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 ε-Poly-l-Lysine Peptide Chain Length Regulated by the Linkers Connecting the Transmembrane Domains of ε-Poly-l-Lysine Synthetase

2014 ◽  
Vol 80 (16) ◽  
pp. 4993-5000 ◽  
Author(s):  
Yoshimitsu Hamano ◽  
Naoko Kito ◽  
Akihiro Kita ◽  
Yuuki Imokawa ◽  
Kazuya Yamanaka ◽  
...  
Keyword(s):  
Chain Length ◽  
Catalytic Mechanism ◽  
Random Mutagenesis ◽  
Transmembrane Domains ◽  
Polymerization Reaction ◽  
Amino Acid Residues ◽  
Amino Groups ◽  
Content Type ◽  
Peptide Synthetases ◽  
Lysine Residues

ABSTRACTε-Poly-l-lysine (ε-PL), consisting of 25 to 35l-lysine residues with linkages between the α-carboxyl groups and ε-amino groups, is produced byStreptomyces albulusNBRC14147. ε-PL synthetase (Pls) is a membrane protein with six transmembrane domains (TM1 to TM6) as well as both an adenylation domain and a thiolation domain, characteristic of the nonribosomal peptide synthetases. Pls directly generates ε-PL chain length diversity (25- to 35-mer), but the processes that control the chain length of ε-PL during the polymerization reaction are still not fully understood. Here, we report on the identification of Pls amino acid residues involved in the regulation of the ε-PL chain length. From approximately 12,000 variants generated by random mutagenesis, we found 8 Pls variants that produced shorter chains of ε-PL. These variants have one or more mutations in two linker regions connecting the TM1 and TM2 domains and the TM3 and TM4 domains. In the Pls catalytic mechanism, the growing chain of ε-PL is not tethered to the enzyme, implying that the enzyme must hold the growing chain until the polymerization reaction is complete. Our findings reveal that the linker regions are important contributors to grasp the growing chain of ε-PL.

scholarly journals Relationship between O-antigen chain length and resistance to colicin E2 in Shigella flexneri

Microbiology ◽  
2014 ◽  
Vol 160 (3) ◽  
pp. 589-601 ◽  
Author(s):  
Elizabeth Ngoc Hoa Tran ◽  
Magdalene Papadopoulos ◽  
Renato Morona
Keyword(s):  
Chain Length ◽  
Shigella Flexneri ◽  
Outer Membrane Proteins ◽  
Bacterial Proteins ◽  
O Antigen ◽  
Repeat Units ◽  
Colicin E2 ◽  
Chain Lengths ◽  
The Impact ◽  
Short Type

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 WzzBpHS2. Colicins are bacterial proteins produced by some Escherichia coli strains to kill related strains. While the presence of Oag chains has been shown to shield outer-membrane proteins from colicins, the impact of Oag chain length against colicins is unknown. In this study, initial testing indicated that a Shigella flexneri Y wzz : : kanr mutant was more sensitive to colicin E2 compared with the WT strain. Plasmids encoding Wzz mutant and WT PCP1a proteins conferring different Oag modal chain lengths were then expressed in the mutant background, and tested against purified colicin E2. Analysis of swab and spot sensitivity assays showed that strains expressing either S-type or long (L)-type Oag chains (16–28 Oag RUs) conferred greater resistance to colicin E2 compared with strains having very-short-type (2–8 Oag RUs), intermediate-short-type (8–14 Oag RUs) or VL-type (>80 Oag RUs) Oag chains. These results suggest a novel role for LPS Oag chain length control that may have evolved due to selection pressure from colicins in the environment.

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

scholarly journals Influence of Shigella flexneri 2a O Antigen Acetylation on Its Bacteriophage Sf6 Receptor Activity and Bacterial Interaction with Human Cells

2020 ◽  
Vol 202 (24) ◽  
Author(s):  
Min Yan Teh ◽  
Axel Furevi ◽  
Göran Widmalm ◽  
Renato Morona
Keyword(s):  
Shigella Flexneri ◽  
Limited Range ◽  
World Health ◽  
Bacteriophage Therapy ◽  
Chemical Modifications ◽  
Content Type ◽  
O Antigen ◽  
Shigella Flexneri 2A ◽  
Bacterial Interaction ◽  
Health Organization

ABSTRACT Shigella flexneri is a major causative agent of bacillary dysentery in developing countries, where serotype 2a2 is the prevalent strain. To date, approximately 30 serotypes have been identified for S. flexneri, and the major contribution to the emergence of new serotypes is chemical modifications of the lipopolysaccharide (LPS) component O antigen (Oag). Glucosylation, O-acetylation, and phosphoethanolamine (PEtN) modifications increase the Oag diversity, providing benefits to S. flexneri. LPS Oag acts as a primary receptor for bacteriophage Sf6, which infects only a limited range of S. flexneri serotypes (Y and X). It uses its tailspike protein (Sf6TSP) to establish initial interaction with LPS Oags that it then hydrolyzes. Currently, there is a lack of comprehensive study on the parent and serotype variant strains from the same genetic background and an understanding of the importance of LPS Oag O-acetylations. Therefore, a set of isogenic strains (based on S. flexneri 2457T [2a2]) with deletions of different Oag modification genes (oacB, oacD, and gtrII) that resemble different naturally occurring serotype Y and 2a strains was created. The impacts of these Oag modifications on S. flexneri sensitivity to Sf6 and the pathogenesis-related properties were then compared. We found that Sf6TSP can hydrolyze serotype 2a LPS Oag, identified that 3/4-O-acetylation is essential for resistance of serotype 2a strains to Sf6, and showed that serotype 2a strains have better invasion ability. Lastly, we revealed two new serotype conversions for S. flexneri, thereby contributing to understanding the evolution of this important human pathogen. IMPORTANCE The emergence of antibiotic-resistant strains and lack of efficient vaccines have made Shigella a priority organism for the World Health Organization (1). Therefore, bacteriophage therapy has received increasing attention as an alternative therapeutic approach. LPS Oag is the most variable part of LPS due to chemical modifications and is the target of bacteriophage Sf6 (S. flexneri specific). We dissected the evolution of S. flexneri serotype Y to 2a2, which revealed a new role for a gene acquired during serotype conversion and furthermore identified new specific forms of LPS receptor for Sf6. Collectively, these results unfold the importance of the acquisition of those Oag modification genes and further our understanding of the relationship between Sf6 and S. flexneri.

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.

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