scholarly journals 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

Microbiology ◽  
2015 ◽  
Vol 161 (4) ◽  
pp. 774-785 ◽  
Author(s):  
Pratiti Nath ◽  
Renato Morona
Keyword(s):  
Chain Length ◽  
Mutational Analysis ◽  
Shigella Flexneri ◽  
O Antigen

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

2014 ◽  
Vol 197 (1) ◽  
pp. 108-119 ◽  
Author(s):  
Pratiti Nath ◽  
Elizabeth Ngoc Hoa Tran ◽  
Renato Morona
Keyword(s):  
Chain Length ◽  
Mutational Analysis ◽  
Shigella Flexneri ◽  
Random Mutagenesis ◽  
Amino Acid Residues ◽  
Transmembrane Region ◽  
Content Type ◽  
O Antigen ◽  
Important Amino Acid ◽  
Colicin E2

The O-antigen (Oag) component of lipopolysaccharide (LPS) is a major virulence determinant ofShigella flexneriand is synthesized by the O-antigen polymerase, WzySf. Oag chain length is regulated by chromosomally encoded WzzSfand pHS-2 plasmid-encoded WzzpHS2. To identify functionally important amino acid residues in WzySf, random mutagenesis was performed on thewzySfgene in a pWaldo-TEV-GFP plasmid, followed by screening with colicin E2. Analysis of the LPS conferred by mutated WzySfproteins in thewzySf-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. The association of WzySfand WzzSfwas investigated by transforming these mutatedwzySfplasmids into awzySf- andwzzSf-deficient (Δwzy Δwzz) strain. Comparison of the LPS profiles in the Δwzyand Δwzy Δwzzbackgrounds identified WzySfmutants whose polymerization activities were WzzSfdependent. Colicin E2 and bacteriophage Sf6c sensitivities were consistent with the LPS profiles. Analysis of the expression levels of the WzySf-GFP mutants in the Δwzyand Δwzy Δwzzbackgrounds identified a role for WzzSfin WzySfstability. Hence, in addition to its role in regulating Oag modal chain length, WzzSfalso affects WzySfactivity and stability.

scholarly journals The normal chain length distribution of the O antigen is required for the interaction of Shigella flexneri 2a with polarized Caco-2 cells

2012 ◽  
Vol 45 (1) ◽  
pp. 21-26 ◽  
Author(s):  
Anilei Hoare ◽  
Denisse Bravo ◽  
Mara Martinic ◽  
Miguel A Valvano ◽  
Inés Contreras ◽  
...  
Keyword(s):  
Chain Length ◽  
Shigella Flexneri ◽  
Length Distribution ◽  
Chain Length Distribution ◽  
O Antigen ◽  
Shigella Flexneri 2A

scholarly journals Coiled-coil regions play a role in the function of the Shigella flexneri O-antigen chain length regulator WzzpHS2

Microbiology ◽  
2008 ◽  
Vol 154 (4) ◽  
pp. 1104-1116 ◽  
Author(s):  
Leanne Purins ◽  
Luisa Van Den Bosch ◽  
Vanessa Richardson ◽  
Renato Morona
Keyword(s):  
Chain Length ◽  
Shigella Flexneri ◽  
Coiled Coil ◽  
O Antigen

Regulation of O‐antigen chain length is required for Shigella flexneri virulence

1997 ◽  
Vol 23 (4) ◽  
pp. 765-775 ◽  
Author(s):  
Luisa Van Den Bosch ◽  
Paul A. Manning ◽  
Renato Morona
Keyword(s):  
Chain Length ◽  
Shigella Flexneri ◽  
O Antigen

scholarly journals Mutagenesis and Chemical Cross-Linking Suggest that Wzz Dimer Stability and Oligomerization Affect Lipopolysaccharide O-Antigen Modal Chain Length Control

