Mycoplasma hyopneumoniae Surface Proteins Mhp385 and Mhp384

Article pubs.acs.org/jpr

Mycoplasma hyopneumoniae Surface Proteins Mhp385 and Mhp384 Bind Host Cilia and Glycosaminoglycans and Are Endoproteolytically Processed by Proteases That Recognize Different Cleavage Motifs Ania T. Deutscher,†,‡ Jessica L. Tacchi,§ F. Chris Minion,∥ Matthew P. Padula,§ Ben Crossett,⊥ Daniel R. Bogema,†,‡ Cheryl Jenkins,† Tracey A. Kuit,‡ Mark J. Walker,‡,# and Steven P. Djordjevic*,†,§ †

NSW Department of Primary Industries, Elizabeth Macarthur Agricultural Institute, Camden NSW 2567, Australia School of Biological Sciences, University of Wollongong, Wollongong NSW 2522, Australia § The ithree Institute, University of Technology Sydney, Broadway NSW 2007, Australia ∥ Department of Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, Iowa 50011, United States ⊥ School of Molecular Bioscience, University of Sydney, Sydney NSW 2006, Australia # School of Chemistry and Molecular Biosciences and the Australian Infectious Diseases Research Centre, University of Queensland, Brisbane Qld 4072, Australia ‡

S Supporting Information *

ABSTRACT: P97 and P102 paralogues occur as endoproteolytic cleavage fragments on the surface of Mycoplasma hyopneumoniae that bind glycosaminoglycans, plasminogen, and fibronectin and perform essential roles in colonization of ciliated epithelia. We show that the P102 paralogue Mhp384 is efficiently cleaved at an S/T-X-F↓X-D/E-like site, creating P60384 and P50384. The P97 paralogue Mhp385 is inefficiently cleaved, with tryptic peptides from a 115 kDa protein (P115385) and 88 kDa (P88385) and 27 kDa (P27385) cleavage fragments identified by LC−MS/MS. This is the first time a preprotein belonging to the P97 and P102 paralogue families has been identified by mass spectrometry. The semitryptic peptide 752IQFELEPISLNV763 denotes the C-terminus of P88385 and defines the novel cleavage site 761L-N-V↓A-V-S766 in Mhp385. P115385, P88385, P27385, P60384, and P50384 were shown to reside extracellularly, though it is unknown how the fragments remain attached to the cell surface. Heparin- and cilium-binding sites were identified within P60384, P50384, and P88385. No primary function was attributed to P27385; however, this molecule contains four tandem R1 repeats with similarity to porcine collagen type VI (α3 chain). P97 and P102 paralogue families are adhesins targeted by several proteases with different cleavage efficiencies, and this process generates combinatorial complexity on the surface of M. hyopneumoniae. KEYWORDS: Mycoplasma hyopneumoniae, paralogue, endoproteolytic cleavage, cilium adhesin, heparin-binding protein

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is derived.3,4 Analysis of the M. hyopneumoniae strain 232 genome showed that 182 of 692 (∼26%) open reading frames could be assigned to a paralogous gene family.1 Prominently featured paralogue families include ABC transporters with ATP binding site motifs and the P97 and P102 paralogue families. Ciliostasis, cilial shedding, and epithelial cell death are pathological hallmarks in the development of PEP.5−8 Adherence to target host cells is a critical, early step in pathogenesis and is a complex multifactorial process involving adhesins that directly target host cell receptors, extracellular matrix (ECM) components, and host molecules that act as a bridge between microbe and host receptors.9−12 Members of the P97 and P102 paralogue families in M. hyopneumoniae play

INTRODUCTION M. hyopneumoniae is a genome-reduced, geographically widespread pathogen that causes porcine enzootic pneumonia (PEP), a chronic, nonfatal respiratory disease that inflicts significant economic losses on swine production worldwide. The M. hyopneumoniae genome is approximately 900 kb (∼700 coding sequences) and has limited coding capacity compared to other medically significant Gram-positive pathogens to which it is phylogenetically most closely related, such as Clostridium and Streptococcus.1 Mycoplasmas are thought to have evolved by a process of degenerative evolution from the low guanine and cytosine (G + C) Firmicutes.2 Genes that encode enzymes for cell wall biosynthesis, the tricarboxylic acid cycle, and many anabolic biosynthetic pathways are absent from the genome. In M. hyopneumoniae, glycolysis and the associated “pyruvate roundhouse”, are the principal means by which adenosine triphosphate (ATP) © 2012 American Chemical Society

Received: November 8, 2011 Published: January 9, 2012 1924

dx.doi.org/10.1021/pr201115v | J. Proteome Res. 2012, 11, 1924−1936

Journal of Proteome Research

Article

P97 and P102 paralogues. Finally, we determined whether these proteins play a role in adherence to porcine cilia and GAGs.

