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Bacteriophage tailspike protein based assay to monitor phase variable glucosylations in Salmonella O-antigens

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ABSTRACT

Background: Non-typhoid Salmonella Typhimurium (S. Typhimurium) accounts for a high number of registered salmonellosis cases, and O-serotyping is one important tool for monitoring epidemiology and spread of the disease. Moreover, variations in glucosylated O-antigens are related to immunogenicity and spread in the host. However, classical autoagglutination tests combined with the analysis of specific genetic markers cannot always reliably register phase variable glucose modifications expressed on Salmonella O-antigens and additional tools to monitor O-antigen glucosylation phenotypes of S. Typhimurium would be desirable.

Results: We developed a test for the phase variable O-antigen glucosylation state of S. Typhimurium using the tailspike proteins (TSP) of Salmonella phages 9NA and P22. We used this ELISA like tailspike adsorption (ELITA) assay to analyze a library of 44 Salmonella strains. ELITA was successful in discriminating strains that carried glucose 1-6 linked to the galactose of O-polysaccharide backbone (serotype O1) from non-glucosylated strains. This was shown by O-antigen compositional analyses of the respective strains with mass spectrometry and capillary electrophoresis. The ELITA test worked rapidly in a microtiter plate format and was highly O-antigen specific. Moreover, TSP as probes could also detect glucosylated strains in flow cytometry and distinguish multiphasic cultures differing in their glucosylation state.

Conclusions: Tailspike proteins contain large binding sites with precisely defined specificities and are therefore promising tools to be included in serotyping procedures as rapid serotyping agents in addition to antibodies. In this study, 9NA and P22TSP as probes could specifically distinguish glucosylation phenotypes of Salmonella on microtiter plate assays and in flow cytometry. This opens the possibility for flow sorting of cell populations for subsequent genetic analyses or for monitoring phase variations during large scale O-antigen preparations necessary for vaccine production.

Electronic supplementary material: The online version of this article (doi:10.1186/s12866-016-0826-0) contains supplementary material, which is available to authorized users.

No MeSH data available.


Comparison of 9NATSP and P22TSP O-antigen binding sites. The O6 of galactose pointing to the protein surface is shown in magenta (see arrow), a putative glucose binding groove in cyan. Right: Crystal structure of P22TSP with 2RU oligosaccharide (pdb: 1tyx) [26]. Left: Crystal structure of 9NATSP (pdb: 3riq) [16]. The 2RU oligosaccharide was positioned in the binding site after 3D alignment with P22TSP (rmsd 2.54 Å) using the CEALIGN algorithm implemented in PyMOL [43]
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Fig6: Comparison of 9NATSP and P22TSP O-antigen binding sites. The O6 of galactose pointing to the protein surface is shown in magenta (see arrow), a putative glucose binding groove in cyan. Right: Crystal structure of P22TSP with 2RU oligosaccharide (pdb: 1tyx) [26]. Left: Crystal structure of 9NATSP (pdb: 3riq) [16]. The 2RU oligosaccharide was positioned in the binding site after 3D alignment with P22TSP (rmsd 2.54 Å) using the CEALIGN algorithm implemented in PyMOL [43]

Mentions: In the present study, TSP from siphovirus 9NA and podovirus P22 were used to monitor the glucosylation state of the Salmonella O-antigen of serotype O4. We propose that in the ELITA test, both TSP could distinguish the glucosylation state of strains due to their different specificities. P22TSP could not bind to 1-6 glucosylated O-antigen, these serogroup O1 strains carry the bacteriophage P22 lysogen, the glucosylation of which prevents phage P22 from infection [25]. By contrast, 9NATSP bound to these strains, which implies that it can specifically bind 1-6 linked glucose. Comparing the O-antigen binding sites of 9NA and P22TSP we propose that in the P22TSP binding site, glucose at this position would probably not fit into the pocket (Fig. 6). By contrast, the binding site of 9NATSP contains a large surface cavity which could probably accommodate 1-6 linked glucose. This further corroborates that 9NATSP is specific for serogroup O1 whereas P22TSP cannot recognize the 1-6 glucose modification [21]. We also found O4 strains where 9NATSP was a weak binder while P22TSP showed a high signal, i.e. for S. Agona, S. Brandenburg, S. Chester or S. Derby (Additional file 2: Figure S2). Although we have not analyzed the glucose content in O-antigens of these strains we propose that they carry 1-4 glucosylations (serotype O12-2) because analysis of phage endorhamnosidase activities had shown earlier that phage 9NA in contrast to phage P22 was unable to produce oligosaccharides from O12-2 strains [21]. This was further supported by an ELITA test on S. Typhi, where P22TSP showed good binding on a O12-2 typed strain whereas 9NATSP showed a weak signal on this strain (Additional file 2: Figure S2). P22TSP can bind to O12-2 O-antigens as shown by crystal structure analysis [26]. The overlay of this structure with the 9NATSP binding site could not show why 9NATSP does not tolerate the 1-4 glucosylation and we are yet to solve a crystal structure to analyze why the architecture of the 9NATSP O-antigen interaction site prevents binding of O12-2 O-antigens. Taken together, P22TSP and 9NATSP are thus useful probes to distinguish O1 from non-O1 Salmonella serogroup O4 strains when employed in a comparative ELITA test (cf. Fig. 3). Moreover, we suggest that they could be employed to distinguish O12-2 positive from O12-2 negative strains, although the molecular details for the lacking interaction with 9NATSP remain to be elucidated.Fig. 6


