Journal: Pathogens

Article Title: Recombinant Ixodes scapularis Calreticulin Binds Complement Proteins but Does Not Protect Borrelia burgdorferi from Complement Killing

doi: 10.3390/pathogens13070560

Figure Lengend Snippet: Differential Precipitation of Proteins (DiffPOP) reveals interactions between r Ixs CRT and complement proteins. Affinity-purified r Ixs CRT (10 µg) was pre-incubated with 10% normal human serum (NHS) for 90 min at 37 °C, while NHS and r Ixs CRT served as controls. The reactions were then stabilized and subjected to differential precipitation using escalating amounts of precipitating buffer, as described in the materials and methods section (see ). Different fractions obtained from the precipitation process were analyzed by standard SDS-PAGE and silver staining. ( A ) shows the protein profile of NHS only, ( B ) illustrates the profile of the NHS + r Ixs CRT mixture, and ( C ) displays the profile of r Ixs CRT only. Additionally, the NHS + r Ixs CRT mixture was subjected to Western blotting analysis using an antibody against the histidine fusion tag to track the precipitation of r Ixs CRT ( D ). The distinct protein profile between NHS and NHS + r Ixs CRT, highlighted by the red arrowhead in panels ( A , B ), indicated potential interactions between r Ixs CRT and specific proteins. The precipitation profile depicted in ( D ) guided the selection of fractions for subsequent LCMS/MS analysis, shedding light on the specific complement proteins interacting with r Ixs CRT. L: ladder depicting molecular weight (kDa), Lanes 1–10 = DiffPOP fractions 1–10.

Article Snippet: The test proteins, human complement serum (HCS; Innovative Research, Inc., Novi, MI, USA) were diluted to 10% in the pull-down buffer (6.5 mM sodium phosphate (pH 7.4), 140 mM NaCl, 0.02% Tween-20), added to the bead–bait complex, and incubated on a roller for 2 h at RT.

Techniques: Affinity Purification, Incubation, SDS Page, Silver Staining, Western Blot, Selection, Molecular Weight

Journal: Pathogens

Article Title: Recombinant Ixodes scapularis Calreticulin Binds Complement Proteins but Does Not Protect Borrelia burgdorferi from Complement Killing

doi: 10.3390/pathogens13070560

Figure Lengend Snippet: Reactome analysis depicting the pathways linked with proteins that were abundant ≥1.5-fold in NHS treated with CRT.

Article Snippet: The test proteins, human complement serum (HCS; Innovative Research, Inc., Novi, MI, USA) were diluted to 10% in the pull-down buffer (6.5 mM sodium phosphate (pH 7.4), 140 mM NaCl, 0.02% Tween-20), added to the bead–bait complex, and incubated on a roller for 2 h at RT.

Techniques: Binding Assay, Dissolution, Migration, Activation Assay

Journal: Pathogens

Article Title: Recombinant Ixodes scapularis Calreticulin Binds Complement Proteins but Does Not Protect Borrelia burgdorferi from Complement Killing

doi: 10.3390/pathogens13070560

Figure Lengend Snippet: Differential precipitation of complement proteins in the presence of r Ixs CRT. Fractions obtained from the differential precipitation (DiffPOP) were pooled into group A (fractions 1–5), group B (fraction 6), and group C (fractions 7 and 8). These fractions were subjected to Liquid Chromatography-Mass Spectrometry (LCMS/MS) analysis to investigate the abundance of complement proteins interacting with r Ixs CRT. ( A ) illustrates the abundance of complement proteins interacting with r Ixs CRT in group A, while ( B ) depicts the corresponding interactions in group B. The Y-axis in ( A , B ) illustrates the protein abundance, indicating the levels of proteins co-precipitated with r Ixs CRT (depicted by the red graph) in comparison to NHS only (represented by the blue graph).

Article Snippet: The test proteins, human complement serum (HCS; Innovative Research, Inc., Novi, MI, USA) were diluted to 10% in the pull-down buffer (6.5 mM sodium phosphate (pH 7.4), 140 mM NaCl, 0.02% Tween-20), added to the bead–bait complex, and incubated on a roller for 2 h at RT.

Techniques: Liquid Chromatography, Mass Spectrometry, Comparison

Journal: Pathogens

Article Title: Recombinant Ixodes scapularis Calreticulin Binds Complement Proteins but Does Not Protect Borrelia burgdorferi from Complement Killing

doi: 10.3390/pathogens13070560

Figure Lengend Snippet: Validation of complement protein and r Ixs CRT interactions via Western blotting analysis. To confirm the interactions observed in LCMS/MS analysis (referenced in ), DiffPOP fractions were subjected to standard Western blotting using antibodies specific for complement proteins: C1q, C1r, C1s, C3, C5, and C9, as indicated. Distinctive binding patterns, highlighting differences between NHS + r Ixs CRT and the NHS control, are denoted by red arrowheads. Panels ( A , C , E , G , I , K ) depict immunoblots of NHS-only samples, while panels ( B , D , F , H , J , L ) represent immunoblots of NHS + r Ixs CRT samples.

