A Comprehensive Proteome of Mycoplasma genitalium - Journal of

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A Comprehensive Proteome of Mycoplasma genitalium Noemí Párraga-Niño,† Nuria Colomé-Calls,‡ Francesc Canals,‡ Enrique Querol,† and Mario Ferrer-Navarro*,† †

Institut de Biotecnologia i de Biomedicina (IBB) and Dpt Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona (UAB), E-08193 Cerdanyola del Vallès (Barcelona), Spain ‡ Proteomics Laboratory, Vall d’Hebron Institute of Oncology - VHIO, E-08035 Barcelona, Spain S Supporting Information *

ABSTRACT: Mycoplasma genitalium is a human pathogen associated with several sexually transmitted diseases. Proteomic technologies, along with other methods for global gene expression analysis, play a key role in understanding the mechanisms of bacterial pathogenesis and physiology. The proteome of M. genitalium, model of a minimal cell, has been extended using a combination of different proteomic approaches and technologies. The total proteome of this microorganism has been analyzed using gel-based and gel-free approaches, achieving the identification of 85.3% of the predicted ORFs. In addition, a comprehensive analysis of membrane subproteome has been performed. For this purpose, the TX-114 soluble fraction has been analyzed as well as the surface proteins, using cell-surface protein labeling with CyDye. Finally, the serological response of M. genitalium-infected patients and healthy donors has been analyzed to identify proteins that trigger immunological response. Here, we present the most extensive M. genitalium proteome analysis (85.3% of predicted ORFs), a comprehensive M. genitalium membrane analysis, and a study of the human serological response to M. genitalium. KEYWORDS: Mycoplasma genitalium, total proteome, membrane proteome, surfome, immunoproteome

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diseases as well.5,6 In addition, this microorganism has also been detected by PCR in rectal7 and synovial fluids of the temporomandibular joint,8 respiratory tract,9 and conjunctival swab.10 Moreover, M. genitalium is associated with chronic infection, as highlighted by retrospective longitudinal studies of urethritis11 and cervicitis.12 Such persistence may aggravate the risk of adverse reproductive outcomes, including tubal-factor infertility and pre-term birth, conditions associated with this microorganism.13,14 The development of proteome analysis has made it feasible to analyze protein profiles in a variety of samples.15 Despite rapid advances in the whole-genome sequencing16−18 for a large number of different bacteria, the analysis of bacteria at the proteome level remains fundamental to investigate many prokaryotes in order to provide insights not available from genomic studies alone. The ability of proteomics to look at bacterial protein synthesis on a global scale also allows for the characterization of previously undocumented proteins. Other mycoplasma proteomes have been analyzed,19−21 but they were, for the first time, analyzed some years ago when the state of the art was 2DE followed by the identification by mass spectrometry and gel-free approaches were at their birth. As

INTRODUCTION Mycoplasma genitalium is the smallest member belonging to the Mollicutes class, with a genome size of 580 kb that contains 524 genes . It has been estimated that M. genitalium has the capacity to encode 487 proteins,1 although 12 of these proteins were removed from this list (NCBI reference sequence: NC_000908.2). This microorganism exhibits a remarkable scarcity of genes involved in biosynthetic pathways. These biosynthetic deficiencies make M. genitalium a fastidious microorganism. To overcome these deficiencies, complex and enriched media are required for its cultivation.2 As a selfreplicating organism, it represents the simplest molecular assembly able to independently subsist and generate its own energy requirements. This mollicute is considered one of the most suitable models to achieve a complete understanding of the cellular biology of a single cell. In a recent analysis, it has been estimated that M. genitalium has approximately 100 nonessential genes,3,4 reducing the minimal essential genome to a set of 426 genes. A minimal essential genome is the set of genes essential for cell growth and division.3 Among the essential genes there are several with an unknown function, and there is no experimental evidence of the existence of the proteins encoded by many of them. This microorganism is the etiological agent of non-gonococcal urethritis in men and cervicitis in women, having been associated with other genital © 2012 American Chemical Society

Received: January 27, 2012 Published: May 14, 2012 3305

dx.doi.org/10.1021/pr300084c | J. Proteome Res. 2012, 11, 3305−3316

Journal of Proteome Research

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reported by Wasinger et al. 12 years ago, the proteome of M. genitalium, was analyzed by two-dimensional electrophoresis in the exponential and stationary growth phases within the gradient of pH 2.3−12 in four windows (2.3−5.0, 4−7, 6−11 and 8.5−12), identifying 158 proteins corresponding to 89 distinct ORFs. 22 Additionally, Su et al. identified 22 phosphoproteins, 14 of them undisclosed in the previous analysis by Wasinger et al., by specific phosphoprotein staining.23 Since then, no additional proteomic analysis of this microorganism has been published. Combining the results of both studies, we can conclude that the identified proteome of M. genitalium at the starting point of this work consisted of 103 of the predicted ORFs (21.7%). For most of the rest of the ORFs there is no experimental evidence of the existence of the corresponding proteins. Despite the accelerated rate of genomic sequencing having led to an abundance of completely sequenced genomes, annotation of the ORFs in these genomes is an important task and is most often performed computationally. There are now some studies in the literature where predicted genes have been validated at the protein level. For example, Jaffe et al. detected 81% of the predicted ORFs of Mycoplasma pneumoniae.24 More recently, Nagaraj et al. have indentified 63% of the predicted ORFs of yeast.25 Moreover, LC−MS/MS-based methods have revealed the existence of several new ORFs in the M. tuberculosis26 and M. pneumoniae24 genomes that were not originally predicted by genomic methods. Additionally, hypothetical genes usually represent a significant proportion of all genes in a genome, and the rapidly growing number of hypothetical proteins within each newly sequenced genome is one of the challenges of modern biology. Since the prediction of these genes is exclusively based on the presence of putative start and stop codons, the probability of erroneous predictions is high and, consequently, needs experimental validation. In M. genitalium there are 108 proteins currently annotated as being hypothetical or putative lipoproteins. The existence of these proteins needs to be validated. About one-quarter to one-third of all bacterial genes encode membrane proteins. Mycoplasmas have evolved a parasitic lifestyle, and membrane transporters, consequently, play a key role in the uptake of nutrients and growth factors. Additionally, envelope proteins are particularly accessible to host immune responses and to drug therapy and thus could be used to control this human pathogen. Because of the importance of membrane proteins and specifically cell surface proteins in antibacterial resistance, transport of nutrients, facilitation of cell−cell-signaling, attachment to host cells, and virulence, we have performed a comprehensive analysis of membrane proteins in M. genitalium. Our goals in this work are to extend the total proteome of M. genitalium and to perform a comprehensive analysis of the membrane proteins with special focus on those present on the cell surface as well as those proteins on the cell surface that trigger the immune response in the host.

