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

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.

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

Journal of Bacteriology ◽

10.1128/jb.01885-14 ◽

2014 ◽

Vol 197 (1) ◽

pp. 108-119 ◽

Cited By ~ 5

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.

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Impact of Molecular Chain Structure of Suspension Phase on Giant Electrorheological Performance

Smart Materials and Structures ◽

10.1088/1361-665x/ac4875 ◽

2022 ◽

Author(s):

Hanqi Xu ◽

Jinbo Wu ◽

Yaying Hong ◽

Weijia Wen

Keyword(s):

Yield Stress ◽

Chain Length ◽

Colloidal Stability ◽

Chain Structure ◽

Solid Particles ◽

High Yield ◽

Suspension Stability ◽

Chain Lengths ◽

The Impact ◽

Oil Particles

Abstract We demonstrate the impact of diester structure, in particular the alkyl chain length and branching structure, on the giant electrorheological (GER) effect and suspension stability. The existence of oil-particles interaction is of critical importance to induce the GER effect. To quantify GER performance and colloidal stability, we examine the yield stress, current density, field-off viscosity and sedimentation ratio with respect to the variation of chain length and branching structure. The oil-particles interaction is quantitatively analyzed by investigating the cluster size of particles in different diesters by a multiple light scattering analyzer, along with the wettability of different chain lengths of diesters and solid particles by the Washburn method. Our results indicate that long chain lengths favor the formation of particle agglomerates, thereby enhancing the GER effect (such as high yield stress). The attachment of branches on diester causes the formation of electronic correlation between branches and main chain, depending on the position of branches located, and hence results in superior GER performance and favorable suspension stability. An optimal GER fluid constituted by bis(2-ethylhexyl) sebacate is acquired with the achieved yield stress of 113 kPa at electric field strength of 4 kV/ mm and the prominent integrated GER properties.

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Bacteriophage-encoded glucosyltransferase GtrII of Shigella flexneri: membrane topology and identification of critical residues

Biochemical Journal ◽

10.1042/bj20050102 ◽

2005 ◽

Vol 389 (1) ◽

pp. 137-143 ◽

Cited By ~ 18

Author(s):

Adele M. LEHANE ◽

Haralambos KORRES ◽

Naresh K. VERMA

Keyword(s):

Sequence Similarity ◽

Repeat Unit ◽

Shigella Flexneri ◽

Membrane Topology ◽

Transmembrane Helices ◽

O Antigen ◽

Repeat Units ◽

Related Group ◽

Critical Residues ◽

O Antigens

The Shigella flexneri serotypes differ in the nature of their O-antigens. The addition of glucosyl or O-acetyl groups to the common backbone repeat units gives rise to the different serotypes. GtrII glucosylates rhamnose III of the O-antigen repeat unit, thus converting serotype Y (which has no modifications to the basic O-antigen repeat unit) into serotype 2a, the most prevalent serotype. In the present study, the topology of GtrII has been determined. GtrII has nine transmembrane helices, a re-entrant loop and three large periplasmic regions. Four critical residues (Glu40, Phe414, Cys435 and Lys478) were identified in two of the periplasmic regions. Despite the lack of sequence similarity between GtrII and the Gtrs from other serotypes, three of the critical residues identified are conserved in the remaining Gtrs. This is consistent with some degree of mechanistic conservation in this functionally related group of proteins.

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

Biological Research ◽

10.4067/s0716-97602012000100003 ◽

2012 ◽

Vol 45 (1) ◽

pp. 21-26 ◽

Cited By ~ 5

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

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Altering the Length of the Lipopolysaccharide O Antigen Has an Impact on the Interaction of Salmonella enterica Serovar Typhimurium with Macrophages and Complement

Journal of Bacteriology ◽

10.1128/jb.188.7.2735-2739.2006 ◽

2006 ◽

Vol 188 (7) ◽

pp. 2735-2739 ◽

Cited By ~ 100

Author(s):

Gerald L. Murray ◽

Stephen R. Attridge ◽

Renato Morona

Keyword(s):

