INTRODUCTION

Top Abstract Introduction Materials and Methods Results and Discussion References

Worldwide, tuberculosis causes nearly 3 million deaths per year, the highest mortality rate of any single disease caused by an infectious organism (28). Approximately one-third of the world's population is thought to be infected with Mycobacterium tuberculosis, which is a facultative intracellular pathogen that resides within the macrophages of its host. For a great majority of these infected individuals, the tubercle bacilli are contained within granulomas by the immune system. The interior of the granuloma is characterized by an acidic pH, the presence of toxic fatty acids, and the low availability of oxygen (7). The survival of M. tuberculosis within macrophages and granulomas is likely to depend upon its ability to mount an effective genetic response to these hostile environments.

Several in vitro model systems have been developed using two-dimensional (2-D) gel electrophoresis to examine the protein level response of M. tuberculosis to environmental stress and intracellular residence within macrophages (13, 17, 32, 38, 40). Additional studies have shown that expression of the 16-kDa M. tuberculosis -crystallin (Acr) protein (encoded by the acr [also known as hspX] gene) is strongly induced during stationary-phase growth (11, 41) and during growth in unagitated, submerged cultures in which oxygen is believed to be limiting (5, 34, 35). We have recently shown, using a green fluorescence protein reporter promoter fusion assay, that the M. tuberculosis acr promoter is also upregulated in M. bovis BCG grown in shallow, standing cultures, compared to cultures constantly agitated on a rocking platform (A. Purkayastha, L. A. McCue, and K. McDonough, submitted for publication).

A growing family of acr-coregulated genes (ACG) is being identified and includes genes whose promoters are upregulated under shallow standing growth conditions and during intracellular growth within macrophages (Purkayastha et al., submitted for publication). The present study was done to compare overall mycobacterial protein expression profiles in response to growth under standing and shaking culture conditions and to identify additional ACG candidate genes. We found that standing and shaking culture conditions significantly affect protein expression in mycobacteria. Sequence-based analyses showed that Rv2623, one of the proteins whose expression was most affected by these differential culture conditions, is a member of a novel class of putative ATP-binding proteins that may be involved in environmental gene regulation. These findings indicate that shaking and standing growth conditions significantly impact mycobacterial protein expression and may affect the subsequent outcome of experiments using these bacteria.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results and Discussion References

Bacterial growth and labeling conditions. M. bovis BCG (Pasteur strain; Trudeau Institute) was grown in mycomedia (liquid 7H9 medium [Difco] supplemented with 0.5% [vol/vol] glycerol, 10% [vol/vol] oleic acid-albumin-dextrose-catalase [Difco] and 0.05% [vol/vol] Tween 80), as previously described (18). Standing cultures were grown undisturbed in 10 ml of mycomedia (approximately 2 mm deep) in 75-cm² flat-bottom tissue culture flasks (catalog no. 353083; Falcon) with the caps tightly sealed, lying flat at 37°C. Shaking cultures were grown on a gently rocking platform (Model 55 Rocking Platform; Reliable Scientific, Inc.) at 24 cycles per minute.

Sample preparation and 2-D gel electrophoresis of M. bovis BCG proteins. Bacteria were harvested by centrifugation and washed three times with ice-cold DPBS (Dulbecco's phosphate-buffered saline [10 mM sodium phosphate; 126 mM NaCl, pH 7.2]) plus 0.2% (wt/vol) EDTA (disodium EDTA dihydrate) containing a protease inhibitor cocktail (catalog no. P8340; Sigma) (DPBS-I). Cells were then resuspended in Tris-sodium dodecyl sulfate (Tris-SDS) buffer (0.3% [wt/vol] SDS and 50 mM Tris-HCl, pH 8.0) and lysed by several rounds of sonication and freeze-thawing as described previously (17).

