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

Abstract

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 ...

Mutational analysis of the major periplasmic loops of Shigella flexneri Wzy: identification of the residues affecting O antigen modal chain length control, and Wzz-dependent polymerization activity

“…LPS was prepared as described previously (Nath et al, 2015). Cells (1610 9 ) were harvested and resuspended in lysing buffer and incubated with proteinase K for approximately 16 h. The LPS samples were then separated by SDS-PAGE on 15 % (w/v) gels for 16.5 h at 12 mA.…”

“…The procedure used to examine Wzy Sf -GFP expression in S. flexneri has been described previously (Nath et al, 2015). Cells were harvested from the 50 ml Larabinose induced culture.…”

“…Site-directed mutagenesis on wzy Sf in plasmid pRMPN1 (Nath et al, 2015;Waldo et al, 1999) was performed using the QuikChange Lightning Site-Directed Mutagenesis kit (catalogue no. 210518; Stratagene) following the manufacturer's instructions.…”

“…It has 12 transmembrane (TM) segments and two large periplasmic (PL) domains (PL3 and PL5) (Daniels et al, 1998;Morona et al, 1994). Previously we were able to identify some of the key functional amino acid residues of Wzy Sf , providing insight into Wzy Sf structure and function (Nath et al, 2015). Wzy Sf has an RX 15 G motif in both PL3 and PL5 starting from R164 in PL3 and from R289 in PL5.…”

“…However, there is a lack of direct evidence on the association of Wzz and Wzy in Oag polymerization and chain length control. Recently, we identified the Wzz Sf -dependent Wzy Sf mutants, and showed that Wzz Sf has a novel role in the stability of Wzy Sf and also in the Oag polymerization activity of Wzy Sf (Nath et al, 2015).…”