Portal de Periódicos da CAPES

The PorX/PorY system is a virulence factor of Porphyromonas gingivalis and mediates the activation of the type IX secretion system

2021; Elsevier BV; Volume: 296; Linguagem: Inglês

10.1016/j.jbc.2021.100574

1083-351X

Dezhi Yang, Chizhou Jiang, Bo Ning, Wei Kong, Yixin Shi,

Salivary Gland Disorders and Functions

PorX/PorY is a two-component system (TCS) of Porphyromonas gingivalis that governs transcription of numerous genes including those encoding a type IX secretion system (T9SS) for gingipain secretion and heme accumulation. Here, an in vitro analysis showed that the response regulator PorX specifically bound to two regions in the promoter of porT, a known PorX-regulated T9SS gene, thus demonstrating that PorX/PorY can directly regulate specific target genes. A truncated PorX protein containing the N-terminal receiver and effector domains retained a wild-type ability in both transcription regulation and heme accumulation, ruling out the role of the C-terminal ALP domain in gene regulation. The PorX/PorY system was the only TCS essential for heme accumulation and concomitantly responded to hemin to stimulate transcription of several known PorX-dependent genes in a concentration-dependent manner. We found that PorX/PorY activated the sigH gene, which encodes a sigma factor known for P. gingivalis adaptation to hydrogen peroxide (H2O2). Consistently, both ΔporX and ΔsigH mutants were susceptible to H2O2, suggesting a PorX/PorY-σH regulatory cascade to confer resistance to oxidative stress. Furthermore, the ΔporX mutant became susceptible to high hemin levels that could induce oxidative stress. Therefore, a possible reason why hemin activates PorX/PorY is to confer resistance to hemin-induced oxidative stress. We also demonstrated that PorX/PorY was essential for P. gingivalis virulence because the ΔporX mutant was avirulent in a mouse model. Specifically, this TCS was required for the repression of proinflammatory cytokines secreted by dendritic cells and T cells in the P. gingivalis–infected mice. PorX/PorY is a two-component system (TCS) of Porphyromonas gingivalis that governs transcription of numerous genes including those encoding a type IX secretion system (T9SS) for gingipain secretion and heme accumulation. Here, an in vitro analysis showed that the response regulator PorX specifically bound to two regions in the promoter of porT, a known PorX-regulated T9SS gene, thus demonstrating that PorX/PorY can directly regulate specific target genes. A truncated PorX protein containing the N-terminal receiver and effector domains retained a wild-type ability in both transcription regulation and heme accumulation, ruling out the role of the C-terminal ALP domain in gene regulation. The PorX/PorY system was the only TCS essential for heme accumulation and concomitantly responded to hemin to stimulate transcription of several known PorX-dependent genes in a concentration-dependent manner. We found that PorX/PorY activated the sigH gene, which encodes a sigma factor known for P. gingivalis adaptation to hydrogen peroxide (H2O2). Consistently, both ΔporX and ΔsigH mutants were susceptible to H2O2, suggesting a PorX/PorY-σH regulatory cascade to confer resistance to oxidative stress. Furthermore, the ΔporX mutant became susceptible to high hemin levels that could induce oxidative stress. Therefore, a possible reason why hemin activates PorX/PorY is to confer resistance to hemin-induced oxidative stress. We also demonstrated that PorX/PorY was essential for P. gingivalis virulence because the ΔporX mutant was avirulent in a mouse model. Specifically, this TCS was required for the repression of proinflammatory cytokines secreted by dendritic cells and T cells in the P. gingivalis–infected mice. One of the most frequently occurring infectious diseases in humans is a chronic inflammatory disease of the periodontium that leads to erosion of the attachment apparatus and supporting the bone of teeth and is associated with increased risk for certain systemic disorders (1Armitage G.C. Periodontal diseases: Diagnosis.Ann. Periodontol. 