Abstract
BackgroundIn 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.ResultsA 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.ConclusionsTogether, 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.
Highlights
In our previous studies on lipoprotein secretion in the Lyme disease spirochete Borrelia burgdorferi, we used monomeric red fluorescent protein 1 fused to mutated outer surface protein A (OspA) N-terminal lipopeptides to gather first insights into lipoprotein sorting determinants
Design of a fluorescence-based screen for lipoprotein localization in B. burgdorferi In our recent studies, the use of fusions of red fluorescent monomeric red fluorescent protein 1 (mRFP1) to various N-terminal fragments and point mutants of B. burgdorferi surface lipoprotein OspA led to an initial assessment of the sequence requirements for proper surface display [4,21]
Our starting point was a previously described OspA-mRFP1 fusion, OspA20:mRFP1, which could be redirected from the inner membrane (IM) to the bacterial surface by mutagenesis of two adjacent negatively charged amino acids (Glu-Asp) at the N-terminus of mRFP1 to two Ala residues
Summary
In our previous studies on lipoprotein secretion in the Lyme disease spirochete Borrelia burgdorferi, we used monomeric red fluorescent protein 1 (mRFP1) fused to 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. Fusion of the first five residues of the mature outer surface lipoprotein OspA was sufficient to target the red fluorescent reporter protein mRFP1 to the surface of the Borrelia cell [4]. Trafficking may depend on specific biophysical properties of the tether polypeptide such as hydrophobicity, charge, or secondary structure propensity, rather than strict amino acid identity alone [8,9]
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