Molecular characterization and T and B cell epitopes prediction of Mycoplasma synoviae 53 strain VlhA hemagglutinin

Mycoplasma sinoviae is a major pathogen of poultry causing synovitis and respiratory infection. M. synoviae hemagglutinin (VlhA) is a lipoprotein encoded by related multigene families that appear to have arisen by horizontal gene transfer. It is an abundant immunodominant surface protein involved in host-parasite interaction mediating binding to host erythrocytes. Herein, we have performed in silico analysis of the vlhA gene product from the Mycoplasma synoviae 53 strain and compared it to the VlhA protein of M. synoviae WUV1853 strain. The VlhA of the M. synoviae 53 strain possesses 569 amino acids and showed 85% identity with the VlhA protein of the M. synoviae WUV1853 strain. Further, a signal peptide was identified from amino acid M1 to D28 and a cleavage site between D28 and Q29, both located in the N-terminal domain of the molecule. Additionally, an insertion of PAPT amino acids was observed between T30-P35 and a deletion of the amino acids GTPGNP within the PRR region of the VlhA from the M. synoviae 53 strain, which may be related to its reduced virulence. Finally, we have identified 17 B cell epitopes and 22 T cells epitopes within the VlhA from the M. synoviae 53 strain. The B cell epitope S263-D277 and the T cell epitopes N45-N54 and G58-N67 showed 100% and 87-100% identity, respectively, with regions of VlhA protein of tested Mycoplasma synoviae and Mycoplasma galisepticum strains. Thus, these peptides represent new candidate molecules for the development of efficient diagnostic assays and new subunit vaccines.


Introduction
Mycoplasma synoviae is one of the smallest and simplest bacteria lacking a cell wall known to exist, and it is a major pathogen of chickens and turkeys, causing respiratory tract infection and arthritis worldwide (Kleven, 1997).Although the basis of mycoplasma pathogenicity remains unknown, it is widely accepted that most of the damage resulting from mycoplasma infections in humans and animals is due to host immune and inflammatory responses rather than to direct toxic effects of mycoplasma virulence factors (Razin et al., 1998).
M. synoviae isolates differ in their infectivity, tissue tropism and pathogenicity (Rottem, 2003).Many animal mycoplasmas depend on adhesion to host tissues for colo-nization and infection.In these mycoplasmas, adherence is the major virulence factor and adherence-deficient mutants are avirulent (Baseman and Trully, 1997).Current theory holds that mycoplasma remains attached to the surface of epithelial cells, although some mycoplasmas have evolved mechanisms for entering host cells that are not naturally phagocytic (Razin et al., 1998).The intracellular localization is obviously a privileged niche, well protected from humoral mechanisms of the host immune system and from the action of many antibiotics.The finding that some mycoplasmas can reside intracellularly opens up new horizons to the study of the role of mycoplasma and host surface molecules in invasion.Although, the ability of internalized mycoplasmas to multiply within the host cell remains to be convincingly demonstrated, reports describing mycoplasma invasive phenotypes have offered new insights into the potential virulence strategies employed by these bacteria (Rottem, 2003).
Escaping the host immune system is of critical importance to mycoplasma survival within its host.The major survival mechanisms that have been extensively studied are molecular mimicry and phenotype plasticity which ensure that mycoplasmas are not fully nor efficiently recognized by the host immune system (Markham et al., 1994;Wren, 2000).Molecular mimicry refers to antigenic epitopes that are shared by different mycoplasmas and host cells and they are considered as putative factors involved in the evasion of host defense mechanisms (Rottem, 2003).Mycoplasmas are also endowed with phenotypic plasticity defined as the ability of a single genotype to change its antigenic make-up to produce more than one morphology, physiological state, and/or behavior in response to environmental conditions (Rottem, 2003).The common way to achieve phenotype plasticity in mycoplasma is by antigenic variation.Additionally, membrane lipoproteins are the major components of intact mycoplasmas and are able to activate macrophages, thus playing an important role in cytokine production and consequently in the inflammatory response during infection (Chambaud et al., 1999).
VlhA is a variable protein encoded by the vlhA gene in M. synoviae that is post-translationally cleaved into the N-terminal lipoprotein fragment MSPB (Major Surface Protein B) and the C-terminal fragment MSPA (Major Surface Protein A) which is directly involved in hemadherence (Noormohammadi et al., 1997).There is only one copy of the complete vlhA gene in the genome of the M. synoviae WUV1853 strain.Other copies are not functional genes and lack the 5' end of the expressed gene (Noormohammadi et al., 1997(Noormohammadi et al., , 2000)).Comparing different M. synoviae strains, it was possible to observe differences in length and antigenic determinants of MSPB proteins (Noormohammadi et al., 1997).The complete genome sequence of Mycoplasma synoviae 53 strain revealed the organization of hemagglutinin genes with a single locus comprising 70 coding DNA sequences (CDS) (Vasconcelos et al., 2005).In this study, we have characterized the vlhA gene product from the Mycoplasma synoviae 53 strain and compared it to the VlhA protein of the Mycoplasma synoviae WUV1853 strain (Noormohammadi et al., 1997).The VlhA of the Mycoplasma synoviae 53 strain possesses 569 amino acids, a signal peptide and a cleavage site located in the Nterminal domain of the molecule.Additionally, using bioinformatic search tools, we have identified 17 B cell epitopes and 22 T cells epitopes that may be involved in host immune response against this microorganism.

