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.
Neorickettsia spp. are unique environmental, Gram-negative, obligate intracellular bacteria maintained in nature through vertical transmission in trematodes (16, 17, 39, 42). Neorickettsia spp. are polymorphic cocci belonging to the family Anaplasmataceae within the order Rickettsiales in the class Alphaproteobacteria (7). Neorickettsia sennetsu (formerly called Rickettsia sennetsu or Ehrlichia sennetsu) is the first human pathogen in the family Anaplasmataceae to have been isolated and cultured (11, 34). N. sennetsu infects human monocytes and macrophages and causes the disease Sennetsu neorickettsiosis (10, 34, 41). Epidemiologic studies of Sennetsu neorickettsiosis show a strong link between the human ingestion of metacercaria-infested gray mullet fish and acquisition of the disease (11). Symptoms are similar to those of infectious mononucleosis and include swelling of the lymph nodes, pyrexia, inappetence, lethargy, sleeplessness, and overall malaise (10, 34, 41). Geographically, N. sennetsu infections have been reported mainly in western and southern Japan, although antibodies to N. sennetsu have also been found in humans in Malaysia, and one strain of N. sennetsu has been isolated from Malaysia (10, 19, 44). Recently, N. sennetsu infection was found in Laos (35). Treatment of Sennetsu neorickettsiosis involves tetracycline therapy and is normally highly successful at resolving the symptoms (10).
Gram-negative bacteria generally have porins spanning their outer membranes. These proteins enable the transport of hydrophilic molecules, such as amino acids, sugars, and other nutrients (36). As seen in other members of the Anaplasmataceae, N. sennetsu is limited in its ability to synthesize necessary compounds, including amino acids and enzymes for intermediary metabolism and glycolysis (20). Therefore, porins are an absolute necessity for the survival of this bacterium. To date, the only porins defined for the order Rickettsiales are major outer membrane proteins of Anaplasma phagocytophilum named P44s (22) and OMP-1F and P28 in Ehrlichia chaffeensis (26). These porins contain 16 (P44s) or 12 (OMP-1F and P28) transmembrane passes, and some are large enough to allow the slow diffusion of tetrasaccharides. P44/Msp2 and OMP-1/P28/P30 proteins belong to the family pfam01617, and the N. sennetsu genome was reported to encode only one hypothetical protein from this family (GenBank accession no. NSE_0875) (20), later named Neorickettsia surface protein 3 (Nsp3) (29).
In the present study, surface-exposed proteins of N. sennetsu were isolated from cell culture and identified by proteomics. We first isolated the outer membrane fraction from host cell-free N. sennetsu and examined porin activity by using an in vitro proteoliposome swelling assay. Second, we used antibodies against the dominant protein P51 to examine neutralization of the porin activity. Third, we purified native P51 and Nsp3 from the isolated outer membrane fraction by high-pressure liquid chromatography (HPLC) and tested whether these proteins have porin activity. Identification of surface-exposed proteins and the protein with major porin activity will help in understanding N. sennetsu and the disease that it causes.
MATERIALS AND METHODS
N. sennetsu.
N. sennetsu MiyayamaT (34) was cultured in P388D1 cells in RPMI 1640 (Mediatech, Herndon, VA) supplemented with 5 to 10% fetal bovine serum (U.S. Biotechnologies, Inc., Pottstown, PA) and 4 to 6 mM l-glutamine (Invitrogen, Carlsbad, CA) at 37°C under a 5% CO2-95% air atmosphere.
Biotin-affinity purification and analysis of N. sennetsu surface proteins.
N. sennetsu was isolated as previously described, with some modifications (15). P388D1 cells (>80% of cells were infected) were harvested and homogenized on ice 50 to 100 times in a 40-ml Dounce homogenizer (Kimble/Kontes, Vineland, NJ). Host cell debris and unbroken cells were pelleted by centrifugation at 448 × g for 5 min. The supernatant containing released bacteria was filtered through a 5-μm-pore-size nylon microfiber syringe filter (Whatman, Florham Park, NJ), followed by a 2.7-μm-pore-size glass microfiber syringe filter (Whatman).
