'Paleontological Institute at The University of Kansas'
Abstract
Borrelia spirochetes are the causative agents of Lyme disease and relapsing fever, two common vector-borne diseases. Early experimental evidence, gained from development of genetic tools in the Lyme disease spirochete, Borrelia burgdorferi, emphasized the importance of outer surface lipo-proteins (Osps) during the infectious cycle. Although the functions of these lipoproteins and the complex mechanism of differential regulation is known in increasing detail, it remains to be understood how these virulence factors reach the spirochetal surface. We observed in previous studies that monomeric red fluorescent protein 1 (mRFP1) fused to specifically mutated outer surface protein A (OspA) lipopeptides could be detected by epifluorescence microscopy in both the periplasm and on the bacterial surface. These findings supported the notion that Borrelia spirochetes do not adhere to the +2/+3/+4 sorting rules established in other eubacteria. Rather, borrelial lipoproteins seem to contain a disordered `tether' peptide located at the extreme N-terminus of the mature lipoprotein that influences sorting within the envelope. One facet of this study utilized an N-proximal tandem negative charge (Glu-Asp) that served as an inner membrane retention signal in OspA20:mRFP1 as a target for mutagenesis. 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 subsurface OspA:mRFP1 fusions. We successfully recovered several mutants that mislocalized the lipo-mRFP1 fusions to the periplasm, adding to our database of peptide sequences that are not permissive for surface export. We then broadened our studies to include the structurally and functionally distinct dimeric OspC-Vsp family lipoproteins and identified their requirements for surface localization. As for OspA, tether sequences influence the localization of OspC-Vsp lipoproteins within the envelope. Interestingly, OspC-Vsp lipoproteins appear to be translocated across the outer membrane as monomers. This suggests that they assume their final oligomeric state only when reaching the spirochetal surface. Additionally, lower molecular weight variants of OspC and Vsp1 were detected indicating cleavage that was exacerbated upon addition of C-terminal epitope tags or mislocalization of the untagged proteins to the periplasm. C-terminal proteolysis of OspC was attributed to a carboxy-terminal protease, CtpA. To date, known substrates of CtpA include the 13-kDa outer membrane porin, P13, and a periplasmic lipoprotein BB0323. C-terminal proteolysis of OspC and Vsp1 suggests CtpA may also function as a periplasmic housekeeping protease. In turn, released C-terminal peptides may play a role in initiation of an envelope stress response. Another aspect of this work examined the subcellular localization pattern of Braun's lipoprotein (Lpp) from E. coli using B. burgdorferi as a surrogate expression host. Surprisingly, Lpp was localized to the B. burgdorferi inner membrane. On the other hand, B. burgdorferi OspA mutants were sorted by E. coli according to E. coli rules. This dataset confirmed that host factors are setting the rules for localization of lipoproteins within the bacterial envelope. Taken together, this work revealed several factors, such as the composition of the lipoprotein tether and the folding state of the lipoprotein, which influences trafficking within the spirochetal cell envelope, and also provided important insights into periplasmic lipoprotein processing of B. burgdorferi. These findings will broaden our understanding of spirochetal lipoprotein transport as well as cell envelope biogenesis. Ultimately, this work may lead to novel treatments and/or vaccination strategies that will be extremely helpful in combating Lyme disease and relapsing fever in the years and decades to come
