The peptidoglycan-associated protein NapA plays an important role in the envelope integrity and in the pathogenesis of the lyme disease spirochete.

The bacterial pathogen responsible for causing Lyme disease, Borrelia burgdorferi, is an atypical Gram-negative spirochete that is transmitted to humans via the bite of an infected Ixodes tick. In diderms, peptidoglycan (PG) is sandwiched between the inner and outer membrane of the cell envelope. In many other Gram-negative bacteria, PG is bound by protein(s), which provide both structural integrity and continuity between envelope layers. Here, we present evidence of a peptidoglycan-associated protein (PAP) in B. burgdorferi. Using an unbiased proteomics approach, we identified Neutrophil Attracting Protein A (NapA) as a PAP. Interestingly, NapA is a Dps homologue, which typically functions to bind and protect cellular DNA from damage during times of stress. While B. burgdorferi NapA is known to be involved in the oxidative stress response, it lacks the critical residues necessary for DNA binding. Biochemical and cellular studies demonstrate that NapA is localized to the B. burgdorferi periplasm and is indeed a PAP. Cryo-electron microscopy indicates that mutant bacteria, unable to produce NapA, have structural abnormalities. Defects in cell-wall integrity impact growth rate and cause the napA mutant to be more susceptible to osmotic and PG-specific stresses. NapA-linked PG is secreted in outer membrane vesicles and augments IL-17 production, relative to PG alone. Using microfluidics, we demonstrate that NapA acts as a molecular beacon-exacerbating the pathogenic properties of B. burgdorferi PG. These studies further our understanding of the B. burgdorferi cell envelope, provide critical information that underlies its pathogenesis, and highlight how a highly conserved bacterial protein can evolve mechanistically, while maintaining biological function.

A simple method to detect Borrelia burgdorferi sensu lato proteins in different sub-cellular compartments by immunofluorescence.

Spirochaetes constitute a unique phylum of bacteria, many of which cause severe clinical diseases. Borrelia burgdorferi sensu lato (B. burgdorferi s.l.)-the primary agent of Lyme borreliosis (LB)-is a quintessential member of this poorly understood phylum and the leading cause of tick-borne illness throughout most of the northern hemisphere. Despite its importance in human health, we lack a fundamental understanding of how B. burgdorferi s.l. is able to accomplish basic physiological tasks, such as DNA replication/segregation, and cell elongation or division. Recent advances in molecular tools to probe these essential cellular processes are great strides forward but require genetic manipulation. The latter is important since not all agents of LB are genetically tractable. Here, we describe a single method that is capable of fluorescently labeling B. burgdorferi s.l. proteins in different sub-cellular compartments. A comparative analysis of six different methods indicates that our optimized procedure outperforms all others and is the first to localize a cytoplasmic protein in B. burgdorferi s.l. by immunofluorescence. We contend that this strategy could be easily adapted to study the localization of any protein, in many Borrelia genospecies, information that will yield functional insights into the complex biology of this fascinating group of bacteria. In addition, it may provide new avenues of research in both in situ studies and in Lyme diagnostics.