Proteomic survey of the pathogenic Mycoplasma hyopneumoniae strain 7448 and identification of novel post-translationally modified and antigenic proteins

Abstract

Mycoplasma hyopneumoniae is an important pathogen for pigs, being the causative agent of enzootic pneumonia. Recently, the genome sequences of three strains, J, 7448 and 232 have been reported. Here, we describe the results of a proteomic analysis, based on two-dimensional gel electrophoresis of soluble protein extracts, immunoblot and mass spectrometry, which was carried out aiming the identification of gene products and antigenic proteins from the M. hyopneumoniae pathogenic strain 7448. A preliminary M. hyopneumoniae proteome map in two pH ranges (3–10 and 4–7) was produced. A total of 31 different coding DNA sequences (CDSs), including three hypothetical ones, were experimentally verified with the identification of the corresponding protein products by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. According to the Clusters of Orthologous Groups (COG) functional classification, the identified proteins were assigned to the groups of metabolism (13), cellular processes (5) and information and storage processing (4). Nine of the identified proteins were not classifiable by COG, including some related to cytoadherence and possibly involved in pathogenicity. Moreover, at least five highly antigenic proteins of M. hyopneumoniae were identified by immunoblots, including four novel ones (a heat shock protein 70, an elongation factor Tu, a pyruvate dehydrogenase E1-beta subunit and the P76 membrane protein). The now available proteome map is expected to serve as a reference for comparative analyses between M. hyopneumoniae pathogenic and non-pathogenic strains, and for methabolic studies based on cells cultured under modified conditions.

Introduction

Mycoplasma hyopneumoniae is highly infective for swines and is the causative agent of enzootic pneumonia (EP), a disease characterized by sporadic, dry and non-productive cough, retarded growth and inefficient food conversion (Maes et al., 1996). EP is responsible for major economical losses in pig industry. Despite presenting low direct mortality rates, it causes increased susceptibility to secondary respiratory infections, due to the M. hyopneumoniae-associated deactivation of mucociliary functions.

There have been several efforts to identify and characterize M. hyopneumoniae antigens (see, for example, Subramaniam et al., 2000, Yang et al., 2005, Castro et al., 2006, Chen et al., 2006, Conceição et al., 2006, Meens et al., 2006). The recently published genome sequences of three M. hyopneumoniae strains, two pathogenic (232 and 7448) and one non-pathogenic (J) (Minion et al., 2004, Vasconcelos et al., 2005), will contribute to such efforts, accelerating the process of identification of novel antigenic proteins. These antigens, besides being potentially useful for immunodiagnosis and/or vaccination, may be relevant as virulence factors (Wassenaar and Gaastra, 2001, Ferreira and Castro, 2007), and their characterization may help to elucidade mechanisms of pathogenicity. Regarding immunodiagnosis, there is an obvious need for better antigens, in order to improve the serological methods currently used for pig herd surveying, an important step prior to preventive or therapeutical measures (Thacker, 2004, Kim et al., 2006, Lorenzo et al., 2006).

A proteomics approach based on two-dimensional gel electrophoresis (2DE), mass spectrometry (MS) and genomic data is a powerful tool for genome expression profiling. Proteome analysis has proven to be useful for bacteria, as a complementary method to support genome annotation and protein identification (Ueberle et al., 2002, Jaffe et al., 2004), and to characterize antigenic, differentially expressed or post-translationally modified proteins (Lebeau et al., 2005). 2DE mapping of M. hyopneumoniae proteins will improve genome annotation and help to characterize the extent of post-translational modifications and to identify potential virulence factors.

In this work, a preliminary 2DE map of M. hyopneumoniae is described and the repertoire of proteins identified by tandem MS is discussed. Moreover, we identified novel antigenic proteins from the M. hyopneumoniae pathogenic strain 7448 with potential for use in diagnosis.