N. sennetsu isolated from a total of 44 75-cm2 flasks was surface biotinylated using sulfo-NHS-SS-biotin (Pierce Biotechnology, Rockford, IL) at 4°C for 30 min (15). Biotinylated bacteria were lysed by sonication for 2 min in 1 ml of radioimmunoprecipitation buffer (25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% sodium dodecyl sulfate [SDS]) containing a 1:100 dilution of protease inhibitor cocktail set II (Calbiochem, San Diego, CA), followed by 30 min of incubation on ice, with vortexing every 5 min. The supernatant, which contained biotinylated N. sennetsu proteins, was collected by centrifugation at 16,000 × g for 10 min at 4°C and stored at −80°C in 10% glycerol (final concentration) until future usage.
Biotinylated N. sennetsu proteins were purified using a streptavidin-agarose gel (Pierce) and then separated by 12% SDS-polyacrylamide gel electrophoresis (PAGE). The gel was fixed, stained with GelCode blue (Pierce), and submitted to the Mass Spectrometry & Proteomics Facility, Campus Chemical Instrument Center, The Ohio State University, for protein identification by capillary liquid chromatography-nanospray tandem mass spectrometry (nano-LC/MS/MS) (15). Nano-LC/MS/MS-identified N. sennetsu proteins were analyzed for similarity to other proteins by use of the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool protein-protein algorithm (BLASTP) with the nonredundant protein sequence database (search was performed in August 2010). Identified N. sennetsu proteins were further examined for motifs by using NCBI BLAST searches against deposited conserved domains (performed in August 2010), with a default E value threshold of 0.01 (30). PSORTb (v.2.0) (12) and LipoP (v.1.0) (24) analyses were also performed on the entire N. sennetsu genome sequence (GenBank accession no. NC_007798).
P51 secondary structure prediction.
The secondary structure of N. sennetsu P51 was predicted using a combination of the programming algorithm in the PRED-TMBB web server (1), the hydrophobicity and hydrophobic movement profile (23), and DNAStar MegAlign (DNAStar, Madison, WI) alignment and analysis of the P51 sequences of the N. sennetsu type strain (20), Neorickettsia risticii strain Illinois (type strain) (29), and the Stellantchasmus falcatus agent (SF agent) Hirose strain (44).
Anti-Nsp3 sera, recombinant Nsp3 (rNsp3), and anti-rNsp3 sera.
Based on DNAStar Protean analysis (DNAStar), a 15-mer peptide was chosen from N. sennetsu Miyayama Nsp3 (amino acids [aa] 107 to 120, with a C-terminal cysteine added [KKDTKLRTKVPASNC]), and rabbit antisera were produced at EZBiolabs (Carmel, IN). Sera were ammonium sulfate precipitated by EZBiolabs to enrich immunoglobulins. Antisera for Nsp3 (anti-Nsp3107-120) and preimmune sera were reconstituted with distilled H2O.
Full-length nsp3 without the signal sequence (aa 25 to 246) was PCR amplified from N. sennetsu genomic DNA and cloned into the pET33b(+) vector (EMD, Madison, WI), using SalI and NotI sites. The recombinant plasmid was amplified by transformation into DH5α cells (Invitrogen), and the insert was confirmed by sequencing. The recombinant plasmid was then transformed into Escherichia coli BL21(DE3) (EMD). A concentration of 1 or 0.1 mM isopropyl β-d-thiogalactopyranoside was used to induce protein expression, and cells were sonicated for 30 s on ice to separate proteins into supernatant and pellet fractions. Production of rNsp3 was confirmed through staining of 10% SDS-PAGE gels with GelCode blue and through Western blotting with horseradish peroxidase (HRP)-conjugated anti-His (Sigma-Aldrich, St. Louis, MO).
Mouse antisera to rNsp3 were produced by inoculating 6-week-old female BALB/c mice with 7 to 10 mg/kg of body weight of His-tag-purified rNsp3 treated with 50 to 100 column volume washes of 0.1% Triton X-114 to remove endotoxin (40) (<5 endotoxin units/kg, according to a ToxinSensor gel clot endotoxin assay kit [GenScript, Piscataway, NJ]). Recombinant protein was suspended in 1× phosphate-buffered saline, pH 7.4 (PBS), with 10 μg Quil A (Accurate Chemicals, Westbury, NY) saponin/mouse, and was injected subcutaneously. Mice were inoculated three times at 2-week intervals and sacrificed 2 weeks after the last inoculation for harvest of immune sera. All mice were housed and treated according to Institutional Animal Care and Use Committee (IACUC) protocol 2008A0066 and IACUC rules and regulations.
