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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.


Related in: MedlinePlus

Analysis of Salmonella strains with ELISA-like tailspike adsorption (ELITA) assay using Strep-tag®II-labelled 9NATSP as a probe. One sample shows strain DB7136 incubated with 9NATSP wild type prior to ELITA (red bar). Strain E. coli HTD2158 [38] and bovine serum albumin (BSA) were used as a negative control. Numbers refer to the Wernigerode collection (cf. Table 3). Error bars represent standard deviation from four replicate experiments
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Fig1: Analysis of Salmonella strains with ELISA-like tailspike adsorption (ELITA) assay using Strep-tag®II-labelled 9NATSP as a probe. One sample shows strain DB7136 incubated with 9NATSP wild type prior to ELITA (red bar). Strain E. coli HTD2158 [38] and bovine serum albumin (BSA) were used as a negative control. Numbers refer to the Wernigerode collection (cf. Table 3). Error bars represent standard deviation from four replicate experiments

Mentions: To rapidly select for bacterial cultures binding to bacteriophage tailspike proteins (TSP) we developed a microtiter-plate based assay. For this, the bacteria were adsorbed to the surfaces of the wells. We then used Strep-tag®II-labelled 9NATSP to probe binding to 44 Salmonella strains of the Wernigerode collection and read out the signal with a Strep-Tactin® labeled horseradish peroxidase. The resulting ELISA-like tailspike adsorption (ELITA) procedure was highly specific (Fig. 1). 9NATSP recognized strains of O-serogroups O2, O4,(5) and O9. These are the serogroups found in the host range of bacteriophage 9NA [21]. By contrast, no signals were observed for non-host strains and for an E. coli strain defined as the false positive control. The host strain S. Typhimurium DB7136 used to propagate bacteriophage 9NA was used to normalize the binding signal and served as false negative control. The test was repeated four times and standard deviation from the independent experiments was less than 10 %. In the ELITA test we used a TSP that was mutated to inactivate its enzymatic cleavage of the O-polysaccharide in order to obtain a stable binding signal [16]. If we pre-incubated the bacteria with an enzymatically active TSP, no binding was detectable with the Strep-tag®II-labelled 9NATSP probe afterwards (Fig. 1). This further confirmed that the 9NATSP binding signal in the ELITA was clearly O-antigen specific. However, within the subset of binding strains, 9NATSP showed signal intensities varying from strain to strain. We purified several LPS from strains with different signals in the ELITA assay and did not observe varying chain lengths which might account for different numbers of TSP binding sites and therefore varying ELITA signals (data not shown). Rather, it is plausible that the ELITA test reflected the heterogeneous adsorption behavior of different strains to the microtiter-plate surface.Fig. 1


Bacteriophage tailspike protein based assay to monitor phase variable glucosylations in Salmonella O-antigens
Analysis of Salmonella strains with ELISA-like tailspike adsorption (ELITA) assay using Strep-tag®II-labelled 9NATSP as a probe. One sample shows strain DB7136 incubated with 9NATSP wild type prior to ELITA (red bar). Strain E. coli HTD2158 [38] and bovine serum albumin (BSA) were used as a negative control. Numbers refer to the Wernigerode collection (cf. Table 3). Error bars represent standard deviation from four replicate experiments
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig1: Analysis of Salmonella strains with ELISA-like tailspike adsorption (ELITA) assay using Strep-tag®II-labelled 9NATSP as a probe. One sample shows strain DB7136 incubated with 9NATSP wild type prior to ELITA (red bar). Strain E. coli HTD2158 [38] and bovine serum albumin (BSA) were used as a negative control. Numbers refer to the Wernigerode collection (cf. Table 3). Error bars represent standard deviation from four replicate experiments
Mentions: To rapidly select for bacterial cultures binding to bacteriophage tailspike proteins (TSP) we developed a microtiter-plate based assay. For this, the bacteria were adsorbed to the surfaces of the wells. We then used Strep-tag®II-labelled 9NATSP to probe binding to 44 Salmonella strains of the Wernigerode collection and read out the signal with a Strep-Tactin® labeled horseradish peroxidase. The resulting ELISA-like tailspike adsorption (ELITA) procedure was highly specific (Fig. 1). 9NATSP recognized strains of O-serogroups O2, O4,(5) and O9. These are the serogroups found in the host range of bacteriophage 9NA [21]. By contrast, no signals were observed for non-host strains and for an E. coli strain defined as the false positive control. The host strain S. Typhimurium DB7136 used to propagate bacteriophage 9NA was used to normalize the binding signal and served as false negative control. The test was repeated four times and standard deviation from the independent experiments was less than 10 %. In the ELITA test we used a TSP that was mutated to inactivate its enzymatic cleavage of the O-polysaccharide in order to obtain a stable binding signal [16]. If we pre-incubated the bacteria with an enzymatically active TSP, no binding was detectable with the Strep-tag®II-labelled 9NATSP probe afterwards (Fig. 1). This further confirmed that the 9NATSP binding signal in the ELITA was clearly O-antigen specific. However, within the subset of binding strains, 9NATSP showed signal intensities varying from strain to strain. We purified several LPS from strains with different signals in the ELITA assay and did not observe varying chain lengths which might account for different numbers of TSP binding sites and therefore varying ELITA signals (data not shown). Rather, it is plausible that the ELITA test reflected the heterogeneous adsorption behavior of different strains to the microtiter-plate surface.Fig. 1

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.


Related in: MedlinePlus