1996; 1: 37-215Crossref PubMed Scopus (232) Google Scholar, 2Hajishengallis G. Periodontitis: From microbial immune subversion to systemic inflammation.Nat. Rev. Immunol. 2015; 15: 30-44Crossref PubMed Scopus (1184) Google Scholar, 3Irfan U.M. Dawson D.V. Bissada N.F. Epidemiology of periodontal disease: A review and clinical perspectives.J. Int. Acad. Periodontol. 2001; 3: 14-21PubMed Google Scholar, 4Page R.C. Offenbacher S. Schroeder H.E. Seymour G.J. Kornman K.S. Advances in the pathogenesis of periodontitis: Summary of developments, clinical implications and future directions.Periodontology. 1997; 2000: 216-248Crossref Scopus (682) Google Scholar, 5Papapanou P.N. Epidemiology of periodontal diseases: An update.J. Int. Acad. Periodontol. 1999; 1: 110-116PubMed Google Scholar). Gram-negative anaerobic bacterium Porphyromonas gingivalis is the major causative agent of chronic periodontitis, which often colonizes in deep periodontal pockets of humans (6Holt S.C. Ebersole J.L. Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia: The "red complex", a prototype polybacterial pathogenic consortium in periodontitis.Periodontology. 2005; 2000: 72-122Crossref Scopus (634) Google Scholar). On blood agar, P. gingivalis cells formed colonies with black pigment, which was derived from the protoheme in erythrocytes, and accumulated as a heme complex, i.e., μ-oxo oligomers or dimeric heme comprising two Fe(III) protoporphyrin IX moieties bridged by an oxygen atom (7Smalley J.W. Silver J. Marsh P.J. Birss A.J. The periodontopathogen Porphyromonas gingivalis binds iron protoporphyrin IX in the mu-oxo dimeric form: An oxidative buffer and possible pathogenic mechanism.Biochem. J. 1998; 331: 681-685Crossref PubMed Scopus (98) Google Scholar). P. gingivalis produces a broad array of virulence factors including cysteine proteinases (namely gingipains) for tissue colonization and destruction, as well as host defense perturbation (8Gallagher A. Aduse-Opoku J. Rangarajan M. Slaney J.M. Curtis M.A. Glycosylation of the Arg-gingipains of Porphyromonas gingivalis and comparison with glycoconjugate structure and synthesis in other bacteria.Curr. Protein Pept. Sci. 2003; 4: 427-441Crossref PubMed Scopus (33) Google Scholar, 9Mikolajczyk-Pawlinska J. Kordula T. Pavloff N. Pemberton P.A. Chen W.C. Travis J. Potempa J. Genetic variation of Porphyromonas gingivalis genes encoding gingipains, cysteine proteinases with arginine or lysine specificity.Biol. Chem. 1998; 379: 205-211Crossref PubMed Scopus (62) Google Scholar, 10Nakayama K. Domain-specific rearrangement between the two Arg-gingipain-encoding genes in Porphyromonas gingivalis: Possible involvement of nonreciprocal recombination.Microbiol. Immunol. 1997; 41: 185-196Crossref PubMed Scopus (68) Google Scholar, 11Okamoto K. Misumi Y. Kadowaki T. Yoneda M. Yamamoto K. Ikehara Y. Structural characterization of argingipain, a novel arginine-specific cysteine proteinase as a major periodontal pathogenic factor from Porphyromonas gingivalis.Arch. Biochem. Biophys. 1995; 316: 917-925Crossref PubMed Scopus (83) Google Scholar, 12Okamoto K. Kadowaki T. Nakayama K. Yamamoto K. Cloning and sequencing of the gene encoding a novel lysine-specific cysteine proteinase (Lys-gingipain) in Porphyromonas gingivalis: Structural relationship with the arginine-specific cysteine proteinase (Arg-gingipain).J. Biochem. 