HcpR of Porphyromonas gingivalis is required for growth under nitrosative stress and survival within host cells

Abstract

Although the Gram-negative, anaerobic periodontopathogen Porphyromonas gingivalis must withstand nitrosative stress, which is particularly high in the oral cavity, the mechanisms allowing for protection against such stress are not known in this organism. In this study, microarray analysis of P. gingivalis transcriptional response to nitrite and nitric oxide showed drastic upregulation of the PG0893 gene coding for hybrid cluster protein (Hcp), which is a putative hydroxylamine reductase. Although regulation of hcp has been shown to be OxyR dependent in Escherichia coli, here we show that in P. gingivalis its expression is dependent on the Fnr-like regulator designated HcpR. Growth of the isogenic mutant V2807, containing an ermF-ermAM insertion within the hcpR (PG1053) gene, was significantly reduced in the presence of nitrite (P < 0.002) and nitric oxide-generating nitrosoglutathione (GSNO) (P < 0.001), compared to that of the wild-type W83 strain. Furthermore, the upregulation of PG0893 (hcp) was abrogated in V2807 exposed to nitrosative stress. In addition, recombinant HcpR bound DNA containing the hcp promoter sequence, and the binding was hemin dependent. Finally, V2807 was not able to survive with host cells, demonstrating that HcpR plays an important role in P. gingivalis virulence. This work gives insight into the molecular mechanisms of protection against nitrosative stress in P. gingivalis and shows that the regulatory mechanisms differ from those in E. coli.

Fig 1

Sensitivity of P. gingivalis to nitrosative stress. P. gingivalis W83 was grown to midlogarithmic phase under anaerobic conditions in mycoplasma broth without hemin and was diluted to an OD₆₆₀ of 0.1. Various concentrations of nitrite or nitrate (A) (amount given in mM) or GSNO (B) (amount given in mM × 10⁻³) were added to the cultures. Unsupplemented cultures served as controls. The cultures were then grown for an additional 48 h at 37°C under anaerobic conditions. Means from the experiment performed in triplicate are shown.

Fig 2

Bioinformatics analysis of HcpR. (A) Genomic locus of hcpR. Genes and their orientations are denoted by arrows. IGR, intergenic region. (B) Comparison of amino acid sequences of P. gingivalis HcpR (HcpR), Thermatoga maritima CRP-like regulator TM1171 (TM1171), Lactococcus lactis Fnr-like regulator CAB53581 (LAFNR), and Pseudomonas stutzeri DNR CAB40908 (PSDNR). Amino acids identical in other proteins to that found in HcpR are indicated in red. Two amino acids in the helix-turn-helix (HTH) confer specificity of the protein binding to DNA targets (R₁₈₀ correlates with G₃ and Q₁₈₁ with G₆ in HcpR). (C) Structural model of P. gingivalis HcpR. Amino acids 25 to 135 form a CAP motif (ligand-binding motif) and are shown in green, and the HTH region (DNA-binding domain; residues 170 to 210) is designated in yellow. The two domains are separated by a dimerization helix. (D) Structural overlay of HcpR (blue) with PADNR (3dkw) (orange).

Fig 3

Roles of HcpR and hemin on P. gingivalis growth in the presence of nitrate or nitrite. P. gingivalis strains were anaerobically grown to midlogarithmic phase in mycoplasma broth without hemin. The cultures were then diluted to an OD₆₆₀ of 0.1 in mycoplasma minus hemin (−Hm) (A and B) and mycoplasma containing 5 μg/ml hemin (+Hm) (C and D). Various concentrations of nitrite or nitrate (A) were then added to the cultures. Unsupplemented cultures served as controls. The cultures were anaerobically grown for an additional 25 h at 37°C. Means and standard deviations from the experiment performed in triplicate are shown (n = 3; results are ± standard error of the mean). *, P < 0.002, W83 versus V2807 under 1 mM nitrite (Student's t test). Parental W83 strain (A) and HcpR-deficient mutant V2807 strain (B) grown in low hemin conditions (−Hm). W83, V2807, and V2835 (complemented HcpR mutant strain) grown in the presence of high hemin concentrations (+ Hm) with 2 mM (C) and 4 mM (D) nitrite. n = 3; results are ± standard error of the mean.

Fig 4

Role of HcpR on P. gingivalis growth in the presence of nitric oxide. P. gingivalis strains were anaerobically grown to midlogarithmic phase in mycoplasma broth without hemin. The cultures were then diluted to an OD₆₆₀ of 0.1 in mycoplasma minus hemin, and 1 mM nitrite or 30 nM GSNO was then added to the cultures. Unsupplemented cultures served as controls. The cultures were grown for an additional 18 h at 37°C under anaerobic conditions. Means and standard deviations from the experiment performed in triplicate are shown (n = 3; results are ± standard error of the mean). Parental W83 strain (A), HcpR-deficient mutant strain (B). *, P < 0.001, W83 versus V2807 under 30 nM GSNO (Student's t test).

Fig 5

P. gingivalis HcpR regulates hcp expression. (A) Organization of the hcp (PG0893) locus. Intergenic sequences (IGS) flank the hcp gene. The HcpR binding site is shown as an orange oval. (B) Bioinformatics analysis of the hcp promoter. The HcpR binding site is shown in orange. The transcriptional start site (ts) is located 29 bp upstream of the translational start site (Met). The primer positions used for the generation of the EMSA probe are shown in italics and are underlined. Positions of primers used for qRT-PCR designated 1R, 2R, and 3R are shown in green, and their direction is indicated by an arrow. (C) EMSA. Lysates prepared from IPTG induced E. coli-pET30 hcpR (lane 2, +i) and IPTG-uninduced E. coli-pET30 hcpR (lane 3,+u), and increasing amounts of rHcpR (lanes 5 to 9) were incubated with a 200-bp DNA fragment containing the hcp promoter (DNA). Reaction mixtures containing DNA only were run as reference controls (lanes 1 and 4). Unlabeled hcp promoter DNA (UDNA) was used as a specific competitor (lanes 7 and 9) (10-fold excess over labeled DNA). rHcpR without (lane 5) and reconstituted with (lanes 6 to 9) hemin was used. Lane 5 contained 30 μg of rHcpR, lanes 6 and 7 contained 10 μg of rHcpR, and lanes 8 and 9 had 30 μg of rHcpR.

Fig 6

Schematic representation of P. gingivalis mechanisms mediating response to nitrosative stress.