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

Abstract

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.

Non-invasive Analysis of Peptidoglycan from Living Animals

“…WGA, also known to bind GlcNAc, was recently shown to bind to sacculi of Borrelia burgdorferi . 42 Treatment of the fecal sacculi with fluorescent WGA resulted in a 120-fold increase in fluorescence, which suggests that the material has GlcNAc ( Figure 2D ), similar to that of Lactobacilli cultured in vitro ( Figure S3 ).…”

Gram-Negative Bacterial Envelope Homeostasis under Oxidative and Nitrosative Stress

“…Although the effects of oxidative and nitrosative stress on peptidoglycan are poorly understood, a few studies reported links between oxidative or nitrosative stress and proteins associated with peptidoglycan or its synthesis [ 67 , 68 , 69 , 70 ]. The clearest demonstrated effect of oxidative stress on peptidoglycan comes from a recent study by Giacomucci et al This study showed that the absence of the protein ElyC of unknown function induces the overproduction of OH • in the periplasm, which leads to a direct or indirect interruption of peptidoglycan synthesis [ 71 ].…”

Borrelia burgdorferi, the Lyme disease spirochete, possesses genetically-encoded responses to doxycycline, but not to amoxicillin

“…Notably, expression of napA increased after 24h exposure to doxycycline (Table 1). This transcript encodes a periplasmic protein (also called BicA) that can bind copper and manganese, and associates with cell wall peptidoglycan [52][53][54]. The predicted sequence of NapA is similar to the Dps proteins of other bacterial species, which are involved with protecting DNA from stresses [55], although borrelia NapA lacks the Dps sequences that are involved with DNA-binding [52].…”

“…The predicted sequence of NapA is similar to the Dps proteins of other bacterial species, which are involved with protecting DNA from stresses [55], although borrelia NapA lacks the Dps sequences that are involved with DNA-binding [52]. NapA derives its name from neutrophil attracting protein A, and has been demonstrated to enhance immune responses [52,54,[56][57][58]. It remains to be seen whether differential expressionof NapA occurs during doxycycline treatment in the context of mammalian infection.…”