Increasing the Affinity of an O‐Antigen Polysaccharide Binding Site in
            Shigella flexneri
            Bacteriophage Sf6 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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Increasing the Affinity of an O-Antigen Polysaccharide Binding Site in Shigella Flexneri Bacteriophage Sf6 Tailspike Protein

10.26434/chemrxiv.11410125 ◽

2019 ◽

Author(s):

R. Sonja Kunstmann ◽

Olof Engström ◽

Marko Wehle ◽

Göran Widmalm ◽

Mark Santer ◽

...

Keyword(s):

Molecular Dynamics ◽

Surface Plasmon Resonance ◽

Binding Site ◽

Shigella Flexneri ◽

Md Simulations ◽

Conformational Space ◽

Binding Affinities ◽

The Impact ◽

Tailspike Protein ◽

Good Agreement

We analysed the tailspike from bacteriophage Sf6 in complex with the O-polysaccharide of the pathogen Shigella flexneri. The conformational space populated by the polyrhamnose backbone of the S. flexneri O-polysaccharide as studied by an octasaccharide in complex with Sf6TSP could be well described with 2D 1H,1H-trNOESY NMR, utilizing a combination of methine-methine and methine-methyl correlations. The results are in good agreement with the conformations obtained from molecular dynamics (MD) simulations. To examine the impact of amino acid exchanges in the glycan binding site of Sf6TSP, MD simulations were used to predict increased O-polysaccharide binding affinities. We used surface plasmon resonance on S. flexneri O-polysaccharide surfaces to measure affinity increases in the obtained mutants. <br>

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Increasing the Affinity of an O-Antigen Polysaccharide Binding Site in Shigella Flexneri Bacteriophage Sf6 Tailspike Protein

10.26434/chemrxiv.11410125.v1 ◽

2019 ◽

Author(s):

R. Sonja Kunstmann ◽

Olof Engström ◽

Marko Wehle ◽

Göran Widmalm ◽

Mark Santer ◽

...

Keyword(s):

Molecular Dynamics ◽

Surface Plasmon Resonance ◽

Binding Site ◽

Shigella Flexneri ◽

Md Simulations ◽

Conformational Space ◽

Binding Affinities ◽

The Impact ◽

Tailspike Protein ◽

Good Agreement

We analysed the tailspike from bacteriophage Sf6 in complex with the O-polysaccharide of the pathogen Shigella flexneri. The conformational space populated by the polyrhamnose backbone of the S. flexneri O-polysaccharide as studied by an octasaccharide in complex with Sf6TSP could be well described with 2D 1H,1H-trNOESY NMR, utilizing a combination of methine-methine and methine-methyl correlations. The results are in good agreement with the conformations obtained from molecular dynamics (MD) simulations. To examine the impact of amino acid exchanges in the glycan binding site of Sf6TSP, MD simulations were used to predict increased O-polysaccharide binding affinities. We used surface plasmon resonance on S. flexneri O-polysaccharide surfaces to measure affinity increases in the obtained mutants. <br>

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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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A Novel Glucosyltransferase Involved in O-Antigen Modification of Shigella flexneri Serotype 1c

Journal of Bacteriology ◽

10.1128/jb.00628-09 ◽

2009 ◽

Vol 191 (21) ◽

pp. 6612-6617 ◽

Cited By ~ 40

Author(s):

Robert M. Stagg ◽

Swee-Seong Tang ◽

Nils I. A. Carlin ◽

Kaisar A. Talukder ◽

Phung D. Cam ◽

...

Keyword(s):

Gene Cluster ◽

Shigella Flexneri ◽

Functional Expression ◽

Transposon Mutagenesis ◽

The Other ◽

Bacterial Chromosome ◽

Gene Encoding ◽

O Antigen ◽

Species Analysis

ABSTRACT The O antigen of serotype 1c differs from the unmodified O antigen of serotype Y by the addition of a disaccharide (two glucosyl groups) to the tetrasaccharide repeating unit. It was shown here that addition of the first glucosyl group is mediated by the previously characterized gtrI cluster, which is found within a cryptic prophage at the proA locus in the bacterial chromosome. Transposon mutagenesis was performed to disrupt the gene responsible for addition of the second glucosyl group, causing reversion to serotype 1a. Colony immunoblotting was used to identify the desired revertants, and subsequent sequencing, cloning, and functional expression successfully identified the gene encoding serotype 1c-specific O-antigen modification. This gene (designated gtrIC) was present as part of a three-gene cluster, similar to other S. flexneri glucosyltransferase genes. Relative to the other S. flexneri gtr clusters, the gtrIC cluster is more distantly related and appears to have arrived in S. flexneri from outside the species. Analysis of surrounding sequence suggests that the gtrIC cluster arrived via a novel bacteriophage that was subsequently rendered nonfunctional by a series of insertion events.

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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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