1996; 120: 398-406Crossref PubMed Scopus (96) Google Scholar). Two cysteine proteinase genes, rgpA and kgp, also encode hemagglutinins and the hemoglobin receptor at the 3'-terminal region of their transcripts (12Okamoto K. Kadowaki T. Nakayama K. Yamamoto K. Cloning and sequencing of the gene encoding a novel lysine-specific cysteine proteinase (Lys-gingipain) in Porphyromonas gingivalis: Structural relationship with the arginine-specific cysteine proteinase (Arg-gingipain).J. Biochem. 1996; 120: 398-406Crossref PubMed Scopus (96) Google Scholar, 13Nakayama K. Ratnayake D.B. Tsukuba T. Kadowaki T. Yamamoto K. Fujimura S. Haemoglobin receptor protein is intragenically encoded by the cysteine proteinase-encoding genes and the haemagglutinin-encoding gene of Porphyromonas gingivalis.Mol. Microbiol. 1998; 27: 51-61Crossref PubMed Scopus (100) Google Scholar, 14Pavloff N. Pemberton P.A. Potempa J. Chen W.C. Pike R.N. Prochazka V. Kiefer M.C. Travis J. Barr P.J. Molecular cloning and characterization of Porphyromonas gingivalis lysine-specific gingipain. A new member of an emerging family of pathogenic bacterial cysteine proteinases.J. Biol. Chem. 1997; 272: 1595-1600Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Gingipains were essential for the colony pigmentation of P. gingivalis (15Shi Y. Ratnayake D.B. Okamoto K. Abe N. Yamamoto K. Nakayama K. Genetic analyses of proteolysis, hemoglobin binding, and hemagglutination of Porphyromonas gingivalis. Construction of mutants with a combination of rgpA, rgpB, kgp, and hagA.J. Biol. Chem. 1999; 274: 17955-17960Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar) and secreted via a unique Por protein secretion system, now referred to as the type IX secretion system (T9SS) ((16Sato K. Naito M. Yukitake H. Hirakawa H. Shoji M. McBride M.J. Rhodes R.G. Nakayama K. A protein secretion system linked to bacteroidete gliding motility and pathogenesis.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 276-281Crossref PubMed Scopus (237) Google Scholar, 17Nonaka M. Shoji M. Kadowaki T. Sato K. Yukitake H. Naito M. Nakayama K. Analysis of a Lys-specific serine endopeptidase secreted via the type IX secretion system in Porphyromonas gingivalis.FEMS Microbiol. Lett. 2014; 354: 60-68Crossref PubMed Scopus (15) Google Scholar), also see a review (18Lasica A.M. Ksiazek M. Madej M. Potempa J. The type IX secretion system (T9SS): Highlights and recent insights into its structure and function.Front. Cell. Infect. Microbiol. 2017; 7: 215Crossref PubMed Scopus (147) Google Scholar)). T9SS plays an essential role in the accumulation of iron source and gingipain secretion since a mutant of the T9SS component gene, porT, exhibited beige colonies and could not secret gingipains (19Sato K. Sakai E. Veith P.D. Shoji M. Kikuchi Y. Yukitake H. Ohara N. Naito M. Okamoto K. Reynolds E.C. Nakayama K. Identification of a new membrane-associated protein that influences transport/maturation of gingipains and adhesins of Porphyromonas gingivalis.J. Biol. Chem. 2005; 280: 8668-8677Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Two-component systems (TCSs) provide the most ubiquitous signal transduction systems among a wide range of regulatory mechanisms developed in bacteria. Multiple TCSs ensure that bacteria can regulate transcription of a range of genes in response to a variety of environmental and intracellular stimuli and control an array of physiological traits, particularly bacterial virulence. Most TCSs consist of a sensor histidine kinase (HK), and a response regulator (RR), which usually functions as a DNA-binding protein. This design allows bacteria to directly sense and interact with a specific extracellular signal, which stimulates the kinase activity of the sensor protein to transfer a phosphoryl group to the cognate regulator, thus modulating transcription of particular genes. Although P. gingivalis seems to experience a number of environmental conditions in the oral cavity and therefore should monitor and respond to a variety of environmental cues, only four HK/RR pairs, one orphan HK, three orphan RRs, and one chimeric HK/RR systems have been characterized from different P. gingivalis genomes so far (20Mattos-Graner R.O. Duncan M.J. Two-component signal transduction systems in oral bacteria.J. Oral Microbiol. 