Double immunofluorescence labeling.
N. sennetsu-infected P388D1 cells (approximately 60 to 90% were infected) were affixed to glass slides by use of a Shandon Cytospin 4 cytocentrifuge (Thermo Fisher Scientific) and were fixed with 4% paraformaldehyde in 1× PBS for 15 min at room temperature. The fixed cells were washed with Tris-buffered saline, pH 7.4 (TBS), and incubated with antibodies to P51 and Nsp3 for 1 h in 1× PBS containing 0.1% gelatin and 0.3% saponin (PGS), including rabbit anti-N. risticii recombinant P51 (anti-rP51; previously shown to react with N. sennetsu P51) (44), anti-Nsp3107-120 preabsorbed with P388D1 cells, and preabsorbed anti-rNsp3. After being washed in 2× PBS containing 0.05% Tween 20 (PBST; Sigma), the bacteria were incubated with preabsorbed horse anti-N. sennetsu from pony 41 (anti-N. sennetsu; collected 24 July 1986) (43) in PGS for 1 h. After being washed in 2× PBST, the bacteria were incubated in a combination of Alexa Fluor 488-conjugated goat anti-rabbit IgG (Invitrogen) or Alexa Fluor 488-conjugated goat anti-mouse IgG (Invitrogen) and Cy3-conjugated goat anti-horse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) in PGS for 30 min.
N. sennetsu organisms were isolated from infected P388D1 cells, affixed to glass slides, and fixed with 4% paraformaldehyde in 1× PBS for 15 to 30 min at room temperature. The fixed bacteria were washed with TBS and incubated with antibodies against outer membrane proteins as described above for 1 h in 1× PBS. After being washed in 2× PBST, the bacteria were incubated with anti-N. sennetsu for 1 h. After being washed in 2× PBST, the bacteria were incubated with a combination of Alexa Fluor 488-conjugated goat anti-rabbit IgG or Alexa Fluor 488-conjugated goat anti-mouse IgG and Cy3-conjugated goat anti-horse IgG in PBS for 30 min. As negative controls, labeling was performed with normal horse serum (N. sennetsu and N. risticii negative), normal rabbit serum, or normal mouse serum. For anti-rNsp3 and normal mouse serum labeling, 1% bovine serum albumin (Sigma) was added to the PBS for each incubation.
Outer membrane fraction and isolation of native P51 and Nsp3.
N. sennetsu was purified from P388D1 cells by sonication at setting 2 for 32 s and by 5-μm filtration. A 0.1% (wt/vol) Sarkosyl-insoluble outer membrane fraction was prepared, and outer membrane proteins were solubilized with 2% (wt/vol) octyl-β-glucoside (OGC; Pierce) (22, 26). For antibody neutralization, 25 μg of pelleted outer membrane was treated for 1 h with 20 μl of anti-rP51 or control rabbit serum and washed with 10 mM Tris-HCl (pH 8.0) before solubilization with 2% OGC.
HPLC was performed as previously described (22), and the fractions were tested by GelCode blue staining and Western blotting with respective antibodies for P51 and Nsp3. The combined P51 and Nsp3 fractions were concentrated by evaporation and dialyzed against 50 mM Tris-HCl, pH 8.0, with 1% OGC. Protein amounts were determined by bicinchoninic acid (BCA) protein assay (Pierce).
Western blotting.
Western blotting was performed against rNsp3, the OGC-solubilized N. sennetsu outer membrane fraction, and HPLC-separated P51 and Nsp3 fractions. Antisera used were anti-rP51, anti-Nsp3107-120, and anti-rNsp3. When Western blotting was performed against rNsp3 with anti-Nsp3107-120, the primary antibody was first preabsorbed against nontransformed E. coli BL21(DE3) proteins separated by SDS-PAGE and transferred to a nitrocellulose membrane. The secondary antibody used was HRP-conjugated goat anti-rabbit or HRP-conjugated goat anti-mouse (Cell Signaling Technology, Inc., Danvers, MA). Protein bands were visualized by enhanced chemiluminescence (ECL) by incubating the membrane with LumiGLO chemiluminescence reagent (Pierce). Images were captured using an LAS3000 image documentation system (Fujifilm Medical Systems USA, Stamford, CT).
Proteoliposome swelling assay.
