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Journal of Proteomics & Bioinformatics
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Proteomic Characterisation of Two Strains of Mycoplasma mycoides Subsp. Mycoides of Differing Pathogenicity

Ivanka Krasteva1, Neil F Inglis2, Flavio Sacchini1, Robin Nicholas3, Roger Ayling3, Colin P Churchward3#,
John March4, Alex Lainson2, Kevin Mclean2, Val Hughes2, Lisa Imrie2, Erin Manson2, Jason Clark4,
Attilio Pini1* and David GE Smith2,5*
1Istituto Zooprofilattico Sperimentale of Abruzzo and Molise “G Caporale”, Teramo, Italy
2Moredun Research Institute, Pentlands Science Park, Penicuik, Midlothian, UK
3Animal and Plant Health Agency, Bacteriology Department,, Weybridge, Addlestone, Surrey, UK
4BigDNA, Wallace Building, Roslin BioCentre, Roslin, Midlothian, UK
5Institute of Infection Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, UK
#Current address Kingston University, Penrhyn Road, Kingston upon Thames, UK
Corresponding Author : David G E Smith
C/o Moredun Research Institute
Pentlands Science Park, Penicuik
Midlothian, UK, EH26 0PZ
Tel: +44 131 445 6251
Fax: +44 131 445 6111
E-mail: David.G.E.Smith@moredun.ac.uk; David.G.Smith@glasgow.ac.uk
  Attilio Pini
Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”
Via Campo Boario 1 Teramo, Italy, OIE Reference Laboratory for CBPP
Tel: +39 0861332481
Fax: +39 0861332251
E-mail: a.pini@izs.it
Received August 26, 2014; Accepted September 29, 2014; Published October 03, 2014
Citation: Krasteva I, Inglis NF, Sacchini F, Nicholas R, Ayling R, et al. (2014) Proteomic Characterisation of Two Strains of Mycoplasma mycoides subsp. mycoides of Differing Pathogenicity. J Proteomics Bioinform S13:001. doi: 10.4172/jpb.S13-001
Copyright: © 2014 Krasteva I, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Abstract

Mycoplasma mycoides subsp mycoides (Mmm) is a major pathogen of cattle, causing contagious bovine pleuropneumonia (CBPP), a severe–frequently fatal-disease that is widespread in sub-tropical countries. Despite recognition as a pathogen in the 19th century and advances made through genome sequencing and synthetic biology, many of the molecular characteristics of Mmm remain poorly characterized. Using a proteomics approach, this study aimed to define Mmm major protein determinants, including surface membrane proteins and those involved in host colonization and pathogenicity. For this purpose, both whole cells and a Triton X-114 enriched membrane fraction of a highly pathogenic African field strain (N6) and a vaccine strain (KH3J) were analysed and compared using gelbased methodologies i.e. 2-DGE and SDS-PAGE in combination with MALDI-MS/MS and LC-ESI-MS/MS.

A total of 318 proteins, (around 31% of the predicted proteome) were identified, comprising lipoproteins, prolipoproteins and hypothetical proteins with predicted membrane locations and possible roles in pathogenicity. One hundred and forty-five of these proteins, with different predicted sub-cellular locations and functions but also including uncharacterized proteins, have not previously been reported as expressed by Mmm. Many more proteins were detected by LC-ESI-MS/MS (n=315) than by 2-DGE and MALDI-MS (n=52), however 3 proteins were detected only by the latter approach.

Most proteins were observed to be common to both strains although a substantial number of proteins were detected exclusively in only one strain with possible implications for fitness and pathogenicity. To our knowledge, this study represents the most extensive proteomics characterization of Mmm to date and has identified characteristics on which to base future studies of the physiology and pathogenicity of this prokaryote.

Keywords
Contagious bovine pleuropneumonia; Mycoplasma mycoides subsp. Mycoides; 2-DGE; LC-ESI-MS/MS; Protein identification
Abbreviations
Mmm: Mycoplasma mycoides subsp. mycoides; CBPP: Contagious Bovine Pleuropneumonia; OIE: Organisation Internationale de la Sante Animal (World Organisation for Animal Health)
Introduction
Members of the class Mollicutes are recognised as the simplest prokaryotic organisms capable of culture outside their host species. Although capable of growth on laboratory media, these organisms are highly fastidious and dependent on their hosts to supply many essential nutrients as a result of loss of many metabolic pathways during adaptation to their hosts; correspondingly, these organisms have reduced genome–and hence, proteome–content. Major genera of Mollicutes include Acholeplasma, Spiroplasma, Ureaplasma and Mycoplasma, which all show dependence upon their hosts to provide an environment suitable for their survival. Among these diverse groups, Mycoplasma species are common causes of infection of animals where they can colonise mucous membranes of respiratory or urogenital tracts. Typically infection is persistent although some Mycoplasmas are associated with severe, acute diseases often resulting in a high rate of mortality. Mycoplasmas belonging to the “Mycoplasma mycoides cluster” represent a group of pathogens affecting ruminants that share phenotypic and genetic characteristics. Within these is Mycoplasma mycoides subsp. mycoides small-colony type (Mmm) which is the aetiological agent of contagious bovine pleuropneumonia (CBPP), a highly contagious respiratory disease affecting cattle and buffaloes [1].
