Research in Veterinary Science 95 (2013) 321–325

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Research in Veterinary Science

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Membrane proteins of Mycoplasma bovis and their role in pathogenesis

0034-5288/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.rvsc.2013.05.016

⇑ Corresponding author. Tel.: +61 406454923; fax: +61 383447374.E-mail address: adamuyaz@yahoo.com (J.Y. Adamu).

James Y. Adamu ⇑, Nadeeka K. Wawegama, Glenn F. Browning, Philip F. MarkhamAsia-Pacific Centre for Animal Health, Faculty of Veterinary Science, The University of Melbourne, Parkville, Victoria 3010, Australia

a r t i c l e i n f o

Article history:Received 26 October 2012Accepted 28 May 2013

Keywords:Mycoplasma bovisMembrane proteinsProtein expression

a b s t r a c t

Mycoplasma membrane proteins influence cell shape, cell division, motility and adhesion to host cells,and are thought to be integrally involved in the pathogenesis of mycoplasmoses. Many of the membraneproteins predicted from mycoplasma genome sequences remain hypothetical, as their presence in cellu-lar protein preparations is yet to be established experimentally. Recent genome sequences of severalstrains of Mycoplasma bovis have provided further insight into the potential role of the membrane pro-teins of this pathogen in colonisation and infection. This review highlights recent advances in knowledgeabout the influence of M. bovis membrane proteins on the pathogenesis of infection with this species andidentifies future research directions for enhancing our understanding of the role of these proteins.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

The name Mycoplasma was first used by Frank in 1889 to de-scribe structures within the root nodules of legumes because oftheir morphological similarities to fungi (mykes-fungus; plasma-formed), but the term was later adopted by Nowak to describethe agent causing contagious bovine pleuropneumonia becauseof its morphology in culture (Krass and Gardner, 1973). In 1898Nocard and Roux reported the first successful cultivation of amycoplasma, Mycoplasma mycoides subspecies mycoides, and in1956 Edward and Freundt recommended the taxonomic nameMycoplasma because it drew attention to the plasticity of the pleu-ropneumonia-like organisms. There are now over 100 recognisedMycoplasma species, distributed over several taxonomic groupswithin the class Mollicutes (Brown et al., 2007), including at least20 species that can infect cattle. Mycoplasma bovis, one of the moresignificant species infecting cattle, was first isolated in 1961 (andinitially named M. agalactiae subspecies bovis) from a cow with se-vere mastitis in the United States.

Their relative structural simplicity and lack of a cell wall havebeen major factors favouring the use of mycoplasmas as modelsin studies for membrane structure and function. However thisapparent simplicity belies the complexity of the relationship be-tween pathogenic mycoplasmas and their hosts, one of the surpris-ing features of mycoplasmoses given the apparently unprotectednature of their cell surface. One of the factors that appears to beparticularly important in the ability of mycoplasmas to establishchronic infections is their genomic flexibility, which allows themto generate highly variable surface antigens (Bailao et al., 2007;

Buchenau et al., 2010). Lysnyansky et al. (1996, 1999) have shownthat the variable surface proteins of M. bovis undergo spontaneousphase variation at a very high frequency. The switch between theON and OFF expression states results from site specific DNA rear-rangements in the M. bovis chromosome, with a highly homolo-gous phase variation mechanism also found in the closely relatedpathogen of small ruminants, M. agalactiae (Citti et al., 2010). Shenet al. (2000) and Rocha and Blanchard (2002) have shown similargene rearrangements are responsible for phase variation in the var-iable surface antigens of M. pulmonis.

M. bovis is an important and emerging cause of a variety of dis-ease syndromes in feedlot cattle, and dairy and veal calvesthroughout the world. Infections can cause pneumonia, arthritis,mastitis, keratoconjunctivitis, otitis media and infertility (Caswelland Archambault, 2007; Buchenau et al., 2010; Wise et al., 2011),all of which are regarded as M. bovis-associated diseases (Maunselland Donovan, 2009). The pathogenicity of M. bovis is highly depen-dent on its cytoplasmic membrane, and in particular on the pro-teins within or tethered to the outer surface of it, whichcontribute to division and the shape of the organism, its motilityand its adhesion to host cells. However, our knowledge of manyof these elements remains limited. With the completion of the gen-ome sequence of the M. bovis type strain PG45, as well as the Hu-bei-1 and HB0801 strains, our understanding of the genetic andpathogenic mechanisms might be expected to increase and thisknowledge is likely to assist in improving diagnosis and in thedevelopment of better vaccine candidates.