2017; 9: 1400858Crossref PubMed Scopus (34) Google Scholar). Interestingly, a TCS identified in a P. gingivalis strain may not be functional in other strains. For example, PG0719 (HK) and PG0720 (RR) in a clinical isolate P. gingivalis W83 strain formed a TCS, HaeS-HaeR, which was activated by hemin to regulate genes for hemin acquisition including gingipains (21Scott J.C. Klein B.A. Duran-Pinedo A. Hu L. Duncan M.J. A two-component system regulates hemin acquisition in Porphyromonas gingivalis.PLoS One. 2013; 8e73351Crossref PubMed Scopus (18) Google Scholar). However, the homologous region encoding this TCS in the less virulent 33277 strain contained PGN_0753 (RR), but carries a 2.5 kb deletion, causing a defective histidine kinase (PGN_0752) and a hemin-dependent growth phenotype (21Scott J.C. Klein B.A. Duran-Pinedo A. Hu L. Duncan M.J. A two-component system regulates hemin acquisition in Porphyromonas gingivalis.PLoS One. 2013; 8e73351Crossref PubMed Scopus (18) Google Scholar). The porX and porY genetic loci are annotated as PGN_1019 and PGN_2001 genes in 33277 strain and PG0928 and PG0052 genes in W83 strain, respectively. It has been demonstrated that these genes, which are located far apart on the P. gingivalis chromosomes, encode a TCS in which PorX is the RR, and PorY is the cognate sensor kinase (16Sato K. Naito M. Yukitake H. Hirakawa H. Shoji M. McBride M.J. Rhodes R.G. Nakayama K. A protein secretion system linked to bacteroidete gliding motility and pathogenesis.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 276-281Crossref PubMed Scopus (237) Google Scholar, 22Kadowaki T. Yukitake H. Naito M. Sato K. Kikuchi Y. Kondo Y. Shoji M. Nakayama K. A two-component system regulates gene expression of the type IX secretion component proteins via an ECF sigma factor.Sci. Rep. 2016; 6: 23288Crossref PubMed Scopus (49) Google Scholar, 23Vincent M.S. Durand E. Cascales E. The PorX response regulator of the Porphyromonas gingivalis PorXY two-component system does not directly regulate the type IX secretion genes but binds the PorL subunit.Front. Cell. Infect. Microbiol. 2016; 6: 96Crossref PubMed Scopus (16) Google Scholar). The PorX/PorY system was able to stimulate transcription of numerous genes, including many of those genes including porK, porL, porM, porN, porP, porT, and sov to encode the T9SS components (16Sato K. Naito M. Yukitake H. Hirakawa H. Shoji M. McBride M.J. Rhodes R.G. Nakayama K. A protein secretion system linked to bacteroidete gliding motility and pathogenesis.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 276-281Crossref PubMed Scopus (237) Google Scholar). Consistently, a mutation at the porX locus, in a manner similar to disruption of T9SS, resulted in the accumulation of unprocessed gingipain proproteins, resulting in the reduction of Rgp and Kgp activities in bacterial cultures and a nonpigmented phenotype (24Sato K. Por secretion system of Porphyromonas gingivalis.J. Oral Biosci. 2011; 53: 187-196Crossref Google Scholar). It was once suggested that a chimeric HK-RR GppX also contributed to maturation and proper localization of gingipains to the outer membrane, thus influencing the activity of gingipains (25Hasegawa Y. Nishiyama S. Nishikawa K. Kadowaki T. Yamamoto K. Noguchi T. Yoshimura F. A novel type of two-component regulatory system affecting gingipains in Porphyromonas gingivalis.Microbiol. Immunol. 