Twenty-five micrograms of outer membrane protein preincubated with anti-rP51 or normal rabbit serum was incorporated into proteoliposomes to test the porin activity of the outer membrane fraction of N. sennetsu, as previously performed (22, 26). In brief, lipid films were created utilizing egg phosphatidylcholine (Avanti Polar Lipids, Alabaster, AL) and dicetyl phosphate (Sigma), membrane protein was added to the lipid film, the protein-lipid mixture was sonicated to incorporate the protein into the proteoliposomes, and the proteoliposomes were reconstituted with 15% dextran. The same molar concentrations of proteins, i.e., 10 μg and 4.8 μg of P51 and Nsp3, respectively, or 5 μg and 2.4 μg of P51 and Nsp3, respectively, were also reconstituted into proteoliposomes. Porin activity of the outer membrane fraction, P51, and Nsp3 was determined through proteoliposome swelling assays utilizing different solutes (22, 26, 37, 38).
Statistical analysis.
Unpaired Student's t test was applied to determine the differences among swelling levels. P values of <0.05 were considered significant.
RESULTS
Nano-LC/MS/MS of streptavidin-affinity-purified surface proteins and in silico analysis of N. sennetsu.
To experimentally identify bacterial surface-exposed proteins, N. sennetsu was biotinylated with sulfo-NHS-SS-biotin, and biotinylated proteins were affinity purified. GelCode blue protein staining of the SDS-PAGE gel detected multiple protein bands. Forty-two of the total of 936 (4.5%) N. sennetsu ORFs were identified by nano-LC/MS/MS, in eight bands. The majority of proteins identified were demonstrated to be expressed proteins for the first time. Table 1 summarizes the number of peptides identified for each protein, the percentage of amino acids covered by the identified peptides for each protein, and protein properties, including predicted molecular mass, isoelectric point, and presence of a signal peptide. Through analysis of the entire N. sennetsu genome, seven proteins were predicted to be outer membrane proteins by PSORTb (v.2.0) (12), and nano-LC/MS/MS demonstrated the surface expression of five of these proteins: P51, an OMP85 family protein (NSE_0718), an outer membrane efflux protein (NSE_0798), NSE_0456, and NSE_0498. Nine lipoproteins were predicted by LipoP (v.1.0) (24), and a single lipoprotein, NSE_0591, was found to be surface exposed by nano-LC/MS/MS.
P51 and Nsp3, found so far only in Neorickettsia spp., had the largest numbers of identified peptides (138 and 95, respectively), and Nsp3 and Nsp2 (another unique protein of Neorickettsia spp.) had the largest percentages of protein coverage (63.0% and 61.8%, respectively). P51 is a major antigen in N. sennetsu, N. risticii, and the SF agent and is predicted to be an outer membrane protein (9, 44). Although three nsp paralogs are present in the N. sennetsu genome (29), Nsp1 was not identified in the current analysis.
Proteomic analysis further characterized unique and hypothetical Neorickettsia proteins. This is the first time that NSE_0908, known as strain-specific antigen 3 (Ssa3) (29), was found to be expressed. Ssa proteins of N. risticii were previously proposed to be a potential cause of vaccine failure (5). One of three N. sennetsu Ssa paralogs, Ssa3 (29), was found to be surface exposed, although no signal peptide for secretion was predicted for Ssa3 or any other Ssa (SignalP, v.3.0). A strongly identified protein, NSE_0456, is conserved in Neorickettsia (20, 29), has no homology to other bacterial proteins, and has an unknown function. Other hypothetical proteins that were identified by proteomics, conserved in Neorickettsia, and not found in other bacteria include NSE_0687, NSE_0591 (no BLASTP results other than that for N. risticii), and NSE_0732.
Several proteins identified as surface exposed by proteomics have homologs in Ehrlichia and Anaplasma. Proteins identified with E. chaffeensis and/or A. phagocytophilum and also identified in N. sennetsu were GroEL (NSE_0642), DnaK (NSE_0019), the OMP85 family protein NSE_0664 (renamed OmpH-like outer membrane protein), a rotamase family protein (NSE_0645), a serine protease of the DO/DeqQ family (NSE_0166), VirB9-1 (NSE_0210), a pentapeptide repeat domain protein (NSE_0725), and translation elongation factor G (FusA; NSE_687) (14, 15, 21). Two M16 peptidases (NSE_0913 and NSE_0914) were detected in N. sennetsu. Although homologs are present in Ehrlichia and Anaplasma spp. (E values of ≤6e−37), these proteins were not previously identified as surface exposed (14, 15, 21). NSE_0498 and NSE_0900 also have homologs in Ehrlichia and Anaplasma, yet this is the first time that surface protein expression was identified.