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phenol red was used to monitor the growth of the culture. Whereas SP4 medium is a rich, undefined medium, MM1428 is a minimal, defined medium that does not contain serum, yeast extract, or other undefined components. M. genitalium was grown in MM14 medium at 37 °C, 5% CO2, for 142 h (stationary phase). In order to standardize the harvesting point, growth curves for each medium were obtained (see Figure S1, Supporting Information) by measuring ATP concentration using the ATP bioluminescence Assay Kit HS II, from Roche, following the manufacturer’s recommendations. Total Protein Extraction for SDS-PAGE

M. genitalium cells from a single 150-cm2 flask culture were washed three times with PBS. The culture was scraped and centrifuged for 15 min at 15,800g. The pellet was resuspended in 500 μL of PBS, and loading buffer was added to the sample. The sample was then boiled for 20 min. For sample preparation of M. genitalium grown in MM14, six washing steps were carried out in order to completely remove the bovine serum albumin from the medium. Total Protein Extraction for 2DE

M. genitalium cells from a single 150-cm2 flask culture were washed three times in PBS and immediately resuspended in 550 μL of lysis solution (8 M urea, 2 M thiourea, 2.5% CHAPS, 2% ASB-14, 60 mM DTT, 40 mM Tris-HCl pH 8.8 and protease inhibitor cocktail (manufacturer’s recommendations were followed)). The sample was disrupted by sonication for 5 min at 4 °C in ice−water and centrifuged 20 min at 15,800g to discard any insoluble particles. The protein sample was immediately used or stored in aliquots at −80 °C until use. Protein concentration was measured with RC DC Protein Assay kit, from Bio-Rad. About 3 mg of protein was obtained from a single 150-cm2 flask with 40 mL of medium. Whole Proteome Labeled with Biotin-streptavidin

A flask containing M. genitalium culture was washed three times with cold PBS 1x. After 30 min of incubation at 4 °C with 2.4 mg/mL Ez-LinkTM Sulfo-NHS-SS-Biotin in cold PBS x1, tris was added to quench the excess of biotin. The culture was scraped and centrifuged for 15 min at 15,800g. The sample was disrupted by sonication for 5 min at 4 °C in ice−water and centrifuged for 10 min at 15,800g to discard insoluble debris and the non-lysed cells. The labeled proteins were purified with a sepharose column with immobilized streptavidin to capture the biotinylated proteins. The proteins captured in the column were eluted after 1 h of incubation with 50 mM DTT in SDS 1%. The elute fraction was concentrated using a Microcon centrifugal filter device with a 10-kDa cutoff. Phase Separation in TX-114

Phase separation in TX-114 was performed following the method of Bordier et al.29 Briefly, flasks containing M. genitalium cells were washed three times with cold PBS 1x. Cells were scraped with TX-114 1%, 1 mM PMSF in cold PBS, and the sample was incubated for 3 h at 4 °C. The sample was centrifuged 20 min at 15,800g to remove the non-disrupted cells and the debris. After 10 min of incubation at 37 °C to condense the micelles containing hydrophobic proteins and detergent, the sample was centrifuged for 3 min at 8000g to separate the two phases. The aqueous phase (upper) was removed, and the volume of the detergent phase (below) was re-established to the original volume with 1 mM PMSF in cold PBS. After 10 min of incubation at 4 °C, the sample was

MATERIAL AND METHODS

Strain and Growth Conditions

M. genitalium G-37 was obtained from the American Type Culture Collection (ATCC Number 33530) and was routinely grown in Spiroplasma (SP)-4 medium27 at 37 °C, 5% CO2, for 96 h (stationary phase). The cells were harvested from 150-cm2 flasks containing 40 mL of SP4 medium. The color range of 3306



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incubated for 10 min at 37 °C and was then centrifuged again for 3 min at 8000g. The aqueous phase was discarded, and this washing step was repeated three times. After the last wash, the proteins in the detergent phase were precipitated using the methanol/chloroform protocol, and the proteins were resuspended in a modified Lysis Solution (8 M urea, 2 M thiourea, 4% CHAPS, 2% ASB-14, 60 mM DTT, 40 mM TrisHCl pH 8.8), more suitable for hydrophobic proteins. Protein concentration was measured with RC DC Protein Assay kit, from Bio-Rad.

acid for 30 min each time. Sensitizing was carried out for 30 min in 0.02% (w/v) sodium thiosulfate. After the gels were washed three times for 5 min with distilled water, they were incubated in 0.1% (w/v) silver nitrate for 20 min. The gels were then washed twice for one minute with distilled water and then developed with 3% (w/v) sodium carbonate and 0.025% (v/v) formaldehyde until the desired contrast was reached. Reaction was stopped with 1.5% (w/v) EDTA-Na2 for 45 min, after which the gels were washed twice with distilled water.

Cell Surface Protein Labeling with CyDye

Once electrophoresis was carried out, the gel was submerged for 15 min in cold transference buffer (25 mM Tris, 192 mM Glycine, 20% ethanol, pH 8.3). A nitrocellulose membrane and filter papers were cut to the same gel size and were submerged in cold transference buffer. Transferences were carried out with the Semidry Unit (Bio-Rad). Transference efficiency was confirmed by Ponceau staining. Blood was collected in a tube without anticoagulant and incubated for 1 h at 37 °C and for 12−16 h at 4 °C to favor the coagulum formation. The blood was then centrifuged for 20 min at 4,000 rpm at 4 °C. The serum was removed and preserved at −20 °C. Nitrocellulose membranes were incubated for 1 h with the pool of sera (control or patient) in a dilution of 1:100 in PBS with 1% blocking reagent (Roche). Detection was performed using a 1:10,000 dilution of a rabbit anti-human antibody, and then the membranes were developed using the Immobilon Western (Millipore) for chemiluminescent peroxidase substrate.