critical roles in binding porcine cilia, highly sulfated glycosaminoglycans (GAGs), fibronectin, and plasminogen.11,13−21 Interactions with heparinase sensitive GAGs displayed on the surface of porcine epithelial cilia provide a mechanism by which M. hyopneumoniae overcomes the mucociliary escalator during the early stages of colonization.16 In addition, interactions with various host molecules enable the pathogen to remain within the host as infection proceeds and epithelial shedding occurs. Reverse transcriptase−polymerase chain reaction (RT-PCR) studies performed on M. hyopneumoniae taken from bronchoalveolar lavage fluid of experimentally M. hyopneumoniaechallenged pigs confirmed that mRNA encoding almost all members of the P97 and P102 paralogue families are simultaneously expressed.22 Members of the P97 and P102 paralogue families are targets for post-translational endoproteolytic cleavage by unknown proteases,13−15,17−19,21,23 a feature that distinguishes them from all other previously described gene families in the Mollicutes. All P97 and P102 paralogues are the products of gene fusion events.1 The processing of these proteins generates endoproteolytic cleavage fragments comprising novel C-terminal sequences and N-terminal sequences that share sequence similarity to other paralogue family members. All of these fragments are displayed on the surface of M. hyopneumoniae cells.14,15,17−19,23 Initial studies of the archetype cilium adhesin P97 indicate that this molecule undergoes extensive processing,23 but further work is required to understand this complexity. Recently, we showed that several members of the P97 and P102 paralogue families possess a sequence motif S/T-X-F↓X-D/E that delineates the site of proteolytic cleavage.15 Cleavage occurs after the phenylalanine (F) residue. A negatively charged amino acid [glutamic acid (E) or aspartic acid (D)] typically occupies the +2 position, and an alcohol-containing residue (S/T) is often found in the −3 position. Cleavage efficiency seems to be influenced by the cleavage motif residing within disordered regions spanning at least 40 amino acids.15 Our prevailing hypothesis is that a single protease is responsible for cleavage at the S/T-X-F↓X-D/E site. However, at least one other protease may play an important role in processing members of the P97 and P102 paralogue families. In strain J, an inefficient cleavage event, which separates the C-terminal 230 amino acids that comprise P28 from the P97 homologue MHJ_0194, occurs at the site 862T↓ NTNTSKF869.23 This sequence is structurally unrelated to the one described above; therefore, its cleavage is thought to be performed by a different protease. Our proteome studies indicate that most representatives of the P97 and P102 paralogue families are very highly expressed during broth culture. Given the limited capacity of M. hyopneumoniae to generate ATP, it is significant that considerable energy is expended to express, secrete, and extensively proteolytically process members of the P97 and P102 paralogue families. To gain a better understanding of the surface topography of M. hyopneumoniae, it is important to precisely map the cleavage events in the P97 and P102 paralogue families and identify the proteases responsible for their cleavage. Here we examine the expression patterns of Mhp385 and Mhp384, P97 and P102 paralogues, respectively, whose genes reside in a two-gene operon. We show that Mhp384 undergoes one major cleavage event at an S/T-X-F↓X-D/E-like motif while Mhp385 undergoes inefficient endoproteolytic cleavage at a unique site. We precisely mapped cleavage sites in Mhp384 and Mhp385 and examined the association between the location of proteolytic cleavage sites and protein disorder in these and other

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EXPERIMENTAL PROCEDURES

Bacterial Strains

M. hyopneumoniae strain 232 cells were grown and harvested as described previously.24 The Escherichia coli strains TOP10 (Invitrogen, USA) and XL1-Blue (Stratagene, USA) were used for plasmid maintenance, while E. coli BL21 Star (DE3) (Invitrogen) was used for protein expression. E. coli were routinely grown at 37 °C on Luria−Bertani (LB) agar plates or cultured in LB broth with shaking at 200 rpm, supplemented with antibiotics where appropriate. Cloning of mhp385 and mhp384 Gene Fragments

Gene fragments were amplified from M. hyopneumoniae strain 232 DNA, using the oligonucleotide primers (Sigma-Aldrich, Australia) listed in Supporting Information Table S1. TGA codons encode tryptophan in M. hyopneumoniae; therefore, all in-frame TGA codons were changed to TGG by PCR using mutated primers or site-directed mutagenesis. The mhp385 and mhp384 PCR amplified gene fragments were cloned into the pET100/D-TOPO and pET160/GW/DTOPO vectors (Invitrogen), respectively, and transformed into E. coli TOP10 cells according to the manufacturer’s instructions. For binding studies (see below), mhp384 gene fragments were subcloned into pET100/D-TOPO. All cloned gene fragments were sequenced prior to performing expression studies. Expression and Purification of Recombinant Fragments of Mhp385 and Mhp384

For protein expression, each plasmid construct was transformed into E. coli BL21 Star (DE3) cells according to the manufacturer’s instructions. Expression and purification of the recombinant proteins was as described by Deutscher et al.,16 except E. coli BL21 Star (DE3) cells transformed with plasmid construct pET100/D-TOPO:f1384 or pET100/D-TOPO:f2385 were induced with 1 mM isopropyl-β-galactopyranoside (IPTG) overnight at 22 °C or for 8 h at 30 °C, respectively. Antiserum to each of the F1384-F3384 (pET160/GW/D-TOPO) and F1385F4385 (pET100/D-TOPO) fusion proteins was generated in New Zealand White rabbits as detailed previously.20 Bioinformatics

To identify transmembrane domains, the Tied Mixture Hidden Markov Model (TMHMM) Server v. 2.025,26 and Phobius,27 a combined transmembrane topology and signal peptide predictor, were used. The ProtParam algorithm28 was used to calculate the theoretical mass and pI of a protein. Basic Local Alignment Search Tool (BLAST) searches, performed using the nonredundant protein database at the National Center for Biotechnology Information (NCBI), were used to identify proteins that share regions of identity or similarity. Protein disorder was predicted using the Predictor of Naturally Disordered Regions (PONDR) algorithms VSL129,30 and PONDR-FIT,31 and the Intrinsically Unstructured Proteins Predictor (IUPred) algorithm32,33 [Access to PONDR was provided by Molecular Kinetics (6201 La Pas Trail, Ste 160, Indianapolis, IN 46268; 317-280-8737; E-mail: [email protected]). PONDR is copyright 1999 by the Washington State University Research Foundation, all rights reserved]. Amino acid scores above 0.5 are considered to be disordered. 1925



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by Seymour et al.,14 using a 1:100 dilution of anti-F4385 serum and antirabbit peroxidase conjugate (Sigma-Aldrich) diluted 1:3000.

The search for coiled-coils was conducted by means of the COILS2 algorithm34 with window widths of 14, 21, and 28 and the MTIDK matrix. The outputs from both weighted and unweighted scans were compared. In addition, the Paircoil2 algorithm,35 with a minimum window size of 21 and a P-score cutoff of 0.025, was used.

1D Liquid Chromatography−Tandem Mass Spectrometry (LC−MS/MS) Using Q-TOF

LC−MS/MS was performed as described previously37 with minor modifications. The peptides were washed off the trap at 300 nL/min onto a PicoFrit column (75 μm × 100 mm) (New Objective, USA) packed with Magic C18AQ resin (Michrom Bioresources, USA), and the following program was used to elute peptides from the column and into the source of a QSTAR Elite hybrid Q-TOF mass spectrometer (AB Sciex, USA): 5−30% MS buffer B (98% acetonitrile−0.2% formic acid) over 30 min, 30−80% MS buffer B over 3 min, 80% MS buffer B for 2 min, 80−85% MS buffer B for 3 min. The mass of the precursor peptide was then excluded for 120 s. Peak list files were generated by MSConvert from ProteoWizard38 release 2.1.2674.

