The D3 Bacteriophage α-Polymerase Inhibitor (Iap) Peptide Disrupts O-Antigen Biosynthesis through Mimicry of the Chain Length Regulator Wzz in Pseudomonas aeruginosa

Taylor, Véronique L.; Udaskin, Molly L.; Islam, Salim T.; Lam, Joseph S.

doi:10.1128/jb.00903-13

Cited by 22 publications

(26 citation statements)

References 60 publications

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“…Islam et al suggested that the chain length of the Oag is determined by the interaction of Wzz and Wzy (40). Recent work of Taylor et al also suggested the direct interaction of Wzz and Wzy in the Oag biosynthesis pathway (41). In this study, for the first time, we were able to provide insight into the association of Wzy Sf and Wzz Sf in Oag biosynthesis.…”

Section: Discussionsupporting

confidence: 58%

Mutational Analysis of the Shigella flexneri O-Antigen Polymerase Wzy: Identification of Wzz-Dependent Wzy Mutants

2015

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The O-antigen (Oag) component of lipopolysaccharide (LPS) is a major virulence determinant of Shigella flexneri and is synthesized by the O-antigen polymerase, Wzy Sf . Oag chain length is regulated by chromosomally encoded Wzz Sf and pHS-2 plasmidencoded Wzz pHS2 . To identify functionally important amino acid residues in Wzy Sf , random mutagenesis was performed on the wzy Sf gene in a pWaldo-TEV-GFP plasmid, followed by screening with colicin E2. Analysis of the LPS conferred by mutated Wzy Sf proteins in the wzy Sf -deficient (⌬wzy) strain identified 4 different mutant classes, with mutations found in periplasmic loop 1 (PL1), PL2, PL3, and PL6, transmembrane region 2 (TM2), TM4, TM5, TM7, TM8, and TM9, and cytoplasmic loop 1 (CL1) and CL5. S higella flexneri lipopolysaccharide (LPS) is crucial for pathogenesis (1). LPS is located exclusively in the outer leaflet of the outer membrane (OM) and has three domains: (i) lipid A, a hydrophobic domain that anchors LPS to the OM; (ii) the core oligosaccharides, a nonrepeating oligosaccharide domain; and (iii) the O-antigen (Oag) polysaccharide, an oligosaccharide repeat domain (1, 2). The complete LPS structure with Oag chains is termed smooth LPS (S-LPS). However, the LPS structure lacking the Oag is termed rough LPS (R-LPS), and LPS with a single Oag tetrasaccharide repeat unit (RU) attached to the lipid A and core sugar is termed semirough LPS (SR-LPS) (3). S. flexneri is subdivided into various serotypes depending on the differences in the composition of the LPS Oag. So far, there are 17 known serotypes of S. flexneri (4). Except for S. flexneri serotype 6, the Oags of all the serotypes have the same polysaccharide backbone containing three L-rhamnose residues (Rha) and one N-acetylglucosamine (GlcNAc). This basic Oag structure is known as serotype Y. Addition of either glucosyl, O-acetyl, or phosphoethanolamine (PEtN) groups by various linkages to the sugars of the Y serotype tetrasaccharide repeat creates different S. flexneri serotypes (5-7). Oag is the protective antigen, as immunity to S. flexneri infection is serotype specific (8, 9). S-LPS confers resistance to complement (10) and colicins (11,12), and Y serotype Oag acts as a receptor to bacteriophage Sf6 (13).S. flexneri Oag biosynthesis occurs by the Wzy-dependent pathway. Most of the Oag biosynthesis genes (except wecA) of S. flexneri are located in the Oag biosynthesis locus between galF and his (6, 14). S. flexneri Oag biosynthesis occurs on either side of the inner membrane (IM). Initially, N-acetylglucosamine phosphate (GlcNAc-1-P) is transferred from UDP-GlcNAc by WecA to undecaprenol phosphate (Und-P) at the cytoplasmic side of the IM (5, 15, 16). RfbG and RfbF then add Rha residues from dTDPrhamnose (dTDP-Rha) to the GlcNAc (3, 17) to form the O unit. In the Wzy-dependent model of LPS assembly, the flippase protein Wzx translocates this O unit to the periplasmic side. At the periplasmic side, the O units are polymerized at the nonreducing end by the Oag polymerization protein Wzy via a block ...

