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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.


MALDI-MS of the two repeat unit fraction oligosaccharides obtained from 9NATSP cleavage of polysaccharide isolated from aS. Heidelberg 586, bS. Brancaster, and cS. Paratyphi B 2924. Putative oligosaccharide structures are given in CFG notation [42] with abequose symbolized as red hexagon. Asterisks mark peaks that lost abequose, diamonds mark acetylated peaks, respectively. All theoretical and experimental masses are given in Table 1
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Fig2: MALDI-MS of the two repeat unit fraction oligosaccharides obtained from 9NATSP cleavage of polysaccharide isolated from aS. Heidelberg 586, bS. Brancaster, and cS. Paratyphi B 2924. Putative oligosaccharide structures are given in CFG notation [42] with abequose symbolized as red hexagon. Asterisks mark peaks that lost abequose, diamonds mark acetylated peaks, respectively. All theoretical and experimental masses are given in Table 1

Mentions: About half of the strains tested were of O-serogroup O4 or O4,(5). These O-antigens have the trisaccharide backbone structure α-d-Manp-1-4-α-l-Rhap-1-3-α-d-Galp-α-1-2 [22]. The didesoxyhexose abequose α-1-3-linked to mannose determines the serotype O4, if the abequose is acetylated, the serotype O4,(5) results [9]. We chose several of these strains that showed similar binding signals in ELITA with 9NATSP and examined the molecular composition of the O-antigen. For this, we analyzed oligosaccharides purified from 9NATSP-O-polysaccharide digests with MALDI-MS (Fig. 2 and Table 1). The mass spectra showed peaks corresponding to fragments of two O-antigen repeat units (2RU) as main digestion products in all samples in agreement with precedent studies on O4 lipopolysaccharides [16]. For several strains acetylations were detected. Additionally, some strains showed peaks at higher masses corresponding to 2RU fragments with two additional hexoses, for example in case of S. Brancaster (Fig. 2b). The analysis of the monosaccharide composition of S. Brancaster O-polysaccharide with high performance anion exchange with pulsed amperometric detection (HPAEC-PAD) confirmed the presence of glucose in the O-antigen of this strain (Additional file 1: Figure S1). Glucosylated oligosaccharides were also detected with polysaccharide from S. Paratyhi B 2924, from S. Kalamu and S. Heidelberg 308. By contrast, polysaccharide from strain S. Heidelberg 586 only showed non-glycosylated oligosaccharides. From this we conclude that 9NATSP tolerates a glucosylated O-antigen.Fig. 2


Bacteriophage tailspike protein based assay to monitor phase variable glucosylations in Salmonella O-antigens
MALDI-MS of the two repeat unit fraction oligosaccharides obtained from 9NATSP cleavage of polysaccharide isolated from aS. Heidelberg 586, bS. Brancaster, and cS. Paratyphi B 2924. Putative oligosaccharide structures are given in CFG notation [42] with abequose symbolized as red hexagon. Asterisks mark peaks that lost abequose, diamonds mark acetylated peaks, respectively. All theoretical and experimental masses are given in Table 1
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC5015238&req=5

Fig2: MALDI-MS of the two repeat unit fraction oligosaccharides obtained from 9NATSP cleavage of polysaccharide isolated from aS. Heidelberg 586, bS. Brancaster, and cS. Paratyphi B 2924. Putative oligosaccharide structures are given in CFG notation [42] with abequose symbolized as red hexagon. Asterisks mark peaks that lost abequose, diamonds mark acetylated peaks, respectively. All theoretical and experimental masses are given in Table 1
Mentions: About half of the strains tested were of O-serogroup O4 or O4,(5). These O-antigens have the trisaccharide backbone structure α-d-Manp-1-4-α-l-Rhap-1-3-α-d-Galp-α-1-2 [22]. The didesoxyhexose abequose α-1-3-linked to mannose determines the serotype O4, if the abequose is acetylated, the serotype O4,(5) results [9]. We chose several of these strains that showed similar binding signals in ELITA with 9NATSP and examined the molecular composition of the O-antigen. For this, we analyzed oligosaccharides purified from 9NATSP-O-polysaccharide digests with MALDI-MS (Fig. 2 and Table 1). The mass spectra showed peaks corresponding to fragments of two O-antigen repeat units (2RU) as main digestion products in all samples in agreement with precedent studies on O4 lipopolysaccharides [16]. For several strains acetylations were detected. Additionally, some strains showed peaks at higher masses corresponding to 2RU fragments with two additional hexoses, for example in case of S. Brancaster (Fig. 2b). The analysis of the monosaccharide composition of S. Brancaster O-polysaccharide with high performance anion exchange with pulsed amperometric detection (HPAEC-PAD) confirmed the presence of glucose in the O-antigen of this strain (Additional file 1: Figure S1). Glucosylated oligosaccharides were also detected with polysaccharide from S. Paratyhi B 2924, from S. Kalamu and S. Heidelberg 308. By contrast, polysaccharide from strain S. Heidelberg 586 only showed non-glycosylated oligosaccharides. From this we conclude that 9NATSP tolerates a glucosylated O-antigen.Fig. 2

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.