2. Membrane proteins of mycoplasmas

Mycoplasmas are generally thought to be more dependent ontheir hosts than most other bacteria because they lack a cell wall.

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They have a morphologically simple cellular structure, comprisinga nucleoid, ribosomes and a cytoplasmic membrane about 10 nmthick. Their cytoplasmic membrane is composed of lipids, includ-ing, uniquely, cholesterol, but has a high total protein content –up to 50% by mass (Demina et al., 2009). These membrane andmembrane-associated proteins are believed to play an importantrole in providing nutrition to the cell for its growth and function.They include cytadhesins, which in some species also have a rolein motility (Browning et al., 2011) and clearly contribute to viru-lence (Indikova et al., 2013; Tseng et al., 2013). Scanning electronmicroscopy of mutant strains has revealed that the developmentof the attachment organelle in Mycoplasma genitalium absolutelyrequires expression of the P140 and P110 cytadhesins (Burgoset al., 2006). Among the other proteins associated with the cyto-plasmic membrane are many lipoproteins, most of which are be-lieved to be exposed to the extracellular milieu, their acyl groupsanchoring them in the cytoplasmic membrane. Several of theselipoproteins have been shown to be virulence factors or to be tar-gets of growth inhibitory antibody (Washburn et al., 2003; Schmidtet al., 2004). Genes that encode many of the lipoproteins appear tobe within the operons encoding the ATP-binding cassette (ABC)transporters and their products are thought to be associated withtransport of nutrients into the cell. With the recent availability ofthe genome sequences of M. bovis strains PG45, HB0801 and Hu-bei-1 (Li et al., 2011; Wise et al., 2011; Qi et al., 2012) our under-standing of, and identification of, novel antigenic proteins in thispathogen will be accelerated, but many of these predicted surfacemembrane proteins are yet to be definitively identified and thefunctions of most of them have not been determined.

Past efforts have been centred on identifying and characterisingthe variable surface proteins (Vsps) in M. bovis (Behrens et al.,1994; Rosengarten et al., 1994; Lysnyansky et al., 1996; Beieret al., 1998; Poumarat et al., 1999; McAuliffe et al., 2004; Rifatbeg-

Table 1Some Mycoplasma bovis membrane proteins of interesta.

Organism Protein number Position ingenome

Aminoacids

Paralogues

PG45 MBOVPG45_0316 355,469–357,175 568 mbv:MBOVPG45_024mbv:MBOVPG45_024mbv:MBOVPG45_033

PG45 MBOVPG45_0481 552,647–557,194 1515 mbv:MBOVPG45_003mbv:MBOVPG45_075mbv:MBOVPG45_086

PG45 MBOVPG45_0790 914,655–915,407 250 mbv:MBOVPG45_031mbv:MBOVPG45_007

PG45 MBOVPG45_0864 989,140–997,266 2708 mbv:MBOVPG45_034mbv:MBOVPG45_048mbv:MBOVPG45_064

HB0801 Mbov_0174 197,851–199,839 662 mbi:Mbov_0016mbi:Mbov_0350

HB0801 Mbov_0305 358,653–360,440 595 mbi:Mbov_0115mbi:Mbov_0492

HB0801 Mbov_0579 683,802–685,988 728 mbi:Mbov_0260mbi:Mbov_0274mbi:Mbov_0739

Hubei-1 MMB_0167 197,288–199,348 686 mbh:MMB_0017mbh:MMB_0657

a The National Center for Biotechnology Information (NCBI) and Kyoto Encyclopedia ostrain PG45; mbi:Mbov, Mycoplasma bovis stain HB0801; mbh:MMB, Mycoplasma bovis stagalactiae strain PG2.

ovic et al., 2009). Although the Vsps clearly play an important rolein the pathogenesis of mycoplasma infections, their variableexpression and structures complicate both definition of their func-tion and their use as serological markers of infection. Variations inthe Vsps may contribute to host specificity and to the chronicity ofthe disease caused by M. bovis (Shen et al., 2000). The vsp genecluster was not identified in the annotated genome of Hubei-1strain, but it is possible that this has resulted from difficulties inunambiguously identifying highly repetitive sequences, such asthe vsp genes, and assembling them for incorporation into the gen-ome sequence. Examples of functionally characterised myco-plasma membrane proteins include the P65 lipoprotein ofMycoplasma hyopneumoniae, which is lipolytic (Schmidt et al.,2004), GapA of Mycoplasma gallisepticum, which is a cytadhesin(Goh et al., 1998), lipoprotein LppC of Mycoplasma mycoides sub-species mycoides SC which is pore forming (Pilo et al., 2003) andP40 of Mycoplasma agalactiae, which is a cytadhesin (Fleury et al.,2002).

