Gathering proteomic information on prokaryotic membranes is a challenging task, due to difficulties in cell fractionation and to the intrinsic chemical properties of membrane proteins in general. Therefore, both systematic and differential proteomic information on prokaryotic membranes is generally lacking. In this work, we approached the systematic characterization of what is believed to be one of the simplest bacterial pathogen membranes, in an attempt to move a step forward in our understanding of its composition, complexity, and function. In addition to its lower complexity, investigating membrane composition and plasticity in mycoplasmas is of particular interest since surface proteins are subjected to size and phase variation, and information on the extent and level of such variation is crucial in studies targeting identification of common immunogens, evaluation of immunological escape mechanisms, and adaptation of the bacterium to its host. All six variable surface lipoproteins encoded in the PG2T genome  were detected by 2-D PAGE, although one of these (VpmaY) was not expressed in a field isolate examined by 2D DIGE. Triplicate experiments showed that the two-dimensional expression pattern of each field isolate is relatively stable under laboratory conditions, and that there is a reproducible differential expression of several protein spots in the field isolates compared to the type strain PG2. Interestingly, these differences are being detected in bacteria which were grown in culture media, where all protein variants should theoretically be expressed . It was already demonstrated that the switching mechanism is so fast that it can be pointed out in a single colony on solid culture . This might suggest that the lack of VpmaY in the isolate Nurri could result from a local genetic mutation. A large-scale study performed on a higher number of field isolates might enable the detection of constantly expressed proteins, which might be useful as targets for the development of vaccines and diagnostic tools for CA.
Mycoplasmas have evolved a parasitic lifestyle, and membrane transporters are consequently very important for uptake of nutrients and growth factors. The genome of M. agalactiae PG2T encodes 18 ABC transporters, but proteins from only 10 of these were identified in this study. We also failed to identify all the components of a complete membrane transporter complex; however, it is possible that expression of all sequences encoded by the transporter gene operon may not necessarily take place at the same time. ABC transporters components encoded by different operons may likely interact to form functional transporters, producing the further advantage of creating many different combinations that can help evasion of host defense mechanisms. For instance, the genome of M. agalactiae PG2T encodes for two oligopeptide (Opp) ABC transporters, one typical of the hominis group and one probably transferred by means of horizontal gene transfer mechanisms from M. mycoides subsp. mycoides and M. capricolum subsp. capricolum. We identified the substrate binding protein (OppA) from one operon, and the permease (OppC) and the ATP-binding protein (OppF) from another operon; notably, these proteins create a functional transporter. Moreover, OppA could be more than a simple substrate binding protein, since it was demonstrated to play an important role in pathogenicity in M. hominis by inducing ATP release and cell death of HeLa cells in vitro and by mediating adhesion to host cells [38–40]. Other authors reported a different pattern of expression of these operons: in the study by Nouvel and co-workers , only OppA, OppF, and OppD were detected. These apparently controversial results could be due to technical issues, or be dependent on variations in expression of Opps within the PG2T strain. This will need to be elucidated in future studies.
Upon analysis of all MS data, the proteins putatively assigned by the GO software as cytoplasmic accounted to 36%. Among these, many hydrolases were present. However, lipases, peptidases, and nucleases might be associated to the membrane compartment and assist in reducing macromolecules to simple 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 was reported that hydrolytic enzymes are surface-located in mycoplasmas, and that they can be associated with ABC transporters in order to digest macromolecules before uptake of simpler components, or play major roles in pathogenicity . Interestingly, in the M. agalactiae genome, the genes coding for many of these hydrolases are also located close to ABC transporter operons.
Several other proteins have a predicted cytoplasmic localization, but could be membrane-associated in mycoplasmas, such as the elongation factor tu (EF-Tu) and the E1 beta subunit of the pyruvate dehydrogenase complex. Traditionally, these are considered to be cytoplasmic proteins involved in protein synthesis and energy production, respectively, but it was demonstrated that in M. pneumoniae they are surface exposed and interact with host fibronectin, mediating adhesion [42, 43]. It was also demonstrated that many "cytoplasmic" proteins such as the EF-Tu are strong antigens in many mycoplasma species [22, 44, 45].
Ribosomal proteins represent a significant proportion of the mycoplasma liposoluble proteome. This might appear inconsistent, but in spite of their traditionally cytoplasmic localization, it was already demonstrated that ribosomes interact with the bacterial protein export complex . Moreover, it is well known that in eukaryotes ribosomes are associated with endoplasmic reticulum, where they participate in the protein secretion pathway . Several proteins that take part in other metabolic pathways were also identified in the liposoluble fraction of M. agalactiae PG2T. We could speculate that many proteins involved in nutrient metabolism might associate with proteins devoted to internalization of precursors in metabolizing complexes, and be co-purified with these. Nonetheless, a pre-fractionation of membranes was not performed because of inherent technical difficulties, and we cannot rule out that enzymes with high hydrophobicity might be present as cytoplasmic contaminants.
The recent work by Sirand-Pugnet and coworkers revealed the occurrence of horizontal gene transfer (HGT) events in M. agalactiae. The expression of proteins acquired by HGT highlights the importance of horizontal gene flow for the evolutionary plasticity of mycoplasmas; for instance, by allowing changes in host and/or tissue tropism through acquisition of traits enabling colonization and survival in new niches [24, 48]. In total, an impressing 11.7% of proteins expressed on the M. agalactiae membrane are coming from other bacteria, reinforcing the view that an important part in the evolution of mycoplasmas might be driven by genetic exchange with bacteria sharing the same host districts, probably in order to compensate the concurrent process of gene loss . Another interesting observation was the detection of MAG_2340, a hypothetical lipoprotein which is apparently the result of an horizontal gene transfer event with mycoplasmas of the mycoides cluster (Additional file 8), which was not detected by Nouvel et al. in the PG2T liposoluble proteome .
Hypothetical proteins were of particular interest; since these did not have an assigned function, similarity searches were conducted with BLAST tools in order to infer their possible role in the biology of mycoplasmas. Among these, the hypothetical lipoprotein MAG_1670 belongs to the mycoides cluster LppA/P72 family, and it is an antigen recognized early and persistently in infection . The hypothetical protein MAG_0250 has an indigoidine synthase A (IdgA)-like domain similar to Clostridium spp. IdgA is involved in the biosynthesis of indigoidine, a blue pigment synthesized by Erwinia chrysanthemi and implicated in pathogenicity and protection from oxidative stress by scavenging oxygen radicals . Indigoidine production increases tolerance to oxidative stress and contributes to aggressiveness, and might therefore act as a virulence factor.