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

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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Molecular Modeling of the Shigella flexneri Serogroup 3 and 5 O-Antigens and Conformational Relationships for a Vaccine Containing Serotypes 2a and 3a

Vaccines ◽

10.3390/vaccines8040643 ◽

2020 ◽

Vol 8 (4) ◽

pp. 643

Author(s):

Jason Hlozek ◽

Sara Owen ◽

Neil Ravenscroft ◽

Michelle M. Kuttel

Keyword(s):

Diarrheal Disease ◽

Shigella Flexneri ◽

Quadrivalent Vaccine ◽

Slight Effect ◽

Work Support ◽

O Antigen ◽

The Common ◽

O Antigens ◽

Chain Conformations ◽

Shared Epitopes

The pathogenic bacterium Shigella flexneri is a leading global cause of diarrheal disease. The O-antigen is the primary vaccine target and distinguishes the 30 serotypes reported. Except for serotype 6, all S. flexneri serotypes have a common backbone repeating unit (serotype Y), with variations in substitution creating the various serotypes. A quadrivalent vaccine containing serotypes 2a and 3a (as well as 6 and Shigella sonnei) is proposed to provide broad protection against non-vaccine S. flexneri serotypes through shared epitopes and conformations. Here we model the O-antigen (O-Ag) conformations of serogroups 3 and 5: a continuation of our ongoing systematic study of the S. flexneri O-antigens that began with serogroup 2. Our simulations show that S. flexneri serogroups 2, 3, and 5 all have flexible O-Ags, with substitutions of the backbone altering the chain conformations in different ways. Our analysis suggests three general heuristics for the effects of substitution on the Shigella O-Ag conformations: (1) substitution on rhamnose C reduces the extension of the O-Ag chain; (2) substitution at O-3 of rhamnose A restricts the O-Ags to predominantly helical conformations, (3) substitution at O-3 of rhamnose B has only a slight effect on conformation. The common O-Ag conformations across serotypes identified in this work support the assumption that a quadrivalent vaccine containing serotypes 2a and 3a could provide coverage against S. flexneri serotype 3b and serogroup 5.

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Model for the Controlled Synthesis of O-Antigen Repeat Units Involving the WaaL Ligase

mSphere ◽

10.1128/msphere.00074-15 ◽

2015 ◽

Vol 1 (1) ◽

Cited By ~ 7

Author(s):

Yaoqin Hong ◽

Peter R. Reeves

Keyword(s):

Repeat Unit ◽

Feedback Mechanism ◽

Growth Defect ◽

Controlled Synthesis ◽

Inner Membrane ◽

O Antigen ◽

Repeat Units

ABSTRACT The Wzx/Wzy O-antigen pathway involves synthesis of a repeat unit (O unit) consisting of 3 to 8 sugars on an inner-membrane-embedded lipid carrier. These O units are translocated across the membrane to its periplasmic face by Wzx, while retaining linkage to the carrier, and then polymerized by Wzy to O-antigen polymer, which WaaL ligase transfers to a lipopolysaccharide precursor to complete lipopolysaccharide synthesis, concomitantly releasing the lipid carrier. This lipid carrier is also used for peptidoglycan assembly, and sequestration is known to be toxic. Thus, O-unit synthesis must involve precise regulation to meet demand but avoid overproduction. Here we show that loss of WaaL reverses a known growth defect in a Salmonella mutant that otherwise accumulates O-unit intermediates and propose that WaaL is also involved in a novel feedback mechanism to regulate O-unit synthesis, based on the availability of O units on the periplasmic face of the membrane.

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Molecular detection of all 34 distinct O-antigen forms of Shigella

Journal of Medical Microbiology ◽

10.1099/jmm.0.000794-0 ◽

2009 ◽

Vol 58 (1) ◽

pp. 69-81 ◽

Cited By ~ 40

Author(s):

Yayue Li ◽

Boyang Cao ◽

Bin Liu ◽

Dan Liu ◽

Qili Gao ◽

...

Keyword(s):

Shigella Flexneri ◽

Bacterial Species ◽

Milk Powder ◽

Shigella Dysenteriae ◽

Detection Sensitivity ◽

Clinical Samples ◽

Shigella Boydii ◽

O Antigen ◽

Probe Concentration ◽

O Antigens

Shigella is the cause of shigellosis or bacillary dysentery, the occurrence of which is estimated to be 165 million cases per year worldwide, resulting in 1.1 million deaths. Rapid and reliable assays for detecting and identifying Shigella in food, environmental and clinical samples are therefore necessary. Shigella species are traditionally identified by their O antigens. This study developed a DNA microarray targeting O-serotype-specific genes to detect all 34 distinct O-antigen forms of Shigella, including Shigella boydii types 1–18, Shigella dysenteriae types 1–13, Shigella flexneri types 1–5 and 6, and Shigella sonnei. A total of 282 strains were used to test the specificity of the microarray, including 186 Shigella and Escherichia coli representative strains, 86 Shigella clinical isolates and ten strains of other bacterial species that are commonly isolated from food or clinical stool specimens. The oligonucleotide probes were printed on the microarray in concentrations from 1 to 100 μM, and 10 μM proved to be the optimal probe concentration. The detection sensitivity for each serotype was 50 ng genomic DNA or 1 c.f.u. in 25 g milk powder sample following a 6 h enrichment in broth. The microarray is specific, sensitive and reproducible, and, to our knowledge, is the first report of a microarray for serotyping all O-antigen forms of Shigella.

