Fig 1.
Strain validation and LCMS analysis of PG isolated from B31-5A3/bb0605.
(A) Schematic of the putative bb0605 operon, as well as the downstream bb0608 locus, mapped from the B. burgdorferi B31 type strain genome. The transposon integration site (+300, 5’ to 3’) in bb0605 is shown (triangle) as are the length, and location, of predicted non-coding regions. (B) Relative mRNA levels of target loci to validate B31-5A3/bb0605 mutant. RT-PCR was performed on RNA isolated from both parental (B31-5A3, charcoal) and mutant (B31-5A3/bb0605, light gray) strains using locus-specific primers and normalized relative to constitutively expressed flaB. Values are the mean + /- SD. n.d., not detected after 45 cycles. (C) Identifying changes in the peptidoglycan composition of B31-5A3/bb0605. Liquid chromatogram of mutanolysin treated PG isolated from B31-5A3 (top, in black) and B31-5A3/bb0605 (bottom, in green). Each numbered peak corresponds to a muropeptide found in Tables 1 and 2, respectively. Peak 5 (shaded gray) corresponds to the muropeptide GlcNAc-MurNAc-Ala-Glu-Orn-[Gly]-Ala-Ala). (D) MS (top) and MS/MS (bottom) of the GlcNAc-MurNAc-Ala-Glu-Orn-[Gly]-Ala-Ala muropeptide. MS/MS fragmentation (inset) data confirms the pentapeptide(-Gly) structure.
Table 1.
Muropeptide analysis of peptidoglycan purified from B. burgdorferi B31-5A3.
Table 2.
Muropeptide analysis of peptidoglycan purified from B. burgdorferi B31-5A3/bb0605.
Fig 2.
Cell cycle and PG synthesis analysis of the carboxypeptidase mutant dacA.
(A) Cell length analysis as a proxy for defects in cell cycle progression. B. burgdorferi B31-5A3 (gray, n = 410) and B31-5A3/dacA (green, n = 724) strains were cultured to mid-log exponential growth, imaged by phase-contrast microscopy, and micrograph data collected was analyzed with Oufti to determine the length of each cell in the population with sub-pixel resolution [88]. Shown are the mean + /- SD, as well as the coefficient of variation (CV). (B) Analysis of zonal PG synthesis. Phase-contrast (upper), Epifluorescence (middle), and merge (lower) images are shown for both strains incubated with 0.25 mM HADA for 4 hrs. Scale bar = 5 µm. (C) Demograph of HADA signal intensity derived from B. burgdorferi B31-5A3 parental (top, n = 410) and B31-5A3/dacA mutant (bottom, n = 724) strains, attained from individual cells, organized by length. Signal intensity is shown in arbitrary units (A.U) as a heatmap. (D) Population-level HADA fluorescent signal intensity analysis of data collected in C, B. burgdorferi B31-5A3 (gray) and B31-5A3/dacA (green). **** p < 0.0001, unpaired Student’s t-test. (E) Swarm assay to assess motility. A single soft agar plate was inoculated with 7.5 µL of 109 cells/mL suspensions of both strains B. burgdorferi B31-5A3 (gray) and B31-5A3/dacA (green), on opposite sides, and colony diameters were measured after 5 days. Data shown are the mean of 3 replica plates, + /- SD. Statistical significance was assessed using an unpaired Student’s t-test.
Fig 3.
dacA mutant bacteria do not cause arthritis in a murine model of Lyme disease.
Mice injected with B31-5A3/dacA exhibit severely attenuated Lyme arthritis. (A) Both wild-type B31-5A3 and B31-5A3/dacA bacteria were needle inoculated (105 cells) into C3H/HeJ mice (n = 25 per group), and arthritis severity was assessed by visual arthritis scoring every two days. Two and four-weeks post injection, five and ten animals, respectively, were sacrificed (dotted lines) for histopathological analysis and qPCR (see Figs 4 and S4). Mean + /- SEM are shown. (B) Representative histopathology of HE-stained sections of tarsal joints from mice infected with B. burgdorferi B31-5A3 (left) and B31-5A3/dacA (right) 14-, 28-, and 84-days post injection. A single PBS control tissue is also shown. Arthritis scores for tissues shown in this panel were as follows: * = 0.0, ** = 0.25, # = 1.0, ## = 2 – 3. (C) Representative ankles from C3H/HeJ mice infected with B. burgdorferi B31-5A3 (upper) or B31-5A3/dacA (lower), 4-weeks after infection. (D) Blind histopathological scores for individual mice in each group at 14-, 28-, and 84--days post PG administration. Statistical significance was determined using Welch’s t-test, * p ≤ 0.05 (*).
