Identification and characterization of a novel stress-responsive outer membrane protein Lip40 from Actinobacillus pleuropneumoniae

Xuehe Hu, Hao Yan, Ke Liu, Jiansheng Hu, Chao Qi, Jihong Yang, Yanli Liu, Jin Zhao, Jinlin Liu

Actinobacillus pleuropneumoniae, a Gram-negative bacterium, is the causative agent of porcine pleuropneumonia, a highly contagious and often fatal disease. Because current vaccines confer limited protection against A. pleuropneumoniae infection, the development of more effective vaccines is urgently required. The identification of immunogenic and protective antigens, such as an outer-membrane lipoprotein, will advance this purpose. Sixty putative lipoproteins were predicted from the genomic sequence of A. pleuropneumoniae using multiple algorithms. Here, we focused on the characteristics of the putative lipoprotein Lip40 from A. pleuropneumoniae strain SLW01 (serovar 1). Lip40 shares sequence similarity with many bacterial lipoproteins, and the structural prediction of Lip40 suggests that it is similar to A. pleuropneumoniae TbpB. The N-terminus of Lip40 contains an interesting tandemly repeated sequence, Q(E/D/P)QPK. Real-time RT-PCR indicated that the expression of lip40 was significantly upregulated at 42 °C, at 16 °C, and under anaerobic conditions. Recombinant Lip40 (rLip40) produced in Escherichia coli BL21(DE3) was specifically recognized by porcine convalescent serum directed against A. pleuropneumoniae. Lip40 was confirmed to localize at the bacterial outer membrane, and its expression was significantly stimulated when A. pleuropneumoniae was cultured under various stress conditions. Lip40 also protected 75 % of mice from fatal virulent A. pleuropneumoniae infection. The immunogenic outer-membrane protein Lip40 is stress responsive, protects mice against infection, and might be a virulence determinant. Further investigation of Lip40 should expedite vaccine development and provide insight into the pathogenesis of A. pleuropneumoniae.