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 ...
The O antigen (Oag) component of Shigella flexneri lipopolysaccharide (LPS) is important for virulence and a protective antigen. It is synthesized by the Wzy-dependent mechanism. S. flexneri Wzy has 12 transmembrane segments and two large periplasmic loops. The modal chain length of the Oag is determined by Wzz. Experimental evidence supports multi-protein interactions in the Wzy-dependent pathway. However, evidence for direct interaction of Wzy with the other proteins of the Wzy-dependent pathway is limited. Initially, we purified Wzy-GFP-His 8 and detected the presence of a dimeric form. In vivo cross-linking was then performed in an S. flexneri wzy mutant strain carrying plasmids encoding Wzy-GFP-His 8 and untagged Wzz. Following solubilization with n-dodecyl-b-D-maltopyranoside (DDM) and affinity purification of Wzy-GFP-His 8 , Western immunoblotting with Wzz antibody detected copurification of Wzz; this was supported by MS analysis. To the best of our knowledge, this is the first reported isolation of a complex between Wzy and Wzz. Wzy mutants (WzyR164A, WzyV92M, WzyY137H, and WzyR250K) whose properties are affected by Wzz were able to form complexes with Wzz. Their mutational alterations did not affect the interaction of Wzy with Wzz. Thus, the interaction may involve many regions of Wzy.
Microbial pathogens use hydrolases as a virulence strategy to spread disease through tissues and colonize medical device surfaces; however, visualizing this process is a technically challenging problem. To better understand the role of secreted fungal hydrolases and their role in Candida albicans virulence, we developed an in situ model system using luminescent Re(I) and Ir(III) containing probe molecules embedded in a biodegradable (poly(lactic-coglycolic acid), PLGA) polymer and tracked their uptake using epifluorescent imaging. We found that secretion of esterases can explain how physically embedded probes are acquired by fungal cells through the degradation of PLGA since embedded probes could not be liberated from nonbiodegradable polystyrene (PS). It was important to verify that epifluorescent imaging captured the fate of probe molecules rather than naturally occurring fungal autofluorescence. For this, we exploited the intense luminescent signals and long spectral relaxation times of the Re and Ir containing probe molecules, resolved in time using a gated imaging system. Results provide a visual demonstration of a key virulence trait of C. albicans: the use of hydrolases as a means to degrade materials and acquire hydrolysis products during fungal growth and hyphal development. These results help to explain the role of nonspecific hydrolases using a degradable material that is relevant to the study of fungal pathogenesis on biotic (tissues) surfaces. Additionally, understanding how fungal pathogens condition surfaces by using nonspecific hydrolases is important to the study of fungal attachment on abiotic surfaces, the first step in biofilm formation on medical devices.
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