Table 3.
Organ outgrowth from infected mice sacrificed 4 weeks post-infection.
Fig 4.
Alterations to PG peptides and cross-links affect tissue tropism.
(A) qPCR analysis of heart (A), ear (B), and ankle (C) tissue 4-weeks post injection. The number of copies of recA were quantified using a standard curve and then normalized to murine nidogen. The black bars indicate the average normalized bacterial load for each group of tissues, + /- SD. Statistical significance was calculated using an unpaired Student’s t-test p ≤ 0.01 (**). (D) IgG response to infection. Total, B. burgdorferi-specific IgG levels were quantified by incubating plasma samples from mice injected with B31-5A3, B31-5A3/dacA or PBS in 96-well microplates coated with B. burgdorferi lysate and determining absorbance at 450 nm.
Fig 5.
Immunodominant antigen p83/100 is a PG-associated protein (PAP) whose interactions are impacted by alterations in cell wall chemistry.
(A) ELISA-based approach to assess PG-p83/100 interaction(s). Sacculi from B. burgdorferi B31-5A3, B31-5A3/dacA, B31-ML23/p83/100 strains were purified and one-half was treated with proteinase K (+PK) while the other was incubated with buffer (-PK). A serial titration of each purified PG preparation was immobilized on microtiter plates using r-mAb2G10, and the amount of PG and p83/100 was quantified using biotin-r-mAb2G10/streptavidin:HRP, anti-p83/100 coupled with anti-rat:IgG:HRP, respectively. The PG value attained for each strain and preparation was used to normalize each p83/100 value, which were plotted as a function of absorbance (450 nm). (B) Lysates from each strain were prepared and total protein loading was optimized using immunoblots with anti-FlaB. After optimization, each immunoblot was performed with the exact same amount of lysate to detect putative PAP (anti-NapA, anti-p83/100), as well as Outer surface protein A (anti-OspA), and the FlaB loading control was repeated (anti-FlaB). (C) The same samples described in ‘A’ were used to assess the amount and localization of p83/100 in purified PG. Each PG preparation and treatment were immobilized on optical glass using poly-Lys and probed using PG-binding lectin Wheat Germ Agglutinin conjugated to Oregon Green 488 (PG, green, upper panel) and anti-p83/100 conjugated to anti-rat IgG:Alexa647 (p83/100, magenta, middle panel). A merge of both micrographs described above is also shown using the color scheme described above (lower panel). Scale bar 5 µm. (D) Total integrated p83/100 signal intensity for each sample and preparation described in ‘A’ and ‘C’. The signal intensity of each pixel, inside each sacculus was measured, and the average was calculated for each, then normalized by area (one dot). This was repeated for > 100 sacculi per sample and the median of each is shown (black line). One-way analysis of variance was used to determine significance between P83-derived signal present in B31-5A3 wild-type sacculi (-PK), relative to B31-5A3/dacA sacculi (-PK), **** p < 0.0001, ANOVA. Note that all other comparisons to wild-type (-PK) sacculi were also statistically lower (****) but not shown for simplicity. n values for each were as follows: B31-5A3 (-PK), 227; B31-5A3 (+PK), 132; B31-5A3/dacA (-PK), 255; B31-5A3/dacA (+PK), 133; B31-ML23/p83/100 (-PK), 178; B31-ML23/p83/100 (+PK), 101. (E) Population-level line scans of values attained in studies described in ‘C’. P83-derived signal intensity present in purified sacculi from B31-5A3 (-PK, gray) and B31-5A3/dacA (-PK, green) was determined by measuring the signal intensity of each pixel along the midline of the cell wall, for each sacculus, and plotted as a function of normalized length (0 – 1). Black lines show the mean of each, SD are shaded. (F) Demograph analysis of p83/100 signal intensity in sacculi purified from B31-5A3 (-PK).