Preparation of Whole Cell Lysates from M. hyopneumoniae Cells

Whole cell lysates from M. hyopneumoniae strain 232 cells were prepared using two different methods. For the analysis of M. hyopneumoniae proteome by “slice and dice” of a one-dimensional (1D) sodium dodecyl sulfate (SDS)-polyacrylamide gel, or by two-dimensional (2D) SDS-polyacrylamide gel electrophoresis (SDS-PAGE), a 0.1 g pellet of M. hyopneumoniae strain 232 cells was prepared as described by Seymour et al.14 For all other experiments, a 0.1 g pellet of M. hyopneumoniae strain 232 cells was resuspended in 5 mL of 0.5% (v/v) Triton X-100 in phosphate-buffered saline (PBS). Trypsin Digest of M. hyopneumoniae Cells

MS/MS Data Analysis

Trypsin digestion of M. hyopneumoniae cells and Western blotting were performed as described previously.16

The MS/MS data files were searched using Mascot v.2.2.0739 through the UNified user InTErface (UNITE) interface provided by the Australian Proteomics Computational Facility (APCF, http://www.apcf.edu.au/) against the LudwigNR database (comprised of the UniProt, plasmoDB, and Ensembl databases; http://www.ludwig.edu.au/archive/LudwigNR/ LudwigNR.pdf). The MS/MS results from the M. hyopneumoniae proteome “slice and dice” experiment, and the polyacrylamide gel bands containing the Mhp385 preprotein and its N-terminal cleavage fragment, were searched against version Q111 with 14359729 sequences and 4875314780 residues. The MS/MS results for the protein gel spots containing the C-terminal cleavage fragment of Mhp385 were searched against version Q311 with 16981479 sequences and 5747323621 residues. The MS/MS results for the polyacrylamide gel bands containing the Mhp384 cleavage fragments were searched against version Q211 with 12931687 sequences and 4483500103 residues. The following parameter settings were used in all searches: fixed modifications, none; variable modifications, propionamide, oxidized methionine, deamidated asparagine; enzyme, semiTrypsin; number of allowed missed cleavages, 3; precursor ions mass tolerance, 100 ppm; fragment ions mass tolerance, 0.2 Da; charge state, 2+ and 3+. The results of the search were then filtered by including only protein hits with at least one unique peptide and excluding peptide hits with P values of >0.05. Peptides identified by Mascot were further validated by manual inspection of the MS/MS spectra for the peptide to ensure that the b and y ion series were sufficiently extensive for an accurate identification.

Protein Separation by Polyacrylamide Gel Electrophoresis and Extraction

One-dimensional SDS-PAGE was performed as described by Deutscher et al.16 or carried out in 4−12% Bis-Tris gels using a Criterion XT gel system (Bio-Rad Laboratories, USA). For “slice and dice” experiments, whole cell lysates from M. hyopneumoniae strain 232 were separated by 1D SDS-PAGE, and then either they were sliced equally into 15 pieces across the molecular mass range or a single slice was cut from the gel as indicated in the Results section. They were then destained and trypsin digested as described by Seymour et al.17 PageRuler Prestained Protein Ladder (Thermo Fisher Scientific Inc., USA) or Unstained Precision Plus Protein Standards (Bio-Rad Laboratories) were run on all polyacrylamide gels. Gels were stained with either GelCode Blue (Thermo Fisher Scientific Inc.), Coomassie Blue G-250, or Flamingo Fluorescent Gel Stain (BioRad Laboratories). 2D SDS-PAGE and Western Blotting

Two-dimensional gel electrophoresis (2DGE) combined with Western blotting was employed to identify the C-terminal cleavage fragment of Mhp385. M. hyopneumoniae strain 232 cells whole cell extracts were passed through a Nanosep 300K omega centrifugal filter (Pall, USA) prior to isoelectric focusing (IEF). Bio-Lyte 3/10 ampholytes (Bio-Rad Laboratories) were added to a final concentration of 0.2% (v/v). The proteins (250 μg in 75 μL) were cup loaded onto an 11 cm pH 6−11 Immobiline DryStrip (GE Healthcare, USA) according to manufacturer’s instructions. IEF was carried out using a Protean IEF cell (Bio-Rad Laboratories) at a constant 20 °C and 50 μA current limit per strip with a 3-step program: slow ramp to 4000 V for 4 h, linear ramp to 10,000 V for 4 h, then 10,000 V until 115 kVh was reached. The strips were equilibrated with 5 mL of equilibration solution [6 M urea, 2% (w/v) SDS, 250 mM Tris-HCl (pH 8.5), 0.0025% (w/v) bromophenol blue] for 20 min before 2DGE using the Bio-Rad Laboratories Criterion gel system. Two-dimensional polyacrylamide gels were visualized by staining with Coomassie Blue G-250, or alternatively, proteins were transferred to PVDF using a semidry transfer method.36 Membranes were blocked, probed, and developed as described

Cilia Adherence Assay

Cilia adherence assays and statistical analysis were performed as described previously.16 Heparin-Binding Assays

Heparin microtiter plate binding assays and dot blots were performed and analyzed as described by Deutscher et al.,16 with minor modifications. Flat bottom Linbro/Titertek 96-well microtiter plates (MP Biomedicals Inc., USA) were used for microtiter plate binding assays, and dot blot membranes were washed with TBS [10 mM Tris-HCl, 0.9% (w/v) NaCl, pH7.4] containing 0.05% (v/v) Tween 20 twice for 5 min, followed by one 5 min wash in TBS only. 1926



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Article

RESULTS

tandem R1 repeats in both P97 and Mhp271 can vary between strains of M. hyopneumoniae;16,41−44 however, alignment of the R1 region in Mhp385 with homologues in M. hyopneumoniae strains J (GenBank accession number AAZ44457), 7448 (AAZ53744), and 168 (ADQ90625) suggests that this region is not highly variable (data not shown). On the basis of the number of repeats, this region is expected to be recognized by the cilia adherence-blocking monoclonal antibody (mAb) F2G5.41 The Mhp385 amino acid sequence shares greater than 96% identity with its respective homologues in M. hyopneumoniae strains J, 7448, and 168, and it possesses a hexapeptide “NNNLRL” that is repeated twice in strain 232 but not in any of its homologues. Outside the M. hyopneumoniae proteome, Mhp385 shows greatest similarity to LppS, a putative adhesin of Mycoplasma conjunctivae45 (GenBank accession number CAC87274; the Mhp385 amino acids 39−399 share 30% identity and 50% positives; and the amino acids 360−735 share 25% identity and 42% positives). The majority of the homology that Mhp385 shares with P97 and its paralogues is within its first 780 amino acids. Apart from the similarity of the R1 repeat region with P97 and Mhp271, the C-terminal sequence of Mhp385 only shares some similarity with collagen alpha-3(VI) chain isoform 1 (Sus scrofa) (NCBI accession number XP_001928122; 25% identity, 42% positives with Mhp385 amino acids 723−930). The last 58 amino acids of Mhp385 do not share any significant similarity to proteins in the NCBI protein database.

