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Self-made phage libraries with heterologous inserts in the Mtd of Bordetella bronchiseptica.

ABSTRACT: Phage display libraries are widely used as tools for identifying, dissecting and optimizing ligands. Development of a simple method to access greater library diversities could expedite and expand the technique. This paper reports progress toward harnessing the naturally occurring diversity generating retroelement used by Bordetella bronchiseptica bacteriophage to alter its tail-fiber protein. Mutagenesis and testing identified four sites amenable to the insertion of <19-residue heterologous peptides within the variable region. Such sites allow auto-generation of peptide libraries surrounded by a scaffold with additional variations. The resultant self-made phage libraries were used successfully for selections targeting anti-FLAG antibody, immobilized metal affinity chromatography microtiter plates and HIV-1 gp41. The reported experiments demonstrate the utility of the major tropism determinant protein of B.bronchiseptica as a natural scaffold for diverse, phage-constructed libraries with heterologous self-made phage libraries.

SUBMITTER: Overstreet CM

PROVIDER: S-EPMC3302436 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

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Self-made phage libraries with heterologous inserts in the Mtd of Bordetella bronchiseptica.

Overstreet Cathie M CM Yuan Tom Z TZ Levin Aron M AM Kong Calvin C Coroneus John G JG Weiss Gregory A GA

Protein engineering, design & selection : PEDS 20120127 4

Phage display libraries are widely used as tools for identifying, dissecting and optimizing ligands. Development of a simple method to access greater library diversities could expedite and expand the technique. This paper reports progress toward harnessing the naturally occurring diversity generating retroelement used by Bordetella bronchiseptica bacteriophage to alter its tail-fiber protein. Mutagenesis and testing identified four sites amenable to the insertion of <19-residue heterologous pept ...[more]

PMID: 22286238

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Project description:The respiratory pathogenic bacterium Bordetella bronchiseptica can persistently survive in terrestrial and aquatic environments, providing a source of infection. However, the environmental lifestyle of the bacterium is poorly understood. In this study, expecting repeated encounters of the bacteria with environmental protists, we explored the interaction between B. bronchiseptica and a representative environmental amoeba, Acanthamoeba castellanii, and found that the bacteria resisted amoeba digestion and entered contractile vacuoles (CVs), which are intracellular compartments involved in osmoregulation, to escape amoeba cells. In prolonged coculture, A. castellanii supported the proliferation of B. bronchiseptica. The avirulent Bvg- phase, but not the virulent Bvg+ phase, of the bacteria was advantageous for survival in the amoebae. We further demonstrate that two Bvg+ phase-specific virulence factors, filamentous hemagglutinin and fimbriae, were targeted for predation by A. castellanii. These results are evidence that the BvgAS two-component system, the master regulator for Bvg phase conversion, plays an indispensable role in the survival of B. bronchiseptica in amoebae. IMPORTANCE The pathogenic bacterium Bordetella bronchiseptica, which causes respiratory diseases in various mammals, exhibits distinct Bvg+ and Bvg- phenotypes. The former represents the virulent phase, in which the bacteria express a set of virulence factors, while the role of the latter in the bacterial life cycle remains to be understood. In this study, we demonstrate that B. bronchiseptica in the Bvg- phase, but not the Bvg+ phase, survives and proliferates in coculture with Acanthamoeba castellanii, an environmental amoeba. Two Bvg+ phase-specific virulence factors, filamentous hemagglutinin and fimbriae, were targeted by A. castellanii predation. B. bronchiseptica turns into the Bvg- phase at temperatures in which the bacteria normally encounter these amoebae. These findings demonstrate that the Bvg- phase of B. bronchiseptica is advantageous for survival outside mammalian hosts and that the bacteria can utilize protists as transient hosts in natural environments.

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Self-made phage libraries with heterologous inserts in the Mtd of Bordetella bronchiseptica.

Publications

Self-made phage libraries with heterologous inserts in the Mtd of Bordetella bronchiseptica.

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