2003; 47: 849-858Crossref PubMed Scopus (42) Google Scholar). Contrastingly, a recent study showed that both gppX deletion and insertion mutants retained the ability to exhibit black-pigmented colonies on the blood agar plates and produce wild-type-level gingipains (26Naito M. Tominaga T. Shoji M. Nakayama K. PGN_0297 is an essential component of the type IX secretion system (T9SS) in Porphyromonas gingivalis: Tn-seq analysis for exhaustive identification of T9SS-related genes.Microbiol. Immunol. 2019; 63: 11-20Crossref PubMed Scopus (23) Google Scholar). This similar phenotype between the wild-type strain and the gppX mutant ruled out the possibility that GppX played a role in modulation of the T9SS activity. It was demonstrated that the PorY and PorX proteins could interact with each other, and autophosphorylation of a truncated PorY protein and a subsequent phosphotransfer to PorX in vitro were stimulated by divalent cation Mn2+ (22Kadowaki T. Yukitake H. Naito M. Sato K. Kikuchi Y. Kondo Y. Shoji M. Nakayama K. A two-component system regulates gene expression of the type IX secretion component proteins via an ECF sigma factor.Sci. Rep. 2016; 6: 23288Crossref PubMed Scopus (49) Google Scholar). The PorX/PorY system was shown to regulate the T9SS-mediated protein secretion via an extracytoplasmic function sigma factor SigP, which was encoded by PGN_0274 in 33277 strain (22Kadowaki T. Yukitake H. Naito M. Sato K. Kikuchi Y. Kondo Y. Shoji M. Nakayama K. A two-component system regulates gene expression of the type IX secretion component proteins via an ECF sigma factor.Sci. Rep. 2016; 6: 23288Crossref PubMed Scopus (49) Google Scholar, 27Fujise K. Kikuchi Y. Kokubu E. Okamoto-Shibayama K. Ishihara K. Effect of extracytoplasmic function sigma factors on autoaggregation, hemagglutination, and cell surface properties of Porphyromonas gingivalis.PLoS One. 2017; 12e0185027Crossref PubMed Scopus (8) Google Scholar). However, another study reached a conclusion in contradiction to the first observation by showing that the PorX protein could only bind to the cytoplasmic domain of PorL subunit of the T9SS apparatus, but not directly bind to the promoters of the T9SS encoding genes (22Kadowaki T. Yukitake H. Naito M. Sato K. Kikuchi Y. Kondo Y. Shoji M. Nakayama K. A two-component system regulates gene expression of the type IX secretion component proteins via an ECF sigma factor.Sci. Rep. 2016; 6: 23288Crossref PubMed Scopus (49) Google Scholar, 23Vincent M.S. Durand E. Cascales E. The PorX response regulator of the Porphyromonas gingivalis PorXY two-component system does not directly regulate the type IX secretion genes but binds the PorL subunit.Front. Cell. Infect. Microbiol. 2016; 6: 96Crossref PubMed Scopus (16) Google Scholar). Therefore, the molecular mechanism for how the PorX/PorY system participated in the regulation of P. gingivalis genes remained largely unclear. P. gingivalis was able to interact with different types of host cells to manipulate immune response and finally escape killing from immune cells. For example, P. gingivalis infection modulated the release of chemokines and cytokines from fibroblasts (28Palm E. Khalaf H. Bengtsson T. Porphyromonas gingivalis downregulates the immune response of fibroblasts.BMC Microbiol. 2013; 13: 155Crossref PubMed Scopus (37) Google Scholar). Particularly, P. gingivalis disarmed a host-protective pathway via proteasomal degradation of myeloid differentiation primary response 88 (MYD88), the key molecule in Toll-like receptor 2 (TLR2)-mediated immune responses (29Maekawa T. Krauss J.L. Abe T. Jotwani R. Triantafilou M. Triantafilou K. Hashim A. Hoch S. Curtis M.A. Nussbaum G. Lambris J.D. Hajishengallis G. Porphyromonas gingivalis manipulates complement and TLR signaling to uncouple bacterial clearance from inflammation and promote dysbiosis.Cell Host Microbe. 