Molecular Analysis of Mhp385 and Mhp384

mhp385 shares a two-gene operon with a gene encoding the P102 paralogue Mhp384 (GenBank accession number AAV27855) (Figure 1A). A possible ribosome binding sequence (AGGAG) is located within the twelve nucleotides that separate the genes. Mhp385 (GenBank accession number AAV27856) consists of 988 amino acid residues and has a theoretical molecular weight and pI of 114.8 kDa and 8.87, respectively. A putative transmembrane region resides between residues 22−44 (score 0.99993, TMHMM 2.0). No signal peptide is predicted. A feature of the mhp385 gene locus is the presence of a homopolymeric tract of 22 thymine residues located 100 base pairs upstream of the start codon of mhp385. This repeat region is potentially placed between a Pribnow box (TATAAT) and a possible −35 region (TTGAGA). In addition, a homopolymeric tract of 19 adenines is located 25 base pairs downstream of the start codon for mhp385. Such regions are known hot spots for slipped-strand mispairing,40 which could result in frameshift mutations affecting the expression of mhp385. The presence of thymine and adenine tracts in very close proximity of each other may also lead to hairpin formation, which would interfere with gene transcription. Mhp385 is one of three M. hyopneumoniae proteins that contain tandem R1 pentapeptide repeats. Cilium-binding occurs in the presence of eight of these repeats.41 Mhp385 contains four tandem R1 repeats (AAKV-AAKPS-AAKPVAAKPE) within the C-terminus of the protein. The number of

Figure 1. Cloning and expression of Mhp385 and Mhp384 recombinant proteins. (A) Schematic representation of mhp385 and mhp384 in the chromosome of M. hyopneumoniae strain 232. The arrows show the direction of translation, and sizes are in base pairs. (B) Schematic of recombinant constructs relative to the Mhp385 and Mhp384 proteins. Numbers refer to amino acid positions. * indicates the presence of a TGA codon. (C) GelCode Blue stained SDS−12% polyacrylamide gel of purified recombinant Mhp385 and Mhp384 6xHis-tagged fragments. Molecular weight markers are indicated on the left. 1927



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232 cells probed with anti-F4385 serum identified two spots with mass of approximately 27 kDa (Figure 2C). The identities of these proteins were confirmed to be the C-terminal fragment of Mhp385 by LC−MS/MS (Figure 2D and Supporting Information Tables S2, S5, and S6). Currently all cleavage sites in the P97 and P102 paralogue families, except for the cleavage event that generates P28 from the C-terminus of the P97 homologue in strain J,23 adhere to the motif S/T-X-F↓X-D/E.15 Analysis of M. hyopneumoniae strain 232 proteins separated by 1D SDS-PAGE, extracted and characterized by LC−MS/MS, identified a semitryptic peptide with the sequence 752IQFELEPISLNV763, matching to Mhp385 (Figure 2D and Supporting Information Figure S1 and Table S7). This peptide falls within the 38 amino acids 752IQFELEPISLNVAVSQEKTNPNNNLRLNNNLRLKYWYK789 that separate the peptide matches that map to P88385 and P27385. Evidence of an S/T-X-F↓X-D/E-like motif was not observed in this stretch of amino acids. The semitryptic cleavage at the C-terminus of the peptide indicates that the cleavage site that separates P88385 from P27385 in Mhp385 is 761L-N-V↓A-V-S766. This cleavage site does not adhere to the previously established cleavage motif, suggesting that a different protease is responsible for this event. Consistent with this view, cleavage at this site is comparatively inefficient because we identified the Mhp385 preprotein.

Mhp384 (GenBank accession number AAV27855) consists of 957 amino acids and has a theoretical molecular weight of 109.2 kDa and a pI of 8.97. Between residues 9−31 lies a putative transmembrane region (score 0.99876, TMHMM 2.0), and no signal peptide is predicted. The Mhp384 amino acid sequence shares greater than 96% identity with its respective homologues in M. hyopneumoniae strains J, 7448, and 168. Outside the M. hyopneumoniae proteome, Mhp384 shows greatest similarity to the M. conjunctivae putative adhesin LppT46 (GenBank accession number CAC87275; the Mhp384 amino acid residues 13−501 share 23% identity and 44% positives). The genes of the putative adhesins LppS and LppT reside in a two-gene operon.45,46 The similarity Mhp384 shares with P102 and its paralogues is within the first 324 amino acids, while the last approximately 450 amino acids of the protein do not share significant sequence similarity to proteins in the NCBI protein database. Both Mhp385 and Mhp384 protein sequences do not contain any cysteine residues. Cloning and Expression of Mhp385 and Mhp384 Recombinant Protein Fragments

Lack of genetic t ools to mut at e and transform M. hyopneumoniae and reliable growth on agar medium are major impediments to generating knockout mutants in this species. To characterize the functions of Mhp385 and Mhp384, nonoverlapping fragments of the mhp385 and mhp384 genes were cloned and expressed in E. coli. Due to the high adenine and thymine (A+T) content of mhp385 (72%) and mhp384 (71%) and the potential difficulty in expressing A+T rich genes in E. coli,21,47 the entire genes were not cloned. Fragments that span mhp385 and mhp384, apart from the transmembrane domains (Figure 1B), were PCR amplified, cloned, and expressed as N-terminal hexahistidine (6xHis)-tagged fusion proteins (F1385-F4385, F1384-F3384). One-dimensional SDS-PAGE analysis of the 6xHis-tagged proteins (Figure 1C) confirmed predicted molecular masses for F1385 (41.1 kDa); F2385 (32.4 kDa); F3385 (25.9 kDa); F4385 (26.9 kDa); F1384 (40.4 kDa); and F3384 (38.7 kDa). F2384 traveled slightly more slowly than its theoretical mass of 39.2 kDa, but this aberrant migration may be due to a cluster of acidic and charged amino acids within its sequence (NEEVKQQESQVKDQAKQEKSSKDSQSKQ), since abnormal migration occurs with proteins enriched in acidic residues.21,48

