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

Abstract

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

Engineered Bacteriophages for Practical Applications

“…As mentioned for tuberculosis, even though there are PCR-based assays faster than the standard method, they cannot differentiate among live and dead cells, which is critical for relevant results, and it is solved with this assay. 43) Similar techniques have been also reported for the detection of Salmonella, 44,45) E. coli, 46) and Clostridium 47,48) in different types of samples.…”

The genera of bacteriophages and their receptors are the major determinants of host range

“…A phylogenetic analysis of the RBPs of the Jerseyvirus phages showed two distinct clades with similarity to either phage 9NA or P22 tail spikes at the C‐terminal (Fig. A), which were shown to have different affinities towards O‐antigen phenotypes (Anany et al ., ; Schmidt et al ., ). Yet, our Jerseyvirus phages had not identical, but highly similar host ranges irrespective of RBP allele (Fig.…”

Salmonella Phage S16 Tail Fiber Adhesin Features a Rare Polyglycine Rich Domain for Host Recognition

“…The phage tail contracts leading to phage DNA ejection into the host. The inherent ability of S16 LTF and other phage RBPs to recognize specific pathogens has been leveraged in the development of an emerging array of biocontrol agents (Waseh et al, 2010) and affinity molecules for rapid detection and serotyping of bacteria (Arutyunov et al, 2014;Denyes et al, 2017;Javed et al, 2013;Schmidt et al, 2016;Simpson et al, 2016;Singh et al, 2010Singh et al, , 2013. Our understanding of the assembly and function of LTFs derives from studies on T4 phage (Hu et al, 2015;Leiman et al, 2010;Taylor et al, 2016;Té tart et al, 1996Té tart et al, , 1998 despite the T4 LTF composition representing only a minority of phages ( Figure 1).…”