P51 surface distribution and predicted secondary structure.
Procedures used to isolate bacteria from infected host cells may strip off bacterial surface proteins and/or alter their distribution patterns. To examine P51 expression and localization among individual bacteria within infected cells with minimal manipulation, N. sennetsu-infected P388D1 cells were prefixed with paraformaldehyde and permeabilized with saponin. Although it was difficult to discern individual bacteria within infected cells because they were densely packed and overlapped, the majority of intracellular bacteria were labeled with anti-rP51, in a rosary-like pattern (Fig. 1A) .
To confirm the bacterial surface exposure and distribution of P51, host cell-free N. sennetsu was prefixed with paraformaldehyde, which does not allow antibody penetration across biological membranes (48), and incubated with anti-rP51. Approximately 50% of isolated bacteria were strongly labeled with anti-rP51. This labeling was noted in bacteria ranging in size from 0.6 to 1.2 μm in diameter; the same size range was noted for nonlabeled bacteria. Individual bacteria were often labeled in a distinct and uniform punctate ring-like surface staining pattern, suggesting that P51 antigen exists as multiple clusters along the entire circumference of a bacterium (Fig. 1B). Representative negative controls demonstrated the specificity of the antibodies and the lack of bleedthrough from other antibody labeling in double immunofluorescence labeling (Fig. 1C and D).
We then examined the two-dimensional structure of P51, using PRED-TMBB (1). The discrimination value for the P51 amino acid sequence was 2.908, which is below the threshold value of 2.965, making P51 more likely to be a β-barrel protein localized to the outer membrane. We next examined whether P51 meets the established criteria for a porin structure, including a length between 7 and 16 amino acids, with the periplasmic turns having fewer than 8 amino acids, an even number of strands, N and C termini of the protein facing the periplasm, lipophilic amino acids at the ends of each β-strand, and amphipathic β-strands being in conserved regions (23, 36, 46). P51 was predicted to have 18 amphipathic and antiparallel transmembrane β-strands (Fig. 1E). These predicted transmembrane domains of P51 are highly conserved among the type strains of N. sennetsu and N. risticii and the first isolate of the SF agent (Table 2).
Nsp3 surface distribution and predicted secondary structure.
Both anti-Nsp3107-120 and anti-rNsp3 specifically recognized rNSP3 and native Nsp3 by Western blot analysis (Fig. 2A). To determine the pattern of labeling within infected cells, N. sennetsu-infected P388D1 cells were prefixed with paraformaldehyde and permeabilized with saponin. Approximately 5 to 25% of bacteria were labeled with anti-Nsp3107-120 and anti-rNsp3 within infected cells (Fig. 2B). In the absence of saponin permeabilization, approximately 15 and 20% of isolated bacteria were strongly labeled with anti-Nsp3107-120 and anti-rNsp3, respectively, confirming the bacterial surface exposure of Nsp3. The pattern of Nsp3 surface labeling appeared mainly as polar clusters, and labeling on smaller bacteria (0.6 to 0.9 μm in diameter) seemed more condensed and stronger than that on larger bacteria (>0.9 μm in diameter) (Fig. 2C). By PRED-TMBB, the discrimination value for Nsp3 was 2.903, suggesting that it is likely a β-barrel protein containing eight transmembrane β strands and is localized to the outer membrane. Nsp1 and Nsp2 had discrimination values of 2.915 and 2.953, respectively, with 9 and 12 predicted transmembrane β strands, respectively.
Porin activity of isolated N. sennetsu outer membrane fraction.