Immunoassay

The flask containing M. genitalium cells was washed three times with cold HBSS 1x (137 mM NaCl, 5.4 mM KCl, 0.25 mM Na2HPO4, 0.44 mM KH2PO4, 1.3 mM CaCl2, 1 mM MgSO4, 4.2 mM NaHCO3, pH 8.5). The sample was centrifuged for 20 min at 15,800g, and the pellets were resuspended with labeling buffer (1 M urea in HBSS, pH 8.5). Next, 1.5 μL of CyDye5 (600 pmol) was added, the sample was incubated for 30 min on ice in the dark, and 20 μL of lysine 20 mM were added to quench the excess of CyDye. The cells were washed with HBSS and were resuspended in lysis solution. The cells were disrupted by sonication for 5 min at 4 °C in ice−water and centrifuged for 20 min at 15,800g to discard insoluble debris. To compare the above sample on a DIGE analysis, a total protein extract labeled with CyDye3 was prepared; 1.5 μL of working solution (600 pmol) was added to 75 μg of protein sample. The sample was incubated on ice for 30 min in darkness, 1 μL of 10 mM lysine was added to quench the reaction, and the sample was then incubated for 10 min on ice, in darkness.

Image Analysis

Electrophoresis

The silver- and Coomassie-stained gels were immediately scanned using a GS-800 scanning device (Bio-Rad), and digitalized images were evaluated using ImageMaster 2D 5.0 (GE Healthcare). Fluorescence images of the gels were obtained on a Typhoon 9400 scanner (GE Healthcare). Cy3 and Cy5 images were scanned at excitation/emission wavelengths of 532/580 nm and 633/670 nm, respectively, at a resolution of 100 μm. Molecular mass was automatically determined with ImageMaster 5.0 using a Benchmark protein ladder (Invitrogen) covering the range of 10 kDa-220 kDa during the second dimension. For Western blot, a prestained molecular weight marker was used. The isoelectric point was also automatically determined by bilinear interpolation between landmark features on each image by ImageMaster 5.0. Images from Western blotting were captured with a VersaDoc Imaging system (Bio-Rad). Volumes and % volume for each spot were also calculated automatically by the software.

For GeLC−MS/MS analysis, 10% acrylamide SDS-PAGE was performed in all of the cases. Two-dimensional electrophoresis with immobilized pH gradients was carried out according to Görg et al.,30 with some minor modifications. Briefly, firstdimension isoelectric focusing was performed on immobilized pH gradient strips (24 cm, pH 3−10 and 4−7 for TX-114 and 3−10 for fluorescence-labeled surface proteins and immunome) using an Ettan IPGphor System. Samples were applied near the basic end of the strips by cup-loading, after being incubated overnight in 450 μL of rehydration buffer (7 M urea, 2 M thiourea, 2.5% w/v CHAPS, 2% ASB-14 w/v, 0.5% pharmalytes, pH 3−10, 100 mM DeStreak reagent). After focusing at 70 kVh, strips were equilibrated, first for 15 min in 10 mL of reducing solution (6 M urea, 100 mM Tris-HCl, pH 8, 30% v/v glycerol, 2% w/v SDS, 5 mg/mL dithiothreitol [DTT]) and then in 10 mL of alkylating solution (6 M urea, 100 mM Tris-HCl, pH 8, 30% v/v glycerol, 2% w/v SDS, 22.5 mg/mL iodoacetamide) for 15 min, on a rocking platform. Second dimension SDS-PAGE was performed by laying the strips on 12.5% isocratic Laemmli gels (24 cm × 20 cm), cast in low-fluorescence glass plates, on an Ettan DALT Six system. Gels were run at 20 °C at a constant power of 2.5 W per gel for 60 min, followed by 17 W per gel until the bromophenol blue tracking front had run off the end of the gel. The sample amount loaded for TX-114, fluorescence-labeled surface proteome and immunome gels was 200 μg.

In-Gel Tryptic Digestion

The Coomassie-stained lanes were cut into 10 equal bands, while the silver-stained protein spots were excised from the acrylamide gel with a cut tip and immediately distained and digested as described elsewhere.31 Briefly, the Coomassiestained lanes were washed twice with water for 20 min and distained with 200 μL of 50 mM ammonium bicarbonate/50% acetonitrile. Silver-stained spots were destained with 200 μL of 30 mM potassium ferricyanide and 100 mM sodium thiosulfate (1:1) for 20 min in the dark, and then spots were washed with Milli-Q water until they were completely clear. Before tryptic digestion, reduction and alkylation with DTT/IAA was performed by incubating samples with 200 μL of 10 mM DTT in 50 mM ammonium bicarbonate for 1 h at 56 °C,

Gel Staining and Detection of Proteins

All SDS-PAGEs were stained with 0.1% Coomassie blue R-250, while 2-D gels were silver stained as described elsewhere.20 Briefly, the gels were fixed twice in 40% ethanol and 10% acetic 3307



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Figure 1. SDS-PAGE of all protein extracts of M. genitalium used in this work. (A) Total protein extract. (B) Biotin-labeled protein extract. (C) Total protein extract in MM14 medium, Passage 1. (D) Total protein extract in MM14 medium, Passage 2. (E) TX-114 soluble protein extract.

followed by alkylation with 200 μL of 55 mM IAA in 50 mM ammonium bicarbonate for 30 min, protected from light. Gel pieces were digested overnight with 6 ng/μL trypsin at 37 °C. The peptide extraction was carried out with three consecutive washes with 0.2% TFA for MALDI-TOF identifications or with 1% formic acid for ESI. The eluted peptides were dried in a SpeedVac and stored at −20 °C until they were analyzed by mass spectrometry.