2014; 15: 768-778Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). In addition, P. gingivalis secreted gingipains that inactivated several key proinflammatory mediators made by dendritic cells (DCs) and T cells by selectively inactivating most proinflammatory cytokines (30Abdi K. Chen T. Klein B.A. Tai A.K. Coursen J. Liu X. Skinner J. Periasamy S. Choi Y. Kessler B.M. Palmer R.J. Gittis A. Matzinger P. Duncan M.J. Singh N.J. Mechanisms by which Porphyromonas gingivalis evades innate immunity.PLoS One. 2017; 12e0182164Crossref PubMed Scopus (15) Google Scholar). By far, very little information is available on the contribution of TCSs to P. gingivalis virulence and signal transduction in response to the host environment. In this study, we established the role of the PorX/PorY system in the regulation of gene transcription and revealed the ability of PorX in recognition of the porT promoter. We also provided evidence that the PorX/PorY system is an essential regulator for P. gingivalis virulence. A porX null mutant of P. gingivalis 33277 strain displayed a nonpigmented phenotype due to disruption of T9SS, which subsequently reduced the accumulation of heme (24Sato K. Por secretion system of Porphyromonas gingivalis.J. Oral Biosci. 2011; 53: 187-196Crossref Google Scholar). We addressed the possibility of whether other TCSs could also influence the T9SS activity and then heme accumulation in P. gingivalis. Besides the porX gene, there were other six genetic loci encoding characterized and putative TCS RRs, i.e., PGN_0012, PGN_0753, PGN_0775, PGN_0903 (i.e., fimR), PGN_1186 (rprY), and PGN_1768 (gppX) from a P. gingivalis 33277 genome (Uniprot, https://www.uniprot.org). We constructed single mutants in which one of the seven RR genes was disrupted by an erythromycin resistance cassette and observed their growth on a blood agar plate with enriched brain heart infusion medium (BHI). Consistent with the previous result (24Sato K. Por secretion system of Porphyromonas gingivalis.J. Oral Biosci. 2011; 53: 187-196Crossref Google Scholar), the porX deletion mutant (ΔporX mutant) produced a nonpigmented bacterial lawn (shown as beige, Fig. 1A). On the contrary, other regulator mutants retained the ability to form pigmented lawns as the wild-type strain (Fig. 1A), suggesting that these TCSs were not required for the heme accumulation. To further confirm that the nonpigmented phenotype was solely due to disruption of the porX gene, we introduced a complementary plasmid pT-COW-PPGN_1016-porX (referred to as p-porX, hereinafter) into the ΔporX mutant strain. In this bacterial cell, the PorX protein was produced in trans from p-porX, which carried the wild-type porX coding sequence controlled by the promoter region of the porX containing operon PGN_1016-PGN_1021 (illustrated in Fig. S1). A wild-type phenotype was completely restored in this ΔporX mutant by p-porX because it was grown into a pigmented lawn like the wild-type strain while a ΔporX mutant carrying the parental plasmid pT-COW remained to form a beige lawn (Fig. 1B). This phenotypic analysis demonstrated that the PorX/PorY system was the major TCS essential for heme accumulation in P. gingivalis. Hence, it is reasonable to postulate that the PorX/PorY system is the major TCS for modulation of the T9SS activity. The porX gene product predicted as a 518-amino acid (aa) protein was shown to be phosphorylated by its cognate sensor kinase PorY (16Sato K. Naito M. Yukitake H. Hirakawa H. Shoji M. McBride M.J. Rhodes R.G. Nakayama K. A protein secretion system linked to bacteroidete gliding motility and pathogenesis.