Vibrio Cholerae ◽

Salmonella Enterica ◽

Double Mutant ◽

Salmonella Enterica Serovar Typhimurium ◽

Shigella Flexneri ◽

O Antigen ◽

Repeat Units ◽

Complement Resistance ◽

Serovar Typhimurium ◽

First Time

ABSTRACT A panel of isogenic Salmonella enterica serovar Typhimurium strains that vary only in the length of the O antigen was constructed through complementation of a wzz double mutant (displaying unregulated O-antigen length) with one of two homologous (wzz ST and wzz fepE) or three heterologous (wzz O139 of Vibrio cholerae and wzz SF and wzz pHS-2 of Shigella flexneri) wzz genes. Each gene was functional in the S. enterica serovar Typhimurium host and specified production of O-antigen polymers with lengths typical of those synthesized by the donor bacteria (ranging from 2 to >100 O-antigen repeat units). By use of this panel of strains, it was found that O-antigen length influences invasion/uptake by macrophage cells; this is the first time this has been shown with Salmonella. O-antigen length was confirmed to be related to complement resistance, with a minimum protective length of >4 and <15 repeat units. O antigen of 16 to 35 repeat units was found to activate complement more efficiently than other lengths, but this was unrelated to complement resistance. No evidence was found to suggest that modifying the length of the O-antigen polymer affected expression of the O1, O4, or O5 antigenic factors.

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

Microbiology ◽

10.1099/mic.0.2007/014225-0 ◽

2008 ◽

Vol 154 (4) ◽

pp. 1104-1116 ◽

Cited By ~ 24

Author(s):

Leanne Purins ◽

Luisa Van Den Bosch ◽

Vanessa Richardson ◽

Renato Morona

Keyword(s):

Chain Length ◽

Shigella Flexneri ◽

Coiled Coil ◽

O Antigen

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Influence of different rol gene products on the chain length of Shigella dysenteriae type 1 lipopolysaccharide O antigen expressed by Shigella flexneri carrier strains.

Journal of Bacteriology ◽

10.1128/jb.179.7.2421-2425.1997 ◽

1997 ◽

Vol 179 (7) ◽

pp. 2421-2425 ◽

Cited By ~ 24

Author(s):

S R Klee ◽

B D Tzschaschel ◽

K N Timmis ◽

C A Guzman

Keyword(s):

Chain Length ◽

Shigella Flexneri ◽

Shigella Dysenteriae ◽

Gene Products ◽

O Antigen ◽

Rol Gene ◽

Shigella Dysenteriae Type

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Identification of a region in Shigella flexneri WzyB disrupting the interaction with Wzz pHS2

Journal of Bacteriology ◽

10.1128/jb.00413-21 ◽

2021 ◽

Author(s):

Vincenzo Leo ◽

Min Yan Teh ◽

Elizabeth N.H Tran ◽

Renato Morona

Keyword(s):

Shigella Flexneri ◽

Direct Interaction ◽

Site Directed Mutagenesis ◽

Physical Interaction ◽

Significant Implication ◽

O Antigen ◽

Repeat Units ◽

Inner Membrane Protein ◽

First Time ◽

Dependent Pathway

Shigella flexneri can synthesise polysaccharide chains having complex sugars and a regulated number of repeating units. S. flexneri lipopolysaccharide O antigen (Oag) is synthesised by the Wzy-dependent pathway which is the most common pathway used in bacteria for polysaccharide synthesis. The inner membrane protein WzyB polymerizes the Oag repeat units into chains, while the polysaccharide co-polymerases WzzB and Wzz pHS2 determine the average number of repeat units or “the modal length”, termed short-type and very long-type. Our data show for the first time a direct interaction between WzyB and Wzz pHS2 , with and without the use of the chemical cross-linker dithiobis (succinimidyl propionate) (DSP). Additionally mutations, generated via random and site directed mutagenesis, identify a region of WzyB that caused diminished function and significantly decreased very-long Oag chain polymerisation, and that affected the aforementioned interaction. These results provide insight into the mechanisms underlying the regulation of Oag biosynthesis. Importance Complex polysaccharide chains are synthesised by bacteria, usually at a regulated number of repeating units, which has broad implications for bacterial pathogenesis. One example is the O antigen (Oag) component of lipopolysaccharide that is predominantly synthesised by the Wzy-dependent pathway. Our findings show for the first time a direct physical interaction between WzyB and Wzz pHS2 . Additionally, a set of Wzy mutant constructs were generated revealing a proposed active site/switch region involved in the activity of WzyB and the physical interaction with Wzz pHS2 . Combined, these findings further the understanding of the Wzy-dependent pathway. The identification of a novel interaction with the polysacchraride co-polymerase Wzz pHS2, and the region of WzyB that is involved in this aforementioned interaction and its impact on WzyB Oag synthesis activity, have significant implication for the prevention/treatment of bacterial diseases, and discovery of novel biotechnologies.

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