CBPP is currently absent from Europe and the USA but outbreaks have occurred in southern Italy (1990-1993), Spain (1994) and Portugal (1999). CBPP is now considered the most significant disease of cattle in Africa–particularly sub-Saharan regions where it can cause high morbidity and mortality and great economic losses [2,3]. CBPP is included in the list of animal diseases that must be reported to the World Organization for Animal Health (OIE) and international regulations impose restrictions on cattle trade from infected territories. CBPP is therefore a disease of considerable socioeconomic importance in developing countries.
Despite the significance of CBPP, much is unknown about the molecular determinants of colonisation, pathogenesis and virulence of Mmm. Approaches for the control of CBPP remain dependent upon serodiagnostic tools and vaccines which are considered unreliable. Development of better control tools can be gained through increasing the understanding of genome and proteome content of these pathogens and related investigations of immune responses [4,5]. The sequencing of the Mmm type strain PG1 genome [6] opened the way to post-genomic research and a better understanding of Mmm function and pathogenic mechanisms. The Mmm PG1 genome is characterized by a single circular chromosome of 1,211,703 bp with a very low G + C content (24 mole %) and encodes 1017 putative proteins (NC_005364.2; http://www.ncbi.nlm.nih.gov/bioproject/10616). Recently, a second Mmm genome of the Australian strain Gladysdale has been annotated (Accession No. CP002107; http://www.ncbi.nlm.nih.gov/bioproject/27713). This genome is 1,193,808 bp, encoding for 1068 putative proteins and it is highly similar to Mmm PG1 strain, differing mainly in single nucleotide polymorphisms [7]. The Mmm genome contains a very high density of insertion sequences (13% of the genome size) and this has given rise to some genetic variation between strains and duplication of regions. Of note, the Mmm genomes possess several large repeated regions which confer some inter-strain variability [8] resulting in duplication and attrition of some genes leading to some genomic divergence particularly in regions adjacent to insertion sequences.
A variety of potential fitness, immune evasion and virulence factors have been identified through genomic and functional analyses. Defining the proteome of an organism is pivotal to defining its biological activities and interactions with its host species.
Previous studies applied proteomics approaches to characterize differences in protein content during adherent compared to planktonic growth of Mmm [9]. In a more recent study Krasteva et al. [10] used polyacrylamide gel electrophoresis and mass spectrometry to characterize the Triton X-114 soluble proteome of nine Mmm strains including T1/44 vaccine, PG1 reference and field strains from Europe and Africa: a total of 250 proteins were identified. Further, immunoproteomics approaches looking at seroreactive proteins, identified a total of thirty seven proteins [11]. Mmm recombinant protein arrays have also been used to systematically survey immunogenicity of selected proteins [12]. Even if providing relevant information, none of these studies incorporated a highly detailed survey of Mmm proteomes and hence this work aimed to provide a more extensive characterization of the protein profiles of Mmm strains.
The study focused on two Mmm strains KH3J and N6 originating from African countries (Sudan and Botswana respectively) and isolated five decades apart (1940 and 1996 respectively). KH3J has been used as a vaccine strain and is of low pathogenicity whilst N6 is a field isolate of high pathogenicity [13-17]. Complementary gel-based and liquid chromatography-based approaches were utilized for characterisation of the entire proteomes of two Mmm strains differing in virulence.
Materials and Methods
Strains and growth conditions
The two Mmm African strains N6 and KH3J were obtained from laboratory stock cultures stored at -70°C in Eaton broth and grown in Eaton broth at 37°C in an atmosphere containing 5% CO2 as described previously [9]. Cells from 1,000 ml cultures were harvested by centrifugation at 8,000×g for 40 min at 4°C. Cell pellets were subsequently washed 3 times with a semi-defined medium [18] without serum to remove any remaining medium components-mainly serum proteins. Mycoplasma cells were harvested by centrifugation and the cell pellet was re-suspended in 200 ml of fresh semi-defined medium then incubated at 37°C overnight into log phase. The cells were separated from this medium by centrifugation at 8,000×g for 40 min at 4°C. The supernatant was carefully removed and placed in a sterile bottle; the pellets were washed 3 times with PBS then whole cell pellets and supernatants were stored at -70°C until required.
Two-dimensional gel electrophoresis and Image analysis
Whole cell lysates of N6 and KH3J were prepared by mechanical disruption of Mycoplasma pellets resuspended in PBS using a Ribolyser™ (Hybaid) and 2-DGE was carried out using Ettan IPGphor and Multiphor II systems (GE Healthcare). Samples were subjected to 2-D Clean-Up Kit (GE Healthcare) according to the manufacturer’s instructions. Protein samples were thoroughly solubilised in isoelectric focusing buffer consisting of 8 M urea, 4% v/v CHAPS, 40 mM Tris Base, Protease inhibitor cocktail (Roche), 1% DTT w/v, 1% v/v IPG buffer pH 3-10 non linear (GE Healthcare) and 0.025% w/v bromophenol blue. Insoluble material was removed by centrifugation at 15,000×g for 15 min.