accepted with a Mascot score higher than that corresponding to a P value of 0.05. For GeLC−MS/MS analysis, the digests of the SDS-PAGE lanes were analyzed on an Esquire HC-Ultra Ion Trap mass spectrometer (Bruker), coupled to a nano-HPLC system (Proxeon). Peptide mixtures were first concentrated on a 300 mm i.d., 1 mm PepMap nanotrapping column and then loaded onto a 75 mm i.d., 15 cm PepMap nanoseparation column (LC Packings). Peptides were then eluted by a 0.1% formic acid/ acetonitrile gradient (0−40% in 100 min; flow rate ca. 300 nL/ min) through a nanoflow ESI Sprayer (Bruker) onto the nanospray ionization source of the Ion Trap mass spectrometer. MS/MS fragmentation (3 × 0.3 s, 100−2800 m/z) was performed on three of the most intense ions, as determined from a 0.8 s MS survey scan (310−1500 m/z), using a dynamic exclusion time of 1.2 min for precursor selection and excluding single-charged ions. An automated optimization of MS/MS fragmentation amplitude, starting from 0.60 V, was used. Proteins were identified using Mascot (Matrix Science) to search the NCBInr database. Searches were restricted to Mycoplasma (36726 sequences) taxon, but first, common contaminants were removed using the contaminants database available in the Mascot search engine. MS/MS spectra were searched with a precursor mass tolerance of 1.5 Da, fragment tolerance of 0.5 Da, trypsin specificity with a maximum of one missed cleavage, and cysteine carbamidomethylation set as fixed modification, with methionine oxidation as the variable modification.

Mass Spectrometry Analysis

For MALDI analysis, 1 μL of sample was mixed with the same volume of a solution of α-cyano-4-hydroxy-trans-cinnamic acid matrix (0.5 mg/mL in ethanol/acetone 6:3) and spotted onto a MALDI target plate (Bruker). The drop was air-dried at room temperature. MALDI-mass spectra were recorded in the positive ion mode on an Ultraflex Extreme time-of-flight instrument. Ion acceleration was set to 25 kV. All mass spectra were externally calibrated using a standard peptide mixture containing angiotensin II (1046.54), angiotensin I (1296.68), substance P (1347.74), bombesin (1619.82), rennin substrate (1758.93), adrenocorticotropic hormone 1-17 (2093.09), adrenocorticotropic hormone 18-39 (2465.20), and somatostatin 28 (3147.47). Calibration was considered good when a value below 1 ppm was obtained. For PMF analysis, the MASCOT search engine (Matrix Science) was used with the following parameters: one missed cleavage permission, 50 ppm measurement tolerance, and at least four matching peptide masses. Cysteine carbamidomethylation was set as fixed modification when appropriate, with methionine oxidation as the variable modification. Common contaminants were removed using the contaminants database available in the Mascot search engine. Searches were carried out using the NCBInr NC_000908.2 database. Positive identifications were

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RESULTS AND DISCUSSION

1. Total Proteome

1.1. Total Proteome by GeLC−MS/MS. Currently, proteomes inferred from genome sequence data are extremely 3308



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accessible but often remain unverified.32 Only proteomics can unambiguously determine if the expressed gene is translated into a protein. High-throughput LC−MS/MS-based proteomics approaches measure protein fragments directly, and the resulting peptide sequences confirm the existence of a protein from a specific genome. Peptides that map to genomic regions outside the boundaries of previously annotated genes represent evidence of novel genes or extensions of their predicted termini. We have used the GeLC−MS/MS approach, widely used in shotgun proteomics, in order to identify the proteome of M. genitalium. The proteome was separated on a SDS-PAGE, and the entire lane was divided into 10 equal fragments. These were tryptically digested, and each digestion was analyzed using an Ion Trap (Figure 1A). Independent replicas of all of the analyses have been performed, and the identified proteins in all analyses have also been classified according to COG (Figure S2, Supporting Information) . Using this approach, 269 proteins were identified (Table 1, Supporting Information), corresponding to the 56.6% of the predicted ORFs. Nineteen of the 20 COG groups are represented (Figure S2.A, Supporting Information). Among the 269 proteins, 73 are annotated as uncharacterized, putative, or unknown proteins with an unidentified function. Here, we have demonstrated the existence of all of these proteins. This result emphasizes the deficiency of knowledge available for this bacterium. One of the identified proteins is a new protein, previously undisclosed in the genome. This protein was identified as “Uncharacterized protein MPN_687”, from the closely related microorganism Mycoplasma pneumoniae. After a careful search in the M. genitalium genome, we found that this protein maps to an intergenic region between the MG469 and MG470 genes, codified between the 579234 bp and the 578581 bp. This gene is transcribed from the antisense strand. 1.2. Total Proteome Analysis by Biotin Labeling. In order to identify the cell-surface proteins, one common approach is the labeling of the cell surface proteins with biotin and the subsequent purification of the labeled proteins with a sepharose column with immobilized streptavidin. Our idea was to use this approach in order to identify the surfome of M. genitalium, but surprisingly, we found all of the proteins of M. genitalium labeled. Taking advantage of this phenomenon, proteins labeled with biotin and purified with immobilized stretavidin were analyzed by GeLC−MS/MS (Figure 1B). Using this approach, we have indentified 369 proteins, corresponding to 77.5% of the predicted ORFs (see Table 2, Supporting Information). This represents an important increase in the number of identified proteins. The greater number of proteins identified in this analysis can be attributed to a higher purity of the protein sample, as a result of the purification with immobilized streptavidin, which would effectively remove contaminants that can obscure protein identification in the mass spectrometer. Only 12 proteins were identified in the classical GeLC−MS/MS approach and not identified in the biotin-labeled approach. A control was performed to validate the results and to confirm the absence of biotinylation processes in M. genitalium. Briefly, a total protein extract of M. genitalium was incubated in the sepharose column with immobilized streptavidin, omitting the biotin labeling of proteins. The eluted fraction was loaded into a 10% SDSPAGE, and only one band corresponding to streptavidin was observed (data not shown).