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 276-281Crossref PubMed Scopus (237) Google Scholar, 22Kadowaki T. Yukitake H. Naito M. Sato K. Kikuchi Y. Kondo Y. Shoji M. Nakayama K. A two-component system regulates gene expression of the type IX secretion component proteins via an ECF sigma factor.Sci. Rep. 2016; 6: 23288Crossref PubMed Scopus (49) Google Scholar). According to a previous analysis (31Galperin M.Y. Structural classification of bacterial response regulators: Diversity of output domains and domain combinations.J. Bacteriol. 2006; 188: 4169-4182Crossref PubMed Scopus (358) Google Scholar), the C-terminal 231- to 518-aa region of this regulator formed an alkaline phosphatase-like core domain with a PglZ motif (ALP), which thus was unlikely to be directly involved in gene regulation. It has been well established that most TCS RRs possess a basic modular architecture built by a receiver domain (normally located in their N termini) with a conserved and phosphorylatable aspartate residue and a variable effector domain (in their C termini) to interact with the target promoters or specific cellular factors (32Gross R. Arico B. Rappuoli R. Families of bacterial signal-transducing proteins.Mol. Microbiol. 1989; 3: 1661-1667Crossref PubMed Scopus (115) Google Scholar, 33Gao R. Mack T.R. Stock A.M. Bacterial response regulators: Versatile regulatory strategies from common domains.Trends Biochem. Sci. 2007; 32: 225-234Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 34Mascher T. Helmann J.D. Unden G. Stimulus perception in bacterial signal-transducing histidine kinases.Microbiol. Mol. Biol. Rev. 2006; 70: 910-938Crossref PubMed Scopus (506) Google Scholar). Our result from a multiple sequence alignment (COBALT, https://www.ncbi.nlm.nih.gov/tools/cobalt/re_cobalt.cgi) revealed that the N-terminal 29- to 122-aa region of PorX shared similarities with the receiver domains of multiple RR proteins from Gram-negative bacterium Escherichia coli including those belonging to the OmpR/PhoB family (Sequences with a brace and illustrated as R, Fig. 2A). Concomitantly, its aspartate residue 58 (D58, marked with an asterisk, Fig. 2A) was equivalent to the aspartate residue conserved in all RR proteins, which had been shown to be phosphorylated by the cognate sensor kinases (35Sanders D.A. Gillece-Castro B.L. Stock A.M. Burlingame A.L. Koshland Jr., D.E. Identification of the site of phosphorylation of the chemotaxis response regulator protein, CheY.J. Biol. Chem. 1989; 264: 21770-21778Abstract Full Text PDF PubMed Google Scholar, 36Egger L.A. Park H. Inouye M. Signal transduction via the histidyl-aspartyl phosphorelay.Genes Cells. 1997; 2: 167-184Crossref PubMed Scopus (154) Google Scholar). Based on these observations, we hypothesized that the first ∼223-aa sequence of PorX could form a modular architecture similar to those OmpR/PhoB-family RRs such as OmpR (239-aa), PhoB (229-aa), and BasR (222-aa) in E. coli (37Martinez-Hackert E. Stock A.M. The DNA-binding domain of OmpR: Crystal structures of a winged helix transcription factor.Structure. 1997; 5: 109-124Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 38Makino K. Amemura M. Kawamoto T. Kimura S. Shinagawa H. Nakata A. Suzuki M. DNA binding of PhoB and its interaction with RNA polymerase.J. Mol. Biol. 1996; 259: 15-26Crossref PubMed Scopus (139) Google Scholar, 39Lou Y.C. Weng T.H. Li Y.C. Kao Y.F. Lin W.F. Peng H.L. Chou S.H. Hsiao C.D. Chen C. Structure and dynamics of polymyxin-resistance-associated response regulator PmrA in complex with promoter DNA.Nat. Commun. 