Protein concentration was determined using a Bicinchoninic acid (BCA) [19] assay, reading absorbance at 562 nm using a Dynatech MR 5,000 plate reader. BSA was used to prepare a concentration standard curve.
Proteins (500 μg) were separated by IEF on pH 3-10 non linear IPG strips (18 cm) (GE Healthcare), which were rehydrated for 12 h and focused for 30,000 Vh. Prior to electrophoresis in the second dimension, strips were equilibrated for 15 min in equilibration buffer (50 mM Tris-HCl, pH 8.8, 6 M urea, 30% v/v glycerol, 2% w/v SDS, bromphenol blue w/v 0.002%) containing 1% w/v DTT. This step was repeated using equilibration buffer supplemented with 4% v/v iodoacetamide. Strips were applied to 12-14% pre-cast (245 mm×180 mm×0.5 mm) gels (GE Healthcare). Electrophoresis was performed at 600V/20 mA/40W for 15-20 min then at 40 mA per gel at 12°C until the dye front reached the bottom of the gel. Gels were fixed in 40% v/v methanol, 10% v/v acetic acid for 1 h and then stained overnight in Coomassie Brilliant Blue R (Sigma). Gels were destained in 25% v/v methanol for 1 h. Triplicate biological samples (i.e. different batches of cultures) for each strain were run and analysed using Phoretix 2D software version 2004 (Non-linear Dynamics).
MALDI-MS and MS/MS
For identification of proteins by MALDI-MS/MS spots were excised from the gels and washed three times for 15 min at room temperature in 50 mM ammonium bicarbonate in 50% acetonitrile (ACN) using a vortex mixer. The solution was removed and the gel pieces covered with 100% ACN to dehydrate for 10 min. Supernatant was removed and the gel pieces vacuum-dried for 20 min. The gel spots were then rehydrated in trypsin digest solution (10 ng/μL sequencing grade modified trypsin) in 25 mM ammonium bicarbonate and incubated at 37°C for at least 16 h. Tryptic peptides were applied to a polished stainless steel target plate in a solution of 10 mg/ml α-Cyano-4-hydroxycinnamic acid (CHCA) in 0.1% trifluoroacetate (TFA) and 50% ACN. MS spectra were obtained using an Ultraflex-II TOF/TOF instrument (Bruker Daltonics) operated in the reflectron mode for both MS and MS/MS analyses. Each spectrum was produced by accumulating data from 10×100 consecutive laser shots. Peptides were identified by matching the measured monoisotopic masses to theoretical monoisotopic masses generated using the MASCOT search engine (see below). From protein samples that remained unidentified, selected peptides were fragmented in MS/MS mode. The search parameters were: maximum of one missed cleavage by trypsin, variable modification of: oxidation of methionine, modification of cysteine by propionamidation and carbamidomethylation and a peptide tolerance of ± 50 ppm. Using these parameters and searching the Mmm PG1 database, probability scores greater than 43 were considered significant (p<0.05).
Phase separation of integral membrane proteins in Triton X-114 solution
Triton X-114 detergent phase fractionation of broth-grown Mmm N6 and KH3J was performed [20]. Protein concentrations of Mycoplasma cell pellets resuspended in PBS were determined using a Bicinchoninic acid (BCA) as described above. An aliquot of Mycoplasma suspension containing 1 mg of protein, was collected, centrifuged at 8,000×g for 40 min at 4°C and the cell pellets suspended in 900 μl of cold TS buffer (154 mM NaCl, 10 mM Tris, pH 7.4) containing protease inhibitor cocktail (Roche) and 100 μl of cold 10% (v/v) Triton X-114. The solution was incubated at 37°C for 5 min during which time the solution became cloudy, indicating the condensation of detergent. The samples were then centrifuged for 3 min at 8,000×g at room temperature and the aqueous phase removed and discarded. Cold TS buffer was added to the detergent phase and incubated for 3 to 5 min on ice and then incubated at 37°C for 5 min to condense detergent. Samples were centrifuged at 8,000×g at room temperature for 3 min and the upper aqueous phase was discarded and detergent phase retained and washes were repeated 3 times. Following the final wash the upper aqueous phase was removed and 900 μl cold methanol was added to the condensed detergent phase. Samples were placed at -70°C overnight, then centrifuged at 12,000×g for 10 min in the cold to precipitate proteins; the methanol phase was removed completely and protein pellets dried prior to storing at -70°C.
