Enrichment and separation of B. quintana membrane proteins resulted in identification of distinct membrane fractions by 1D and 2D SDS-PAGE.
OMP were isolated from B. quintana
JK31 using lauryl sarcosine fractionation. This method has been used to enrich OMP from a number of bacterial species (3
) and was also utilized to separate the OMP from the IMP of B. henselae
). We initially compared the protein profiles of the subcellular fractions of B. quintana
after separation by 1D SDS-PAGE, followed by Coomassie blue staining (Fig. ). Enrichment of the OMP and IMP from the TMP preparation was evident when the proteins were compared with proteins from the cytosolic preparation. There were prominent bands in the OMP preparation at approximately 116, 93, 45, 40, and 34 kDa (Fig. ). These prominent OMP bands either were observed exclusively with the OMP fraction or were highly enriched in the OMP fraction compared with the IMP fraction. Coomassie blue staining of proteins separated on a gel containing a lower percentage of acrylamide revealed that the 116-kDa bands in the OMP and IMP fractions were actually at slightly different molecular masses (data not shown).
FIG. 1. Subcellular fractions of proteins from B. quintana wild-type strain JK31 visualized on a Coomassie blue-stained 1D SDS-PAGE gel. Proteins were fractionated using a lauryl sarcosine method, separated on a 10% acrylamide gel, and stained with Coomassie (more ...) Mass spectrometry analysis identified distinct proteins that comprise the TMP subproteome of B. quintana.
The TMP and OMP preparations were resolved by 2D SDS-PAGE, followed by silver staining. The resolution of individual spots from gel to gel and for different protein preparations was highly reproducible. We visualized more than 300 distinct protein spots in the TMP preparation in a pI range from 4.5 to 9.5 and in a molecular mass range from 14 to 220 kDa (Fig. ). Protein spots were numbered, excised from Coomassie blue-stained gels, subjected to in-gel tryptic digestion, and submitted for protein identification by PMF. We excised 137 spots and identified 110 separate protein spots (Table ). Some spots were not positively identified because of the low concentration of protein and/or contamination with human keratin. The 110 spots identified by PMF correspond to 60 B. quintana genes. With the exception of a few spots that stained intensely with silver stain but not with Coomassie blue, most protein spots that were visualized were identified.
FIG. 2. 2D map of the total membrane subproteome of B. quintana wild-type strain JK31. Membrane proteins were separated by isoelectric point in the first dimension and then by molecular mass in the second dimension. Proteins were visualized by silver staining, (more ...)
B. quintana proteins identified in this study by PMF using matrix-assisted laser desorption ionization-time of flight mass spectrometry
A number of the B. quintana membrane proteins identified appeared as protein isoforms or families. The protein product of a single gene can appear as several protein spots on a 2D gel due to posttranslational modifications; these isoforms are usually visualized as a horizontal pattern of spots at the same molecular weight. The modifications of bacterial proteins can include phosphorylation, glycosylation, methylation, deamidation, and biotinylation, each of which can affect the charge and the isoelectric point. Of the 60 unique proteins that we identified, 18 had at least two isoforms, and these proteins included proteins that play a role in energy metabolism (AcnA, AtpA, AtpD, PpdK, SucB, and SucD), protein fate (ClpB and MopA), protein synthesis (FusA, RpsA, and Tuf1), transcription (Pnp and Rho), purine ribonucleotide synthesis (GuaB and GlyA), and virulence (HbpE, HbpD, VompA, VompB, and VompC).
Twenty-four percent (26/110) of the proteins identified were predicted by PSORTb to localize to the outer membrane, 65% (72/110) of the proteins were predicted to localize to the cytoplasm, and the localizations of 11% (12/110) of the proteins are not known. The 26 proteins localized to the outer membrane correspond to 10 distinct gene products, including three hemin-binding proteins (HbpA, HbpD, and HbpE), Omp43, Omp89, peptidyl-prolyl cis-trans-isomerase (Ppi), BQ08370 (a putative OMP), and three adhesins (VompA, VompB, and VompC). The spots with the greatest apparent protein concentration correspond to GroEL (MopA) (at 57.6 kDa; spots JB51, JB52, JB74, JB78, JB109, and JB121), EF-Tu (Tuf1) (at 42.9 kDa; spots JB1/108, JB37, JB38, JB79, and JB115), HbpA (at 29.3 kDa; spot JB87), HbpD (at 32.7 kDa; spots JB2, JB3/31, JB30, JB88, JB119, and JB137), HbpE (at 33 kDa; spots JB4/107, JB5/113, and JB114), VompA (97.0 kDa), VompB (100.5 kDa), and VompC (99.8 kDa). Pfam predictions and grand average of hydropathy values for all of the spots identified are shown in Tables and .
