Fig 1.
Sequence analysis of BpiP (BB_0167) and its predicted site of interaction with Peptidoglycan (PG) of Bb.
(A) The location of bpiP (bb_0167) between malQ and dksA is conserved in borrelial species examined by bioinformatics analysis of the published genomes. A conserved peptidoglycan-interacting domain between amino acid positions 314 and 342 is present in all BpiP homologs in different borrelial species examined. (B) The PG of Bb is unique in having L-Ornithine as the third amino acid in the pentapeptide bridge cross-linking N-acetyl glucosamine (GlcNAc) and N-acetyl muramic acid (MurNAc) polymers compared to the presence of meso-DAP or L-Lysine in the PG of Gram-negative and Gram-positive bacteria, respectively. Structural analysis of BB_0167 revealed the presence of a Glutamic acid as amino acid at position 315 (E315) mediating interaction with L-Ornithine in the pentapeptide bridge. Fig 1B is adapted from references [25, 30].
Fig 2.
Cellular Localization of BpiP.
Fractionation of (A) Bb wild type strain B31- A3 and (C) bpiP mutant expressing Flag-tagged BpiP, in trans, transformed with E) plasmid pYC153. pYC153 was derived from pTM61 that constitutively express green fluorescent protein (GFP) under the control of the PflaB promoter. The construct also carries determinants for replication in both E. coli and B. burgdorferi, gentamicin resistance (aacC1) cassette, upstream regions of bpiP and bpiP fused to 3x- FLAG tag. Spirochetes were fractionated into outer membrane vesicle (OMV) and protoplasmic cylinder (PC) fractions as described in the Materials and Methods section. Fractions were separated on 12% SDS-PAGE gel stained with Coomassie brilliant blue (A, C) or transferred to PVDF membranes and subjected immunoblot analysis (B, D) using specific antisera indicated to the right of each blot. (F). Periplasmic and spheroplasmic fractions were separated on SDS-PAGE, transferred to PVDF membrane as described in the Materials and Methods section and subjected to immunoblot analysis with anti-FLAG tag, anti-Green Fluorescent Protein (gfp) or anti-FlaB antibodies. Blots were developed using goat-anti-mouse or anti-rabbit IgG conjugated with HRPO as secondary antibody and Enhanced Chemiluminescence. Lanes 1) Outer membrane vesicle fraction (OMV) 2) Periplasmic cylinder (PC). Note detection of BpiP or BpiP-FLAG only in the PC fraction using either anti-BpiP or anti-FLAG antibody. P66 localized to OMV and PotA to PC fraction in both wild type and bpiP mutant complemented with pYC153. Molecular masses in kDa are indicated to the left.
Fig 3.
(A) Schematic representation of recombinant BpiP proteins with site-specific changes at (1) position E315 or (2) with 7 site directed mutations (7SDMs) replacing amino acids of PG-interacting motif of BpiP with alanines or glycine, (3) wild type BpiP, (4) borrelial PG and (5–7) cross-linked complexes of various recombinant BpiP proteins with borrelial PG that were subjected immunoblot analysis. (B) Recombinant BpiP proteins (Lane 1–3), purified borrelial PG (Lane 4) and complexes of recombinant BpiP proteins crosslinked with borrelial PG were precipitated (pulled down) by centrifugation, dissociated via boiling and supernatants were separated as described in the flow chart in S3 Fig (Lanes 5–7) on 12% SDS-PAGE gel, transferred to PVDF membranes. Immunoblot analysis was performed using anti-BpiP serum followed by goat-anti-mouse IgG conjugated with HRPO and blots developed using Enhanced Chemiluminescence. Lanes 1) rE315ABpiP, 2) r7SDM BpiP 3) rBpiP, 4) PG only, 5) rE315A-BpiP cross-linked to PG, 6) r7SDM cross-linked to PG, 7) rBpiP cross-linked to PG. Molecular masses (M) in kilo Daltons are shown to the left. (C) Schematic representation of PG from E. coli, B. subtilis, B. burgdorferi either alone or cross-linked with BpiP and pulled down by centrifugation as complexes. Samples were subjected to boiling and supernatants were separated on 12% SDS-PAGE gel, transferred to PVDF membrane and detected for the presence of BpiP by immunoblot analysis (D) Immunoblot analysis was performed using anti-BpiP serum followed by goat-anti-mouse IgG conjugated with HRPO and blots developed using Enhanced Chemiluminescence. Lane 1) E. coli PG alone, 2) B. subtilis PG alone, 3) B. burgdorferi PG alone, 4) BpiP cross-linked to E.coli PG, 5) BpiP cross-linked to B.subtilis PG, 6) BpiP cross-linked to Bb PG. Molecular masses (M) in kilo Daltons are shown to the left.