Combining the results obtained in the GeLC−MS/MS and the biotinylation protocol, 381 different proteins were identified, corresponding to 80.2% of the total predicted ORFs. These results give us a global picture of the proteome of M. genitalium grown in SP4 and harvested during the stationary phase. 1.3. Total Proteome in Minimal Medium MM14. In order to extend the number of identified proteins, an analysis of the total proteome of M. genitalium grown in different conditions was performed. For this purpose, M. genitalium was grown in another medium, the MM14 medium. Whereas the SP4 medium is a rich, undefined medium and also one of the most effective media to culture this bacterium, MM14 is a minimal, defined medium without serum that can be also used to culture M. genitalium, but with lower rates. A growth period of 142 h was required to obtain cells in a stationary phase, whereas in SP4 medium only 96 h were required. In order to get the bacteria used to the MM14 medium, a second passage was performed to consolidate the adaptation of M. genitalium to the MM14 medium. Interestingly, we observed that the bacterium aggregates in the second passage, and cells lose the adherence to the plastic surface of the flask (data not shown). As a result of these differences between the first and the second passages, the proteome of M. genitalium was analyzed in the two conditions: during the first passage, when the microorganism is getting used to the MM14 medium and after that, the second passage, when the microorganism is completely adapted to the MM14 medium. Samples were analyzed using the GeLC−MS/MS technique. Total protein extracts were obtained for the first and the second passages (Figure 1C and D). In the first passage, 305 different proteins were identified (64.2% of the total predicted ORFs) (see Table 3, Supporting Information). It is expected that in different growth conditions the microorganism would express different proteins. In this condition three new proteins have been identified. These are MG098, MG123, and MG437. MG123 and MG437 are noncharacterized proteins, while MG098 is a tRNA methyltransferase. For the second passage, 192 proteins were identified (40.4% of the total predicted ORFs) (see Table 4, Supporting Information). During this second passage a drastic reduction in the number of identified proteins is observed. There is a clear change in the expression profile of M. genitalium once this has been adapted to the minimal medium MM14. In the second passage, 191 proteins are no longer expressed. Among these 191 proteins there are a lot of key enzymes for cell division, DNA replication, protein synthesis, and other important enzymatic activities. These proteins must remain at very low expression levels in these growth conditions because it is obvious that, in these conditions, this microorganism is also capable of synthesizing proteins, nucleic acids and others. Interestingly, a new protein has been identified, MG052, which was not identified in either the SP4 analysis or in the MM14 Passage 1. This protein is a cytidine deaminase involved in pyrimidine salvaging, used to recover bases and nucleosides that are formed during degradation of RNA and DNA. Additionally, another seven proteins (MG025, MG118, MG130, MG150, MG234, MG289, and MG387) have been found in MM14 Passage 2 but not in Passage 1. Among the identified proteins, we have identified some proteins involved in adhesion (MG191, MG192, and MG318), but as explained before, during the second passage a clear 3309



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Figure 2. (A) 2DE of TX-114 soluble fraction of M. genitalium, pH gradient from 3 to 10. (B) 2DE of TX-114 soluble fraction of M. genitalium, pH gradient from 4 to 7.

aggregation of the cells was observed, and the cells did not adhere to the plastic surface of the culture flask. Although it has been demonstrated that the presence of MG191 and MG192 is absolutely mandatory for proper adhesion,33 the mere presence of these proteins is not enough to keep the cells adhered to the plastic surface. Among all of the identified proteins there are two proteins (MG199 and MG333) whose genes are currently annotated like pseudogenes. Herein, we have demonstrated the existence of the polypeptidic products of these genes, but further experiments would be necessary in order to demonstrate if these proteic products are really functional.

The combination of the results of the different approaches used to analyze the proteome of M. genitalium has allowed us to identify 85.3% of the predicted ORFs. This is the most extensive proteomic analysis ever performed in M. genitalium. However, there are still 14.1% of the predicted ORFs that have not been found. The absence of these proteins can be explained by two reasons: first, they may have extreme physical and chemical properties, like low mass or high hydrophobicity, which makes them hard to be detected by proteomics techniques, and second, those proteins may be at very low expression levels, which would be beyond equipment sensitivity, or may be expressed in different conditions than 3310



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those tested in this work. A list of the 14.1% of the predicted ORFs that have not been identified is provided (Table 5, Supporting Information). Among these unidentified proteins, 31 are conserved hypothetical proteins or proteins with unknown function, threeare ribosomal proteins with a very low mass, and five are annotated ABC transporters.

not identify peptides of either N-terminal or C-terminal sequence ends. These proteins are MG185, MG421, and MG260 (Spot 9, Figure 2B). 2.2. TX-114 Soluble Fraction Analysis by GeLC−MS/ MS. Although well-suited for lipoprotein analysis, the 2-D PAGE strategy presents some drawbacks in analysis of highly hydrophobic proteins. Moreover, these protein classes may undergo selective loss during precipitation/resolubilization steps. In order to increase the membrane protein coverage and minimize selective protein loss, GeLC−MS/MS was performed on the same samples analyzed by 2-D PAGE (Figure 1E). As expected, the GeLC−MS/MS approach allowed for the identification of more proteins. A total of 246 proteins (51.8% of the total proteome) were identified (see Table 7 of Supporting Information). Interestingly, eight proteins identified by 2-D PAGE were not found by GeLC− MS/MS. Among the identified proteins there are many involved in the transport as well as being involved in membrane components synthesis. The main surface protein known to be involved in adhesion, and also immunodominant MgPa has been identified as well as other components known to be involved in adhesion. Ribosomal proteins represent a significant proportion of the mycoplasma liposoluble proteome. This might appear inconsistent, but despite their traditionally cytoplasmic localization, it was already demonstrated that ribosomes interact with the bacterial protein export complex.37 About one-fifth of the TX-114 soluble fraction identified proteins using the GeLC−MS/MS approach are uncharacterized proteins. We have predicted the subcellular location of these 58 proteins, and 49 of them are predicted to be located in the membrane according to PSORT38 (Table 7 of Supporting Information). Several identified proteins are predicted to be cytoplasmic. Among these, many hydrolases are present. However, lipases, peptidases, and nucleases might be associated with the membrane compartment and assist in reducing macromolecules to simpler components, enabling their uptake. In fact, mycoplasmas lack many biosynthetic pathways and rely on internalization of nucleotides, amino acids, sugars, and lipids from their external environment. Recently, it has been reported that hydrolytic enzymes are surface-located in mycoplasmas and that they can be associated with ABC transporters in order to digest macromolecules before the uptake of simpler components or play major roles in pathogenicity.39 Moreover, proteins that have been traditionally considered to be cytoplasmic, such as elongation factor TU and the E1 beta subunit of the pyruvate dehydrogenase complex, are surface-exposed in mycoplasma and are also strong antigens in many mycoplasma species.40−42