In-gel proteolytic digestion of proteins. Protein spots of interest were isolated from Coomassie blue-stained 2-D PAGE gels, destained, and partially dehydrated with 0.1 M Tris (pH 8.0)-50% (vol/vol) acetonitrile for 30 min at 37°C, and this was followed by 5 min in a sonicating water bath to ensure that all visible Coomassie stain was removed from the gel pieces. Gel pieces were then dried in a Speed-Vac at ambient temperature under vacuum for approximately 30 min and rehydrated in 0.1 M Tris (pH 8.0)-0.05% (wt/vol) n-octylglucoside (Sigma) containing modified trypsin (0.1 µg/µl; Boehringer Mannheim) or endoproteinase Glu-C (protease V8) (Roche Molecular Biochemicals). Proteins were digested for 18 to 24 h at 37°C.

MS analysis. The peptides extracted from gel slices were desalted using a C₁₈ ZipTip (Millipore, Bedford, Mass.) and eluted onto a matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) sample plate with 60% (vol/vol) acetonitrile, using 1-µl wash volumes. After partial air drying of the wash volumes on the sample plate, 0.5 µl of MALDI matrix solution containing alpha-4-hydroxy-cinnamic acid (10 mg/ml; Sigma) was added to each sample spot.

Database searches. Genomic databases were searched using MS-FIT (http://prospector.ucsf.edu). The MALDI-TOF MS peptide mass fingerprint data were used to search the National Center for Biotechnology Information (NCBI) nonredundant database using a species-limited search filter, which restricted the search to Mycobacterium species. The search parameters were protein molecular mass ranging from 1,000 to 100,000 Da, trypsin digests (one missed cleavage), peptide mass tolerance of ±50 ppm, monoisotopic peptide masses, and cysteines modified by carbamidomethylation. Positive protein identification required matched peptides to cover a minimum of 15% of the protein sequence if the predicted molecular weight and pI of the identified protein were consistent with those of the observed protein spot isolated from the gel. A second search was routinely performed using only unassigned peptide masses to identify additional proteins that may be present in the isolated protein spot (12). MS/MS spectra data sets were used to query the complete M. tuberculosis H37Rv genome (NCBI accession no. NC_000962) with the SEQUEST protein identification software (15).

Western analysis. Approximately 50 µg of total mycobacterial protein was separated on an SDS-12.5% PAGE gel for each bacterial growth condition. After electroblotting onto polyvinylidene difluoride membranes (Millipore), the blots were blocked with buffer containing 5% (wt/vol) milk and 0.05% (vol/vol) Tween 20 (Sigma) in Tris-buffered saline (50 mM Tris, 0.5 M NaCl, pH 7.6). Rabbit antisera generated against M. tuberculosis isocitrate lyase (ICL) and malate synthase (both kindly provided by D. Russell) and catalase-peroxidase (KatG) (a generous gift of S. Cole) were diluted 1:10,000 in blocking buffer. The ICL antibody was generated against the whole recombinant protein from M. tuberculosis (10). The malate synthase antibody was generated against amino acids 424 to 439 (QNTMKIGIMDEERRTT) of the M. tuberculosis H37Rv malate synthase protein. Primary antibodies were detected using goat anti-rabbit immunoglobulin G antibodies conjugated to horseradish peroxidase (Jackson Labs) at a 1:5,000 dilution. Chemiluminescence detection was performed using the ECL detection kit (Amersham). Western blots were analyzed using Zero-Dscan (version 1.0; Scanalytics).

Sequence analysis. Pairwise local sequence alignments were performed with BLAST (1). Database mining and multiple sequence alignment of the Rv2623 family was performed with PROBE (16, 23). Given a single protein query sequence, PROBE performs a transitive BLAST search to identify a set of related sequences in a protein database and then proceeds to align only functionally constrained regions of the proteins, creating a "superlocal" multiple sequence alignment model consisting of individual motifs. The alignment model is then used to search the database for additional sequences. PROBE iterates between refining the alignment model and database searching until no additional sequences are recruited. PROBE calculates maximum a posteriori (MAP) scores for the model as well as for each motif. MAP scores are a measure of how well the alignment model describes the data, relative to unaligned random background (MAP scores represent the log of the probability ratio). A MAP score above zero indicates that the motif is more likely to belong to the alignment model than to unaligned random background.

