Dataset Information

Time-resolved DNA release from an O-antigen-specific Salmonella bacteriophage with a contractile tail.

ABSTRACT: Myoviruses, bacteriophages with T4-like architecture, must contract their tails prior to DNA release. However, quantitative kinetic data on myovirus particle opening are lacking, although they are promising tools in bacteriophage-based antimicrobial strategies directed against Gram-negative hosts. For the first time, we show time-resolved DNA ejection from a bacteriophage with a contractile tail, the multi-O-antigen-specific Salmonella myovirus Det7. DNA release from Det7 was triggered by lipopolysaccharide (LPS) O-antigen receptors and notably slower than in noncontractile-tailed siphoviruses. Det7 showed two individual kinetic steps for tail contraction and particle opening. Our in vitro studies showed that highly specialized tailspike proteins (TSPs) are necessary to attach the particle to LPS. A P22-like TSP confers specificity for the Salmonella Typhimurium O-antigen. Moreover, crystal structure analysis at 1.63 Å resolution confirmed that Det7 recognized the Salmonella Anatum O-antigen via an ?15-like TSP, DettilonTSP. DNA ejection triggered by LPS from either host showed similar velocities, so particle opening is thus a process independent of O-antigen composition and the recognizing TSP. In Det7, at permissive temperatures TSPs mediate O-antigen cleavage and couple cell surface binding with DNA ejection, but no irreversible adsorption occurred at low temperatures. This finding was in contrast to short-tailed Salmonella podoviruses, illustrating that tailed phages use common particle-opening mechanisms but have specialized into different infection niches.

SUBMITTER: Broeker NK

PROVIDER: S-EPMC6682738 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Time-resolved DNA release from an O-antigen-specific <i>Salmonella</i> bacteriophage with a contractile tail.

Broeker Nina K NK Roske Yvette Y Valleriani Angelo A Stephan Mareike S MS Andres Dorothee D Koetz Joachim J Heinemann Udo U Barbirz Stefanie S

The Journal of biological chemistry 20190612 31

Myoviruses, bacteriophages with T4-like architecture, must contract their tails prior to DNA release. However, quantitative kinetic data on myovirus particle opening are lacking, although they are promising tools in bacteriophage-based antimicrobial strategies directed against Gram-negative hosts. For the first time, we show time-resolved DNA ejection from a bacteriophage with a contractile tail, the multi-O-antigen-specific <i>Salmonella</i> myovirus Det7. DNA release from Det7 was triggered by ...[more]

PMID: 31189652

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Project description:Bacteriophage-based assays and biosensors rival traditional antibody-based 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 105 CFU · ml-1, and they yielded equivalent recovery rates (93% ± 5%, n = 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 peroxidase-conjugated 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 102 and 107 CFU · ml-1 as determined by spectrophotometric quantification. The ELLTA assay took 2 h to complete and detected as few as 102 CFU · ml-1S 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.IMPORTANCE The 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.

Dataset Information

Time-resolved DNA release from an O-antigen-specific Salmonella bacteriophage with a contractile tail.

Publications

Time-resolved DNA release from an O-antigen-specific <i>Salmonella</i> bacteriophage with a contractile tail.

Similar Datasets

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