2. TX-114 Soluble Fraction Analysis

2.1. TX-114 Soluble Fraction Analysis by 2DE. In this study, the membrane proteome of M. genitalium has been characterized by means of triton X-114 fractionation. The TX114 soluble fraction has been analyzed by both 2-D PAGE-MS and GeLC−MS/MS. Total protein extract and the triton X-114 soluble fraction of M. genitalium were subjected to 2D-PAGE separation in order to evaluate the extent of enrichment in the basic and liposoluble proteins. Upon comparison, the 2-D PAGE map generated with the triton X-114 soluble fraction showed a significant enrichment in basic proteins, with an excellent resolution also in high-abundance spots (Figure 2). In order to achieve a better resolution in the pH range from 4 to 7, 2D-PAGE was also obtained in this pH range (Figure 2B). This protocol has allowed for the identification of 49 proteins (10.3% of total ORFs) (Tables 6A and 6B, Supporting Information). After spot-volume calculation, the most prominent spots in the 2-D PAGE map were pyruvate dehydrogenase component E1 β subunit (MG273; 2.43% vol), inorganic pyrophosphatase (MG351; 2% vol), uncharacterized lipoprotein (MG338; 1.83% vol), and putative lipoprotein (MG260; 1.52% vol). The first two proteins, MG273 and MG351, according to PSORT34 prediction, are cytoplasmic proteins, while MG338 and MG260 are membrane proteins. In the closely related bacterium M. pneumoniae, the presence of the pyruvate dehydrogenase β subunit in the cell surface has been demonstrated, and it has been also found that this protein binds fibronectin.35,36 The molecular weight (MW) and the isoelectric point (pI) of the identified spots were experimentally determined with ImageMaster 5.0 Software (GE Healthcare) and were compared with gene-deduced MW/pI coordinates obtained from MASCOT (for a detailed report, see Tables 6A and 6B of Supporting Information). The majority of gel-estimated and theoretical Mr/pI fitted quite well. Lower MW values could be due to post-translational processing or proteolysis, while higher MW values could be the result of covalent binding of chemical groups. There were also proteins which were identified in different spots. This is an indicator that these proteins coexist as different isoforms. These isoforms can vary in the pI and/or in the MW. The proteins with more noticeable pI differences are MG040, MG067, MG095, MG252, MG260, MG307, MG309, MG338, MG412, MG453, MG458, MG460, MG469, and MG470. All of these proteins showed a more acidic pI than expected. We have detected that there are several proteins that show an experimental MW much lower than that calculated from the database sequence, in some cases as low as 10% of the predicted MW. For some of these proteins no peptides were identified in the C-terminal region of the sequence: MG040, MG191 (only the isoform detected in Spot 20, Figure 2A), MG260, MG307, and MG309. Conversely, for other proteins only C-terminal peptides have been identified: MG095, MG321 (Spot 79, Figure 2A), and MG338. There were three proteins whose mass reduction could be the result of the proteolysis of both extremes because we could

3. Cell-Surface Protein Labeling with CyDye

The cell surface of a bacterial pathogen is the interface between the cell and the environment and, thus, is the initial mediator for infection, providing an important reservoir for components that may be used for novel vaccine development as well as the characterization of new drug targets. The study of such biological molecules has, however, been under-represented in proteomics studies due to the difficulty involved in their analysis. Cell-envelope proteins in bacteria are typically difficult to characterize due to their low abundance, poor solubility, and the problematic isolation of pure surface fractions with only minimal contamination. There are several approaches in order to identify and characterize these proteins; one of the most 3311



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In order to pick the spots, the gels were silver-stained (Figure 3B). About 200 spots differentially observed in the cell-surface sample were detected in the gels. From these, a total of 61 different spots could be identified (Table 8 of Supporting Information), and these correspond to 37 different ORFs. Among the most abundant proteins were a putative lipoprotein (MG039), excinuclease ABC subunit B (MG073), MgPa adhesion protein (MG191), proline-rich P65 protein (MG217), uncharacterized protein MG218.1, conserved hypothetical protein (MG269), HMW3 cytadherence accessory protein (MG317), OsmC-like protein (MG427), and elongation factor Ts (MG433). Note that two of the most abundant proteins in the surface are proteins with an unknown function, indicating the lack of knowledge regarding the cell surface of this pathogen. All of these proteins identified on the surface of the cell, because of their localization, constitute potential targets for clinical diagnostics or, from a more general point of view, tools for bacterial monitoring. Among the indentified proteins there are some that have already been pointed out as such, such as MgPa (MG191)33 or P65 (MG217).48 Chaperonine GroEL, originally depicted as cytoplasmic, plays a key role in the proper folding of a large number of proteins, but it has been reported in other microorganisms to play a crucial role in bacterial adhesion and has also been identified on the cell surface.49,50 The present work constitutes an original and complementary contribution to identify cell-surface proteins of M. genitalium. Within this population, we have found proteins which were expected to be found in the compartment studied but also a great number of proteins that had never been experimentally described as cell-surface proteins.

used in Gram-positive pathogen bacteria is cell-surface shaving. This methodology has been successfully used in some human pathogens;43,44 however, it still remains unclear if this methodology is useful for Gram-negative bacteria because the Gram-negative cell envelope is not as rigid and resistant as those that are Gram-positive, which leads to increased cell lysis and thus identification of more false-positive cytoplasmic proteins. Furthermore, the abundance of these contaminating proteins may completely obscure peptides belonging to surface proteins. A popular method for the examination of bacterial surface proteins is the use of tags or other molecules to label surfaceexposed proteins. A major advantage of this method over others is that it can be applied not only to Gram-positive organisms but also to the Gram-negative cell envelope, since it does not digest or compromise cell integrity.45−47 As explained before, our intention was to use biotin in order to label the surfaceexposed proteins, but we found that all of the proteins were labeled, allowing for the identification of the most extended proteome of M. genitalium. Another method used to label the surface proteins is labeling with CyDyes. The surface proteins of M. genitalium were labeled with Cy5, while Cy3 was used to label a total M. genitalium extract. Labeled samples were mixed and separated by 2-D PAGE (Figure 3A).