Expression of Mhp384 in M. hyopneumoniae

Whole cell lysates from M. hyopneumoniae strain 232 cells probed with anti-F1384 serum identified a 60 kDa protein (P60384), while anti-F3384 serum detected a 50 kDa protein (P50384) (Figure 3A). No evidence of the 109 kDa Mhp384 preprotein was observed by probing blots with F1384-F3384 antisera (data not shown), indicating that the single endoproteolytic cleavage event that generates P60384 and P50384 is efficient. Since anti-F2384 serum detected both P60384 and P50384, the cleavage site of Mhp384 is likely to occur within the sequence of this fragment. LC−MS/MS was performed on trypsin digests of proteins found within gel slices containing P60384 and P50384 (Figure 3B). Tryptic peptides from P60384 matched to the N-terminal sequence of Mhp384, while peptides from P50384 matched to the C-terminal sequence of Mhp384 (Figure 3C and Supporting Information Tables S2, S8, and S9). Two semitryptic peptides, with sequences 530NEEVKQQESQVK541 and 530NEEVKQQESQVKDQAK545, were identified in a global analysis of the M. hyopneumoniae strain 232 proteome by 1D SDS-PAGE followed by LC−MS/MS (Supporting Information Figure S2 and Table S7). These semitryptic peptides define the N-terminus of P50384 (Figure 3C). These data indicate that the cleavage site for Mhp384 spans the sequence 527I-L-F↓N-E-E532 with a phenylalanine residue (F) in the −1 position of the cleavage site and a glutamic acid residue (E) in the position +2. This cleavage site is similar to the cleavage motif S/T-X-F↓X-D/E suggested by Bogema et al.15

Expression of Mhp385 in M. hyopneumoniae

To detect the expression of Mhp385 by M. hyopneumoniae cells, whole cell lysates of M. hyopneumoniae strain 232 cells were probed with rabbit immune sera raised against each of the Mhp385 recombinant fragments (Figure 2A). All anti-Mhp385 sera detected a protein with an approximate molecular weight of 115 kDa (P115385), which corresponds to the theoretical molecular weight of the Mhp385 preprotein. Anti-F1385, antiF2385, and anti-F3385 sera also detected an 88 kDa protein (P88385), while anti-F3385 and anti-F4385 sera also detected a 27 kDa protein (P27385). P88385 and P27385 are most likely N-terminal and C-terminal cleavage fragments of P115385, respectively. Since anti-F3385 serum detected all three bands, the cleavage site of Mhp385 is likely to fall within the sequence of F3385. To confirm the identities of the 115 and 88 kDa proteins, the respective protein bands were cut from a 1D SDS-7% polyacrylamide gel, digested with trypsin, and analyzed by LC−MS/MS (Figure 2B). Peptides unique to Mhp385 were found in both the 115 kDa and 88 kDa bands (Figure 2D and Supporting Information Tables S2, S3, and S4). Blots of a 2D gel (pI range 6−11) of whole cell lysates from M. hyopneumoniae strain

Mapping the Cleavage Site in the P97 Paralogue Mhp107

To further refine the cleavage sites in the P97 and P102 paralogue families, we also looked for the presence of a semitryptic peptide within another P97 paralogue, Mhp107. Previously we were unsuccessful in attempts to determine if the multifunctional adhesin Mhp107 undergoes endoproteolytic processing.18 Immunoblotting studies showed that Mhp107 and homologues in different strains of M. hyopneumoniae migrate with a mass of approximately 100 kDa, although the predicted molecular weight of Mhp107 is approximately 120 kDa.18 LC− MS/MS analysis of M. hyopneumoniae strain 232 proteins 1928



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Figure 2. Expression of Mhp385 by M. hyopneumoniae. (A) Whole cell lysates from M. hyopneumoniae strain 232 cells were separated on a 1D SDS− 12% polyacrylamide gel and stained with GelCode Blue or transferred onto PVDF membrane. The membranes were incubated with preimmune (PI) or immune (I) antisera raised against each Mhp385 recombinant protein (F1385−F4385), followed by sheep antirabbit serum. Identified proteins are indicated on the right by arrowheads, and molecular weight markers are indicated on the left. (B) A 1D SDS−7% polyacrylamide gel of M. hyopneumoniae strain 232 cells whole cell lysates stained or transferred onto PVDF membrane and reacted with anti-F1385 serum. The arrows indicate proteins recognized by the antiserum, which were excised, digested, with trypsin and subjected to LC−MS/MS. (C) Section of a 2DGE gel (Bio-Safe Coomassie stained) of whole cell lysates from M. hyopneumoniae strain 232 cells. A corresponding gel was transferred onto PVDF and probed with anti-F4385 serum. Two prominent protein spots were detected (circled), excised from the gel, and subjected to trypsin digestion followed by LC−MS/MS. (D) Protein sequence of Mhp385. Peptides identified by LC−MS/MS of the 115 kDa band shown in panel B are underlined. Highlighted in bold are the peptides identified by LC−MS/MS of the 88 kDa band in panel B. Peptides identified by LC−MS/MS of both protein spots boxed in panel C are in bold and italicized. Highlighted in bold and dash underlined is the Mhp385 semitryptic peptide identified in a global analysis of the M. hyopneumoniae strain 232 proteome by 1D SDS-PAGE followed by LC−MS/MS. The transmembrane domain is boxed.

separated by 1D SDS-PAGE that migrated within the mass range of approximately 25−30 kDa identified tryptic peptides that match to the N-terminal 199 amino acids of Mhp107 (Figure S3

and Supporting Information Tables S2 and S10). In this mass range a semitryptic peptide with the sequence 184QINENILNIGNFTTNF199 matched to Mhp107 (Supporting Information 1929



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Figure 3. Expression of Mhp384 by M. hyopneumoniae. (A) Whole cell lysates from M. hyopneumoniae strain 232 cells were separated on a 1D SDS− 12% polyacrylamide gel and stained with GelCode Blue or transferred onto a PVDF membrane. Membranes were treated with preimmune (PI) or immune (I) antisera raised against each Mhp384 recombinant protein (F1384−F3384), followed by sheep antirabbit serum. Arrows point to cleavage fragments of Mhp384. Molecular weight markers are indicated on the left. (B) A 1D SDS−10% polyacrylamide gel of whole cell lysate of M. hyopneumoniae strain 232 cells, GelCode Blue stained or transferred onto PVDF membrane and reacted with anti-F1384 and anti-F3384 sera. The arrows indicate proteins recognized by the antisera that were excised, digested with trypsin, and subjected to LC−MS/MS. (C) Amino acid sequence of Mhp384. Peptides identified by LC−MS/MS of the 60 kDa band shown in panel B are in bold. Highlighted in bold and underlined are the peptides identified by LC−MS/MS of the 50 kDa band in panel B. Highlighted in bold and dash underlined is one of the two Mhp384 semitryptic peptides identified in a global analysis of the M. hyopneumoniae strain 232 proteome by 1D SDS-PAGE followed by LC−MS/MS. The transmembrane domain is boxed.

