Isolation and characterization of mini-Tn<i>10</i> lipopolysaccharide mutants of <i>Actinobacillus pleuropneumoniae</i> serotype 1

Abstract

Lipopolysaccharide (LPS) has previously been identified as the major adhesin of Actinobacillus pleuropneumoniae involved in adherence to porcine respiratory tract cells. The purpose of the present study was to isolate and characterize mutants in LPS biosynthesis by using a mini-Tn10 transposon mutagenesis system. Seven mutants appeared to possess a rough LPS (among which two had similar Southern blot profiles) while one mutant (#5.1) expressed the high-molecular-mass LPS, but as visualized by Tricine SDS-PAGE, showed an additional band in the core-lipid A region. The LPS mutants showed sensitivity to pig serum to various degrees, while the parent strain was serum-resistant. Use of piglet frozen tracheal sections indicated that, surprisingly, the rough LPS mutants adhered similarly or in greater numbers than the parent strain. However, the LPS mutant #5.1 adhered significantly less than the parent strain and was also less virulent in pigs. The gene affected by mini-Tn10 in LPS mutant #5.1 is galU, the structural gene for UTP-alpha-D-glucose-1-phosphate uridylyltransferase, involved in LPS core biosynthesis. Complementation analysis confirmed that the phenotypic characteristics of LPS mutant #5.1 are the result of the inactivation of the galU gene. Our data suggest that although the presence of O-antigen does not seem to be essential, an intact core-lipid A region might be required for adherence of A. pleuropneumoniae to porcine respiratory tract cells. To the best of our knowledge, these mutants represent the first isogenic mutants of A. pleuropneumoniae defective in LPS biosynthetic genes.

Transcriptional profiling of Actinobacillus pleuropneumoniae during the acute phase of a natural infection in pigs

“…Finally, the kpsF gene, coding for an arabinose-5-phosphate isomerase, should also be mentioned since it is involved in the pathway responsible for the synthesis of 2-keto-3deozyoctulosonic acid (Kdo), which is present in the A. pleuropneumoniae LPS core oligosaccharide [52] and capsule of serotype 5 [53]. Studies in our laboratory have shown that it is the core oligosaccharide that is responsible for the previously observed LPS-associated adhesion mechanism [54,55]. …”

Virulence factors ofActinobacillus pleuropneumoniaeinvolved in colonization, persistence and induction of lesions in its porcine host

“…Glycosphingolipids were identified as receptors in respiratory epithelial cells [1]. Although the involvement of lipopolysaccharides in adherence has been questioned [97], and the adherence of A. pleuropneumoniae to lung epithelial cells has been found to be lipopolysaccharide-independent [18], a study using mutant strains with altered lipopolysaccharide structures confirmed their role in adhesion [93]. The oligosaccharide core of lipopolysaccharides seems to play a role in this phenomenon.…”

“…Capsular polysaccharides and/or lipopolysaccharides of A. pleuropneumoniae exert antiphagocytic properties [61, 97–99, 113, 118, 120]. Furthermore, Ward et al.…”

Immunoprotective Efficacy of Six In vivo-Induced Antigens against Actinobacillus pleuropneumoniae as Potential Vaccine Candidates in Murine Model

“…RnhB ( Itaya, 1990 ) and HflX ( John et al, 2005 ) are ribonuclease H and GTP-binding protein, respectively. GalU, UTP-α- D -glucose-1-phosphate uridylyltransferase is involved in the LPS core biosynthesis ( Rioux et al, 1999 ), and GalU and GalT are enzymes involved in metabolism of galactose ( Ebrecht et al, 2015 ), all essential for the virulence of different bacterial pathogens ( Chai et al, 2012 ; Caboni et al, 2015 ).…”