Since a large amount of P51 was detected on the N. sennetsu bacterial surface, we first examined whether the isolated N. sennetsu outer membrane fraction had porin activity and whether this activity could be reduced by anti-rP51. Porin activity was measured by the proteoliposome swelling assay (37, 38), using the outer membrane fraction derived from N. sennetsu preincubated with either control serum or anti-rP51. When control serum-incubated proteoliposomes were mixed with 33 mM (isosmotic solute concentration) l-glutamine, the monosaccharide arabinose or glucose, or the tetrasaccharide stachyose, swelling was observed, indicating that the N. sennetsu outer membrane fraction has strong porin activity. When the anti-rP51-incubated N. sennetsu outer membrane fraction was utilized in the proteoliposome swelling assay (Fig. 3A and C), swelling was significantly reduced with arabinose, glucose, and stachyose compared to that with the control serum-incubated outer membrane fraction (Fig. 3B and C), suggesting that P51 is the major porin of N. sennetsu. The antibody was more effective at blocking larger solutes (such as stachyose) than smaller solutes (such as l-glutamine), suggesting steric hindrance of solute diffusion (Fig. 3C).
Porin activity of isolated native P51 and Nsp3.
The isolated N. sennetsu outer membrane fraction contained two major proteins: P51 and Nsp3 (Fig. 4A). To experimentally determine if P51 and Nsp3 had porin activity, we isolated native P51 and Nsp3 from the outer membrane fraction by HPLC (Fig. 4A). Proteoliposome swelling assays were performed using the same molar concentration of P51 and Nsp3 proteins, with l-glutamine, glucose, sucrose, and stachyose as solutes. P51 showed statistically greater swelling than Nsp3 with glucose, sucrose, and stachyose (Fig. 4B). Nsp3 did not demonstrate significant swelling compared with the blank 60 s after the addition of any tested solutes. The results showed that P51 has prominent porin activity, whereas Nsp3 does not have significant porin activity.
DISCUSSION
The present study identified surface-expressed proteins in Neorickettsia for the first time. Expression of most of these proteins by Neorickettsia spp. was also established for the first time. Similar to the case in Anaplasma and Ehrlichia spp., heat shock proteins, a type IV secretion apparatus protein, and serine protease (HtrA) were surface exposed (14, 15, 27). Two major β-barrel proteins were confirmed by immunofluorescence labeling: the 51-kDa antigen (P51; NSE_0242) and Neorickettsia surface protein 3 (Nsp3; NSE_0875). P51, a unique protein that has so far been found only in Neorickettsia spp. (44), has long been assumed to be a potential surface-exposed protein. The presence of P51 on the surfaces of Neorickettsia spp. was demonstrated here for the first time. Protein secondary structure analysis revealed that P51 and Nsp3 are predicted to be multispan β-barrel outer membrane proteins.
While N. sennetsu lacks most genes required for glycolysis (20), isolated N. sennetsu can metabolize exogenous l-glutamine and generate ATP and CO2 (49, 50). The present study demonstrated that N. sennetsu does indeed have a porin: P51 is the major surface-exposed protein and is a major porin of N. sennetsu, allowing the diffusion of l-glutamine and sugars. Compared with A. phagocytophilum P44s (22) and E. chaffeensis OMP-1F (26), P51 is larger and predicted to have more transmembrane domains (18 β-strands). The pore size of P51 also appears to be larger than that of P44 or OMP-1, as it allowed diffusion of the tetrasaccharide stachyose at a similar rate to that of glucose or sucrose. Although bacterial porins generally cannot transport sugars larger than disaccharides (∼600-kDa pore size) (25), among the alphaproteobacteria, Omp2a of Brucella abortus can transport the tetrasaccharide maltotetraose when expressed in Escherichia coli (31). In most cases, porin proteins have been demonstrated or predicted to contain 16 β-strands (36). There are several instances of 18-β-strand porins, including a major outer membrane protein of Campylobacter jejuni (28, 51), a lambda phage receptor protein of E. coli called LamB (45), and a plasmid-encoded sucrose channel called ScrY found in E. coli and Salmonella (18). P51 is therefore at the upper end of the size range of known bacterial porins. The size and abundance of porins are expected to be advantageous for Neorickettsia organisms to acquire a variety of nutrients in the host cytoplasm, as they may have less concern for preventing toxic molecule intake within their protected intracellular environments, similar to mitochondria (36).
P51 was initially identified in N. risticii and was predicted to be a surface antigen due to strong antigenic responses to the 51-kDa N. risticii antigen by N. risticii and N. sennetsu immune sera (8, 9, 47), and a P51 ortholog was later sequenced for N. sennetsu (44). This study showed that antibodies against rP51 neutralize porin activity in N. sennetsu. These results corroborate observations from a previous study which showed that immune serum against N. risticii inhibits l-glutamine metabolism of isolated N. risticii (32). There was a small population of bacteria not labeled with anti-rP51 within host cells, possibly indicating differential expression of P51 among N. sennetsu populations. Future studies will be required to confirm this preliminary observation.