SDS-PAGE and LC-MS/MS
These procedures were carried out on both Mycoplasma whole cells and on Triton-X 114 membrane enriched fraction as described previously in similar surveys of bacterial proteomes [21-25] summarized as follows:
Sample preparation: Approximately 15 μg of each Mmm sample material (whole cells and Triton-X 114 membrane enriched fraction) were loaded into single sample wells of a discontinuous Tris/glycine SDS-PAGE mini-gel (4% stacking gel; 12% resolving gel) and separated at 130V (constant voltage) over ~ 90 min using a Mini-ProteanTM II Dual Slab Cell (BioRad). Resolved proteins were visualized using SimplyBlue Safe StainTM (Invitrogen). The stained gel lane was excised and then sliced horizontally from top to bottom to yield a series of ~25 equal gel slices of 2.5 mm deep. Each of the resulting 25 gel slices was then subjected to standard in-gel destaining, reduction, alkylation and trypsin digestion procedures [26].
LC-ESI-MS/MS: Liquid chromatography was performed using an Ultimate 3000 nano-HPLC system (Dionex) comprising a WPS- 3000 well-plate micro auto sampler, a FLM-3000 flow manager and column compartment, a UVD-3000 UV detector, an LPG-3600 dualgradient micropump and an SRD-3600 solvent rack controlled by Chromeleon chromatography software (Dionex). A micro-pump flow rate of 246 μl/min-1 was used in combination with a cap-flow splitter cartridge, affording a 1/82 flow split and a final flow rate of 3 μl/min-1 through a 5 cm×200 m ID monolithic reversed phase column (Dionex) maintained at 50°C. Samples of 4 μl were applied to the column by direct injection. Peptides were eluted by the application of a 15 min linear gradient from 8-45% solvent B (80% v/v acetonitrile, 0.1% (v/v) formic acid) and directed through a 3 nl UV detector flow cell. LC was interfaced directly with a 3-D high capacity ion trap mass spectrometer (Esquire HCTplus™, Bruker Daltonics) via a low-volume (50 μl/min-1 maximum) stainless steel nebuliser (Agilent, cat. no.G1946-20260) and ESI. Parameters for tandem MS analysis were set as previously described [21-25].
Database mining: Deconvoluted MS/MS data was submitted to an in-house MASCOT server and searched against a fully annotated Mmm PG1 genomic database (http://www.ncbi.nlm.nih.gov) using the MASCOT search algorithm http://www.matrixscience.com/. The high extent of genomic similarities among Mmm strains makes the PG1 genome a relevant resource for assigning protein identities. The presentation and interpretation of MS/MS data was performed in accordance with published guidelines [27]. To this end, fixed and variable modifications selected were carbamidomethyl (C) and oxidation (M) respectively and mass tolerance values for MS and MS/MS were set at 1.5 Da and 0.5 Da respectively. Molecular weight search (MOWSE) scores attained for individual protein identifications were inspected manually and considered significant only if: a) two peptides were matched for each protein, and b) each peptide contained an unbroken “b” or “y” ion series of a minimum of four amino acid residues.
Results and Discussion
Contagious bovine pleuropneumonia (CBPP) is recognized as one of the major constraints on cattle welfare and productivity in many sub-tropical countries, especially in Africa. Despite reportedly being first isolated as the etiological agent of CBPP as early as 1896 [28], much about the physiology and pathogenicity of Mmm remains poorly defined. There is, therefore, continuing need for improved phenotypical characterization and understanding of these organisms in order to advance novel control measures. In the present investigation we have taken dual approaches of 2-DGE combined with MALDI-MS/ MS and SDS-PAGE combined with LC-ESI-MS/MS to characterize the entire proteomes of two Mmm strains, this representing the most extensive proteome survey to date of these organisms.
For this purpose we have selected one strain of low pathogenicity (KH3J) and another of high pathogenicity (N6), both originally isolated in African countries. Strain KH3J was originally isolated in Sudan in 1940 and has been used as a vaccine strain on account of its low pathogenicity whereas strain N6 is a more recent field isolate (1996) from Botswana and retains high pathogenicity [17]. Although Mmm strains show some genomic differences-especially between strains of African, Australian and European origins [29,30]-strains from diverse origins remain antigenically similar [31]. Of note, genotypic characteristics of these strains are similar to that of the reference strain PG1 [32] which provides the original genome sequence [6]; furthermore, the recent formal release of a second Mmm genome for strain Gladysdale [7] defines only SNPs as genomic differences between strains. This was further confirmed by recent phylogenetic analyses conducted using next-generation sequencing technologies that compared Mmm strains of different origins [30]. Hence the selected strains are broadly representative of those available in the Mmm research community.
