates from each strain corresponding to 5 μl of bacterial culture at OD₅₉₅ 1.0 were applied to SDS-PAGE and transferred onto a PVDF membrane, followed by immunodetection with anti-SigP antibody. (B) Quantitative real time RT-PCR experiments with total RNA extracted from exponentially growing bacterial cells (OD₅₉₅ 0.6) were performed.

Figure 6. Interaction between PorX and SigP.

(A) Affinity assays between rPorX and rSigP by SPR analysis. rSigP (0–200 μg/ml) were injected into the sensor chip immobilized by rPorX. (B) Co-immunoprecipitation of SigP by anti-PorX antibody. P. gingivalis lysates from sigP/sigP-Myc + complement and porX-deficient strains were incubated with anti-PorX antibody conjugated with Protein G sepharose. The unbound, wash, bound fractions were analyzed by Western blotting with anti-PorX (left), anti-SigP (middle), and anti-c-Myc (right) antibodies. Blue arrows (left and middle) and black arrows (left) indicate the heavy chain derived from antibodies and a nonspecific protein, respectively.