4. Immunoproteome of Mycoplasma genitalium

Our aim in this part of the study was to characterize the M. genitalium immunoproteome, which catalogues those proteins recognized by the host immune response, and possibly to identify novel antigens. By using a combination of 2-D gels of total-protein extracts of M. genitalium, Western blotting, and mass spectrometry, we have identified a set of proteins that are recognized by sera from both M. genitalium-infected patients and healthy donors. This immunoproteomic approach has been applied to a wide range of organisms including M. tuberculosis,51 Streptococcus pneumoniae,52 Candida albicans,53 and Neisseria meningitidis.54 In many cases this has led to the identification of novel antigens. Twenty-five different sera have been collected. Eleven of them were from healthy volunteer donors, and the remaining 14 were obtained from M. genitalium-infected patients from the Basurto Hospital (Bilbao, Spain). Prior to 2-D analysis, all sera were tested individually in a monodimensional SDS-PAGE (Figure S3A for healthy donors and Figure S3B for M. genitalium-infected patients, Supporting Information). All control sera showed signals, but this is not an unexpected result because M. genitalium has been already reported for asymptomatic healthy people.55 The number of proteins recognized by each serum varied considerably. The origin of this diversity of immune response is currently unknown. It can result from innate differences in immunological responsiveness to the antigens, or variations in antigenic exposure in different patients due to variations in treatment times or disease severity. As a result of this great variation, it was decided to perform the Western blotting analysis of 2DPAGEs with pools of both the patient sera and the control sera.

Figure 3. (A) Image obtained for the CyDye surface−protein labeling. Spots in green (Cy5) are the surface-exposed labeled proteins. Spots in red (Cy3) are the total protein extract. (B) Silver-stained gel after CyDye image acquisition. 3312



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On the Western blot, using the M. genitalium-infected patients pooled sera 39 spots corresponding to 19 different ORFs have been indentified, while for the control sera 21 spots were identified corresponding to 13 different ORFs (Table 9 of Supporting Information). Many of the proteins were identified from more than one spot, often with the same molecular weight but with differing pI values. Presumably, these represent isoforms of the same protein that retain their immunogenicity. Some of these isoforms appear to be immunologically different from each other. For instance, the HMW3 cytadherence accessory protein (MG317) is recognized by both sera, patients and controls, but only one isoform (Spot 9, Figure 4A) is recognized by the serum from patients. The immunogenic proteins identified represent a wide range of functions, including chaperones and some proteins involved in the central metabolism. Eight of the 19 proteins identified were indentified in both the TX-114 fractionation and the cellsurface protein labeling with CyDye. Nine were only identified in the TX-114 fractionation and two proteins were only identified in the cell-surface protein labeling with CyDyes. These results corroborate, as expected, that the majority of the immunoreactive proteins are exposed on the cell surface. It is noteworthy that there are proteins that are recognized by both sera (patients and controls): M218.1 (hypothetical protein), MG272 (E2 subunit α-keto dehydrogenase acid), MG317 (cytadherence protein HMW3), and MG392 (GroEL chaperone). Six of the identified proteins are recognized only by the patient sera but not by the control sera. These differential proteins between infected and control are MG191 (MgPa adhesion protein), MG265 (Cof-like hydrolase), MG282 (transcriptional elongation factor GreA), MG299 (phosphotransacetylase), MG305 (molecular chaperone DnaK), and MG438 (restriction/modification DNA domain protein type 1). Some of the identified proteins are of cytosolic origin. The most feasible explanation for these immune responses is that they are generated during episodes of septicaemia when dead M. genitalium cells release their cytoplasmic contents into circulation. Such a scenario would suggest that although these antigens may be involved in the immune response to M. genitalium disease, they are unlikely to be good vaccine candidates. Interestingly, MG191 encodes the adhesin molecule MgPa. This protein is highly immunogenic and necessary for attachment of the organism56 and is recognized only by the infected patients’ serum. A couple of previous works have been published where immunogenic proteins of M. genitalium were characterized using hyperimmune rabbit sera,57,58 but the data presented herein represent the first proteome-wide investigation of the naturally induced, human immune response to M. genitalium infection with implications for its understanding. The antigens identified demonstrate that M. genitaliuminfected patients have highly variable immune responses against a wide range of M. genitalium antigens. The sera tested contain antibodies capable of binding a range of M. genitalium proteins, some of which are, for the first time, shown to be available to IgG on the surface of M. genitalium cells.

Figure 4. (A) Western blot of a total protein extract of M. genitalium, using a pooled serum of M. genitalium-infected patients. (B) Western blot of a total protein extract of M. genitalium, using a pooled serum of healthy donors. (C) Silver-stained 2DE gel of total M. genitalium protein extract.

of M. genitalium obtained from the core proteome of three different Mycoplasmas (Acholeplasma laidawii, Mycoplasma gallisepticum, and Mycoplasma mobile).60 For a detailed comparative table, see Table 10 of Supporting Information. Comparing the results obtained with the calculated core proteome of M. genitalium, it can be observed that almost all proteins predicted to be members of the core proteome in M. genitalium have been identified. There are only 20 proteins that have been predicted to be members of the core proteome that have not been identified in any of the analyses performed. Most of these proteins are membrane proteins that probably have not

5. Proteome Comparison with other Mycoplasmas

We have compared the results obtained in this work with other proteomes from different mycoplasmas. Specifically, the proteome of M. genitalium has been compared with M. pneumoniae,59 M. penetrans,20 and the calculated core proteome 3313



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Notes

been identified due to their high hydrophobicity or as a result of their low molecular weight. The results obtained have also been compared with the previously published proteome of M. penetrans.20 In this work, the authors identified 123 different proteins, but only 70 have a homologue in the M. genitalium proteome. We have looked for those identified proteins of M. penetrans that have a homologue in M. genitalium. There are only two proteins identified in M. penetrans that have not been found in M. genitalium, and they are Pseudouridine synthase (MYPE1310) and Cell division protein FtsZ (MYPE8370). Perhaps, the most interesting comparison is between the close microorganisms M. genitalium and M. pneumoniae. Mycoplasma pneumoniae is a microorganism whose proteome has been analyzed in depth,59 and at the moment 88% of its proteome has been identified experimentally. From a general point of view, there are 17 proteins that have not been identified in either M. genitalium or M. pneumoniae;, six proteins that have been identified in M. genitalium but not in M. pneumoniae, and 59 proteins that have been identified in M. pneumoniae but not in M. genitalium. Most of these proteins are hypothetical proteins with an unknown function or membrane proteins.

