Identification of genes involved in biosynthesis of &lt;I&gt;Actinobacillus pleuropneumoniae&lt;/I&gt; serotype 1 O-antigen and biological properties of rough mutants

Labrie, Josée; Rioux, Stéphane; Wade, Mary Margaret; Champlin, Franklin R.; Holman, Steven C.; Wilson, W. William; Savoye, C.; Kobisch, Marylène; Suquet, Marc; Galarneau, Catherine; Jacques, Mario

doi:10.1179/096805102125000065

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“…The samples were purified on a Bio-Gel P-2 column as described previously (12). Core fractions (fractions 19 -24) eluting off the column slightly after the O-chain (fractions [15][16][17] were combined and lyophilized.…”

Section: Methodsmentioning

confidence: 99%

“…Characterization of the surface properties of these LPS mutants demonstrated that O-antigen deletions in rough mutants have no effect on the adhesion of the bacteria to frozen tracheal sections of pigs, whereas an intact core oligosaccharide region seems to be required for optimal adherence (7,13). Most interestingly, in vivo experiments in pigs showed that the core LPS mutant 5.1 that still produces an O-antigen was less virulent, whereas no difference was observed between the rough mutant 27.1 lacking O-antigen and the parent strain (13)(14)(15). These observations suggest that the LPS core oligosaccharide could play a major role in colonization and pathogenesis of A. pleuropneumoniae in pigs.…”

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confidence: 96%

“…Overall, our data indicate that the LPS of all the mutants tested exhibited the same capacity to stimulate cytokines production as the LPS of the parent strain. Susceptibility to Antimicrobial Peptides-The susceptibility test was carried out with the three LPS core mutants (5.1, CG1, and CG3), and the following two additional mutants of A. pleuropneumoniae serotype 1, previously characterized by our group, were also added to the study: an acapsular mutant 33.2 (34) and a rough LPS mutant 27.1 lacking O-antigen (13,15). The MIC values obtained (TABLE FIVE) showed that the acapsular mutant (33.2), the rough LPS mutant (27.1), and the wild type parent strain exhibited similar MICs for all the cationic peptides shown in TABLE FIVE, suggesting that the absence of O-antigen of the LPS or the absence of the capsule has no effect on the susceptibility of A. pleuropneumoniae serotype 1 to antimicrobial peptides.…”

Section: Structure and Properties Of A Pleuropneumoniae Lps Mutantsmentioning

confidence: 99%

“…We have previously generated four core LPS and seven rough LPS mutants of A. pleuropneumoniae serotype 1 by using a mini-Tn10 mutagenesis system, and the gene affected by the transposon was identified in each of the mutants (13)(14)(15). Characterization of the rough LPS mutants allowed us to identify a cluster of 13 genes involved in O-polysaccharide biosynthesis in A. pleuropneumoniae serotype 1 (15).…”

mentioning

confidence: 99%

“…Characterization of the rough LPS mutants allowed us to identify a cluster of 13 genes involved in O-polysaccharide biosynthesis in A. pleuropneumoniae serotype 1 (15). The genes affected in the core LPS mutants were galU in mutant 5.1 (13), which encodes a UTP-␣-D-glucose-1-phosphate uridylyltransferase, a gene involved in outer core elongation with galactose in mutant CG1 (14) and a gene coding for a D-glycero-D-manno-heptosyltransferase in mutants CG3 and CG5 (14).…”

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confidence: 99%

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Truncation of the Lipopolysaccharide Outer Core Affects Susceptibility to Antimicrobial Peptides and Virulence of Actinobacillus pleuropneumoniae Serotype 1

Ramjeet

Deslandes

Michael

et al. 2005

Journal of Biological Chemistry

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We reported previously that the core oligosaccharide region of the lipopolysaccharide (LPS) is essential for optimal adhesion of Actinobacillus pleuropneumoniae, an important swine pathogen, to respiratory tract cells. Rough LPS and core LPS mutants of A. pleuropneumoniae serotype 1 were generated by using a mini-Tn10 transposon mutagenesis system. Here we performed a structural analysis of the oligosaccharide region of three core LPS mutants that still produce the same O-antigen by using methylation analyses and mass spectrometry. We also performed a kinetic study of proinflammatory cytokines production such as interleukin (IL)-6, tumor necrosis factor-␣, IL1-␤, MCP-1, and IL8 by LPS-stimulated porcine alveolar macrophages, which showed that purified LPS of the parent strain, the rough LPS and core LPS mutants, had the same ability to stimulate the production of cytokines. Most interestingly, an in vitro susceptibility test of these LPS mutants to antimicrobial peptides showed that the three core LPS mutants were more susceptible to cationic peptides than both the rough LPS mutant and the wild type parent strain. Furthermore, experimental pig infections with these mutants revealed that the galactose (Gal I) and D,D-heptose (Hep IV) residues present in the outer core of A. pleuropneumoniae serotype 1 LPS are important for adhesion and overall virulence in the natural host, whereas deletion of the terminal GalNAc-Gal II disaccharide had no effect. Our data suggest that an intact core-lipid A region is required for optimal protection of A. pleuropneumoniae against cationic peptides and that deletion of specific residues in the outer LPS core results in the attenuation of the virulence of A. pleuropneumoniae serotype 1.

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Section: Methodsmentioning

confidence: 99%

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confidence: 96%

Section: Structure and Properties Of A Pleuropneumoniae Lps Mutantsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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Truncation of the Lipopolysaccharide Outer Core Affects Susceptibility to Antimicrobial Peptides and Virulence of Actinobacillus pleuropneumoniae Serotype 1

Ramjeet

Deslandes

Michael

et al. 2005

Journal of Biological Chemistry

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

et al. 2016

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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...

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Identification of genes involved in biosynthesis of <I>Actinobacillus pleuropneumoniae</I> serotype 1 O-antigen and biological properties of rough mutants

Cited by 2 publications

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Truncation of the Lipopolysaccharide Outer Core Affects Susceptibility to Antimicrobial Peptides and Virulence of Actinobacillus pleuropneumoniae Serotype 1

Truncation of the Lipopolysaccharide Outer Core Affects Susceptibility to Antimicrobial Peptides and Virulence of Actinobacillus pleuropneumoniae Serotype 1

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

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