3. M. bovis membrane proteins

Apart from the variable surface lipoproteins, only a few M. bovismembrane proteins have been identified and characterized. A48 kDa membrane lipoprotein P48 of M. bovis, homologous to themacrophage activating lipoprotein (MALP) of M. fermentans, theprototype in mycoplasmas of the basic membrane protein family,which is widely distributed in bacteria, was detected by Robinoet al. (2005). It is structurally and antigenically conserved in M. bo-vis and has potential for use as a specific serological marker ofinfection. A paralogue encoding a hypothetical 68 kDa proteinP68 has been identified in M. bovis PG45 clonal variant 6. P68shares the conserved selective lipoprotein-associated motifs of

Orthologues Remarks

3 mbi:Mbov_0575 This gene is orthologous to the adenosinetriphosphatase of Mbov_0575, and may serve as acoenzyme in intracellular energy transfer

5 mbh:MMB_05367 mal:MAGa5460

8 mbi:Mbov_0399 This protein is similar to the cysteine peptidasefamily forming part of the catalytic triad1 mbh:MMB_0376

4 maa:MAG_3890

6 mbi:Mbov_0778 This protein is in the acyltransferase family andmay be involved in the enzymatic conversion offree cholesterol into cholesteryl esters, which isthen sequestered into the core of a lipoproteinparticle

48 mbh:MMB_0744maa:MAG_6910

3 mbh:MMB_0800 Putative ABC transporter permease protein1 maa:MAG_74406 mal:MAGa8620

mbh:MMB_0167 P48-like surface lipoproteinmal:MAGa1620maa:MAG_0120

mbh:MMB_0283 Closely located to the phosphonate ABCtransporter substrate-binding and permeaseproteins downstream of the gene

mbv:MBOVPG45_0550mal:MAGa2810

mbh:MMB_0540 Belongs to the lipoprotein family and within theglycerol ABC transporters.mbv:MBOVPG45_0311

maa:MAG_5030

maa:MAG_120 Major surface lipoprotein P48mbi:Mbov_0174

f Genes and Genomes (KEGG) were used; mbv:MBOVPG45, Mycoplasma bovis typerain Hubei-1; mal:MAGa, Mycoplasma agalactiae strain 5632; maa:MAG, Mycoplasma

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the MALP-related lipoproteins (Lysnyansky et al., 2008). The P68gene was only detected in some strains of M. bovis, all of which alsocontained the P48 gene. Thus far M. bovis is the only mycoplasmaspecies found to contain multiple homologues of MALP.

The M. bovis homologue of the gene encoding the P40 adhesin ofM. agalactiae has been found to be a pseudogene (Thomas et al.,2004). The presence of a deletion, resulting in a frameshift, in theP40 homologue of M. bovis, may suggest that cattle lack the recep-tor for the P40 adhesin, which is used by M. agalactiae to bind toovine cells (Thomas et al., 2004). However, the identity of theP40 receptor in ovine cells and its expression in different speciesare yet to be elucidated.

Clearly there are many M. bovis membrane and hypotheticalproteins that remain to be investigated and characterized, andthe ease with which this can be achieved has been enhanced bythe publication of genome sequences of several strains of M. bovis.Some membrane proteins that are likely to be of interest in suchinvestigations are listed in Table 1.

Table 2Comparative genome features of Mycoplasma bovis strains.

Features PG45a Hubei-1b

HB0801c

Genome size (bp) 1,003,404 948,121 991,702G + C content (%) 29.3 29.4 29.3Number of protein coding genes including

pseudogenes826 833 808

Average length of CDS (bp) 1089 1058 1097Percentage coding (%) 83 89.5 84.2Predicted lipoproteins 96 96 103Number of insertion sequences 54 26 51Number of tRNA 34 34 34Number of rRNA sets (5S, 16S and 23S) 2 1 2Number of variable surface protein gene

clusters13 0 6

Number of integrative conjugativeelements

2 1 1

CDS – coding domain sequence.a Wise et al. (2011).b Li et al. (2011).c Qi et al. (2012).

