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      Development and validation of a FACS-based lipoprotein localization screen in the Lyme disease spirochete Borrelia burgdorferi

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      1 ,   1 , 2 , 1 , 1 ,
      BMC Microbiology
      BioMed Central

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          Abstract

          Background

          In our previous studies on lipoprotein secretion in the Lyme disease spirochete Borrelia burgdorferi, we used monomeric red fluorescent protein 1 (mRFP1) fused to specifically mutated outer surface protein A (OspA) N-terminal lipopeptides to gather first insights into lipoprotein sorting determinants. OspA:mRFP1 fusions could be detected by epifluorescence microscopy both in the periplasm and on the bacterial surface. To build on these findings and to complement the prior targeted mutagenesis approach, we set out to develop a screen to probe a random mutagenesis expression library for mutants expressing differentially localized lipoproteins.

          Results

          A Glu-Asp codon pair in the inner membrane-localized OspA20:mRFP1 fusion was chosen for mutagenesis since the two negative charges were previously shown to define the phenotype. A library of random mutants in the two codons was generated and expressed in B. burgdorferi. In situ surface proteolysis combined with fluorescence activated cell sorting (FACS) was then used to screen for viable spirochetes expressing alternative subsurface OspA:mRFP1 fusions. Analysis of 93 clones randomly picked from a sorted cell population identified a total of 43 distinct mutants. Protein localization assays indicated a significant enrichment in the selected subsurface phenotype. Interestingly, a majority of the subsurface mutant proteins localized to the outer membrane, indicating their impairment in "flipping" through the outer membrane to the spirochetal surface. OspA20:mRFP1 remained the protein most restricted to the inner membrane.

          Conclusions

          Together, these results validate this FACS-based screen for lipoprotein localization and suggest a rather specific inner membrane retention mechanism involving membrane anchor-proximal negative charge patches in this model B. burgdorferi lipoprotein system.

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          Most cited references28

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          Isolation and cultivation of Lyme disease spirochetes.

          A Barbour (1984)
          The successful isolation and cultivation of Lyme disease spirochetes traces its lineage to early attempts at cultivating relapsing fever borreliae. Observations on the growth of Lyme disease spirochetes under different in vitro conditions may yield important clues to both the metabolic characteristics of these newly discovered organisms and the pathogenesis of Lyme disease. Images FIG. 1
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            Outer-surface protein C of the Lyme disease spirochete: a protein induced in ticks for infection of mammals.

            Environmentally responsive synthesis of surface proteins represents a hallmark of the infectious cycle of the Lyme disease agent, Borrelia burgdorferi. Here we created and analyzed a B. burgdorferi mutant lacking outer-surface protein C (OspC), an abundant Osp that spirochetes normally synthesize in the tick vector during the blood meal and down-regulate after transmission to the mammal. We demonstrate that B. burgdorferi strictly requires OspC to infect mice but not to localize or migrate appropriately in the tick. The induction of a spirochetal virulence factor preceding the time and host in which it is required demonstrates a developmental sequence for transmission of this arthropod-borne pathogen.
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              • Record: found
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              Electrotransformation of the spirochete Borrelia burgdorferi.

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                Author and article information

                Journal
                BMC Microbiol
                BMC Microbiology
                BioMed Central
                1471-2180
                2010
                3 November 2010
                : 10
                : 277
                Affiliations
                [1 ]Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Mail Stop 3029, 3901 Rainbow Boulevard, Kansas City, KS, 66160, USA
                [2 ]University of Bristol, Department of Biochemistry, School of Medical Sciences, University Walk, Bristol BS8 1TD, UK
                Article
                1471-2180-10-277
                10.1186/1471-2180-10-277
                2987989
                21047413
                ee11cb91-fd61-4b6b-bbca-610ebe0112f3
                Copyright ©2010 Kumru et al; licensee BioMed Central Ltd.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 17 July 2010
                : 3 November 2010
                Categories
                Methodology Article

                Microbiology & Virology
                Microbiology & Virology

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