Bacteriophage tailspike protein based assay to monitor phase variable glucosylations in Salmonella O-antigens
Comparison of 9NATSP and P22TSP O-antigen binding sites. The O6 of galactose pointing to the protein surface is shown in magenta (see arrow), a putative glucose binding groove in cyan. Right: Crystal structure of P22TSP with 2RU oligosaccharide (pdb: 1tyx) [26]. Left: Crystal structure of 9NATSP (pdb: 3riq) [16]. The 2RU oligosaccharide was positioned in the binding site after 3D alignment with P22TSP (rmsd 2.54 Å) using the CEALIGN algorithm implemented in PyMOL [43]
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Fig6: Comparison of 9NATSP and P22TSP O-antigen binding sites. The O6 of galactose pointing to the protein surface is shown in magenta (see arrow), a putative glucose binding groove in cyan. Right: Crystal structure of P22TSP with 2RU oligosaccharide (pdb: 1tyx) [26]. Left: Crystal structure of 9NATSP (pdb: 3riq) [16]. The 2RU oligosaccharide was positioned in the binding site after 3D alignment with P22TSP (rmsd 2.54 Å) using the CEALIGN algorithm implemented in PyMOL [43]
Mentions: In the present study, TSP from siphovirus 9NA and podovirus P22 were used to monitor the glucosylation state of the Salmonella O-antigen of serotype O4. We propose that in the ELITA test, both TSP could distinguish the glucosylation state of strains due to their different specificities. P22TSP could not bind to 1-6 glucosylated O-antigen, these serogroup O1 strains carry the bacteriophage P22 lysogen, the glucosylation of which prevents phage P22 from infection [25]. By contrast, 9NATSP bound to these strains, which implies that it can specifically bind 1-6 linked glucose. Comparing the O-antigen binding sites of 9NA and P22TSP we propose that in the P22TSP binding site, glucose at this position would probably not fit into the pocket (Fig. 6). By contrast, the binding site of 9NATSP contains a large surface cavity which could probably accommodate 1-6 linked glucose. This further corroborates that 9NATSP is specific for serogroup O1 whereas P22TSP cannot recognize the 1-6 glucose modification [21]. We also found O4 strains where 9NATSP was a weak binder while P22TSP showed a high signal, i.e. for S. Agona, S. Brandenburg, S. Chester or S. Derby (Additional file 2: Figure S2). Although we have not analyzed the glucose content in O-antigens of these strains we propose that they carry 1-4 glucosylations (serotype O12-2) because analysis of phage endorhamnosidase activities had shown earlier that phage 9NA in contrast to phage P22 was unable to produce oligosaccharides from O12-2 strains [21]. This was further supported by an ELITA test on S. Typhi, where P22TSP showed good binding on a O12-2 typed strain whereas 9NATSP showed a weak signal on this strain (Additional file 2: Figure S2). P22TSP can bind to O12-2 O-antigens as shown by crystal structure analysis [26]. The overlay of this structure with the 9NATSP binding site could not show why 9NATSP does not tolerate the 1-4 glucosylation and we are yet to solve a crystal structure to analyze why the architecture of the 9NATSP O-antigen interaction site prevents binding of O12-2 O-antigens. Taken together, P22TSP and 9NATSP are thus useful probes to distinguish O1 from non-O1 Salmonella serogroup O4 strains when employed in a comparative ELITA test (cf. Fig. 3). Moreover, we suggest that they could be employed to distinguish O12-2 positive from O12-2 negative strains, although the molecular details for the lacking interaction with 9NATSP remain to be elucidated.Fig. 6

View Article: PubMed Central - PubMed

ABSTRACT

Background: Non-typhoid Salmonella Typhimurium (S. Typhimurium) accounts for a high number of registered salmonellosis cases, and O-serotyping is one important tool for monitoring epidemiology and spread of the disease. Moreover, variations in glucosylated O-antigens are related to immunogenicity and spread in the host. However, classical autoagglutination tests combined with the analysis of specific genetic markers cannot always reliably register phase variable glucose modifications expressed on Salmonella O-antigens and additional tools to monitor O-antigen glucosylation phenotypes of S. Typhimurium would be desirable.

Results: We developed a test for the phase variable O-antigen glucosylation state of S. Typhimurium using the tailspike proteins (TSP) of Salmonella phages 9NA and P22. We used this ELISA like tailspike adsorption (ELITA) assay to analyze a library of 44 Salmonella strains. ELITA was successful in discriminating strains that carried glucose 1-6 linked to the galactose of O-polysaccharide backbone (serotype O1) from non-glucosylated strains. This was shown by O-antigen compositional analyses of the respective strains with mass spectrometry and capillary electrophoresis. The ELITA test worked rapidly in a microtiter plate format and was highly O-antigen specific. Moreover, TSP as probes could also detect glucosylated strains in flow cytometry and distinguish multiphasic cultures differing in their glucosylation state.

Conclusions: Tailspike proteins contain large binding sites with precisely defined specificities and are therefore promising tools to be included in serotyping procedures as rapid serotyping agents in addition to antibodies. In this study, 9NA and P22TSP as probes could specifically distinguish glucosylation phenotypes of Salmonella on microtiter plate assays and in flow cytometry. This opens the possibility for flow sorting of cell populations for subsequent genetic analyses or for monitoring phase variations during large scale O-antigen preparations necessary for vaccine production.

Electronic supplementary material: The online version of this article (doi:10.1186/s12866-016-0826-0) contains supplementary material, which is available to authorized users.

No MeSH data available.