Fig 4.
Transcriptional analysis of bpiP.
(A) Total RNA from wt grown at pH7.6/23°C or pH6.8/37°C was converted to cDNA and quantitative real-time PCR was performed as described in the Materials and Methods. The value for each sample was normalized to the value of recA and change in the Ct value for flaB or bpiP shown is an average of three replicates. The ΔΔCt values are shown as fold difference. The asterisk indicates the levels of significance as follows: *p<0.05.
Fig 5.
Generation of the bpiP mutant and complement strains.
(A) Schematic representation of the genetic manipulation performed to generate bpiP (bb_0167) mutant and cis-complemented strains. (B) Total RNA from wt, mt and ct grown at pH7.6/32°C was converted to cDNA and quantitative real-time PCR was performed as described in the Materials and Methods section. The levels of bpiP transcripts were absent as expected in the mt compared the control strains. The change in the Ct value for each sample was subjected to an unpaired t test in GraphPad Prism software. The asterisk indicates the levels of significance as follows: *** p<0.001. Total protein lysates from wt (Lane 1), mt (Lane 2) and ct (Lane 3) were separated on 12% SDS-PAGE gel and (C) stained with Coomassie blue or (D) transferred to PVDF membrane for immunoblot analysis using antibodies specific to streptomycin resistance (α-Strep, mt) or gentamicin resistance (α-Gent, ct) proteins synthesized due to the expression of aadA and gentR resistance genes under the control of borrelial promoter PflgB.
Fig 6.
Levels of pathogenesis-related proteins in bpiP mutant.
Total protein lysates of wt, mt, ct strains grown under conditions mimicking midgut of unfed tick (Lane 1, pH 7.6/ 23°C) and shifted to conditions mimicking midgut of fed tick (Lane 2, pH 6.8/37°C) were separated on a 12% SDS-PAGE gel, transferred to PVDF membrane and subjected to immunoblot analysis using antisera against various borrelial proteins indicated to the right of each blot. Goat anti-mouse HRPO-conjugated antibodies were used as secondary antibodies and blots were developed using ECL system. Molecular masses in kilo Daltons (kDa) are indicated to the left. (*indicate protein samples diluted 1:10 due to quality of the antibody reagent).
Fig 7.
Quantitative real-time PCR analysis of spirochetal burden in C3H/HeN mice infected with bpiP mutant.
Groups (n = 3) of 4-week old C3H/HeN mice were intradermally infected via needle inoculation with wt, mt and ct strains with 105 spirochetes per mouse and data from 3 such independent experiments (total n = 9 mice) were analyzed. Total genomic DNA was isolated from tissue (skin, lymph node, spleen and one joint) using the High Pure PCR template preparation kit and was then subjected to quantitative real-time PCR. Numbers of borrelial flaB copies were normalized against total mouse β-actin. Data analyzed using a one-way analysis of variance test with Tukey’s multiple-comparison test. Statistical significance was accepted when p values were less than 0.05. Tissues samples analyzed were collected at day (A) 14, (B) 28 and (C) 62 post-infection.
Table 1.
Analysis of infectivity in C3H/HeN mice with wt, bpiP mt and bpiP ct strains at day 14, 28 and 62 post infection.
Fig 8.
Quantitative real-time PCR analysis of spirochetal burden in BALB/c or SCID mice infected with bpiP mutant.
Groups (n = 3) of 4-week old immunocompetent BALB/c or immunodeficient SCID mice were intradermally infected via needle inoculation with wt, mt and ct strains with 105 spirochetes per mouse and were analyzed. Total genomic DNA was isolated from tissue (skin, lymph node, spleen and one joint) using the High Pure PCR template preparation kit and was then subjected to quantitative real-time PCR. Numbers of borrelial flaB copies were normalized against total mouse β-actin. Data analyzed using a one-way analysis of variance test with Tukey’s multiple-comparison test. Statistical significance was accepted when p values were less than 0.05. Tissues samples analyzed were collected at day 28 from (A) BALB/c (B) SCID mice.
Table 2.
Analysis of infectivity of immunocompetent BALB/c mice and immunodeficient SCID mice with wt, mt and ct strains at day 28 post infection.
Fig 9.
Quantitative real-time PCR analysis of the spirochetal burden in mice challenged with the bpiP mutant via I. scapularis nymphs.