M. hyopneumoniae strain J, whose cleavage sites were previously identified.13,23 Since several disorder predicting algorithms interpret coiled-coil regions as disordered,49 coiled-coil analyses were also performed. The endoproteolytic cleavage sites in Mhp384 and in MHJ_0195 occur within a long region of disorder (greater than 50 amino acids long) (Supporting Information Figure S5B and E). However, Mhp107 is predicted not to be disordered at the site of cleavage (Supporting Information Figure S5C). The endoproteolytic cleavage site in Mhp385 occurs prior to the start of a short, five amino acid long disordered region (Supporting Information Figure S5A). Although PONDR VSL1 and PONDR-FIT predicted that the endoproteolytic cleavage site of MHJ_0194 occurs within a disordered region (Supporting Information Figure S5D), both Paircoil2 (amino acid residues 173−203; P-score less than 0.025) and COILS2 (residues 175−203; probability greater than 0.9 using weights and a 21-long window) identified a coiled-coil domain at the cleavage site, suggesting that this region is not likely to be disordered.

Figure S4 and Table S7). The semitryptic cleavage at the C-terminus of this peptide indicates that the cleavage site is 197 T-N-F↓S-D-Q202. The cleavage site strongly resembles the cleavage motif suggested by Bogema et al.15 Cleavage at this site would remove 199 amino acids from the N-terminus of Mhp107, generating a mature C-terminal cleavage fragment with a theoretical molecular weight of 96.4 kDa and a 23.4 kDa N-terminal cleavage fragment. The C-terminal cleavage protein has a theoretical molecular weight similar to that of the protein detected by Western blotting with anti-Mhp107 sera in ref 18. The Mhp107 preprotein was not identified in the whole proteome analysis, suggesting that cleavage of Mhp107 is efficient. Prediction of Regions of Disorder among the P97 and P102 Paralogue Families

It was recently shown that the two TTKF↓QE cleavage sites in the P102 paralogue Mhp683 reside within long (greater than 40 amino acids) disordered regions.15 To determine if protein disorder plays a role in the endoproteolytic cleavage of other P97 and P102 paralogues, we applied several protein disorder prediction algorithms, as recommended by Ferron et al.,49 to Mhp385, Mhp384, and Mhp107, and to MHJ_0194 and MHJ_0195, respectively the P97 and P102 homologues in

Cellular Location of Mhp385 and Mhp384

Trypsin digestion of viable Mycoplasma cells has been widely used to demonstrate the surface location of proteins.19,21,42 1930



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300 μg/mL, indicating that the cells remained intact during the digestion procedure (Figure 4F).

Electrophoretic profiles of whole cell lysates from M. hyopneumoniae strain 232 cells, which were exposed to concentrations of trypsin ranging from 0 to 300 μg/mL prior to 1D SDS-PAGE, showed gradual loss of proteins across the entire mass range, but particularly with proteins with masses greater than 80 kDa (Figure 4A).

Mhp385 and Mhp384 Bind Porcine Cilia

To investigate whether Mhp385 and Mhp384 have a role in adherence, the recombinant fragments were assessed for their ability to bind to purified swine tracheal cilia coated on a microtiter plate. F1384, F2384, F3384, F1385, F2385, F3385, and positive control F2P97, which includes the R1 cilium-binding domain and R2 repeat region from P97,20 reproducibly bound to cilia (P < 0.05) (Figure 5). F4385 did not bind cilia.

Figure 5. Adherence of Mhp385 and Mhp384 recombinant proteins to swine cilia. Mhp385 and Mhp384 recombinant protein fragments and the positive control F2P97 protein20 were incubated with purified swine tracheal cilia coated on a microtiter plate. Bound protein was detected using polyclonal antiserum raised against the respective recombinant protein. Each bar represents the mean response above background plus the upper standard error from triplicate wells from two independent experiments (n = 6). Bars marked with an asterisk represent results that are significantly different (P < 0.05) from the background.

Mhp385 and Mhp384 Bind Heparin

P9720 and its paralogues Mhp493,19 Mhp271,16 and Mhp107,18 and the P102 paralogue Mhp68315 bind heparin. Since GAGs sensitive to heparinase are found along the surface of porcine respiratory cilia,50 our prevailing hypothesis is that members of the P97 and P102 paralogue families bind cilia during the early stages of colonization by targeting proteoglycans decorated with highly sulfated heparin-like GAGs. To determine if recombinant proteins spanning Mhp385 and Mhp384 bind heparin, an analogue for the highly sulfated iduronic acid(1→4)-D-glucosamine disaccharide domains of heparan sulfate,51 dot blots and microtiter plate binding studies were performed using biotinylated porcine heparin (Figure 6). F2385, F3385, F2384, and F3384 bound heparin in dot blot and the microtiter plate binding assays (Figure 6A and B). F1384 displayed faint reactivity in heparin-binding dot blot assays and low absorbance values in microtiter plate heparin-binding assays, indicating that it is at best a weak heparin-binding protein. F4385 showed evidence of weak heparin-binding in the dot blot assays and low absorbance values in the microtiter plate heparinbinding assays. In contrast, F1385 bound heparin in microtiter plate binding assays, but only faint reactivity was observed in heparin dot blot assays. Since the absorbances of F1384 and F4385 in the heparin microtiter plate assays were low, indicating weak heparin-binding potential, they were not investigated further.