DNA and amino acid sequences
The DNA and translated amino acid sequences of vlhA genes from Mycoplasma synoviae 53 (Vasconcelos et al., 2005) and Mycoplasma synoviae WUV1853 strains (Noormohammadi et al., 1997) were retrieved from GenBank under accession no.NC007294 and AF035624, respectively.The vlhA gene is located between the nucleotides of number 292135 and 293844 of the genome sequence of the Mycoplasma synoviae 53 strain sequenced by our group (Vasconcelos et al., 2005).

Characterization of M. synoviae VlhA by bioinformatics
These amino acid sequences for VlhA from Mycoplasma synoviae 53 and Mycoplasma synoviae WUV1853 strains were aligned by CLUSTALW Multiple Sequence Alignment available online.SOSUISignal and SOSUI were used to identify motifs in these proteins, such as peptide signal and hydrophobic domains.Additionally, SignalP 3.0 software was used for prediction of cleavage sites.

T and B cell epitopes prediction
The B cell epitope prediction was performed using the program Predicting Antigenic Peptides available online.The software for the detection of antigenic peptides is based on Kolaskar's and Tongaonkar's method previously described (Kolaskar and Tongaonkar, 1990).The T cell epitope prediction was performed using RANKPEP software.This software uses Position Specific Scoring Matrices (PSSMs) or profiles from a set of aligned peptides known to bind to a given MHC molecule as the predictor of MCH-peptide binding.We used the mouse MHC system H-2, as a model for this study and tested the I-A b and I-A k alleles for MHC class II.Herein, we have selected several peptides that had high scores of binding to these MHC class II alleles.Predicted T and B cell epitopes shared between M. synoviae 53 and M. synoviae WUV1853 strains were also analyzed for their identity with other Mycoplasma synoviae strains (Mycoplasma synoviae B133-96, B154-02, B2700, B31-88, B38-96-170, B94-91, J26-85, J151-85, K1, K4, K1968, K2581, K27, MS-H, TN/427, ULB925 and ULB925KF) and Mycoplasma gallisepticum strains (M.gallisepticum R and S6) using the BLAST computer program blastp.