Compilation of Mmm N6 and KH3J proteins identified
In total, 318 Mmm proteins were identified in the two strains through the combined methodologies employed in this study (Supplementary Table 1). One hundred and forty-five out of the 318 proteins identified, with different predicted sub-cellular location and function, have not ever before been reported as being expressed by Mmm. Since the Mmm genome contains several large duplicated regions, several proteins are represented by more than one open reading frame encoding identical or indistinguishable proteins– accordingly the numbers of open reading frames from which proteins were detected ranges from 310 to 318. These proteins represent approximately 31% of the deduced proteome of this organism and this survey greatly extends the proteome coverage compared to earlier investigations. Multiple functional classes were represented among the proteins detected and, as is common among global proteome analyses of prokaryotes, proteins of the class “translation, ribosomal structure and biogenesis” were most common (representing approx 21% of proteins detected), followed by “not classified” (approx 14%). A further 4 classes of proteins involved in “transport” and “metabolism” (COG categories G and F, 8% and 7% respectively), “energy production and conversion” (C, 7%), “replication, recombination and repair” (L, 7%) were next most frequently represented as were proteins of “general function prediction only” (approx. 9%). Using PSORTb v2.0 bacterial localization prediction tools to predict sub-cellular location, the majority of detected proteins were predicted to be cytoplasmic (n=114; 36%) or membrane–associated proteins (n=52; 16%). No location was predicted for the remaining proteins (n=152). As anticipated, many more proteins were detected by LC-ESI-MS/MS (n=315) than by 2-DGE (n=52), nonetheless 3 proteins were detected only by the latter and not the former methodology thus re-emphasizing the benefits of complementary methods in characterizing microbial proteomes.
2-DGE electrophoresis and MALDI-MS/MS analyses of Mycoplasma whole cells: Representative 2-DGE gels for N6 and KH3J whole cell lysates are shown in Figure 1. A comparison of 2-DGE gel spot volumes using the Kruskal-Wallis Test showed that all p-values were greater than p-calculated (Supplementary table 2), indicating that there is no statistical difference between the spots of the six 2-DGE gels (corresponding to three gels from each of two strains); i.e. spots were consistently observed between gels and between strains.
A total of 113 protein spots were detected on the reference gel by 2D Phoretix software and 81 of these spots were identified by MALDI-MS/MS corresponding to 52 different proteins (Table 1) which were mostly soluble cytoplasmic proteins. In 2-DGE, some proteins (namely putative phosphonate ABC transporter (MSC_0079), NADH oxidase (MSC_0263), pyruvate dehydrogenase lipoamide alpha chain (MSC_0265), pyruvate dehydrogenase (lipoamide) beta chain (MSC_0266), PTS system glucose-specific IIA component (MSC_0274), L-lactate dehydrogenase (MSC_0527), hypothetical protein MSC_0587, glyceraldehyde 3-phosphate dehydrogenase (MSC_0679), glycerone kinase (MSC_0747), triosephosphate isomerase (MSC_0823), thymidine phosphorylase (MSC_0830), purine nucleoside phophorylase (MSC_0835), uracil phosphoribosyltransferase (MSC_0893), ribose-phosphate pyrophosphokinase (MSC_0952) and glucose-inhibited division protein A (MSC_1017)) presented isoforms with charge differences, while others, (namely 30S ribosomal protein S6 (MSC_0027), AhpC/ TSA family protein (MSC_0053) and translation elongation factor Tu (MSC_0160)) presented charge and mass variations (Figure 1 and Table 1). In most cases, two or three forms were identified for a given protein, but, in the cases of 30S ribosomal protein S6 (spots 235, 236, 239 and 364), uracil phosphoribosyltransferase (spots 80, 225, 227 and 265), ribose-phosphate pyrophosphokinase (spots 206, 209, 213 and 214), translation elongation factor Tu (spots 159, 163, 165, 365 and 366) and thymidine phosphorylase (spots 28, 144, 148 and 150), a greater number of corresponding spots were found. Migration patterns of isoforms suggest that post-translational modifications could account for observed changes–among these, acylation is widely described in mycoplasmas including Mmm [33], protein phosphorylation and acetylation have been noted as common post translational modifications (PTMs) for M. pneumoniae [34,35] and can be presumed to be PTMs in Mmm. Furthermore, carbamylation of proteins, producing altered isoelectric points (“carbamylation trains”) should also be considered [36]. Three proteins, not previously described, were identified through 2-DGE only: MSC_0051 (hypothetical protein) is an uncharacterized conserved protein of unknown function; MSC_0335 (ribosome binding factor) is a cytoplasmic protein essential for efficient processing of 16S rRNA; MSC_0894 (glycine hydroxymethyl transferase) is also a cytoplasmic protein playing an important role in the biosynthesis of purines, thymidylate, methionine, and other important biomolecules.
SDS-PAGE and LC-ESI-MS/MS analyses of Mycoplasma whole cells and TritonX-114 enriched membrane protein fraction: Whilst the 2-DGE approach is very useful for profiling and comparison of protein content, several classes of proteins - including proteins with extremes of pI and molecular weight, low abundance proteins and integral membrane proteins - tend to be under-represented in 2-DGE analysis [37,38]. The proteins identified in the two Mmm strains reflect this anticipated under-representation hence a complementary “shotgun” approach was taken to identify proteins in whole cell and membrane-associated compartments. Representative SDS-PAGE gels for N6 and KH3J whole cells and Triton X-114 membrane fraction are shown in Supplementary Figure 1. The application of SDS-PAGE combined with LC-ESI-MS/MS greatly extended the proteome coverage and 315 of the proteins detected were identified by this methodology, including many membrane-associated proteins which tend to be under-represented in 2-DGE. Two hundred and eighty proteins were identified in whole cell lysates using SDS-PAGE and LCESI- MS/MS analysis, 49 of which were also identified by 2-DGE and MALDI-MS/MS analysis.