(A) Schematic representation of the experimental plan used in mouse-tick-mouse cycle of Bb infection. Naïve I. scapularis larvae were fed to repletion on C3H/HeN mice infected with wt, mt or ct strains with 105 spirochetes per mouse via needle inoculation. Bb burden in a proportion of fed larvae (3 per mouse) determined by (B) qPCR or (E) examined for the presence of spirochetes by dark field microscopy. Another proportion of fed larvae were allowed to molt to nymphs in an incubator maintained at 22°C with 90% humidity and a 15-hour light /9-hour dark cycle. Groups (n = 3) of 4-week-old naïve C3H/HeN female mice were challenged with 10 infected nymphs and after nymphs fed to repletion. Bb burden was determined from 3 nymphs per mouse by (C) qPCR or (E) examined for the presence of spirochetes in the midgut by dark field microscopy Infectivity analysis was done at day 14 post-infection by propagating several tissues for the presence of spirochetes (Table 3). A portion of spleen, one lymph node, spleen and one joint was used to extract total genomic DNA and (D) Bb burden was determined quantitative real time PCR. The infectivity analysis was done twice and results of both the experiments are reported together for mice infected with nymphs. Numbers of borrelial flaB copies were normalized against total mouse β-actin copies. The differences between the number of wt and mt or mt and ct in mouse tissues were statistically analyzed using a one-way analysis of variance test with Tukey’s multiple-comparison test. Statistical significance was accepted when p values were less than 0.05.
Table 3.
Analysis of infectivity of C3H/HeN mice challenged with wt, mt and ct strains via infected Ixodes scapularis nymphs.
Fig 10.
Sensitivity of bpiP mutant to vancomycin.
All three strains (wt, mt and ct) were propagated at 106/ml in BSKII growth medium with (A) 0, 0.125, 0.25, 0.5, 1 and 2 μg/ml of Vancomycin, and grown at pH7.6/32°C with 1% CO2. (A) Spirochetes were enumerated every 24 hrs by dark-field microscopy and the number of spirochetes/ml at day 5 post-treatment is shown. (B) Colony forming units of cultures with 0 or 0.5 μg/ml of vancomycin at day 5 post-treatment were determined by diluting cultures of all three strains to 1:100,000 in 1X BSKII medium, plated on BSKII agar overlays and the final CFU/ml of the treated cultures were calculated after 10 days of incubation at 32°C with 1% CO2. The CFUs were determined in triplicate, one of three independent experiment is shown. Statistically analysis was done using unpaired t-test. Statistical significance was accepted when p values were less than 0.05. The asterisks indicate the levels of significance as follows: ****, p<0.0001; ***, p<0.001; **, p<0.01.
Fig 11.
Sensitivity of bpiP mutant to osmotic stress.
All three strains (wt, mt or ct) were seeded at a density of 1x106 spirochetes per ml in 5 ml of normal BSKII or BSKII supplemented with 200 or 300 mM of sodium chloride. (A) Cultures were incubated at 32°C, enumerated every 24 hours and number of spirochetes/ml at 5 days post-treatment is shown. (B) Colony forming units of cultures with 0 or 200 mM concentration of sodium chloride at day 5 post-treatment were determined by diluting cultures of all three strains to 1:100,000 in 1X BSKII medium, plated on BSKII agar overlays and the final CFU/ml of the treated cultures were calculated after 10 days of incubation at 32°C with 1% CO2. Data shown are means of three technical replicates with error bars representing Standard Error Mean and is representative of one of the three experiments. Statistical analysis was done using unpaired t-test and the asterisks indicate levels of significance as follows: ***, p<0.001; *, p <0.05.
Fig 12.
Sensitivity of bpiP mt to lysosomal extracts.
All three strains (wt, mt, ct) were seeded at a density of 1×106 spirochetes in 1ml BSKII media with or without 10% lysosomal extract obtained from murine bone marrow derived dendritic cells as described in the Materials and Methods section and incubated at 37°C, 1% CO2 for five days. (A) Spirochetes were enumerated every 24 hours and data shown is for the number of spirochetes/ml after 5 days of treatment. (B) Colony forming units of cultures with 0 or 10% lysosomal extract at day 5 post-treatment were determined by diluting cultures of all three strains to 1:100,000 in 1X BSKII medium, plated on BSKII agar overlays and the final CFU/ml of the treated cultures were calculated after 10 days of incubation at 32°C with 1% CO2. One of two independent experiments is shown. Statistical analysis of differences in the number of spirochetes between treated and untreated cultures were determined by unpaired t test. The asterisks indicate levels of significance as follows: ***, p <0.001; **, p <0.01.
Fig 13.
Sensitivity of bpiP mt to human antimicrobial peptides.
All three strains (wt, mt and ct) were propagated at 105/ml in BSKII growth medium at pH 6.8/32°C with 0 or 100 μg/ml LL37. (A) Spirochetes were enumerated every 24 hours and data shown is for the number of spirochetes/ml after 5 days of treatment. (B) Colony forming units of cultures with 0 or 100 μg/ml of LL37 were determined by diluting cultures of all three strains to 1:100,000 in 1X BSKII medium, plated on BSKII agar overlays and the final CFU/ml of the treated cultures were calculated after 10 days of incubation at 32°C with 1% CO2. The cultures were grown in triplicate and one out of two independent experiments is shown. Statistical analysis of the difference in the number of wt and mt or ct and mt spirochetes was done by unpaired t test. The asterisks indicate levels of significance as follows: **, p<0.01; *, p<0.05.