The present study characterized a new N. sennetsu major surface protein: Nsp3. Using NCBI conserved domain database searches, Nsp1 and Nsp3 were found to belong to the family pfam01617, and Nsp2 showed a weak similarity to members of pfam01617 (E value = 0.18). Yet unlike P44s and OMP-1/P28 members which belong to pfam01617 and have porin activity, Nsp3 has no significant porin activity. This is in agreement with the Nsp3 structure prediction, which showed only eight β-strands. β-Barrel proteins with eight β-strands and similar structures to those of porins, such as β8 proteins, are suggested to serve roles other than forming pores, such as in the invasion of host cells (2, 3, 6, 33). With the smaller percentages (15 to 20%) of smaller-diameter (<0.9 μm) bacteria labeled by anti-Nsp3107-120 and anti-rNsp3, it is possible that Nsp3 might have such a role. The roles of Nsp3 and Nsps in Neorickettsia infection remain to be characterized further.
In conclusion, the present study defined the N. sennetsu surface proteome. Our functional study demonstrated that the hypothesized surface-exposed protein P51 has prominent porin activity which can be blocked by a specific antibody. This study provides new critical data as a basis for future studies, which will contribute toward a better understanding of this unique trematode-borne human pathogen and its environmental persistence.
FIG. 1.
FIG. 2.
FIG. 3.
FIG. 4.
TABLE 1.
a
b Total number of peptides detected for the given protein. Numbers in parentheses show the coverage of proteins by the identified peptides.
c Theoretical isoelectric point of the given protein, as predicted by the ExPASy Compute pI/MW tool (13).
d Determined by SignalP v.3.0 (4). The numbers in parentheses indicate the amino acids between which cleavage is predicted to occur in the given protein.
e For this protein, the neural networks predicted cleavage between amino acids 24 and 25, and HMM predicted cleavage between amino acids 20 and 21.
f Only the hidden Markov model predicts a signal peptide in the given protein.
TABLE 2.
| P51 protein | Amino acid at transmembrane domain (T) positiona: | ||||||||||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| T2 aa 2 | T3 aa 3 | T5 aa 6 | T6 | T7 aa 2 | T8 | T10 | T11 aa 4 | T13 aa 4 | T14 aa 7 | T15 | T18 aa 4 | ||||||||||||||||||||
| aa 3 | aa 6 | aa 5 | aa 6 | aa 3 | aa 6 | aa 2 | aa 6 | ||||||||||||||||||||||||
| NSb | K | K | L | A | I | N | A | N | V | G | A | T | T | M | G | A | |||||||||||||||
| NRc | R | • | F | V | • | S | • | D | M | • | • | S | A | • | • | T | |||||||||||||||
| SFd | • | E | F | • | V | • | T | D | M | A | G | S | A | A | N | T | |||||||||||||||
a
Bullets represent conserved positions relative to the P51 predicted transmembrane domains T1 to T18 of the N. sennetsu Miyayama type strain. T1, T4, T9, T12, T16, and T17 are 100% identical among all strains.
b
N. sennetsu Miyayama type strain P51 protein (GenBank accession no. YP_506136).
c
N. risticii Illinois type strain P51 protein (GenBank accession no. YP_003081464).
d
SF agent Hirose strain P51 protein (GenBank accession no. AAL12490).
Acknowledgments
We thank the members of the Mass Spectrometry and Proteomics Facility, including K. Green-Church, for their assistance. We appreciate H. Nikaido for his valuable advice. Furthermore, we thank M. Lin, H. Niu, T.-H. Lai, and Y. Ge for their technical advice and assistance.
This work was funded by grant R01AI30010 from the National Institutes of Health, and K. Gibson was partially supported by T32 RR0070703-07 from the National Institutes of Health.
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© 2010 American Society for Microbiology.
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Received: 4 June 2010
Accepted: 30 August 2010
Published online: 15 November 2010
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Proteomic Analysis of Neorickettsia sennetsu Surface-Exposed Proteins and Porin Activity of the Major Surface Protein P51. J Bacteriol
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