The membrane is the primary point of contact between Mycoplasma and their host cells. Mmm membrane proteins are recognized by host immune responses and variation in membrane protein content offers a means for evading host immunity [39,40] therefore it is important to determine in detail the protein composition of this bacterial compartment. Membrane-associated proteins were enriched by Triton X-114 fractionation and subsequent SDS-PAGE – LC-ESI-MS/MS analysis identified 81 proteins, 46 of which were also identified in whole cell lysates using the same approach. Among these 46 proteins, common to whole cell lysates and TritonX-114 enriched fraction, 36 were identified only by SDS-PAGE – LC-ESI-MS/MS while 10 proteins were also detected using 2-DGE – MALDI-MS/MS (Supplementary Table 1). Of the 81 proteins identified, twenty were annotated as lipoproteins or prolipoproteins, a number almost equal to that indicated by a recent study [41] in an LC-MS/MS approach however in that study no protein identities were specified. Our results reflect recent findings in Triton X-114 enriched membrane protein fractions of 9 Mmm strains where up to 23 lipoproteins were identified [10], including all of the lipoproteins found in the present study. In the current survey, a further 22 proteins within the membrane-enriched fraction were annotated as “hypothetical”, 10 of which (MSC_0065, MSC_0107, MSC_0374, MSC_0389, MSC_0457, MSC_0606, MSC_0671, MSC_0708, MSC_0757, MSC_0916; have putative membrane locations as predicted by PsortB (Table 2). These proteins may have roles in pathogenicity and represent important targets for future investigations.
As indicated above, a feature of mycoplasmas, including Mmm, is the variable expression of surface proteins. These proteins have been described as important for colonization and adaptation to the host environment at different phases of infection. Phase variation, antigenic variation or epitope masking have been previously described as possible mechanisms used by mycoplasmas to evade the host immune response [42-44].
Among the recognized variable surface proteins of Mmm, the proteomics approaches used in this study identified LppA (MSC_0013), LppB (MSC_0519) and LppQ (MSC_1021). It should be noted that while several proteins are annotated as LppA, these actually differ in sequence; lppB is present in a single copy; and lppQ is present in duplicate in the genome – MSC_1021 and MSC_1046 (both 445 amino acids). Because lipoproteins are known to stimulate the release of proinflamatory cytokines and are strongly immunodominant, they are considered to be prime target antigens for the development of specific and sensitive serological diagnostic tests. LppA is a highly conserved antigen of Mmm capable of stimulating both humoral and cell mediated response and as such is a potential candidate for a future CBPP vaccine [45-47]. LppB is expressed in African and Australian but not in European clusters [10,29]. This protein is able to elicit a strong humoral response while its involvement in disease pathogenesis remains to be elucidated. LppQ is a lipoprotein specific to M. mycoides subsp. mycoides. A combination of specificity and powerful antigenicity saw LppQ exploited in the development of an indirect ELISA [48].
Conversely, immunization of animals with LppQ resulted in an increased susceptibility to CBPP lesion development, suggesting a role for this protein in immunopathology [49].
Noteworthy is the absence of many of the other reported variably expressed membrane proteins including LppC (MSC_0122/ MSC_0177), MSC_0117, MSC_0364, MSC_0390, MSC_0809, MSC_0810, MSC_0812, MSC_0813, MSC_0815, MSC_0816, MSC_0817, MSC_0818, MSC_0847, MSC_1005, MSC_1033, MSC_1058. Similarly, results are described in the recent investigation of 9 Mmm strains by LC-MS/MS, where these proteins were also absent with the exception of MSC_0364 and MSC_1005 which were identified only in 3 strains, while MSC_0847 was found only in T144 strain [10].
Assuming that the absence of these proteins is not attributable to the limitations of the technique used, these results may reflect the lack of immune or other selective pressures on Mmm during growth in vitro. A comparative study of the Mmm proteome following multiple passages in serial culture would be of value for the purposes of demonstrating qualitative shifts in mycoplasma protein expression during in vitro propagation.
Another characteristic feature of the Mmm genome is the presence of insertion sequences (ISs) and duplications. Prominent in the genome are three large repetitive regions flanked by ISs [8] and proteins from each of these large repeat regions were detected: PhnD–MSC_0790/ MSC_0804; PtsG–MSC-0860/MSC_0873; GalE–MSC_0971/ MSC_0978; GlfG–MSC_0977/MSC_0984; AsnA–MSC_1015/ MSC_1040; GidA–MSC_1017/1042; LppQ–MSC_1021/MSC_1046. As a result of the very high sequence conservation in the proteins encoded by these duplicated regions, it is not possible to selectively define from which of the repeated genomic regions the detected proteins are derived. Despite detecting peptides representing these aforementioned repeat region proteins, not all proteins in these regions were detected (Glk (MSC_0863 & MSC_0875), Arc (MSC_0700, MSC_0864 & MSC_0877), MgtA (MSC_0868, MSC_0881, MSC_0907), PncA (MSC_1020 & MAC_1045), OppF (MSC_0183, MSC_0968, MSC_0975, MSC_0983) and PhnB (MSC_0788) and it is therefore unclear whether this represents differential expression of these proteins or a reflection of abundance of these proteins in the proteome milieu.
