Comprehensive Spatial Analysis of the Borrelia burgdorferi Lipoproteome Reveals a Compartmentalization Bias toward the Bacterial Surface

Membrane fractionation of B. burgdorferi strains expressing a His-tagged lipoprotein library. Strains that were found to express proteinase K-resistant recombinant lipoproteins were subjected to membrane fractionation using a hypotonic acidic citrate buffer and sucrose gradient to obtain OMVs. Lipoproteins were then localized based on presence or absence in the OMV fraction relative to control proteins. OMVs and PCs were separated by SDS-PAGE, transferred to nitrocellulose, and analyzed by Western blotting using anti-OspA mouse MAb, anti-OppAIV rabbit polyclonal antiserum, or HisProbe-HRP reagent. Lipoproteins are organized by open reading frame with the common name listed, if applicable. Note that the PC fraction is the equivalent of a whole-cell protein preparation partially depleted of OM proteins (see the text). Parentheses flanking the ORF designation indicate the determined lipoprotein localization as follows: (ORF, inner membrane; ORF), outer membrane. The localization of BBA65 remains undetermined due to multiple isoforms with variable distributions. Determined molecular masses of the His-tagged proteins are indicated in Table 1.

Surface proteolysis of B. burgdorferi strains expressing a His-tagged lipoprotein library using pronase. (A) Intact cells expressing His-tagged lipoproteins that were determined to be proteinase K resistant but enriched in the OM were subjected to surface proteolysis with pronase. Cell lysates were then separated by SDS-PAGE and analyzed by Western blotting, as described in the legend to Fig. 1. Pronase −, untreated mock control; pronase +, pronase-treated sample. Parentheses flanking the ORF designation indicate the determined lipoprotein localization as in Fig. 1: ))ORF, surface; (ORF), periplasmic. (B) Coomassie-stained SDS-PAGE gel of B. burgdorferi strain B31-A3 whole-cell protein preparations obtained before (−) or after (+) incubation with proteinase K or pronase. Protein molecular masses, indicated to the left, were derived from a protein molecular weight marker (Bio-Rad). An asterisk indicates a known proteinase K-resistant, but apparently pronase-sensitive, fragment of integral OM protein P66 (108).

Scatter plot of MudPIT-derived dNSAF ratios of surface and periplasmic lipoproteins. Ratios of dNSAF before treatment with pK to that after proteinase K treatment (dNSAF_−pK/dNSAF_+pK) were calculated for 87 lipoproteins detected by MudPIT and plotted using GraphPad Prism. Horizontal lines indicate the mean dNSAF ratios for surface and subsurface proteins. Note that (i) infinite dNSAF values due to undetectable protein after pK treatment were capped at the highest calculated value of 185.84 for BBA16/OspB and that (ii) surface-assigned proteins that are partially resistant to pK (Fig. 1; see also the text) cluster with subsurface proteins (specific dNSAF values are given in Table 1).

Sequence alignment of B. burgdorferi surface, periplasmic OM, or IM lipoprotein tether peptides. A LogoBar (153) representation of the N-terminal sequence of known or predicted mature B. burgdorferi lipoproteins (54) illustrates the maintained complexity of surface (S), periplasmic OM (P-OM), and periplasmic IM (P-IM) lipoprotein tether peptides. The height of each column, measured in bits, is proportional to the lack of complexity at a given position. The columns are stacked from the bottom starting with the most frequently occurring residue at that position and continuing upward. Below each column are the six most frequently occurring residues at each position, in order of frequency from top to bottom. Colors indicate residues with similar characteristics (e.g., red for negatively charged Asp and Glu residues).