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials and Methods Results and Discussion References

[³⁵S]-L-methionine-cysteine labeling of mid-logarithmic- phase shaking and standing cultures. The expression of M. tuberculosis ACG family promoters is upregulated similarly within macrophages and under shallow standing, undisturbed growth conditions, compared to constantly agitated culture conditions (Purkayastha et al., submitted for publication). We reasoned that the ACG family could belong to a larger set of genes that are differentially expressed under standing and shaking growth conditions.

View larger version (107K): [in this window] [in a new window]   FIG. 1.   2-D gel analysis of metabolically labeled 5-day M. bovis BCG cultures growing under standing (A) or shaking (B) growth conditions. Approximately 5 × 106 dpm of radiolabeled bacterial protein was loaded onto each gel. Proteins were first separated by IEF (pH 4 to 8, left to right) and then by SDS-PAGE (10% polyacrylamide) in the second dimension. Open circles indicate proteins that were more abundant under the standing growth condition, and open squares indicate proteins that were less abundant. Asterisks mark the positions of two highly induced mycobacterial proteins under standing growth conditions. Spots 1 and 4 indicate the positions of two mycobacterial proteins that were identified by 2-D GEMS analysis in Fig. 2. Approximate molecular masses are in kilodaltons.

Identification of differentially regulated proteins in standing and shaking cultures. Steady-state protein levels were examined by comparing total protein lysates of mid-logarithmic-phase shaking and standing cultures on Coomassie blue-stained 2-D protein gels. Comparison of these gels showed 15 protein spots that were upregulated under standing and shaking culture conditions and 2 protein spots that were downregulated. Differences in the protein expression profiles of radiolabeled (Fig. 1) and Coomassie-stained (Fig. 2) gels are largely due to differences in the sensitivity and nature of these two protein detection methods. Coomassie-stained gels detect accumulated, steady-state proteins and have a detection limit of 0.3 to 1 µg per gel spot. In contrast, only de novo-synthesized proteins appear on [³⁵S]-L-methionine-cysteine-labeled protein gels and are detected at a limit of several picograms of protein, depending on amino acid composition (2).

View larger version (80K): [in this window] [in a new window]   FIG. 2.   2-D gel analysis of steady-state levels of M. bovis BCG proteins growing in culture for 5 days under standing (A) or shaking (B) growth conditions. Approximately 500 µg of total protein was loaded onto each gel. Proteins were separated as described for Fig. 1 and stained with Coomassie blue dye. Open circles indicate proteins that were more abundant under standing growth conditions; open squares indicate the position of proteins that were less abundant. Large numbers in boldface type indicate protein spots identified by 2D-GEMS and correspond to the protein spot numbers in Table 1.

(i) Rv2623. Protein gel spot number 1 (Fig. 2A) was identified as Rv2623, a putative M. tuberculosis open reading frame of unknown function. No corresponding protein spot was observed on the Coomassie-stained shaking gel. The equivalent spot was also dramatically increased under standing conditions in radiolabeled 2-D gels (spot 1; Fig. 1A). Eight peptides from the tryptic peptide mixture matched the predicted protein sequence of Rv2623 and provided 25% coverage of this 297-amino-acid hypothetical M. tuberculosis protein (Table 1). Digestion with endoproteinase Glu-C resulted in eight peptide fragments that matched Rv2623 and provided 31% coverage of the predicted protein sequence. Rv2623 has a predicted molecular mass of 31.65 kDa and an isoelectric point of 5.46, which are consistent with the position of protein spot 1 on the 2-D gel (Fig. 2A).

View larger version (51K): [in this window] [in a new window]   FIG. 3.   Sequence logos (30) of the Rv2623 protein family motifs representing the multiple sequence alignment output of PROBE. Below each motif is the corresponding protein sequence for that motif in Rv2623, UspA, and MJ0577. Rv2623 contains two complete sets of these motifs, with N corresponding to the copy in the amino portion of the protein and C denoting the copy closer to the carboxy terminus. The MAP scores for the motifs are 1162.49 (motif 1), 980.12 (motif 2), and 832.65 (motif 3). The underlined sequences indicate amino acids involved in ATP binding in MJ0577 (42). Abbreviations: A, adenine; R, ribose; P, phosphate; D, dimer interface.

