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Patrick da Silva, Fernanda Z Manieri, Carmen M Herrera, M Stephen Trent, Cristiano G Moreira
Salmonella enterica serovars are associated with diarrhea and gastroenteritis and are a helpful model for understanding host-pathogen mechanisms. Salmonella Typhimurium regulates the distribution of O-antigen (OAg) and presents a trimodal distribution based on Wzy polymerase, WzzST (long chain length OAg, L-OAg) and WzzfepE (very long chain length OAg, VL-OAg) co-polymerases; however, several mechanisms regulating this process remain unclear. Here, we report that LPS modifications modulate the infectious process and that OAg chain length determination plays an essential role during infection...
June 4, 2018: Infection and Immunity
N Hegerle, J Bose, G Ramachandran, J E Galen, M M Levine, R Simon, S M Tennant
AIMS: O-polysaccharide (OPS) molecules are protective antigens for several bacterial pathogens, and have broad utility as components of glycoconjugate vaccines. Variability in the OPS chain length is one obstacle towards further development of these vaccines. Introduction of sizing steps during purification of OPS molecules of suboptimal or of mixed lengths introduces additional costs and complexity while decreasing the final yield. The overall goal of this study was to demonstrate the utility of engineering Gram-negative bacteria to produce homogenous O-polysaccharide populations that can be used as the basis of carbohydrate vaccines by overexpressing O-polysaccharide chain length regulators of the Wzx-/Wzy-dependent pathway...
March 30, 2018: Journal of Applied Microbiology
Jared M Hoffman, Shea Sullivan, Erin Wu, Eric Wilson, David L Erickson
RfaH enhances transcription of a select group of operons controlling bacterial surface features such as lipopolysaccharide (LPS). Previous studies have suggested that rfaH may be required for Yersinia pseudotuberculosis resistance to antimicrobial chemokines and survival during mouse infections. In order to further investigate the role of RfaH in LPS synthesis, resistance to host defense peptides, and virulence of Yersinia, we constructed ΔrfaH mutants of Y. pseudotuberculosis IP32953 and Y. pestis KIM6+. Loss of rfaH affected LPS synthesis in both species, resulting in a shorter core oligosaccharide...
September 7, 2017: Scientific Reports
Richard F Collins, Vasileios Kargas, Brad R Clarke, C Alistair Siebert, Daniel K Clare, Peter J Bond, Chris Whitfield, Robert C Ford
Wzz is an integral inner membrane protein involved in regulating the length of lipopolysaccharide O-antigen glycans and essential for the virulence of many Gram-negative pathogens. In all Wzz homologs, the large periplasmic domain is proposed to be anchored by two transmembrane helices, but no information is available for the transmembrane and cytosolic domains. Here we have studied purified oligomeric Wzz complexes using cryoelectron microscopy and resolved the transmembrane regions within a semi-continuous detergent micelle...
May 2, 2017: Structure
Magdalene Papadopoulos, Elizabeth Ngoc Hoa Tran, Gerald Laurence Murray, Renato Morona
The O antigen (Oag) component of lipopolysaccharides (LPS) is crucial for virulence and Oag chain-length regulation is controlled by the polysaccharide co-polymerase class 1 (PCP1) proteins. Crystal structure analyses indicate that structural conservation among PCP1 proteins is highly maintained, however the mechanism of Oag modal-chain-length control remains to be fully elucidated. Shigella flexneri PCP1 protein WzzBSF confers a modal-chain length of 10-17 Oag repeat units (RUs), whereas the Salmonella enterica Typhimurium PCP1 protein WzzBST confers a modal-chain length of ~16-28 Oag RUs...
June 2016: Microbiology
Pratiti Nath, Renato Morona
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...
September 2015: Microbiology
Pratiti Nath, Renato Morona
The O antigen (Oag) component of LPS is a major Shigella flexneri virulence determinant. Oag is polymerized by WzySf, and its modal chain length is determined by WzzSf and WzzpHS2. Site-directed mutagenesis was performed on wzySf in pWaldo-wzySf-TEV-GFP to alter Arg residues in WzySf's two large periplasmic loops (PLs) (PL3 and PL5). Analysis of the LPS profiles conferred by mutated WzySf proteins in the wzySf deficient (Δwzy) strain identified residues that affect WzySf activity. The importance of the guanidium group of the Arg residues was investigated by altering the Arg residues to Lys and Glu, which generated WzySf mutants conferring altered LPS Oag modal chain lengths...
