Proteomic analysis of Neorickettsia sennetsu surface-exposed proteins and porin activity of the major surface protein P51

Abstract

Neorickettsia sennetsu is an obligate intracellular bacterium of monocytes and macrophages and is the etiologic agent of human Sennetsu neorickettsiosis. Neorickettsia proteins expressed in mammalian host cells, including the surface proteins of Neorickettsia spp., have not been defined. In this paper, we isolated surface-exposed proteins from N. sennetsu by biotin surface labeling followed by streptavidin-affinity chromatography. Forty-two of the total of 936 (4.5%) N. sennetsu open reading frames (ORFs) were detected by liquid chromatography-tandem mass spectrometry (LC/MS/MS), including six hypothetical proteins. Among the major proteins identified were the two major β-barrel proteins: the 51-kDa antigen (P51) and Neorickettsia surface protein 3 (Nsp3). Immunofluorescence labeling not only confirmed surface exposure of these proteins but also showed rosary-like circumferential labeling with anti-P51 for the majority of bacteria and polar to diffuse punctate labeling with anti-Nsp3 for a minority of bacteria. We found that the isolated outer membrane of N. sennetsu had porin activity, as measured by a proteoliposome swelling assay. This activity allowed the diffusion of L-glutamine, the monosaccharides arabinose and glucose, and the tetrasaccharide stachyose, which could be inhibited with anti-P51 antibody. We purified native P51 and Nsp3 under nondenaturing conditions. When reconstituted into proteoliposomes, purified P51, but not Nsp3, exhibited prominent porin activity. This the first proteomic study of a Neorickettsia sp. showing new sets of proteins evolved as major surface proteins for Neorickettsia and the first identification of a porin for the genus Neorickettsia.

FIG. 1.

Surface localization and predicted secondary structure of P51. (A) Labeling pattern of P51 on N. sennetsu within a P388D₁ host cell by double-immunofluorescence assay. Infected P388D₁ cells were fixed in paraformaldehyde, permeabilized with PGS, incubated with anti-rP51 and anti-N. sennetsu (α-NS), stained with Alexa Fluor 488 (green)-goat anti-rabbit IgG and Cy3 (red)-goat anti-horse IgG, and visualized by fluorescence microscopy. Note that the majority of bacteria are stained with anti-rP51. Dashed lines indicate the outlines of the cell and nucleus (N). Bar, 5 μm. (B) Surface localization of P51 on N. sennetsu by double-immunofluorescence assay. Host cell-free N. sennetsu was fixed in paraformaldehyde, incubated with anti-rP51 and anti-N. sennetsu, stained with Alexa Fluor 488-goat anti-rabbit IgG and Cy3-goat anti-horse IgG, and visualized by fluorescence microscopy. Note the regular ∼0.1-μm dotted pattern of P51. Bar, 1 μm. Negative controls were cells incubated with anti-rP51 and control horse serum (N. risticii- and N. sennetsu-negative serum [Ctl Horse]) (C) or control rabbit serum (Ctl Rabbit) and anti-N. sennetsu (D). Bars, 5 μm. (E) Hydrophobicity and hydrophobic moment profiles (23) for the P51 sequence. The x axis demonstrates the amino acid number. The y axis depicts the probability of the presence of a transmembrane domain. The black line denotes the presence of normal β-strands. The broken red line indicates twisted β-strands. The N-terminal signal peptide (20 aa) was removed from the structure. The blue numbers demonstrate the locations of the predicted β-strands. (F) Secondary structure of the N. sennetsu P51 mature protein with 18 transmembrane domains, based on the results for panel E, PRED-TMBB analysis (1), and alignment and analysis of the P51 sequences of the N. sennetsu type strain (GenBank accession no. YP_506136), the N. risticii type strain (GenBank accession no. YP_003081464), and the SF agent Hirose strain (AAL12490), using MegAlign (DNAStar). Listed amino acids span the outer membrane, with lipophilic residues labeled in red and charged residues labeled in blue.

FIG. 2.

Reactivities of anti-Nsp3_107-120 and anti-rNsp3 and surface localization of Nsp3. (A) Recombinant Nsp3 (27.7 kDa) was separated by 10% SDS-PAGE and stained with GelCode blue (lane 1) or transferred to a nitrocellulose membrane for Western blot analysis with anti-His (lane 2), anti-Nsp3_107-120 (lane 3), anti-rNsp3 (lane 4), or preimmune serum (lane 5). (B) Labeling pattern of Nsp3 on N. sennetsu within a P388D₁ host cell by double-immunofluorescence assay. Infected P388D₁ cells were fixed in paraformaldehyde, permeabilized with PGS, incubated with anti-Nsp3_107-120 or anti-rNsp3 and anti-N. sennetsu (α-NS) stained with Alexa Fluor 488-goat anti-mouse IgG and Cy3-goat anti-horse IgG, and visualized by fluorescence microscopy. Dashed lines indicate the outlines of the cells and nuclei (N). Arrows indicate anti-rNsp3 staining. Bars, 5 μm. (C) Double-immunofluorescence labeling of host cell-free N. sennetsu fixed in paraformaldehyde, incubated with anti-Nsp3_107-120 or anti-rNsp3 and anti-N. sennetsu, stained with Alexa Fluor 488-goat anti-rabbit IgG and Cy3-goat anti-horse IgG, and visualized by fluorescence microscopy. Note the polar labeling of Nsp3. Bars, 1 μm. (D) Negative control cells were incubated with control mouse serum (Ctl Mouse) and anti-N. sennetsu. Bar, 5 μm.

FIG. 3.

Porin activity of N. sennetsu outer membrane fraction incorporated into liposomes. Optical density (OD) changes in the first 20 s are shown for anti-rP51-treated (A) and control preimmune rabbit serum-treated (B) N. sennetsu outer membrane fraction, using 33 mM l-glutamine (open squares), arabinose (open diamonds), glucose (filled squares), and stachyose (filled diamonds) for representative readings for three independent experiments. (C) Initial swelling over 60 s for anti-rP51-treated and control rabbit serum-treated (control) N. sennetsu outer membrane fraction for three independent experiments. Average blank values (lipid film without protein mixed with 2% OGC in 10 mM Tris-HCl) for three independent experiments with each solute were subtracted from each data point. *, P < 0.05; **, P < 0.01 (unpaired Student's t test).

FIG. 4.

Porin activity of HPLC-separated P51 and Nsp3 fractions incorporated into liposomes. (A) Lane 1, GelCode blue staining of the OGC-solubilized Sarkosyl-insoluble N. sennetsu outer membrane fraction; lane 2, HPLC P51 fraction with GelCode blue stain; lane 3, Western blotting of P51 fraction with anti-rP51; lane 4, HPLC Nsp3 fraction with GelCode blue stain; lane 5, Western blotting of Nsp3 fraction with anti-Nsp3_107-120. (B) Initial swelling over 60 s for P51 and Nsp3 fractions for three independent experiments. Average blank values (lipid film without protein mixed with 1% OGC in 50 mM Tris-HCl) for three independent experiments with each solute were subtracted from each data point. Glucose and sucrose results were obtained using 10 μg P51 and 4.8 μg Nsp3. l-Glutamine and stachyose results utilized 5 μg P51 and 2.4 μg Nsp3. *, P < 0.05; **, P < 0.01 (unpaired Student's t test).