Virginia Research Day 2021

A Peptidoglycan Associating Protein: Neutrophil Attracting Protein A and its Role in the Biology and Pathogenesis of the Lyme Disease Spirochete

Marisela M. Davis 1 , Aaron M. Brock 1 , Tanner G. DeHart 1 , Richard F. Helm 1 , Jun Liu 2 , Brandon L. Jutras 1 1. Department of Biochemistry, Virginia Tech, Blacksburg, VA 2. Yale Microbial Sciences Institute, Yale University, West Haven, CT Neutrophil-Attracting Protein A (NapA) in B. burgdorferi

Introduction

B. Burgdorferi Shed PG Fragments During Growth

A

• A Dps (DNA-binding protein for starved bacteria) homologue; although experimental and bioinformatic analysis indicate that the B. burgdorferi lacks a DNA binding domain. • Sequesters the metal ions copper and iron • Known to stimulate IL-17 from Th17 Cells • Lyme Arthritis patients produce specific antibody to NapA Biological role in B. burgdorferi remains unknown

A

B

The_Gram-negative_spirochete Borrelia burgdorferi is the causative agent of Lyme disease, a bacterial infection transmitted to humans via the bite of an infected Ixodes tick. Lyme disease causes over 300,000 cases per year in the United States, establishing this disease as the most common vector borne illness in the country (Fig 1A). Early localized symptoms commonly manifest as the bullseye rash, and if left untreated can_result_in_late_stage symptoms_including_arthritis, carditis,_and_neurological complications (Fig 1B). It remains largely unknown what bacterial_components_are responsible for these symptoms, although recent discoveries from our lab suggest they may be attributed to the_cell_envelope_component peptidoglycan (PG) (Fig 1C).

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Figure 3 : Structure of B. burgdorferi neutrophil-attracting protein A, image rendered in Pymol

Localization of NapA within the Cell Envelope of B. burgdorfer i

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A

Cellular Location:

Outer Membrane

B

Periplasm

Figure 7:(A) B. burgdorferi lacks the genetic factors needed to undergo the PG recycling pathway, a pathway that involves the recycling of PG fragments during growth. Instead, PG fragments are shed from the cell during growth. (B) Representation of multiple generations of growth of B. burgdorferi and the associating loss of L-Ornithine, an amino acid incorporated into the PG. PG was radio-labeled using 3 H or 14 C. (C) These PG fragments are also pro-arthritic. Mice receiving tail end injections of PG fragments become arthritic within 24 hours, arthritis score, prevelance and ankle histopathological scores are shown.

Cytosol

Unknown localization

Figure 4: (A) Western blot analysis of the cellular components correlating with the potential location of NapA. (A) Incubation of live cells with Proteinase K, which cleaves proteins that are exposed on the surface of the bacteria. Listed is OspA, a known outer surface membrane protein in B. burgdorferi as a control. Cleaved proteins are then process via Western blot and tagged for the presence of OspA or NapA. Cell types are 5A4 parental strain and 5A4 napA mutant. (B) Western blot analysis for the cellular location of NapA. Whole cell lysate (L) and fraction enriched for the periplasmic space (P). Anti-NapA antibodies appear in the periplasmic fraction just as the control FlaB appears. Antibodies used were for OspA, a known outer surface membrane protein, FlaB, a periplasmic flagellar component, and BpuR, a cytosolic transcription factor.

NapA and PG Fragments are Found in B. burgdorferi Outer Membrane Vesicles

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B

NapA is Associated with PG

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Cellular Envelope Integrity

The Peptidoglycan of B. burgdorferi

Figure 6: PG fragments are released during B. burgdorferi growth, leading to the hypothesis that NapA may also be shed during this process, potentially through outer membrane vesicles (OMVs). Layers 5 and 6 of this density gradient show association of both PG and NapA within OMVs. (A) Following a crude collection of supernatant from parental and mutant strain grown in BSK-II media, the samples were ultracentrifuged to enrich for OMV collection . This sample was then again ultracentrifuged through a 45-20% iodixanol density gradient, outer membrane vesicle preparations were blotted for the presence of NapA in each density gradient layer. (B) Density gradient layers blotted against both anti-PG and anti-NapA. NapA Stimulates the Release of Interferon- A B 1500 ✱ 1500 ✱

Host Immune Detection and Response

Cellular Growth and Division

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Major Antibiotic Target

NapA 1 p Rabbit NapA 2 0 Goat Rabbit IgG-AF488 PG: WGA-AF350

200 400 600 800 pg/mL IFN g

200 400 600 800 pg/mL IFN g

10 µm

Fragments of B. burgdorferi PG are released during growth and trigger an inflammatory reaction by the host (Fig 7A). We hypothesize that these proteins are associated with the PG and would also be released during growth. Here, we focus on Neutrophil-attracting protein A (NapA), which our lab has found associating with the PG of B. burgdorferi and that we believe contributes both to the structural integrity of the cell and to the host response.