Among the Mmm proteins detected was PtsG (MSC_0860/ MSC_0873), encoding a glucose phosphotransferase system permease. A previous study [50] did not detect this protein in strain KH3J using a specific antibody and also could not select KH3J variants that did express PtsG at appreciable levels. Therefore the present study is the first to report that this low pathogenicity strain can express this protein. Whether this represents heterogeneity in this strain through storage and passage in different laboratories, or is another example of a protein variably expressed under adaptive selection remains to be defined. Both Mmm strains N6 and KH3J originate from Africa and therefore possess an intact glycerol uptake system encoded by GtsABC (MSC_0516- MSC_0518). However, this component was not detected in this study even though glycerol was a component of the growth medium used in preparing Mmm cultures.
A recent comparative analysis of the genomes of Mmm and related Mycoplasma species within the “mycoides cluster” [41] highlighted the presence of frameshifts, substitutions or insertion sequences resulting in pseudogenes in Mmm. Despite these alterations, peptides representing five of the pseudogene products were detected in that study and in the present investigation peptides of MSC_0103 (47% sequence coverage) and MSC_0710-0711 (40% and 22% respectively) have been detected indicating that these, too, are expressed by both strains N6 and KH3J, at least during culture of Mmm in vitro. In addition, proteins representing additional pseudogene products which were not detected in the study of Thiaucourt et al. [41] were detected in the current study: MSC_0235 (leucyl aminopeptidase; 19% sequence coverage; detected in strain KH3J) and MSC_0242 (Hypothetical NTPase; 55% sequence coverage; detected in strain N6). This raises several possibilities including (i) possible genome sequencing errors leading to artefactual nucleotide substitutions or frameshifts or (ii) potential variation in expression of these genes among strains. Such polymorphisms could lead to functionally relevant inter-strain differences in, for instance, surface lipoprotein (MSC_0103; MSC_0710-0711) expression with consequent effects on pathogenicity and/or immunogenicity. Short sequence polymorphisms are not mutually exclusive with altered protein expression since even single nucleotide alterations can affect protein expression via both mis-sense and non-sense - which of these possibilities operates in Mmm requires further systematic investigation of protein expression under selective pressure.
Comparison of the proteomes of Mmm N6 and KH3J strains
As indicated above, the total number of proteins identified in Mmm strains N6 and KH3J was 318 (including proteins duplicated in the genome). For any given protein, a frequency of occurrence of at least 2 out of 3 biological replicates in any one strain was set as a strict criterion for inclusion. Applying this rule, a total of 239 proteins was shared by both strains of which 53 were detected only in strain N6 and a further 26 only in strain KH3J. However, it should be born in mind that both the Mycoplasma culture and the LC-ESI-MS/MS methods used are subject to variation between replicates. Accordingly, these numbers may over-represent proteins that are specific to, or preferentially expressed, in any particular strain. Thus to reduce the likelihood of falsely assigning “strain-specificity” per se to any given protein, the inclusion criteria for a strain-specific proteins was adjusted such that any protein occurring at a frequency of only 1 out of 3 biological replicates in either strain was excluded. This increased stringency reduced the number of putative strain-specific proteins to 25 and 15 for strains N6 and KH3J respectively.
Proteins unique to each strain represent multiple functional classes. Different metabolic enzymes were particular to each strain although within an individual strain no clear common metabolic role was apparent. It is noteworthy that 12 and 5 hypothetical proteins were differentially detected in strains N6 and KH3J respectively and 3 lipoproteins were detected in N6 only (Table 3). For instance, LppA (MSC_0013), and prolipoproteins MSC_0635 and MSC_0653 were detected only in strain N6-(highly pathogenic) as was an additional hypothetical predicted membrane protein (MSC_0065). Of these (pro)lipoproteins, only LppA has been investigated to any extent and reported as an immunogen as mentioned above [46,47]. One could assume that these differences may correlate with differences in pathogenicity between the two strains, although no role in virulence has been defined for LppA and no information is available on the other three proteins. Furthermore these proteins were recently found not only in African and European pathogenic field strains, but also in the T1/44 vaccine and the PG1 reference strains [10]. Thus, despite the differences observed between the two strains in the current study, it is not possible to outline any correlation between the presence/absence of these proteins and differences in pathogenicity of N6 and KH3J. However, given the significant role of lipoproteins in Mmm virulence, the differential detection of proteins of this class could be a relevant feature with important roles in Mycoplasma-host interactions.