4. Use of M. bovis membrane proteins in diagnosis

Diagnosis of mycoplasmal infection is typically achieved by iso-lation of the bacterium, with confirmation by polymerase chainreaction (PCR) amplification of the 16S rRNA gene followed bysequencing of the PCR product (Alberti et al., 2006). Even thoughisolation is a more definite approach to diagnosis, serological diag-nosis is often more reliable, as detection of specific antibody by en-zyme-linked immunosorbent assay (ELISA) is possible for manymonths after infection and is also more likely to detect infectionon farms on which antibiotics that may inhibit mycoplasmagrowth have been used. ELISAs are less labour-intensive and lesstime-consuming than culture and allow the screening of largenumbers of samples. An indirect ELISA using whole cell proteinshas been used to detect antibodies to M. bovis (Boothby et al.,1981), but the specificity is likely to be improved by targeting spe-cific proteins rather than using whole cell proteins (Boothby et al.,1986; Heller et al., 1993).

Several M. bovis membrane proteins and lipoproteins have beenused to develop diagnostic assays for detection of antibodies spe-cific for M. bovis in sera. Behrens et al. (1996) identified a mem-brane protein (pMB67) that is the predominant antigenrecognized during infection with M. bovis. This protein has attri-butes characteristic of a functionally important variable antigeniccomponent of Vsps but has no antigenic or structural similarityto the Vsps and is not lipid-modified. pMB67 is localized on theorganism’s surface, where it displays a specific monoclonal anti-body-defined epitope, does not contain multiple repeating sub-units like the Vsps and can be independently expressed incombination with Vsps, increasing its capacity to generate andmaintain a structurally, antigenically and functional versatile sur-face (Behrens et al., 1996). Recombinant Vsps have also beenshown to be highly immunoreactive and recombinant Vsp fusionproteins have been suggested as a basis for development of assaysfor rapid identification of cattle infected with M. bovis. Vsps andpMB67 have been shown to exhibit distinct expression patternsof a diverse and highly variable nature in animals infected withM. bovis (Rosengarten et al., 1994; Behrens et al., 1996), suggestingthat a cocktail composed of recombinant forms of these antigensmight be used as the basis of a sensitive immunoassay systemfor specific and rapid identification of M. bovis infected animals(Behrens et al., 1996; Brank et al., 1999). A surface lipoprotein ofM. bovis p48 (homologous to p48 of Mycoplasma agalactiae) hasalso been characterised and evaluated as a potential marker ofinfection by Robino et al. (2005). An antibody response against re-combinant p48 was detected in both experimentally and naturally

infected animals, suggesting it might be used as a specific markerof M. bovis infection.

In addition to use of membrane proteins for serological diagno-sis of M. bovis, a monoclonal antibody against the p26 antigen hasbeen used to develop an antigen capture ELISA (Heller et al., 1993),which enabled detection of M. bovis in bovine milk samples.Although the p26 monoclonal antibody showed cross-reactivitywith M. agalactiae, this would not affect the diagnosis of M. bovismastitis, as M. agalactiae does not occur in cows. The p26 proteinhas been shown to play a major role in adhesion of M. bovis toembryonic bovine lung (EBL) cells (Sachse et al., 1993), with levelsof expression of p26 correlating with the relative levels of adher-ence of different strains and with the inhibitory effect the mono-clonal antibody against p26 has on adherence. The a-enolase ofM. bovis has been shown to be a surface exposed protein and to en-able it to adhere to EBL cells by binding plasminogen (Song et al.,2012). Its ability to serve as a plasminogen receptor suggests thatit may facilitate M. bovis invasion and dissemination in infectedhost. Antibodies raised against recombinant M. bovis a-enolase de-tected the protein in the soluble cytosolic fraction, the cell-mem-brane fraction and in whole-cell proteins. This demonstrates thata-enolase is a membrane-associated protein in M. bovis and is be-lieved to play a role in the invasion of host tissue.

5. Genome sequences of M. bovis

Wise et al. (2011) were the first to publish a genome sequenceof M. bovis, for the type strain PG45, which was initially isolated in1961 from a severe case of mastitis in a cow. The genome is1,003,404 bp in length, is 29.3% G + C and is predicted to encode826 ORFs. Subsequently, the complete genome sequences of strainHubei-1 (Li et al., 2011), which was isolated from a case of severerespiratory disease in Hubei province in China, and of strainHB0801, which was isolated from lung lesions in a cow fromYingcheng in Hubei (Qi et al., 2012), were published. Comparativefeatures of the sequenced M. bovis genomes are shown in Table 2.Their sequences are available under the GenBank accession num-bers CP002188 (PG45), CP002513 (Hubei-1) and CP002058(HB0801). Comparison of their genomes revealed that HB0801and Hubei-1 had a large inversion (of 580 kb), associated withtwo mobile genetic elements, ISMbov3 and ICEB-2, compared toPG45. The HB0801 genome was 43,581 bp longer than that of Hu-bei-1, but 11,702 bp shorter than PG45. The differences betweenPG45 and HB0801 were mostly associated with insertion se-