2010 ◽  
Vol 192 (13) ◽  
pp. 3385-3393 ◽  
Author(s):  
Magdalene Papadopoulos ◽  
Renato Morona
Keyword(s):  
Chain Length ◽  
Shigella Flexneri ◽  
Three Dimensional ◽  
Class Ii ◽  
Cross Linking ◽  
Wild Type ◽  
Class V ◽  
O Antigen ◽  
Class Iv

ABSTRACT In Shigella flexneri, the polysaccharide copolymerase (PCP) protein WzzSF confers a modal length of 10 to 17 repeat units (RUs) to the O-antigen (Oag) component of lipopolysaccharide (LPS). PCPs form oligomeric structures believed to be related to their function. To identify functionally important regions within WzzSF, random in-frame linker mutagenesis was used to create mutants with 5-amino-acid insertions (termed Wzzi proteins), and DNA sequencing was used to locate the insertions. Analysis of the resulting LPS conferred by Wzzi proteins identified five mutant classes. The class I mutants were inactive, resulting in nonregulated LPS Oag chains, while classes II and III conferred shorter LPS Oag chains of 2 to 10 and 8 to 14 RUs, respectively. Class IV mutants retained near-wild-type function, and class V mutants increased the LPS Oag chain length to 16 to 25 RUs. In vivo formaldehyde cross-linking indicated class V mutants readily formed high-molecular-mass oligomers; however, class II and III Wzzi mutants were not effectively cross-linked. Wzz dimer stability was also investigated by heating cross-linked oligomers at 100°C in the presence of SDS. Unlike the WzzSF wild type and class IV and V Wzzi mutants, the class II and III mutant dimers were not detectable. The location of each insertion was mapped onto available PCP three-dimensional (3D) structures, revealing that class V mutations were most likely located within the inner cavity of the PCP oligomer. These data suggest that the ability to produce stable dimers may be important in determining Oag modal chain length.

scholarly journals The cellular level of O-antigen polymerase Wzy determines chain length regulation by WzzB and WzzpHS-2 in Shigella flexneri 2a

Microbiology ◽  
2009 ◽  
Vol 155 (10) ◽  
pp. 3260-3269 ◽  
Author(s):  
Javier A. Carter ◽  
Juan C. Jiménez ◽  
Mercedes Zaldívar ◽  
Sergio A. Álvarez ◽  
Cristina L. Marolda ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Chain Length ◽  
Shigella Flexneri ◽  
Length Distribution ◽  
Direct Interaction ◽  
Cellular Level ◽  
Chain Length Distribution ◽  
Inducible Promoters ◽  
O Antigen

The lipopolysaccharide O antigen of Shigella flexneri 2a has two preferred chain lengths, a short (S-OAg) composed of an average of 17 repeated units and a very long (VL-OAg) of about 90 repeated units. These chain length distributions are controlled by the chromosomally encoded WzzB and the plasmid-encoded WzzpHS-2 proteins, respectively. In this study, genes wzzB, wzz pHS-2 and wzy (encoding the O-antigen polymerase) were cloned under the control of arabinose- and rhamnose-inducible promoters to investigate the effect of varying their relative expression levels on O antigen polysaccharide chain length distribution. Controlled expression of the chain length regulators wzzB and wzz pHS-2 revealed a dose-dependent production of each modal length. Increase in one mode resulted in a parallel decrease in the other, indicating that chain length regulators compete to control the degree of O antigen polymerization. Also, when expression of the wzy gene is low, S-OAg but not VL-OAg is produced. Production of VL-OAg requires high induction levels of wzy. Thus, the level of expression of wzy is critical in determining O antigen modal distribution. Western blot analyses of membrane proteins showed comparable high levels of the WzzB and WzzpHS-2 proteins, but very low levels of Wzy. In vivo cross-linking experiments and immunoprecipitation of membrane proteins did not detect any direct interaction between Wzy and WzzB, suggesting the possibility that these two proteins may not interact physically but rather by other means such as via translocated O antigen precursors.

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