Results and Discussion
Mycoplasma synoviae is a major pathogen of chickens and turkeys that causes great economic losses in intensive poultry production.This bacterium synthesizes hemagglutinin VhlA, an abundant immunodominant surface lipoprotein.In most M. synoviae strains, the hemagglutinin VlhA is cleaved into N-terminal region (MSPB) and a C-terminal region (MSPA), which mediates erythrocytes binding (Bencina et al., 2001).MSPB has been divided into two domains: a conserved and a variable region (Bencina et al., 2001).In different M. synoviae isolates, MSPB proteins differ in length as well as antigenic determinants defined by monoclonal antibodies and also differ in the insertion or deletion of amino acid sequences within the proline-rich re-peats (PRR) region which has been identified in immunodominant surface antigens involved in the interaction between pathogens and host cells (Noormohammadi et al., 1997;Bencina et al., 2001).A longer PRR region has been associated with higher invasiveness for the M. synoviae strain K1968 (Bencina et al., 2001).Further, M. synoviae clonal populations can synthesize size and antigenic variants of MSPB proteins and their expression can be associated with transition from HA + to HA -phenotype (Noormohammadi et al.,1997).
The 569 amino acid sequence of VlhA from the M. synoviae 53 strain and the 785 amino acid sequence of the VlhA protein derived from the M. synoviae WUV1853 strain were retrieved from the GenBank and aligned in order to compare their identity.VlhA protein of M. synoviae 53 strain showed 85% identity with VlhA of the M. synoviae WUV1853 strain, despite the difference in length (Figure 1).Amino acid sequence analysis performed by SOSUISignal and SignalP 3.0 computer programs resulted in the identification of a region encoding a signal peptide from amino acid M 1 to D 28 and a cleavage site between D 28 and Q 29 in the VlhA of the M. synoviae 53 strain that are also present and they correspond to the same positions in the VlhA of the M. synoviae WUV1853 strain.No variation in the signal peptide domains of both proteins was detected.The cleavage site which results in the MSPB and MSPA membrane antigens is located at S 306 in the VlhA of the M. synoviae 53 strain and at S 308 in the VlhA of the M. synoviae WUV1853 strain (Figure 2).Among the differences between the two sequences, there is an insertion of four amino acids (PAPT) after T 30 of VhlA from the M. synoviae 53 strain This insertion is similar to an insertion observed in the M. synoviae FMT strain which is considered mildly pathogenic (Bencina et al., 2001).The MSPB domain of the M. synoviae 53 strain has also a deletion of six amino acids within the proline-rich repeats (PRR) (GTPGNP) in the N-terminal region which correlates to amino acids G 54 to P 59 of the M. synoviae WUV1853 strain (Figures 1 and  2).This deletion may affect bacterial virulence.The live attenuated M. synoviae MS-H strain vaccine has a similar deletion of the amino acids PGNPGT within the PRR region  ( Noormohammadi et al., 2002).Since this deletion was not observed in the VlhA M. synoviae WUV1853 strain, we speculate that the M. synoviae WUV1853 strain may be more virulent than the M. synoviae 53 strain.Identification of immunodominant epitopes within a vaccine candidate antigen is extremely useful, since it is possible to formulate a vaccine composed of relevant epitopes from different antigens.In silico epitope predictions resulted in the identification of 17 B cell epitopes, ranging from 7 to 23 mers (Table 1), and 22 T cell epitopes of 9 mers (Table 2).The predicted epitopes were distributed along the entire protein sequence.Comparing the epitopes predicted from the VlhA protein sequence of the M. synoviae 53 strain with the M. synoviae WUV1853 strain, we observed that 13 of them are shared by both strains as indicated in Figure 1.Additionally, amino acids from S 195 to L 211 , K 345 to A 356 , and N 440 to E 446 represent epitopes that could be recognized by either T and B cells, as shown in Figure 1.These B and T cells epitopes can improve efficacy of these peptides in vaccine design.
B and T cell epitopes comprised in the VlhA protein and shared by M. synoviae 53 and M. synoviae WUV1853 strains were also compared to sequences of other Mycoplasma synoviae and Mycoplasma gallisepticum strains.These analyses revealed that the B cell epitope S 263 -D 277 and the T cell epitopes N 45 -N 54 and G 58 -N 67 showed 100% and 87-100% identity, respectively, with corresponding regions of the VlhA protein of Mycoplasma synoviae and Mycoplasma gallisepticum strains (Table 3  and 4).
Although in vivo or in vitro assays have to be performed to confirm these selected peptides, in silico epitope prediction has been used in many studies in the development of new immunodiagnostic and vaccine formulations (Panigada et al., 2002;Iwai et al., 2003;Fonseca et al., 2004).Identification of T and B cell epitopes on different Mycoplasma strains become even more relevant since evasion mechanisms used by these bacteria to escape host immune response are based on antigen mimicry and antigenic variability (Markham et al., 1994;Wren, 2000).Among B cell predicted epitopes, peptides E 94 -E 101 , T 165 -L 171 , S 195 -L 211 , A 220 -A 237 , S 263 -D 277 , K 345 -A 356 , T 364 -V 376 and M 440 -E 446 are shared between VhlA of M. synoviae 53 and M. synoviae WUV1853 strains (Figure 1).The S 263 -D 277 peptides represent the most conserved B cell epitope which possesses 100% identity with peptides of the VhlA from the M. synoviae K1968, MS-H, ULB925 and ULB925KF strains and also with M. gallisepticum S6 and R strains (Table 3).
Regarding T cells epitopes, N 45 -N 54 and G 58 -N 67 are the most conserved epitopes (with identity ranging from 87-100%) found in the VhlA of the majority of M. synoviae strains tested and also within the M. gallisepticum S6 strain (Table 4).Finally, the analysis performed here demon- *Protein regions containing B and T cell epitopes (Figure 1).strated that there are conserved B and T cell epitopes mainly in the N-terminal region of the VhlA protein from the M. synoviae 53 strain, which may represent potential targets for the development of new diagnostic assays and subunit vaccines.

266
Figure 1 -Comparison of the deduced amino acid sequences of VlhA of Mycoplasma synoviae strain 53 (GenBank accession number NC007294) and Mycoplasma synoviae strain WUV1853 (GenBank accession number AF035624).Asterisks indicate identical residues and gaps are indicated by dashes.Squares with dashed-lines represent T cell epitopes and squares with solid-lines represent B cell epitopes.

Figure 2 -
Figure 2 -Schematic representation of VlhA protein of M. synoviae 53 strain.Signal peptide region (SP) comprises amino acids from M 1 to D 28 .The MSPB region contains two proline-rich repeats region (PRR) and has a PAPT insertion at T 30 and a GTPGNP deletion at P 57 .D 28 and S 306 indicate signal peptide and VlhA cleavage site, respectively.

Table 1 -
B cell epitopes predicted in the VlhA protein of M. synoviae 53 strain.

Table 2
-T cell epitopes predicted in the VlhA protein of M. synoviae 53 strain.
a higher scores represent high affinity binding to MHC class II molecules.