Modified Bacteriophage S16 Long Tail Fiber Proteins for Rapid and Specific Immobilization and Detection of Salmonella Cells

Abstract

Bacteriophage-based assays and biosensors rival traditional antibodybased immunoassays for detection of low-level Salmonella contaminations. In this study, we harnessed the binding specificity of the long tail fiber (LTF) from bacteriophage S16 as an affinity molecule for the immobilization, enrichment, and detection of Salmonella. We demonstrate that paramagnetic beads (MBs) coated with recombinant gp37-gp38 LTF complexes (LTF-MBs) are highly effective tools for rapid affinity magnetic separation and enrichment of Salmonella. Within 45 min, the LTF-MBs consistently captured over 95% of Salmonella enterica serovar Typhimurium cells from suspensions containing from 10 to 10 5 CFU · ml Ϫ1 , and they yielded equivalent recovery rates (93% Ϯ 5%, n ϭ 10) for other Salmonella strains tested. LTF-MBs also captured Salmonella cells from various food sample preenrichments, allowing the detection of initial contaminations of 1 to 10 CFU per 25 g or ml. While plating of bead-captured cells allowed ultrasensitive but time-consuming detection, the integration of LTF-based enrichment into a sandwich assay with horseradish peroxidaseconjugated LTF (HRP-LTF) as a detection probe produced a rapid and easy-to-use Salmonella detection assay. The novel enzyme-linked LTF assay (ELLTA) uses HRP-LTF to label bead-captured Salmonella cells for subsequent identification by HRP-catalyzed conversion of chromogenic 3,3=,5,5=-tetramethylbenzidine substrate. The color development was proportional for Salmonella concentrations between 10 2 and 10 7 CFU · ml Ϫ1 as determined by spectrophotometric quantification. The ELLTA assay took 2 h to complete and detected as few as 10 2 CFU · ml Ϫ1 S. Typhimurium cells. It positively identified 21 different Salmonella strains, with no cross-reactivity for other bacteria. In conclusion, the phage-based ELLTA represents a rapid, sensitive, and specific diagnostic assay that appears to be superior to other currently available tests. IMPORTANCEThe incidence of foodborne diseases has increased over the years, resulting in major global public health issues. Conventional methods for pathogen detection can be laborious and expensive, and they require lengthy preenrichment steps. Rapid enrichment-based diagnostic assays, such as immunomagnetic separation, can reduce detection times while also remaining sensitive and specific. A critical component in these tests is implementing affinity molecules that retain the ability to specifically capture target pathogens over a wide range of in situ applications. The protein complex that forms the distal tip of the bacteriophage S16 long tail fiber is shown here to represent a highly sensitive affinity molecule for the specific enrichment and detection of Salmonella. Phage-encoded long tail fibers have huge potential for development as novel affinity molecules for robust and specific diagnostics of a vast spectrum of bacteria.

Target highlights in CASP13: Experimental target structures through the eyes of their authors

“…Both phages are equipped with baseplate‐attached long tail fibers (LTFs) that mediate receptor‐binding through their distal tips . We recently exploited the Salmonella‐ specific binding of the S16 LTF as a tool for rapid, ELISA‐like detection of Salmonella contaminants in food …”

“…[64][65][66][67][68] We recently exploited the Salmonella-specific binding of the S16 LTF as a tool for rapid, ELISA-like detection of Salmonella contaminants in food. 69 The T4 and S16 LTFs are similar to each except for the structure of their distal tip that interacts with the host cell surface during host recognition. The distal tip of the T4 LTF is formed by the C-terminal domain of gene product 37 (gp37), whereas the S16 LTF carries an additional protein--gp38 that caps gp37.…”

The dawn of phage therapy

“…Phages directly enter eukaryotic host epithelial cells via active transcytosis . This phage uptake can be specific, ie, via eukaryotic cellular receptors or it can be nonspecific . It is said that during this internalization process, phages escape degradation in cellular lysosomal compartments and thus induce cellular immunity .…”

The use of bacteriophages to control and detect pathogens in the dairy industry

“…Microbead‐bound affinity proteins derived from phages incorporated into these enrichments increased the likelihood of capturing viable cells for further identification. Such is the case with a RBP of phage S16, which when immobilised onto microbeads, could recover Salmonella serovars in milk and chocolate milk from an initial inoculum of just 1–10 CFU/25 mL (Denyes et al ).…”