B. quintana proteins identified in this study and predicted to be localized to the outer membrane by PSORTba
Immunoblotting with human sera identified 24 immunoreactive B. quintana membrane proteins.
We identified 24 B. quintana
proteins that are recognized consistently by sera from humans with documented B. quintana
infections. TMP from the same preparation that was used for PMF were separated simultaneously by 2D SDS-PAGE to produce two identical 2D gels, and then TMP from one gel were transferred and immunoblotted with sera from each of 21 patients from whom B. quintana
was isolated and whose sera were positive for Bartonella
antibodies as determined by IFA analysis (10
) (Table ). Each patient's serum was analyzed on a separate immunoblot, and immunoreactive antigens were identified by alignment with a simultaneously prepared silver-stained gel using the 2D Evolution software (Nonlinear Dynamics). For negative controls, two blots with 2D-separated TMP were immunoblotted with sera that were from Bartonella
IFA-negative, culture-negative patients. These control sera detected a few B. quintana
proteins, usually the protein spots that had the highest protein concentrations and were most dense. The proteins that were immunoreactive on these two negative control blots were considered false positives and were not included in the analysis of positive sera.
To identify the B. quintana
antigens most commonly recognized by sera from patients infected with B. quintana
, we established a positive cutoff value of 24, representing the B. quintana
TMP antigens recognized by sera from 24% or more of the patients infected with B. quintana
(at least 5 of the 21 patients analyzed). Using this cutoff value, we identified 24 immunodominant B. quintana
proteins recognized by sera from these patients (Table ). Figure shows a representative 2D immunoblot of B. quintana
TMP probed with serum from patient 4 (Table ). The pI values of these immunoreactive antigens ranged from 4 to 7, and the predicted molecular masses ranged from 20 to 100 kDa. Four of the immunodominant antigens were OMP (VompA, VompB, HbpE, and Ppi). The remainder were predicted to be cytoplasmic proteins, and many of these cytoplasmic proteins have been identified previously in the outer membrane fractions of other gram-negative organisms, including other Bartonella
). Each of the 24 immunoreactive antigens commonly recognized by patients’ sera was labeled in the immunoblot shown in Fig. . Note that the serum from B. quintana
-infected patient 4 recognized all of the 24 immunoreactive proteins whose values were above the 24% cutoff value for all patients. Antibodies in the serum of patient 4 also recognized several B. quintana
protein spots whose values were below the cutoff value of 24%; therefore, although these proteins (e.g., NusA, VompC, CarB, SecA, SdhA, and PdhB) were strongly immunoreactive on the blot, they were not labeled or included in Table . Note that in some cases (e.g., GroEL [MopA], AtpA, EF-Tu [Tuf1], RpsA, HbpE, and Pnp), the most dominant member of a protein family did not meet the inclusion criteria because the spot was so highly concentrated that it was also immunoreactive with the negative sera. Therefore, these proteins were not included in the analysis.
B. quintana proteins found to be immunoreactive with sera from patients infected with Bartonella
FIG. 3. 2D immunoblot of TMP from B. quintana JK31 probed with serum from a B. quintana-infected human. 2D separation of the TMP fraction was performed, and the proteins were transferred and immunoblotted with a 1:50 dilution of serum from patient 4, who had (more ...)
Spots JB15 and JB17 had low sequence coverage as determined by PMF, and spot JB16 had no significant hits in the database search but was found to have monoisotopic peaks similar to those of spots JB15 and JB17. Because these proteins were found to be highly immunogenic, we confirmed their identities by submitting the peptides for mass spectrometry/mass spectrometry analysis. Validation of the liquid chromatography-mass spectrometry/mass spectrometry results confirmed that these three protein spots (spots JB15, JB16, and JB17) were dihydrolipoamide succinyltransferase (SucB) from B. quintana.
In addition to the immunoblot analysis, screening of an expression library was performed to identify antigenic proteins using a λ phage genomic library of B. quintana JK31. The primary screen included ~3,000 plaques per plate and four plates (a total of 12,000 plaques). Twelve plaques were identified as plaques that were reactive with human and rabbit antisera, and they were replaqued and rescreened to ensure clonality. Characterization of two positive clones resulted in identification of SucB and GroEL (MopA), confirming the immunoreactivities of these two proteins observed by immunoblot analysis.