Surface Polysaccharide Mutants Reveal that Absence of O Antigen Reduces Biofilm Formation of Actinobacillus pleuropneumoniae

Abstract

c Actinobacillus pleuropneumoniae is a Gram-negative bacterium belonging to the Pasteurellaceae family and the causative agent of porcine pleuropneumonia, a highly contagious lung disease causing important economic losses. Surface polysaccharides, including lipopolysaccharides (LPS) and capsular polysaccharides (CPS), are implicated in the adhesion and virulence of A. pleuropneumoniae, but their role in biofilm formation is still unclear. In this study, we investigated the requirement for these surface polysaccharides in biofilm formation by A. pleuropneumoniae serotype 1. Well-characterized mutants were used: an Oantigen LPS mutant, a truncated core LPS mutant with an intact O antigen, a capsule mutant, and a poly-N-acetylglucosamine (PGA) mutant. We compared the amount of biofilm produced by the parental strain and the isogenic mutants using static and dynamic systems. Compared to the findings for the biofilm of the parental or other strains, the biofilm of the O antigen and the PGA mutants was dramatically reduced, and it had less cell-associated PGA. Real-time PCR analyses revealed a significant reduction in the level of pgaA, cpxR, and cpxA mRNA in the biofilm cells of the O-antigen mutant compared to that in the biofilm cells of the parental strain. Specific binding between PGA and LPS was consistently detected by surface plasmon resonance, but the lack of O antigen did not abolish these interactions. In conclusion, the absence of the O antigen reduces the ability of A. pleuropneumoniae to form a biofilm, and this is associated with the reduced expression and production of PGA.A ctinobacillus pleuropneumoniae is a Gram-negative bacterium belonging to the Pasteurellaceae family and is the causative agent of porcine pleuropneumonia, a disease causing important economic losses to the swine industry worldwide (1). Several virulence factors of A. pleuropneumoniae have been identified. These factors include the Apx toxins, iron uptake systems, and surface polysaccharides. These polysaccharides are divided into three categories: the lipopolysaccharides (LPS), the capsular polysaccharides (CPS), and the poly-N-acetyl-D-glucosamine polymer (PGA) present in the biofilm matrix (2-4).LPS are large biomolecules composed of three well-defined regions: (i) lipid A, anchored in the outer membrane; (ii) the core oligosaccharide; and (iii) the O antigen, a polysaccharide consisting of repeating units. Altman and coworkers described the structure of the A. pleuropneumoniae serotype 1 O antigen to be branched tetrasaccharide repeating units composed of two Lrhamnopyranosyl residues, one D-glycopyranosyl residue, and one 2-acetamido-2-deoxy-D-glucose residue (5). The LPS inner core oligosaccharide is relatively conserved among A. pleuropneumoniae serotype 1, 2, 5a, and 5b strains and is composed of 2-keto-3-deoxyoctulosonic acid and heptose, whereas the outer core oligosaccharide is variable among serotypes and is generally made of a variable number of branching hexoses (6). The A. pleuropneumoniae LPS has been identified to b...

The Helicobacter pylori biofilm involves a multi-gene stress-biased response including a structural role for flagella

“…To further characterize the EPS that composed the SS1 biofilm matrix, BOBO-3 and FilmTracer SYPRO Ruby biofilm matrix stains were used to stain extracellular DNA (eDNA) and extracellular proteins, respectively as described previously (29,30). Both of these molecules extensively stained the biofilm EPS, consistent with the idea that the H. pylori SS1 biofilm matrix contains significant amount of eDNA and extracellular proteins (Fig.…”

Impact of Actinobacillus pleuropneumoniae biofilm mode of growth on the lipid A structures and stimulation of immune cells

“…pleuropneumoniae was grown in 6-well plates (Costar® 3516, Corning, Corning, NY, USA) as previously described. 30 After 4 hours in culture, media containing non-attached, planktonic bacteria were removed (the planktonic fraction). Attached bacteria were washed twice with phosphate buffered-saline (PBS) to remove remaining planktonic cells.…”

“…[54][55][56] They are frequently associated with chronic disease 25,26,57 and represent a serious therapeutic challenge, since bacteria within biofilm exhibit enhanced antibiotic tolerance and are more resistant to clearance by the immune system. 3,11,24,57 We recently demonstrated that LPS has an important role in biofilm formation in A. pleuropneumoniae, and that a biofilm matrix exopolysaccharide, poly-N-acetyl-Dglucosamine, can interact specifically with LPS 30 . However, the impact on host cells of LPS and particularly lipid A in biofilms remains to be determined.…”

Structural investigation on WlaRG from Campylobacter jejuni: A sugar aminotransferase

“…Likewise, it has been reported that the O‐antigen is essential for full virulence of Yersinia enterocolitica O:8 and, strikingly, its absence affects the expression of other Yersinia virulence factors . As yet another example, a recent study has demonstrated that an O‐antigen mutant of Actinobacillus pleuropneumoniae serotype 1 is severely defective in biofilm formation …”

“…4 As yet another example, a recent study has demonstrated that an O-antigen mutant of Actinobacillus pleuropneumoniae serotype 1 is severely defective in biofilm formation. 5 Through the research efforts of various laboratories over the last fifteen years, many of the biosynthetic pathways for the production of unusual sugars found in bacterial O-antigens have begun to be revealed in exquisite biochemical and structural detail. 6 Some Gram-negative bacteria such as Campylobacter jejuni, however, have a modified LPS lacking the O-antigen, which is referred to as the lipooligosaccharide or LOS.…”