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by Grant Nos. BES-2008-002086 and BIO2007-67904-C02-01 from the MCYT (Ministerio de Ciencia y Tecnologia,́ Spain). We wish to thank and recognize the collaboration of Dr. Ezpeleta of the Basurto Hospital (Bilbao, Spain) for patient sera contribution. The Proteomics Laboratory at VHIO is a member of the ProteoRed-ISCIII network, supported by the Instituto de Salud Carlos III (Ministerio de Sanidad, Spain). This manuscript has been proofread by Mr. Chuck Simmons, a native, English-speaking University instructor of English.

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(1) Fraser, C. M.; Gocayne, J. D.; White, O.; Adams, M. D.; Clayton, R. A.; Fleischmann, R. D.; Bult, C. J.; Kerlavage, A. R.; Sutton, G.; Kelley, J. M.; Fritchman, R. D.; Weidman, J. F.; Small, K. V.; Sandusky, M.; Fuhrmann, J.; Nguyen, D.; Utterback, T. R.; Saudek, D. M.; Phillips, C. A.; Merrick, J. M.; Tomb, J. F.; Dougherty, B. A.; Bott, K. F.; Hu, P. C.; Lucier, T. S.; Peterson, S. N.; Smith, H. O.; Hutchison, C. A., 3rd; Venter, J. C. The minimal gene complement of Mycoplasma genitalium. Science 1995, 270 (5235), 397−403. (2) Razin, S.; Yogev, D.; Naot, Y. Molecular biology and pathogenicity of mycoplasmas. Microbiol. Mol. Biol. Rev. 1998, 62 (4), 1094−156. (3) Hutchison, C. A.; Peterson, S. N.; Gill, S. R.; Cline, R. T.; White, O.; Fraser, C. M.; Smith, H. O.; Venter, J. C. Global transposon mutagenesis and a minimal Mycoplasma genome. Science 1999, 286 (5447), 2165−9. (4) Glass, J. I.; Assad-Garcia, N.; Alperovich, N.; Yooseph, S.; Lewis, M. R.; Maruf, M.; Hutchison, C. A., 3rd; Smith, H. O.; Venter, J. C. Essential genes of a minimal bacterium. Proc. Natl. Acad. Sci. U.S.A. 2006, 103 (2), 425−30. (5) Tully, J. G.; Taylor-Robinson, D.; Cole, R. M.; Rose, D. L. A newly discovered mycoplasma in the human urogenital tract. Lancet 1981, 13 (1), 1288−91. (6) Taylor-Robinson, D.; Horner, P. J. The role of Mycoplasma genitalium in non-gonococcal urethritis. Sex. Transm. Infect. 2001, 77, 229−31. (7) Jensen, J. S. Mycoplasma genitalium: the aetiological agent of urethritis and other sexually transmitted diseases. J. Eur. Acad. Dermatol. Venereol. 2004, 18 (1), 1−11. (8) Kim, S. J.; Park, Y. H.; Hong, S. P.; Cho, B. O.; Park, J. W.; Kim, S. G. The presence of bacteria in the synovial fluid of the temporomandibular joint and clinical significance: preliminary study. J. Oral Maxillofac. Surg. 2003, 61 (10), 1156−61. (9) Baseman, J. B.; Dallo, S. F.; Tully, J. G.; Rose, D. L. Isolation and characterization of Mycoplasma genitalium strains from the human respiratory tract. J. Clin. Microbiol. 1988, 26 (11), 2266−9. (10) Bjornelius, E.; Jensen, J. S.; Lidbrink, P. Conjunctivitis associated with Mycoplasma genitalium infection. Clin. Infect. Dis. 2004, 39 (7), e67−9. (11) Horner, P.; Thomas, B.; Gilroy, C. B.; Egger, M.; TaylorRobinson, D. Role of Mycoplasma genitalium and Ureaplasma urealyticum in acute and chronic nongonococcal urethritis. Clin. Infect. Dis. 2001, 32 (7), 995−1003. (12) Cohen, C. R.; Nosek, M.; Meier, A.; Astete, S. G.; IversonCabral, S.; Mugo, N. R.; Totten, P. A. Mycoplasma genitalium infection and persistence in a cohort of female sex workers in Nairobi, Kenya. Sex. Transm. Dis. 2007, 34 (5), 274−9. (13) Clausen, H. F.; Fedder, J.; Drasbek, M.; Nielsen, P. K.; Toft, B.; Ingerslev, H. J.; Birkelund, S.; Christiansen, G. Serological investigation of Mycoplasma genitalium in infertile women. Hum. Reprod. 2001, 16 (9), 1866−74.

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CONCLUSIONS In this work we have identified 85.3% of the total predicted ORFs of M. genitalium. This is the most extensive proteomic analysis of M. genitalium and represents one of the highest proteome coverages ever reported. Among the identified proteins, we have demonstrated the existence of three proteins annotated as pseudogenes and a protein encoded by an intergenic region between genes MG469 and MG470 and confirmed the existence of 133 proteins previously labeled as hypothetical protein or unknown protein. The use of biotin in order to purify the M. genitalium proteins has allowed considerably increase in the number of identified proteins in a shotgun proteomic experiment. In addition, the total proteome has been analyzed in three different conditions: M. genitalium grown in the rich medium SP4, M. genitalium getting used to the minimal medium MM14 (Passage1), and, finally M. genitalium adapted to the minimal medium MM14. Furthermore, a comprehensive analysis of the M. genitalium membrane has been performed. TX-114 fractionation has been performed, and the TX-114 soluble fraction has been analyzed by both,2DE and by GeLC−MS/MS. Moreover, the identification of those proteins accessible on the surface of the cell has been performed by surface−protein labeling, and proteins triggering the natural immune response in healthy donors and in M. genitalium-infected patients have been disclosed. The work herein presented will establish the basis for a more complete understanding of this human pathogen.

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ASSOCIATED CONTENT

S Supporting Information *

Lists of the proteins identified in each analysis and supplementary figures. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Tel: (+34)93-581 30 28. FAX: (+34)93-581 20 11. E-mail: [email protected] . 3314



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A Comprehensive Proteome of Mycoplasma genitalium - Journal of

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