Fig 14.
Phagocytosis of bpiP mt by activated macrophages and dendritic cells.
All three strains (wt, mt and ct) were labeled with CFSE dye as described in the Materials and Methods sections and incubated with activated macrophages or dendritic cells for 10 mins. The infected cells were then harvested and stained for macrophage positive markers CD45 and F4/80 using antibodies conjugated to the fluorophores PE and APC respectively. Macrophages were identified as CD45+ (Ex(max)/Em(max): 565/578; channel 3) /F480+ (Ex(max)/Em(max): 652/660; channel 11) cells while DC were identified as CD45+/CD11c+ (conjugated to the flurophore BV421, Ex(max)/Em(max): 495/520; channel 7) but F480 negative cells. The samples were analyzed by Image Flow Cytometry with (A) macrophage markers detected using channels 3 and 11 corresponding to excitation wavelengths of 488nm and 642 nm, respectively and (C) DC markers were detected using channel 7 with an excitation wavelength of 405nm. CFSE-labeled Bb was detected using channel 2 (Bb/CFSE Ex(max)/Em(max): 495/520) with an excitation wavelength of 488nm. Data shown are means of three technical replicates with error bars representing Standard Error Mean. Pictures are representative of data obtained from one of three independent experiments. The percentage of internalized Bb within (B) macrophages and (D) dendritic cells was quantified and analyzed statistically using unpaired t test. The asterisks indicate levels of significance as follows: **, p <0.01; *, p <0.05.
Fig 15.
Seroconversion and borrelicidal antibody responses in C3H/HeN mice.
Serological response in C3H/HeN mice infected with wt, mt and ct by needle inoculation at day (A) 14, (B) 28 and (C) 62 post-infection. Antibody responses in serum from wt, mt and ct infected mice were measured using ELISA plates with whole wt Bb lysate coated as antigen. Serial two-fold dilution starting at 1:400 dilution was used and plates developed using HRPO-conjugated anti-mouse whole Ig antibodies and plates developed using ABTS as substrate and absorbance measured at OD410 nm using BIOTEK Synergy ELISA reader. Data shown are means of three technical replicates with error bars representing Standard Error Mean and is representative of one of the three experiments. (D) Wt, mt and ct strains were treated with intact or heat inactivated guinea pig complement in the presence day 14 serum from C3H/HeN mice infected with the mt as described in the materials and Materials and Methods. Survival percentage of spirochetes was determined by number of motile spirochetes from intact complemented treated sample in comparison to heat inactivated sample. The levels of significance were determined using unpaired t test with GraphPad Prism software and a p value of less than 0.5 was considered as significant.
Fig 16.
Verification of the presence of Lp28-1 and Lp25 in wt, mt, and ct strains.
Total genomic DNA from wt, mt, ct and lp28-1- deficient clone of B31-A3 (negative control) was used to determine the presence of Lp28-1 (Lane 1) and Lp25 (Lane 2) by PCR using primers listed in the reference [52, 102]. Amplicons were separated using a 1% agarose gel and molecular size markers in kilobases are shown on both sides of the gel.
Fig 17.
Levels of VlsE in wt, mt and ct strains.
Coomassie blue stained SDS-PAGE gel of proteins from borrelial strains (wt (B31-A3), lp28-1 negative clone (B31-A3), mt and ct) grown under 32°C/pH7.6 in BSKII medium (A). Immunoblot analysis of lysates from the above borrelial strains with anti-VlsE serum (B) and following Proteinase K treatment of borrelial strains (C). Immunoblots using FlaB antibodies served as controls in both B and C. Molecular weight markers in kilodaltons are indicated to the left.
Fig 18.
Graphical representation of role of BpiP.
Structural, biochemical and genetic analysis reveal that BpiP interacts with the pentapeptide of borrelial PG stabilizing the cell wall integrity and most likely anchoring PG to the inner membrane. Absence of BpiP results in a mutant strain with a significant deficit in colonization of the mammalian host and the phenotypic analysis of bpiP mutant indicated increased sensitivity to a variety of environmental stressors as well as to soluble and cellular effectors of host immune response. The effects of pathogen-specific and host-derived PG degrading enzymes on borrelial PG are unknown and the role of BpiP conferring resistance to Bb against these enzymes is bound to open novel strategies to limit the survival of Bb during various stages of the infectious cycle.
Table 4.
Plasmids and strains used in this study.
Table 5.
Primers list.