(ii) CysA2-CysA3. Protein gel spot 2 in Fig. 2A was identified as a putative thiosulfate sulfurtransferase, which is a rhodanese-like protein (4). The amount of protein in this spot was approximately sixfold greater under standing culture conditions than under shaking culture conditions. The peptide mixture contained 7 peptides that matched CysA2 (Rv0815c) and CysA3 (Rv3117), which are 100% identical, providing 23% coverage of these 277 amino acid proteins (Table 1). These proteins have a predicted molecular mass of 31 kDa and a predicted isoelectric point of 5.14, consistent with the gel position of the isolated spot. The M. tuberculosis genome encodes a total of four putative thiosulfate transferases (CysA2, CysA3, SseA [Rv3283], and SseB Rv2291]), suggesting an important role for these proteins in mycobacteria. BLAST analysis showed that M. tuberculosis CysA2-CysA3 shares 50% identity with M. tuberculosis SseA over 271 amino acids and 26% identity with M. tuberculosis SseB over 253 amino acids.

(iii) Gap. Protein gel spot 3 (Fig. 2A) was identified as Gap (Rv1436), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (4). The corresponding spot was not detected under shaking culture conditions. The peptide mixture contained seven peptides that matched the Gap protein sequence (Table 1) and provided 20% coverage of this 339-amino-acid protein.

(iv) GlcB-KatG. Protein gel spot 4 was found to contain two proteins. The amount of protein in this spot was approximately 3- to 14-fold greater under standing culture conditions than under shaking culture conditions in different experiments. Increased de novo synthesis of a protein at this gel position was also observed in radiolabeling experiments (spot 4; Fig. 1A). These proteins were identified as an M. tuberculosis malate synthase (Rv1837c, GlcB) and an M. tuberculosis catalase-peroxidase protein (Rv1908c, KatG) (4). The predicted molecular mass (80.4 kDa) and pI (4.9) for M. tuberculosis GlcB are essentially identical to those for KatG, making it difficult to resolve these two proteins by 2-D PAGE. Eleven tryptic peptide fragments (Table 1) matched the M. tuberculosis GlcB protein and resulted in 18% coverage of this 741-amino-acid protein. KatG was identified as a possible second component with 11% coverage on a second-pass search after the subtraction of all tryptic peptides matching the predicted glcB gene product.

View larger version (28K): [in this window] [in a new window]   FIG. 4.   Western analysis of 5-day M. bovis BCG cultures grown under shaking and standing growth conditions for the expression of ICL, malate synthase, and catalase-peroxidase (KatG) in M. bovis BCG. (A) Coomassie blue-stained SDS-PAGE gel of M. bovis BCG proteins. (A) Arrows indicate the position of the protein band identi- fied as the -crystallin (Acr) protein. Immunoblots were probed with anti-malate synthase (B), anti-catalase-peroxidase (C), or anti-ICL polyclonal antibodies (D). Molecular mass markers are in kilodaltons.

(v) Acr (HspX). In Fig. 4A, we observed a low-molecular- weight protein that was significantly induced in standing mycobacterial cultures. Tryptic peptides from this 16-kDa protein were analyzed by MS-MS. MS-MS spectra of the most abundant protein in each band corresponded to 72% of the amino acid count of the M. tuberculosis HspX (Acr) protein. This finding is consistent with the work of Purkayastha et al. who have shown that the acr promoter is upregulated under shallow standing growth conditions (Purkayastha et al., submitted for publication).

Relevance of standing and shaking culture conditions as a model system. The aim of this study was to begin to provide a more detailed understanding of the differences between mycobacteria grown under shallow standing and shaking culture conditions through the identification of differentially expressed proteins. These studies showed that a number of proteins are differentially regulated in response to growth under shallow standing culture conditions compared to constantly shaking cultures.