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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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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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Bacteriophage Sf6 Tailspike Protein for Detection of Shigella flexneri Pathogens

Viruses ◽

10.3390/v10080431 ◽

2018 ◽

Vol 10 (8) ◽

pp. 431 ◽

Cited By ~ 7

Author(s):

Sonja Kunstmann ◽

Tom Scheidt ◽

Saskia Buchwald ◽

Alexandra Helm ◽

Laurence Mulard ◽

...

Keyword(s):

Mass Spectrometry ◽

Detection System ◽

Shigella Flexneri ◽

Microtiter Plate ◽

Diagnostic Tools ◽

Intensity Increase ◽

O Antigen ◽

Visible Light Range ◽

O Antigens ◽

Tailspike Protein

Bacteriophage research is gaining more importance due to increasing antibiotic resistance. However, for treatment with bacteriophages, diagnostics have to be improved. Bacteriophages carry adhesion proteins, which bind to the bacterial cell surface, for example tailspike proteins (TSP) for specific recognition of bacterial O-antigen polysaccharide. TSP are highly stable proteins and thus might be suitable components for the integration into diagnostic tools. We used the TSP of bacteriophage Sf6 to establish two applications for detecting Shigella flexneri (S. flexneri), a highly contagious pathogen causing dysentery. We found that Sf6TSP not only bound O-antigen of S. flexneri serotype Y, but also the glucosylated O-antigen of serotype 2a. Moreover, mass spectrometry glycan analyses showed that Sf6TSP tolerated various O-acetyl modifications on these O-antigens. We established a microtiter plate-based ELISA like tailspike adsorption assay (ELITA) using a Strep-tag®II modified Sf6TSP. As sensitive screening alternative we produced a fluorescently labeled Sf6TSP via coupling to an environment sensitive dye. Binding of this probe to the S. flexneri O-antigen Y elicited a fluorescence intensity increase of 80% with an emission maximum in the visible light range. The Sf6TSP probes thus offer a promising route to a highly specific and sensitive bacteriophage TSP-based Shigella detection system.

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Functional Characterization of WaaL, a Ligase Associated with Linking O-Antigen Polysaccharide to the Core of Pseudomonas aeruginosa Lipopolysaccharide

Journal of Bacteriology ◽

10.1128/jb.187.9.3002-3012.2005 ◽

2005 ◽

Vol 187 (9) ◽

pp. 3002-3012 ◽

Cited By ~ 84

Author(s):

Priyanka D. Abeyrathne ◽

Craig Daniels ◽

Karen K. H. Poon ◽

Mauricia J. Matewish ◽

Joseph S. Lam

Keyword(s):

Pseudomonas Aeruginosa ◽

Lipid A ◽

Functional Characterization ◽

Wild Type ◽

The Core ◽

O Antigen ◽

Repeat Units ◽

Cytoplasmic Face ◽

O Antigens

ABSTRACT The O antigen of Pseudomonas aeruginosa B-band lipopolysaccharide is synthesized by assembling O-antigen-repeat units at the cytoplasmic face of the inner membrane by nonprocessive glycosyltransferases, followed by polymerization on the periplasmic face. The completed chains are covalently attached to lipid A core by the O-antigen ligase, WaaL. In P. aeruginosa the process of ligating these O-antigen molecules to lipid A core is not clearly defined, and an O-antigen ligase has not been identified until this study. Using the sequence of waaL from Salmonella enterica as a template in a BLAST search, a putative waaL gene was identified in the P. aeruginosa genome. The candidate gene was amplified and cloned, and a chromosomal knockout of PAO1 waaL was generated. Lipopolysaccharide (LPS) from this mutant is devoid of B-band O-polysaccharides and semirough (SR-LPS, or core-plus-one O-antigen). The mutant PAO1waaL is also deficient in the production of A-band polysaccharide, a homopolymer of d-rhamnose. Complementation of the mutant with pPAJL4 containing waaL restored the production of both A-band and B-band O antigens as well as SR-LPS, indicating that the knockout was nonpolar and waaL is required for the attachment of O-antigen repeat units to the core. Mutation of waaL in PAO1 and PA14, respectively, could be complemented with waaL from either strain to restore wild-type LPS production. The waaL mutation also drastically affected the swimming and twitching motilities of the bacteria. These results demonstrate that waaL in P. aeruginosa encodes a functional O-antigen ligase that is important for cell wall integrity and motility of the bacteria.