Transport proteins were also among those differentially detected in the two Mmm strains. Spermidine/putrescine transport ATP-binding component MSC_0224 was detected only in strain N6 although the permease and substrate-binding components of this system (respectively MSC_0225 and MSC_0226) were detected in both N6 and KH3J strains. Conversely, several transport-associated proteins were detected only in strain KH3J: MSC_0008 (galactose/ribose transporter permease component), MSC_0405 (predicted to be a substrate-specific energy-coupling factor (ECF) transporter component-IPR024529), MSC_0434 (ABC transporter ATP-binding permease protein), MSC_0558 (sodium-solute symporter) and MSC_0598 (FtsX-like lipid transport permease protein). Although not defined herein, it is possible that the attenuation of KH3J involves a higher requirement for several substrates hence the over-abundance of transport-associated proteins relative to the more pathogenic N6. The extent to which this is the case will require further systematic evaluation including assessment of additional strains of differing virulence.
Glycerol metabolism has been reported as an important virulenceassociated metabolic activity of Mmm [51] and several enzymes involved in this pathway were detected in only KH3J: notably, MSC_0259 (GlpO) – which metabolises glycerol to release H2O2 and mediate cytoxicity [52-54] in addition, MSC_0577 (PlsC - 1-acyl-snglycerol- 3-phosphate acyltransferase) and MSC_0259 (GlpF - glycerol- 3-phosphate oxidase) were also detected in KH3J but not N6. Since the glycerol uptake system GtsABC (MSC_0516-0518) was detected in neither KH3J nor N6, the presence of these glycerol metabolising proteins in KH3J may represent a futile function. This, together with the presence of multiple substrate uptake mechanisms, could be interpreted as a metabolic dysregulation in strain KH3J which could contribute to its attenuation.
Transcriptional regulators were also differentially detected in the 2 strains examined and, given their role in controlling multiple functions through modification of gene expression, this has potential implications for pathogenicity. Specifically, the regulatory proteins RpiR (MSC_0018) and HrcA (MSC_0612), both of which are negative regulators of gene transcription, were observed in Mmm strain N6 only. A function for RpiR has not been described in Mycoplasma spp. although roles have been demonstrated in regulation of metabolic and virulence activities in diverse bacterial genera [55-57]. HrcA has been shown to contribute to “heat shock” responses in Mycoplasma hyopneumoniae [58] and M. genitalium [59] and has been correlated with resistance to antimicrobial peptides in M. pulmonis [60]. Despite detection of HrcA in only N6, several proteins up-regulated during heat shock and involving HrcA-dependent regulation (i.e. DnaJ (MSC_0609); DnaK (MSC_0610); ClpB (MSC_0613) and Lon (MSC_0454) were detected in both strains of Mmm investigated in this study.
Other proteins detected differentially in the two strains were GidA (MSC_1017) in strain N6 - the sole differential protein encoded on the large repetitive elements and GnsAB (MSC_0032), a deduced membrane protein involved in phospholipid synthesis (PFAM08178, IPR012563) in only strain KH3J. This re-emphasises that a range of protein functional classes may be identified through comparison of prokaryote strains of differing phenotype (such as virulence) and any functional relevance to these differences can only be presumed until proved. Nevertheless, the differences observed in proteomes between strains support the multifactorial nature of Mycoplasmahost interactions and is broadly in keeping with the covert nature of pathogenesis of these prokaryotes.
Concluding Remarks
Herein, the first extensive proteome analysis of Mmm strains has been presented. Two strains of differing pathogenicity have been examined: strains N6 and KH3J of high and low pathogenicity respectively. Our goal was not to provide a comprehensive, indepth comparative analysis of Mmm proteomes but was, rather, to initiate an extensive survey to identify proteins as a basis for further targeted analyses of Mmm physiology, pathogenicity and immunity with this intractable pathogen. Using two simple complementary methodologies, more than 300 proteins, representing approximately 31% of the proteome, were detected, extending current knowledge of Mmm protein expression.
Proteins representing multiple functional categories were identified comprising many metabolic, immunogenic and virulenceassociated factors including several of the membrane lipoproteins which have key roles in Mycoplasma-host interactions. Also present were 59 hypothetical proteins including proteins represented by pseudogenes. Overall, therefore, this proteome analysis re-emphasises the complexity of Mycoplasma expression and adaptation even in the absence of host-imposed selective pressures, and supports the need for further systematic investigations of Mmm expression and function including inter-strain comparisons.
This study also initiated direct comparison between two strains of differing pathogenicity. Although differences in protein content were observed, the biological significance of these observations requires further study. Even though not a definitive appraisal of proteins contributing to virulence/attenuation, the identification of differences in proteome content between these two strains highlights a substantial number of Mmm proteins, including regulators of gene expression and a number of possible proteins that are subject to variable expression. Further investigation of inter-strain heterogeneity and of selected proteins will be necessary to define which or whether any of these correspond to fitness and pathogenicity. Our own studies are now extending the range of Mmm strains to incorporate field strains in order to define conserved proteins and further advance understanding of Mycoplasma mycoides subsp. mycoides interaction with the bovine host.
Acknowledgements
This work was supported by Italian Ministry for Health, (project grant IZS AM 06/08 RC). Moredun Research Institute is supported by program funding from RESAS (Rural and Environment Science and Analytical Services Division) of the Scottish Government.
The authors are aware of no financial, commercial or other conflicts of interest in regard to this work.
 
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