April 2015: Microbiology
Donglei Han, Zhongrui Ma, Min Chen
The O-antigen of Gram-negative bacterium plays an important role in the signal identification, adhesion, immune evasion and other processes. There are three O-antigen polysaccharides biosynthesis mechanisms according to the type of the flippase that is involved. The Wzy-dependent mechanism is more commonly seen. In Wzy-dependent mechanism, the wzz gene is involved in regulating the length of O-antigen polysaccharide chain which can affect antigenicity of the pathogen and immune response of the host. Based on the crystal structure of Wzz (regulator of the O- antigen polysaccharides length) , different length of O-antigen chain can be obtained through molecular modification of the gene wzz...
September 4, 2014: Wei Sheng Wu Xue Bao, Acta Microbiologica Sinica
Salim T Islam, Joseph S Lam
The surfaces of bacteria mediate a multitude of functions in the environment and in an infected host, including adhesion to both biotic and abiotic substrata, motility, immune system interaction and (or) activation, biofilm formation, and cell-cell communication, with many of these features directly influenced by cell-surface glycans. In both Gram-negative and Gram-positive bacteria, the majority of cell-surface polysaccharides are produced via the Wzx/Wzy-dependent assembly pathway; these glycans include heteropolymeric O-antigen, enterobacterial common antigen, exopolysaccharide, spore coat, and capsule in diverse bacteria...
November 2014: Canadian Journal of Microbiology
Pratiti Nath, Elizabeth Ngoc Hoa Tran, Renato Morona
The O-antigen (Oag) component of lipopolysaccharide (LPS) is a major virulence determinant of Shigella flexneri and is synthesized by the O-antigen polymerase, WzySf. Oag chain length is regulated by chromosomally encoded WzzSf and pHS-2 plasmid-encoded WzzpHS2. To identify functionally important amino acid residues in WzySf, random mutagenesis was performed on the wzySf gene in a pWaldo-TEV-GFP plasmid, followed by screening with colicin E2. Analysis of the LPS conferred by mutated WzySf proteins in the wzySf-deficient (Δwzy) 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...
January 1, 2015: Journal of Bacteriology
K O Mironov, A E Platonov, E A Dunaeva, V I Kuseva, G A Shipulin
AIM: Development and application of real-time PCR (RT-PCR) procedure for determination of Streptococcus pneumoniae serotypes. MATERIALS AND METHODS: S. pneumoniae cps-locus wzx, wzy, wzz, wcwV and galU genes were chosen as PCR targets to select serotype-specific oligonucleotide primers and fluorescent labeled probes. 89 samples of cerebrospinal fluid (CSF) obtained in 2007 - 2010 from patients with pneumococcal meningitis diagnosis undergoing therapy in the Infectious Clinical Hospital No...
January 2014: Zhurnal Mikrobiologii, Epidemiologii, i Immunobiologii
Elizabeth Ngoc Hoa Tran, Magdalene Papadopoulos, Renato Morona
The Shigella flexneri polysaccharide co-polymerase class 1a (PCP1a) protein, WzzBSF, regulates LPS O-antigen (Oag) chain length to confer short (S)-type Oag chains of ~10-17 Oag repeat units (RUs). The S-type Oag chains affect Shigella flexneri virulence as they influence IcsA-mediated actin-based motility. However, they do not confer resistance to complement; this is conferred by the very-long (VL)-type Oag chains determined by WzzB(pHS2). Colicins are bacterial proteins produced by some Escherichia coli strains to kill related strains...
March 2014: Microbiology
Salim T Islam, Steven M Huszczynski, Timothy Nugent, Alexander C Gold, Joseph S Lam
O antigen (O-Ag) in many bacteria is synthesized via the Wzx/Wzy-dependent pathway in which Wzy polymerizes lipid-linked O-Ag subunits to modal lengths regulated by Wzz. Characterization of 83 site-directed mutants of Wzy from Pseudomonas aeruginosa PAO1 (WzyPa) in topologically-mapped periplasmic (PL) and cytoplasmic loops (CL) verified the functional importance of PL3 and PL5, with the former shown to require overall cationic properties. Essential Arg residues in the RX10G motifs of PL3 and PL5 were found to be conserved in putative homologues of WzyPa, as was the overall sequence homology between these two periplasmic loops in each protein...
2013: Scientific Reports
Johanna J Kenyon, Peter R Reeves
Lipopolysaccharide is a major immunogenic structure for the pathogen Yersinia pseudotuberculosis, which contains the O-specific polysaccharide (OPS) that is presented on the cell surface. The OPS contains many repeats of the oligosaccharide O-unit and exhibits a preferred modal chain length that has been shown to be crucial for cell protection in Yersinia. It is well established that the Wzz protein determines the preferred chain length of the OPS, and in its absence, the polymerization of O units by the Wzy polymerase is uncontrolled...