Peptidoglycan is a polymer composed of repeating sugar and peptide residues that primarily acts to protect the bacterial cell from osmotic stress. This PG layer lay between the outer and inner membrane in the periplasmic space (Fig 2). The PG is further hypothesized to have linked proteins that provide additional structural support as is seen in many other bacteria.

500 pg/mL IFN g 1000

500 pg/mL IFN g 1000

-200 0

-200 0

10 µm

10 µm

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0

12 hr 72 hr

12 hr 72 hr

Figure 5: (A) Dot blot analysis for the association of NapA with PG. Concentrations of PG were serially diluted and tagged with an anti- PG antibody (left column) and an anti-NapA antibody (right column). This analysis was performed with and without enzymatic trypsin treatment, which cleaves proteins associated with PG. Association of NapA and PG are seen in the Parental, pre-trypsin treatment and not seen in the post trypsin treatment blots. (B) F luorescence using wheat germ agglutinin conjugated to Alexa Fluor 350 (Blue) and immunofluorescence using an anti-NapA antibody conjugated to Alexa Fluor 488 (Orange).

PBS

5A4

5A4/ napA

Figure 6: Purified B. burgdorferi PG (trypsin treated) is known to stimulate an array of proinflammatory cytokines including IL-6, TNF , and IL-1B, but not INF . ELISA data for human peripheral blood mononuclear cells stimulated with digested parental and mutant PG +/- trypsin treatment for INF . (A) Comparison of the overall IFN released in pg/mL between the 72 and 12 hour time period. (B) IFN release from cells stimulated for 72 hours. From left to right the samples were 5A4/ napA , 5A4 wild type, both pre-trypsin treatment, followed by the PBS stimulated cells as a control. 5A4/ napA 5A4 PBS

Susceptibility to Stress and Ultrastructure Defects are Present in napA Mutant

Figure 1: (A) Reported, and confirmed, cases of Lyme disease in the United States of America in 2017. Each blue dot represents a single confirmed case reported to the CDC. There has been around 200% increase in the prevalence of this disease in the United States. (B) Clinical manifestations of untreated Lyme disease. The most common chronic manifestation of Lyme disease is arthritis. (C) The repeating structural motif of peptidoglycan. Repeating glycan strands composed of N-acetyl-glucosamine (GlcNAc) and N- acetylmuramic acid (MurNAc), colored in maroon. The short peptides composed of diamines and D-amino acids are colored in orange.

A

Summary

In summary, our lab has provided evidence for the association of NapA with the PG of B. burgdorferi. We have confirmed these findings in two overarching methods using fluorescent imaging and cryo-EM. These findings support the notion that NapA is involved in maintaining cell envelope integrity. NapA associating with PG may also play a role in exacerbating the host immune response during the course of Lyme disease.

Typical Gram-Negative Bacterial Cell Envelope

Key: IM: Inner Membrane PG: Peptidoglycan OM: Outer Membrane

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IM PG OM

5A4

5A4/ napA

IM PG OM

Outer Membrane

Future Plans

Periplasm

• •

Further assessment of the cell envelope integrity

Peptidoglycan (PG)

Neutrophil attracting ability of PG with and without associating NapA • Quantification of arthritic capacity of PG with and without associating NapA • Determine how NapaA is associated with PG

Inner Membrane

Acknowledgments

Figure 2: The typical cell envelope structure of a Gram-negative bacteria. Although B. burgdorferi is classified as a Gram- negative bacteria, it maintains distinctive attributes including the lack of Lipopolysaccharide, as well as the presence of periplasmic flagella. This figure portrays proteins that are common among both Gram-positive and Gram-negative bacteria which are (PG) associating proteins seen here bound to the PG within the periplasm. These proteins are primarily responsible for contributing structural integrity to the cell envelope.

• Thank you to the Virginia Tech College of Agriculture and Life Sciences for the award of the Carver Fellowship that will allow further work on this project. • Funding for the Jutras lab is provided by the NIH, NIAD, USDA, & the Virginia Tech Fralin Life Sciences Institute

Figure 6: (A) Lysozyme growth and stress test. Both parental and mutant strains were grown to the same density prior to increasing amounts of lysozyme added. Cells were allowed to grow for one week prior to growth analysis using spectrophotometry. (B) Cryogenic electron microscopy images localized at the pole of a single B. burgdorferi cell, zoomed in images show the outer membrane, peptidoglycan layer, and inner membrane. Top left image is the parental strain, and the top right image is of the mutant. Immediate results reveal a ruffled, thinner layer of peptidoglycan in the napA mutant.

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