324 J.Y. Adamu et al. / Research in Veterinary Science 95 (2013) 321–325

quences and also encoded putative lipoproteins and membraneproteins. The differences between Hubei-1 and HB0801 weremainly due to nine distinct insertions in HB0801, many of whichcontained predicted lipoprotein and membrane protein genes. Asurprising finding was the absence of the vsp gene cluster in Hu-bei-1, which is found in both PG45 and HB0801. The adjacent xerCgene, which encodes the integrase-recombinase that mediates site-specific inversion in the vsp gene family, thus generating phasevariation in Vsp expression, was present. Vsps have been identifiedin all strains of M. bovis that have been characterised (Wise et al.,2011; Qi et al., 2012), suggesting that the absence of this clusterin Hubei-1 may be a result of deletion during culture or possiblyan artefact of genome assembly. Definitive resolution of the signif-icance of the absence of vsp genes from the published genome se-quence will require both direct confirmation that this region is notpresent in the strain and assessment of the pathogenicity of the se-quenced strain. Vsp lipoproteins have been shown to be highlyimmunogenic and to contain repetitive domains involved in adhe-sion. Site-specific DNA inversion within the vsp locus results indiversification of Vsp expression within propagating populations(Flitman-Tene et al., 2003).

6. Possible roles of membrane proteins in the pathogenesis ofM. bovis infection

In the absence of the protective cell wall in mycoplasmas themembrane proteins are essential for colonization of and survivalwithin the host. Mycoplasmas are able to modulate the host im-mune system, despite the absence of the classical immunomodula-tors present in other prokaryotes. This immunomodulation hasbeen attributed in part to mycoplasma lipoproteins (Muhlradt,2002), which are the most abundant surface proteins (Razinet al., 1998).

Uptake of specific substrates required for metabolic pathways iscritical for survival of mycoplasmas due to their limited capacity tosynthesize many essential molecules, so they must acquire themfrom exogenous sources through membrane transporters. Productsof metabolism are also likely to be extruded through the transport-ers and these may have deleterious effects on the host. Many genesencoding mycoplasma lipoproteins, such as Mbov_0395 andMBOVPG45_0550, appear to be within operons encoding ABCtransporters and may be associated with acquisition of nutrientsfor transport into the cell (Browning et al., 2011). Some of theselipoproteins can also be targets for growth inhibitory antibody,as seen with the M. hyopneumoniae surface lipoprotein P65, whichis a lipolytic enzyme (Schmidt et al., 2004). Mycoplasmas are ableto stimulate and suppress lymphocytes in a non-specific polyclonalmanner and their cell components can modulate the activities ofmonocyte/macrophages and natural killer cells, triggering the pro-duction of a wide variety of up-regulating and down-regulatingcytokines and chemokines (Razin et al., 1998). Mbov_0575(MBOVPG45_0316; MMB_0536) encodes a predicted membraneadenosine triphosphatase, and may serve as a coenzyme in intra-cellular energy transfer. Another interesting role that mycoplasmamembrane proteins may play is in pulmonary surfactant disrup-tion, as demonstrated during infection of mice with M. pulmonis(Hickman-Davis et al., 2007). Both surfactant protein A and induc-ible nitric oxide synthase are thought to be important in pulmon-ary defence against respiratory pathogens such as mycoplasmas(Hickman-Davis et al., 2007). Using human surfactant protein-A(hSP-A)-coupled Sepharose affinity chromatography and poly-acrylamide gel electrophoresis, Kannan et al. (2005) identified a65 kDa hSP-A binding protein of M. pneumoniae. Experimentalinfection of calves with an aerosolised culture of M. bovis has beenshown to induce goblet cell hyperplasia and metaplasia in the

bronchial epithelium and an increase in the epithelial height,although affects on pulmonary surfactant have yet to be investi-gated (Wawegama et al., 2012).

7. Conclusion

There remains a great deal of work to do to classify the M. bovisORFs that have no match in the current databases and to assignfunctions to these ORFs, as well as to definitively assign functionto those ORFs with predicted roles. While many of the M. bovismembrane-associated proteins are yet to be characterized, withthe completion of the genome sequences of several strains, weare closer to achieving the goal of deciphering the molecular basisof the pathogenicity of M. bovis.

Acknowledgements

JYA is supported by Melbourne International Fee Remission andMelbourne International Research Scholarships.

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