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Membrane Topology of the NixA Nickel Transporter ofHelicobacter pylori: Two Nickel Transport-Specific Motifs within Transmembrane Helices II and III

Journal of Bacteriology ◽

10.1128/jb.182.6.1722-1730.2000 ◽

2000 ◽

Vol 182 (6) ◽

pp. 1722-1730 ◽

Cited By ~ 41

Author(s):

John F. Fulkerson ◽

Harry L. T. Mobley

Keyword(s):

Cytoplasmic Membrane ◽

Transmembrane Domain ◽

Transmembrane Domains ◽

Membrane Topology ◽

Structural Elements ◽

Transmembrane Helices ◽

Translational Initiation ◽

Amino Terminal ◽

Localization Signal ◽

Critical Residues

ABSTRACT NixA, the high-affinity cytoplasmic membrane nickel transport protein of Helicobacter pylori, imports Ni2+into the cell for insertion into the active site of the urease metalloenzyme, which is required for gastric colonization. NixA fractionates with the cytoplasmic membrane, and protein cross-linking studies suggest that NixA functions as a monomer. A preliminary topological model of NixA with seven transmembrane domains was previously proposed based on hydropathy, charge dispersion, and homology to other transporters. To test the proposed topology of NixA and relate critical residues to specific structural elements, a series of 21 NixA-LacZ and 21 NixA-PhoA fusions were created along the entire length of the protein. Expression of reporter fusions was confirmed by Western blotting with β-galactosidase- and alkaline phosphatase-specific antisera. The activities of reporter fusions near to and upstream of the predicted translational initiation demonstrated the presence of an additional amino-terminal transmembrane domain including a membrane localization signal. Activities of fusions immediately adjacent to motifs which have been shown to be requisite for Ni2+ transport localized these motifs entirely within transmembrane domains II and III. Fusion activities localized six additional Asp and Glu residues which reduced Ni2+transport by >90% when mutated within or immediately adjacent to transmembrane domains II, V, VI, and VII. All fusions strongly support a model of NixA in which the amino and carboxy termini are located in the cytoplasm and the protein possesses eight transmembrane domains.

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Stable Chromosomal Expression of Shigella flexneri 2a and 3a O-Antigens in the Live Salmonella Oral Vaccine Vector Ty21a

Clinical and Vaccine Immunology ◽

10.1128/cvi.00181-17 ◽

2017 ◽

Vol 24 (12) ◽

Cited By ~ 8

Author(s):

Madushini N. Dharmasena ◽

Manuel Osorio ◽

Kazuyo Takeda ◽

Scott Stibitz ◽

Dennis J. Kopecko

Keyword(s):

Oral Vaccine ◽

Shigella Flexneri ◽

Serum Antibody ◽

Vaccine Vector ◽

Shigella Dysenteriae ◽

Antigenic Determinants ◽

Content Type ◽

O Antigen ◽

Shigella Flexneri 2A ◽

O Antigens

ABSTRACT We have been exploring the use of the live attenuated Salmonella enterica serovar Typhi Ty21a vaccine strain as a versatile oral vaccine vector for the expression and delivery of multiple foreign antigens, including Shigella O-antigens. In this study, we separately cloned genes necessary for the biosynthesis of the Shigella flexneri serotype 2a and 3a O-antigens, which have been shown to provide broad cross-protection to multiple disease-predominant S. flexneri serotypes. The cloned S. flexneri 2a rfb operon, along with bgt and gtrII, contained on the SfII bacteriophage, was sufficient in Ty21a to express the heterologous S. flexneri 2a O-antigen containing the 3,4 antigenic determinants. Further, this rfb operon, along with gtrA, gtrB, and gtrX contained on the Sfx bacteriophage and oac contained on the Sf6 bacteriophage, was sufficient to express S. flexneri 3a O-antigen containing the 6, 7, and 8 antigenic determinants. Ty21a, with these plasmid-carried or chromosomally inserted genes, demonstrated simultaneous and stable expression of homologous S. Typhi O-antigen plus the heterologous S. flexneri O-antigen. Candidate Ty21a vaccine strains expressing heterologous S. flexneri 2a or 3a lipopolysaccharide (LPS) elicited significant serum antibody responses against both homologous S. Typhi and heterologous Shigella LPS and protected mice against virulent S. flexneri 2a or 3a challenges. These new S. flexneri 2a and 3a O-antigen-expressing Ty21a vaccine strains, together with our previously constructed Ty21a strains expressing Shigella sonnei or Shigella dysenteriae 1 O-antigens, have the potential to be used together for simultaneous protection against the predominant causes of shigellosis worldwide as well as against typhoid fever.

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