Figure 4. Trypsin sensitivity of Mhp385 and Mhp384 in M. hyopneumoniae cells. Whole cell lysates from freshly cultured M. hyopneumoniae strain 232 cells treated with a range of trypsin concentrations (μg/mL), as listed at the top of the gel, were subjected to SDS-PAGE and stained with GelCode Blue (A) or transferred onto PVDF for immunoblot analyses. (B−E) Immunoblots probed with antiserum raised against F1385 (1:500), F4385 (1:1000), F1384 (1:500), and F3384 (1:500), respectively. (F) Control immunoblot probed with antiserum (1:5000) against cytosolic ribosomal protein L7/L12.21 Molecular weight markers are indicated on the left.

Immunoblots of lysates from trypsin digested M. hyopneumoniae cells showed that the Mhp385 preprotein (P115385) and the Mhp385 and Mhp384 cleavage fragments (P88385, P27385, P60384, and P50384) were readily digested by trypsin (Figures 4B−E). P115385 and the C-terminal cleavage fragments P27385 and P50384 were more susceptible to trypsin than the N-terminal cleavage fragments P88385 and P60384. Antiserum against the ribosomal protein L7/L12 was used to control for cell lysis.21 L7/L12 protein was identified at trypsin concentrations up to 1931



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Figure 6. Recombinant protein fragments of Mhp385 and Mhp384 bind heparin. (A) Dot blot of F1−F4385 and F1−F3384 recombinant proteins stained with amido black or probed with biotinylated heparin or streptavidin peroxidase only. (B) Binding curves of biotinylated heparin to F1−F4385 and F1−F3384 recombinant proteins immobilized on a microtiter plate. (C) Specific biotinylated heparin-binding curves to F1385, F2385, F3385, F2384, and F3384 recombinant proteins. (D−H) Competitive-binding assays for F1385, F2385, F3385, F2384, and F3384, respectively, with biotinylated heparin and various unlabeled sulfated polysaccharides: heparin (■), fucoidan (▲), porcine mucin type II (●), chondroitin sulfate A (▼), and chondroitin sulfate B (◆). All data represent the mean and standard error of triplicate readings from two independent experiments (n = 6); except for the competition assays, a representative experiment of two independent experiments is shown (n = 3).

equilibrium binding dissociation constant (KD) for heparin to each recombinant protein was calculated: F1385, 19 ± 2.6 nM; F2385, 2.7 ± 0.15 nM; F3385, 16 ± 1.7 nM; F2384, 26 ± 2.2 nM; and F3384, 8.9 ± 2.6 nM. To provide some indication of the type of interaction occurring between the recombinants and

Heparin-binding to F1385, F2385, F3385, F2384, and F3384 was dose-dependent and saturating (Figure 6C). Unlabeled heparin effectively inhibited biotinylated heparin binding to F1385, F2385, F3385, F2384, and F3384, indicating that the interaction was specific (Figure 6C). From the specific heparin-binding curves, the 1932



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Disordered regions are flexible and therefore more accessible to proteases.53 Notably, however, the cleavage site in Mhp107 does not fall within a predicted region of disorder. The inefficient MHJ_0194 cleavage site that removes the C-terminal 28 kDa R2-containing region of the adhesin occurs within a coiled-coil region. As further cleavage sites are mapped in surface proteins of M. hyopneumoniae, we will examine the association between protein disorder and endoproteolytic cleavage. A P97 paralogue that migrates at a mass equivalent to its predicted mass (of 115 kDa) was identified here for the first time. Western blots of whole cell lysates from M. hyopneumoniae strain 232 cells probed with F1385−F4385 antisera identified a 115 kDa protein, and LC−MS/MS analyses generated peptide coverage spanning the length of P115385. These data confirm that a proportion of the Mhp385 preprotein remains intact and that the cleavage event that generates P88385 and P27385 is inefficient. Trypsin digestion studies show P115385 is accessible on the cell surface of M. hyopneumoniae. The cleavage site in Mhp385 that generates P88385 and P27385 displays the sequence 761LNV↓AVS766, which bears no sequence resemblance to the S/T-X-F↓X-D/E cleavage motif within Mhp384 and other paralogues of P97 and P102.15 This suggests that Mhp385 is processed by a different protease. The cleavage site 859ATNT↓NTNTGFS869 in the C-terminal region of MHJ_019423 also bears no sequence identity to the S/T-X-F↓X-D/E consensus motif or to the Mhp385 cleavage site sequence. Therefore, cleavage at this site is likely to be performed by a different protease again. This cleavage event in MHJ_0194 is likely to be significant in a biological context because it results in the separation of the R1 cilium-binding domain from the R2 repeat region and effectively destroys a heparin-binding site.20 Collectively, our data suggest that endoproteolytic processing of the P97 and P102 paralogues involves at least three proteases. Both Mhp385 and Mhp384 contain several more S/T-X-F↓ X-D/E-like motifs. Further research is required to ascertain if cleavage is occurring at these sites. The cleavage products identified in the current study were the strongly reactive protein bands, other than Mhp385 preprotein (P115385), identified by immunoblotting, whose identities were confirmed by LC−MS/MS. Immunoblots of whole cell lysates of M. hyopneumoniae probed with anti-F4385 serum display a faint ladder suggesting further cleavage of Mhp385, cross reactivity with other R1-containing M. hyopneumoniae proteins, or a combination of both. Future studies examining enriched fractions of the M. hyopneumoniae proteome will assist in the identification and characterization of further cleavage fragments. It is also possible that these cleavage sites are not accessible to the protease(s) due to protein folding or the presence (or absence) of post-translational modifications surrounding cleavage motifs. It is clear from data presented here and with other P97 and P102 paralogue family members13−15,17−19,21,23 that processing is complex and strain-specific. Further research is needed to refine our understanding of surface adhesin processing and the precise role it plays in pathogenesis. F4385 does not bind cilia and only showed evidence of weak heparin-binding. Apart from 20 amino acids missing at the N-terminus of the fragment, F4385 is identical to P27385. Therefore, in this study, we identify only weak heparin-binding as a function of P27385; however, the weak binding suggests that this is not the main function of this cleavage fragment. The release of P27385 from the C-terminus of Mhp385 is likely to be important because the molecule contains a short R1-like motif comprised of four tandem pentapeptide repeats. The cilium

biotinylated heparin, competitive-binding assays were performed using unlabeled heparin, fucoidan, chondroitin sulfates A and B, and porcine intestinal mucin (Figure 6D-H). Consistent with previous studies,15,16 fucoidan, a highly sulfated polysaccharide extracted from seaweed, was most efficient at inhibiting the F2385, F2384, and F3384 interaction with heparin. At 600 μg/mL (30 times the concentration of heparin), porcine intestinal mucin reduced the adherence of heparin to F1385, F3385, F2384, and F3384 by at least 30%. Chondroitin sulfates A and B did not inhibit adherence of F2385, F2384, and F3384 to heparin, and chondroitin sulfate A was a minor inhibitor of heparin binding to F1385 and F3385.