2015; 6: 8838Crossref PubMed Scopus (30) Google Scholar). Consequently, the receiver domain (the 29- to 122-aa region) should coordinate with the downstream 123- to 223-aa region (i.e., a receiver domain, illustrated as E, Fig. 2A) and form a functional structure to carry out transcription regulation. To investigate the regulatory activity conferred by this region, we constructed a 33277 strain (porX-s) in which a stop codon TAA (arrowhead, Fig. S2A) was added between the 223rd and 224th codons of the porX coding region through the insertion of a suicide plasmid (pGEM-ermF-porX-s, illustrated in Fig. S2A). This porX-s strain produced a truncated PorX protein (referred to as PorX-s), which contained the first 223-aa sequence, thus only carrying the receiver domain and the predicted effector domain (Fig. 2A, and Fig. S2B). A quantitative real-time polymerase chain reaction (PCR) analysis (qRT-PCR) was used to determine the transcription of two known PorX-activated genes, i.e., porT (PGN_0778) and PGN_0341 (16Sato K. Naito M. Yukitake H. Hirakawa H. Shoji M. McBride M.J. Rhodes R.G. Nakayama K. A protein secretion system linked to bacteroidete gliding motility and pathogenesis.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 276-281Crossref PubMed Scopus (237) Google Scholar, 22Kadowaki T. Yukitake H. Naito M. Sato K. Kikuchi Y. Kondo Y. Shoji M. Nakayama K. A two-component system regulates gene expression of the type IX secretion component proteins via an ECF sigma factor.Sci. Rep. 2016; 6: 23288Crossref PubMed Scopus (49) Google Scholar), and the sigH (PGN_1740) gene, which was identified as a PorX-activated gene according to an RNA sequencing analysis and a proteomic analysis conducted by our laboratory recently (manuscript in preparation). The mRNA levels of porT, PGN_0341, and sigH in the wild-type strain were similar to those in the porX-s strain, but 3.9-, 10.4-, and 2.2-fold higher than those in the ΔporX mutant (Fig. 2B). The reduced mRNA levels of porT, PGN_0341, and sigH in ΔporX mutant were fully restored to wild-type levels by plasmid p-porX (Fig. 2B), confirming that the deficient expression of these loci indeed resulted from deletion of the porX gene. Our results demonstrated that the first 223-aa sequence still retained the PorX function in transcription regulation in a manner comparable to the full-length PorX protein. In support of this notion, the porX-s strain formed black-pigmented colonies on a blood BHI agar plate like its wild-type parental strain (Fig. 2C). Therefore, our observations clarified that the C-terminal ALP domain was not essential for PorX to control transcription regulation. Unlike the ΔporX mutant, a ΔporY mutant displayed as pigmented colonies on the blood BHI agar plate (Fig. 2C). In fact, a previous study had revealed that the contribution of PorX to gene regulation was greater than that of PorY (16Sato K. Naito M. Yukitake H. Hirakawa H. Shoji M. McBride M.J. Rhodes R.G. Nakayama K. A protein secretion system linked to bacteroidete gliding motility and pathogenesis.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 276-281Crossref PubMed Scopus (237) Google Scholar). Thus, these observations implied that PorX might interact with a sensor kinase other than PorY to modulate gene regulation required for hemin accumulation in P. gingivalis. We studied the expression of the PorX/PorY system in 33277 wild-type strain grown in BHI medium and found that mRNA levels of the porX and porY (PGN_2001) genes from the cultures of 24 and 48 h incubation were 6.6- and 3.0-fold higher than those of 12 h incubation, respectively (Fig. 3A). On the other hand, the mRNA level of the PGN_1571gene (rpoB), which encodes the RNA polymerase subunit beta subunit, remained constant at various growth times (Fig. 3A). Similarly, transcription of the porX and porY genes in a clinical isolate of P. gingivalis, W83 wild-type strain, was also stimulated significantly (2