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

Abstract

BackgroundActinobacillus 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.ResultsSixty 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.ConclusionsThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-015-0199-8) contains supplementary material, which is available to authorized users.

Outer Membrane Lipoprotein Lip40 Modulates Adherence, Colonization, and Virulence of Actinobacillus pleuropneumoniae

“…A putative lipoprotein, Lip40, was identified in our previous study ( Hu et al, 2015 ). It is located on the outer membrane and is stress responsive and immunoprotective.…”

“…Lipoproteins are important in many aspects of virulence of pathogenic bacteria, including adhesion, invasion, colonization, immune evasion, and immunomodulation ( Kovacs-Simon et al, 2011 ). Although many lipoproteins are predicted in A. pleuropneumoniae ( Chung et al, 2007 ; Hu et al, 2015 ), the functions of these putative lipoproteins in the physiological and pathogenic activities have rarely been investigated.…”

Genome-wide screening of lipoproteins in Actinobacillus pleuropneumoniae identifies three antigens that confer protection against virulent challenge

“…Blood samples were collected from animals (the tail vein for mice and the front cavity vein of pigs) 1 d before each immunization and before challenge. The levels of serum antibodies against target proteins were measured as described previously with minor modifications 18 . Enzyme-linked immunosorbent assay (ELISA) plates were coated with appropriate amounts of purified proteins (0.2-0.3 μg/well for each protein), and blocked with PBST (PBS with 0.05% Tween 20) plus 5% skimmed milk for 1 h. Serum samples were serially diluted and added to coated ELISA plates, and incubated at 37 °C for 30 min.…”

“…In addition, many bacterial lipoproteins are protective antigens and good candidates for vaccine development 16 . A number of lipoproteins were predicted from A. pleuropneumoniae in previous studies 17,18 . To evaluate the potential of these lipoproteins as vaccine candidates, 55 lipoproteins were cloned from the genome of A. pleuropneumoniae JL03 (serovar 3) and expressed in Escherichia coli.…”

“…Based on our previous bioinformatics prediction, 60 lipoproteins were identified from the A. pleuropneumoniae JL03 (Table 1) genome for further investigation. Since lipoproteins Lip40 and PalA have been analyzed before 18,19 , here, the remaining 58 open reading frames were amplified by PCRs (Table S1) and E. coli expression vectors were constructed, to generate recombinant proteins. Of the 58 genes cloned, 47 (81%) were expressed successfully.…”