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Section: Discussionsupporting

confidence: 58%

Mutational Analysis of the Shigella flexneri O-Antigen Polymerase Wzy: Identification of Wzz-Dependent Wzy Mutants

2015

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“…LPS is composed of a lipid A membrane anchor, a core oligosaccharide linker and a distal polysaccharide termed O-antigen, in the form of A and B bands (Taylor et al, 2013). Both WT and mutant forms of wzy and mucA genes were simultaneously found in the presently described mutants, suggesting that the mutation can reverse at a high rate.…”

Section: Cross-resistance and Reversibility Of Mutantsmentioning

confidence: 99%

Pseudolysogeny and sequential mutations build multiresistance to virulent bacteriophages in Pseudomonas aeruginosa

Latino

2016

Microbiology

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Coevolution between bacteriophages (phages) and their prey is the result of mutualistic interactions. Here, we show that pseudolysogeny is a frequent outcome of infection by virulent phages of Pseudomonas aeruginosa and that selection of resistant bacterial mutants is favoured by continuous production of phages. We investigated the frequency and characteristics of P. aeruginosa strain PAO1 variants resisting infection by different combinations of virulent phages belonging to four genera. The frequency of resistant bacteria was 10 25 for single phage infection and 10 26 for infections with combinations of two or four phages. The genome of 27 variants was sequenced and the comparison with the genome of the parental PAO1 strain allowed the identification of point mutations or small indels. Four additional variants were characterized by a candidate gene approach. In total, 27 independent mutations were observed affecting 14 genes and a regulatory region. The mutations affected genes involved in biosynthesis of type IV pilus, alginate, LPS and O-antigen. Half of the variants possessed changes in homopolymer tracts responsible for frameshift mutations and these phase variation mutants were shown to be unstable. Eleven double mutants were detected. The presence of free phage DNA was observed in association with exclusion of superinfection in half of the variants and no chromosomal mutation could be found in three of them. Upon further growth of these pseudolysogens, some variants with new chromosomal mutations were recovered, presumably due to continuous evolutionary pressure.

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“…Recent work on the D3 bacteriophage of P. aeruginosa revealed that the serotype switching mechanism is critically dependent on a small hydrophobic 3-kDa protein (Iap), which mimics the N-terminal transmembrane domain of the Wzz proteins and uses it to compete with chain-length regulators for binding to the endogenous Wzy polymerase (Taylor et al, 2013). Recent work on the D3 bacteriophage of P. aeruginosa revealed that the serotype switching mechanism is critically dependent on a small hydrophobic 3-kDa protein (Iap), which mimics the N-terminal transmembrane domain of the Wzz proteins and uses it to compete with chain-length regulators for binding to the endogenous Wzy polymerase (Taylor et al, 2013).…”

Section: Proposed Models Of O-antigen Chainlength Regulation In the Wmentioning

confidence: 99%

Progress in understanding the assembly process of bacterial O-antigen

2014

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The discovery that the surfaces of Gram-negative bacteria often carry unique polysaccharide signatures pre-dates most seminal discoveries of molecular biology and biochemistry of the 20th century. The O-antigen component of the lipopolysaccharide has been one of the most intensely studied bacterial polysaccharide surface structures for over 80 years. Yet, many questions about the mechanism of biosynthesis of the O-antigen and its transport to the cell surface remain unanswered. In this review we provide an overview of how the molecular basis of the O-antigen assembly and trafficking were unraveled in a historical context. We pay particular attention to the emergence of novel technological approaches and how they fueled the elucidation of the O-antigen maturation process. Moreover, we provide a brief perspective on the biosynthesis of enterobacterial common antigen and underline the similarities and differences between the pathways used to assemble these two surface polysaccharides. Finally, we highlight key discoveries that led to the understanding of the mechanistic basis of bacteriophage-induced O-antigen modifications. We place special emphasis on the regulation of the length of O-antigen polymers and provide a detailed overview of the models explaining the O-antigen length determination. Finally, we highlight outstanding questions that need to be addressed both structurally and functionally to advance our understanding of the O-antigen assembly, trafficking and export within cellular and molecular contexts.

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The D3 Bacteriophage α-Polymerase Inhibitor (Iap) Peptide Disrupts O-Antigen Biosynthesis through Mimicry of the Chain Length Regulator Wzz in Pseudomonas aeruginosa

Cited by 22 publications

References 60 publications

Mutational Analysis of the Shigella flexneri O-Antigen Polymerase Wzy: Identification of Wzz-Dependent Wzy Mutants

Mutational Analysis of the Shigella flexneri O-Antigen Polymerase Wzy: Identification of Wzz-Dependent Wzy Mutants

Pseudolysogeny and sequential mutations build multiresistance to virulent bacteriophages in Pseudomonas aeruginosa

Progress in understanding the assembly process of bacterial O-antigen

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