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DISCUSSION Adherence to host tissues is an early and essential step in pathogenesis and a complex molecular interplay between bacterial adhesins and host receptor molecules. Here we show that the P97 and P102 paralogues, Mhp385 and Mhp384, respectively, are expressed during broth culture and undergo endoproteolytic cleavage, generating P88385 and P27385, and P60384 and P50384 on the surface of M. hyopneumoniae cells. On the basis of the binding abilities of the recombinant Mhp385 and Mhp384 fragments expressed in this study, P88385 and P50384 bind heparin and porcine cilia. The sequences of recombinant F1384 and half of F2384 make up P60384; therefore, the location of the heparin-binding site(s) within F2384 will determine the affinity P60384 has for heparin. However, this cleavage fragment has at least one cilium-binding site. P27385 was only shown to be a weak heparin-binder. The affinity of the recombinant fragments F1385, F2385, F3385, F2384, and F3384 to heparin is similar to that reported for other M. hyopneumoniae heparin-binding P97 and P102 paralogues and the Mycoplasma gallisepticum protein MG1142.15,16,18,19,52 The fact that Mhp385 and Mhp384 bind to cilia and heparin, combined with the presence of heparinase sensitive GAGs along the surface of porcine respiratory cilia,50 means that they are both likely to be important to the ability of M. hyopneumoniae to colonize the porcine respiratory tract. The P97 and P102 paralogue families are targets of endoproteolytic processing events.13−15,17−19,21,23 Prior to this study, a P97 or P102 paralogue at a mass consistent with its deduced amino acid sequence had not been found by MS, indicating that, at least in vitro, cleavage is efficient and critical for protein function. Recent studies comparing the endoproteolytic cleavage sites in MHJ_0194 (P97 homologue), MHJ_0195 (P102 homologue), and the P97 and P102 paralogues Mhp493 (P216) and Mhp683 (P135), respectively, showed that most endoproteolytic cleavage events occur at a phenylalanine residue (−1 position) with an alcohol-containing residue in the −3 position and an acidic (D/E) residue in the +2 position.15 Cleavage at these S/T-X-F↓XD/E sites is invariably efficient. Here, we show that cleavage sites in Mhp384 and Mhp107 conform to the S/T-X-F↓X-D/E motif. LC−MS/MS revealed that both cleavage sites have a phenylalanine residue at the −1 position and an acidic residue in the +2 position and possess the sequences 527ILF↓NEE532 and 197TNF↓SDQ202, respectively. The location of the endoproteolytic cleavage site in Mhp384 and MHJ_0195, like Mhp683 and Mhp493,15 was localized within a disordered region spanning more than 40 amino acids. Our inability to find evidence of the Mhp384 preprotein by MS or Western blotting indicates that cleavage is efficient. Our studies show that the primary amino acid sequence and the degree of protein disorder encompassing, or in close proximity to, the site of endoproteolysis appear to influence protease cleavage behavior. 1933



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ACKNOWLEDGMENTS A.T.D. is the recipient of an Australian Postgraduate Award. This work was partly funded by ARC-Linkage Grant LP776711 and a grant from the McGarvie Smith Trust to S.P.D.

adhesins Mhp183 (P97) and Mhp271 are the only other molecules in the four sequenced strains of M. hyopneumoniae to contain a R1 repeat region. Although the R1 repeat region in Mhp385 does not contain a sufficient number of tandem R1 repeats for cilium-binding, the P97 R1 region is an immunogenic domain,54 and R1 repeat regions may also be a source of surface variation with the number of tandem repeats in P97 and Mhp271 varying between field strains.16,42−44 The function of the R1-like domain in Mhp385 is currently unknown, although it contains the minimum number of tandem pentapeptide repeats sufficient to bind the mAb F2G5.41 This repeat region also shares high similarity with porcine collagen type VI (α3 chain), which implicates that molecular mimicry may be utilized as a means of avoiding host immune system recognition. P27385 remains associated with the surface of M. hyopneumoniae cells despite the absence of anchorage motifs and transmembrane domains. Members of the P97 and P102 paralogue families have been shown to be multifunctional proteins.14−21 We add Mhp385 and Mhp384 to that list with their ability to bind cilia and GAGs. Gene families are common among the mycoplasmas, and many encode surface exposed lipoproteins that are purported to play a role in immune evasion.55−57 Most lipoprotein family members are transcriptionally silent, with a single lipoprotein family member dominating the cell surface architecture.58 Phase and antigenic size variation of M. hyopneumoniae lipoproteins have not been reported, and we have not identified evidence for such a mechanism from our proteome analyses. Only a few genes in the M. hyopneumoniae genome display tandem repeat sequences.1,59 Clearly, M. hyopneumoniae does not appear to employ the same evasion strategies as reported for many other Mycoplasma species. The surface architecture of M. hyopneumoniae is potentially quite variable, with P97, P102, and their paralogues being the specific target of several proteases. Genes encoding members of the P97 and P102 paralogue families are derived from gene fusions with unrelated sequences,1 and the C-terminal regions of most P97 and P102 paralogues are novel. Our cumulative data suggest that processing of P97, P102, and their paralogues generates N-terminal endoproteolytic fragments, that retain a transmembrane domain and remain tethered to the membrane, and C-terminal cleavage fragments, often novel in sequence, that retain a presence on the surface of M. hyopneumoniae despite lacking known anchorage motifs. All members of the P97 and P102 paralogue families characterized to date are targets of endoproteolytic cleavage generating fragments that play essential roles in binding cilia, ECM molecules, and circulatory host proteins. Consequently, processing may represent a novel mechanism to generate surface diversity. Further studies are needed to gain an understanding of the proteases involved in adhesin processing and the circumstances that govern the efficiency of cleavage.

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REFERENCES

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