December 2013: FEMS Microbiology Letters
Véronique L Taylor, Molly L Udaskin, Salim T Islam, Joseph S Lam
Lysogenic bacteriophage D3 causes seroconversion of Pseudomonas aeruginosa PAO1 from serotype O5 to O16 by inverting the linkage between O-specific antigen (OSA) repeat units from α to β. The OSA units are polymerized by Wzy to modal lengths regulated by Wzz1 and Wzz2. A key component of the D3 seroconversion machinery is the inhibitor of α-polymerase (Iap) peptide, which is able to solely suppress α-linked long-chain OSA production in P. aeruginosa PAO1. To establish the target specificity of Iap for Wzyα, changes in OSA phenotypes were examined via Western immunoblotting for wzz1 and wzz2 single-knockout strains, as well as a wzz1 wzz2 double knockout, following the expression of iap from a tuneable vector...
October 2013: Journal of Bacteriology
Jennifer K Bender, Thorsten Wille, Kathrin Blank, Anna Lange, Roman G Gerlach
The larvae of the wax moth, Galleria mellonella, have been used experimentally to host a range of bacterial and fungal pathogens. In this study we evaluated the suitability of G. mellonella as an alternative animal model of Salmonella infection. Using a range of inoculum doses we established that the LD₅₀ of SalmonellaTyphimurium strain NCTC 12023 was 3.6 × 10³ bacteria per larva. Further, a set of isogenic mutant strains depleted of known virulence factors was tested to identify determinants essential for S...
2013: PloS One
Kayo Osawa, Katsumi Shigemura, Atsushi Iguchi, Hiroki Shirai, Toru Imayama, Kazuko Seto, Dadik Raharjo, Masato Fujisawa, Ro Osawa, Toshiro Shirakawa
Escherichia coli O157 strains belonging to a distinct lineage and expressing different O-antigen (Oag) lengths were isolated. Although the function of wzz in E. coli has not been adequately investigated, this gene is known to be associated with regulation of Oag length. Using E. coli O157:H7 ATCC43888 (wild-type), several wzz mutants of E. coli O157, including a wzz deletion mutant, were generated and the relationship between the length of Oag modulated by the wzz gene and sensitivities to serum complement investigated...
September 2013: Microbiology and Immunology
Zhang Yang, Xiaohua Li, Xiaoqaing Qi, Junjie Shan, Rong Jiang, Lianhong Guo, Ren Zhang, Yuan Li
Ebosin, a novel exopolysaccharide produced by Streptomyces sp. 139, has obvious antirheumatic arthritis activity in vivo, and its biosynthesis gene cluster (ste), consisting of 27 open reading frames, has been identified. This paper reports our study of the gene functionality of ste8, the predicted protein product of which is homologous to some bacterial chain length determinant Wzz proteins. For characterization of Ste8, ste8 was cloned and expressed in the mutant strain E. coli 086:H2 (Δwzz). The functional complementation of wzz by ste8 was demonstrated by the restoration of wild-type lipopolysaccharide biosynthesis and increased levels of serum resistance of E...
November 28, 2013: Journal of Microbiology and Biotechnology
John F May, Eduardo A Groisman
Chemical modifications of components of the bacterial cell envelope can enhance resistance to antimicrobial agents. Why then are such modifications produced only under specific conditions? Here, we address this question by examining the role of regulated variations in O-antigen length in the lipopolysaccharide (LPS), a glycolipid that forms most of the outer leaflet of the outer membrane in Gram-negative bacteria. We determined that activation of the PmrA/PmrB two-component system, which is the major regulator of LPS alterations in Salmonella enterica serovar Typhimurium, impaired growth of Salmonella in bile...
June 2013: Molecular Microbiology
Mikio Shoji, Hideharu Yukitake, Keiko Sato, Yasuko Shibata, Mariko Naito, Joseph Aduse-Opoku, Yoshimitsu Abiko, Michael A Curtis, Koji Nakayama
The periodontal pathogen Porphyromonas gingivalis has two different lipopolysaccharides (LPSs) designated O-LPS and A-LPS, which are a conventional O-antigen polysaccharide and an anionic polysaccharide that are both linked to lipid A-cores, respectively. However, the precise mechanisms of LPS biosynthesis remain to be determined. In this study, we isolated a pigment-less mutant by transposon mutagenesis and identified that the transposon was inserted into the coding sequence PGN_2005, which encodes a hypothetical protein of P...
June 2013: MicrobiologyOpen
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