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

Abstract

The Lyme disease spirochete Borrelia burgdorferi is unique among bacteria in its large number of lipoproteins that are encoded by a small, exceptionally fragmented, and predominantly linear genome. Peripherally anchored in either the inner or outer membrane and facing either the periplasm or the external environment, these lipoproteins assume varied roles. A prominent subset of lipoproteins functioning as the apparent linchpins of the enzootic tick-vertebrate infection cycle have been explored as vaccine targets. Yet, most of the B. burgdorferi lipoproteome has remained uncharacterized. Here, we comprehensively and conclusively localize the B. burgdorferi lipoproteome by applying established protein localization assays to a newly generated epitope-tagged lipoprotein expression library and by validating the obtained individual protein localization results using a sensitive global mass spectrometry approach. The derived consensus localization data indicate that 86 of the 125 analyzed lipoproteins encoded by B. burgdorferi are secreted to the bacterial surface. Thirty-one of the remaining 39 periplasmic lipoproteins are retained in the inner membrane, with only 8 lipoproteins being anchored in the periplasmic leaflet of the outer membrane. The localization of 10 lipoproteins was further defined or revised, and 52 surface and 23 periplasmic lipoproteins were newly localized. Cross-referencing prior studies revealed that the borrelial surface lipoproteome contributing to the hostpathogen interface is encoded predominantly by plasmids. Conversely, periplasmic lipoproteins are encoded mainly by chromosomal loci. These studies close a gap in our understanding of the functional lipoproteome of an important human pathogen and set the stage for more in-depth studies of thus-far-neglected spirochetal lipoproteins.IMPORTANCE The small and exceptionally fragmented genome of the Lyme disease spirochete Borrelia burgdorferi encodes over 120 lipoproteins. Studies in the field have predominantly focused on a relatively small number of surface lipoproteins that play important roles in the transmission and pathogenesis of this global human pathogen. Yet, a comprehensive spatial assessment of the entire borrelial lipoproteome has been missing. The current study newly identifies 52 surface and 23 periplasmic lipoproteins. Overall, two-thirds of the B. burgdorferi lipoproteins localize to the surface, while outer membrane lipoproteins facing the periplasm are rare. This analysis underscores the dominant contribution of lipoproteins to the spirochete's rather complex and adaptable host-pathogen interface, and it encourages further functional exploration of its lipoproteome.KEYWORDS cell envelope, lipoproteins, localization, membrane biogenesis, membrane proteins, outer membrane, protein secretion, proteomics, spirochetes, surface proteins

Blood treatment of Lyme borreliae demonstrates the mechanism of CspZ ‐mediated complement evasion to promote systemic infection in vertebrate hosts

“…Consistent to previous work (Brooks et al, ; Bykowski et al, ; Tokarz et al, ), CspA levels decreased 3.3‐fold in blood‐treated compared with untreated cells (Figure c). Finally, similar levels of CspZ were detected on permeabilized and unpermeabilized strain B31‐A3 (Figure c), in agreement with this protein localized on the surface (Bykowski et al, ; Dowdell et al, ; Hartmann et al, ). Strikingly, we observed a 5.3‐fold enhancement of CspZ production in human blood‐treated strain B31‐A3 compared to that protein's production in the same strain with no blood treatment.…”

“…Treating B. burgdorferi with proteases has been commonly used to determine a particular spirochete protein's surface localization (El‐Hage et al, ; Exner, Wu, Blanco, Miller, & Lovett, ; Zuckert, Kerentseva, Lawson, & Barbour, ). CspZ remains intact after the treatment of proteinase K and trypsin, suggesting this protein's resistance to digestion by these proteases (Coleman et al, ; Dowdell et al, ; Hartmann et al, ). However, CspZ is eliminated when spirochetes are treated with pronase (Dowdell et al, ), a protease isolated from Streptomyces griseus (Hiramatsu & Ouchi, ).…”

“…CspZ remains intact after the treatment of proteinase K and trypsin, suggesting this protein's resistance to digestion by these proteases (Coleman et al, ; Dowdell et al, ; Hartmann et al, ). However, CspZ is eliminated when spirochetes are treated with pronase (Dowdell et al, ), a protease isolated from Streptomyces griseus (Hiramatsu & Ouchi, ). This finding indicates this protein's surface localization, consistent with our and previous observations using fluorescence‐based methodologies (Bykowski et al, ; Hartmann et al, ; Siegel et al, ; Figure b,c,e,f).…”

“…The asterisk ("*") indicates significantly different protein production (P < 0.05 by Kruskal-Wallis test with Dunn's multiple comparison) between blood-treated and untreated indicated B. burgdorferi strains. Note that the production of FlaB, CspA, or CspZ among different spirochete strains when these strains were in blood-treated or untreated conditions is no different (P > 0.05 by Kruskal-Wallis test with Dunn's multiple comparison) Dowdell et al, 2017;Hartmann et al, 2006).…”

Outer surface protein polymorphisms linked to host‐spirochete association in Lyme borreliae

“…Investigating the host‐specific roles of many Lyme borreliae proteins poses difficult challenges. Borrelia burgdorferi sl encodes nearly 100 outer surface proteins, many with redundant functions and/or expressed in a similar manner (Fraser et al , ; Dowdell et al , ), which makes it difficult to delineate the phenotype promoted by each of these proteins and protein variants during infection. Thus, identifying the appropriate spirochete background strains with required defects, such as susceptibility to different hosts’ sera, lack of infectivity in different hosts or lack of adhesion to different hosts’ cells, is needed to study the influence of the protein variants on host competence.…”