Article Snippet: The test proteins, human complement serum (HCS; Innovative Research, Inc., Novi, MI, USA) were diluted to 10% in the pull-down buffer (6.5 mM sodium phosphate (pH 7.4), 140 mM NaCl, 0.02% Tween-20), added to the bead–bait complex, and incubated on a roller for 2 h at RT.

Techniques: Western Blot, Binding Assay, Control

Journal: Pathogens

Article Title: Recombinant Ixodes scapularis Calreticulin Binds Complement Proteins but Does Not Protect Borrelia burgdorferi from Complement Killing

doi: 10.3390/pathogens13070560

Figure Lengend Snippet: Pull-down assay validated the r Ixs CRT binding of complement proteins. Affinity-purified r Ixs CRT bound to His specific magnetic beads (Dynabeads™, Thermo Fisher Scientific, Waltham, MA, USA) was used to pull down complement proteins from human complement serum (HCS). The beads were washed and the eluted protein complexes were subjected to Western blotting analysis using antibodies for the histidine tag ( A ) and complement proteins C1q ( B ), C1r ( C ), C1s ( D ), C3 ( E ), C5 ( F ), and C9 ( G ). In all the panels ( A – G ), HCS is the human complement serum that was eluted from the empty beads, i.e., without the bait protein (r Ixs CRT), and HCS + r Ixs CRT denotes the human complement proteins that were pulled down and eluted from the beads loaded with r Ixs CRT. Red arrowheads denote the detected complement proteins at their expected molecular weight size (kDa).

Article Snippet: The test proteins, human complement serum (HCS; Innovative Research, Inc., Novi, MI, USA) were diluted to 10% in the pull-down buffer (6.5 mM sodium phosphate (pH 7.4), 140 mM NaCl, 0.02% Tween-20), added to the bead–bait complex, and incubated on a roller for 2 h at RT.

Techniques: Pull Down Assay, Binding Assay, Affinity Purification, Magnetic Beads, Western Blot, Molecular Weight

Journal: Pathogens

Article Title: Recombinant Ixodes scapularis Calreticulin Binds Complement Proteins but Does Not Protect Borrelia burgdorferi from Complement Killing

doi: 10.3390/pathogens13070560

Figure Lengend Snippet: ELISA analysis demonstrates the r Ixs CRT binding of complement proteins. High binding ELISA plates coated with affinity-purified r Ixs CRT (250 ng) were incubated with normal human serum (NHS) followed by antibodies for C1q ( A ), C1r ( B ), C1s ( C ), C3 ( D ), activated C3 ( E ), C5 ( F ), C9 ( G ), and C5b-9 or MAC ( H ) antibodies. Y-axis denotes the absorbance measured at A 450nm , which reflected the intensity of the specific antibody binding to r Ixs CRT. Non-coated wells blocked with 1% BSA were incubated with NHS and used as a negative control (Neg. Cont.). Data represent mean ± SEM of 3 biological replicates. For statistical analysis, t -test was performed on GraphPad Prism 9 and ** represents p < 0.01, *** represents p < 0.001, **** represents p < 0.0001, and ns represents not significant.

Article Snippet: The test proteins, human complement serum (HCS; Innovative Research, Inc., Novi, MI, USA) were diluted to 10% in the pull-down buffer (6.5 mM sodium phosphate (pH 7.4), 140 mM NaCl, 0.02% Tween-20), added to the bead–bait complex, and incubated on a roller for 2 h at RT.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Affinity Purification, Incubation, Negative Control

Journal: Pathogens

Article Title: Recombinant Ixodes scapularis Calreticulin Binds Complement Proteins but Does Not Protect Borrelia burgdorferi from Complement Killing

doi: 10.3390/pathogens13070560

Figure Lengend Snippet: r Ixs CRT apparently enhances membrane attack complex (MAC) deposition in the classical pathway and alternate pathway but inhibits MAC deposition in the lectin pathway. The Wieslab ® Complement System Kit (Svar Life Science AB, Malmo, Sweden) was used to detect the effects of r Ixs CRT on MAC deposition via the classical pathway, alternative pathway, and lectin pathway, as described in the materials and methods section. In brief, r Ixs CRT (4 μM) was incubated with NHS (provided with the kit) at 37 °C for 30 min and then added to wells pre-coated with the antibody for MAC. Diluent and kit-provided reagent served as negative and positive controls, respectively. After washing, the conjugate and substrate were added according to the manufacturer’s instructions for each kit. NC denotes negative control and NHS denotes normal human serum, used as the positive control. % MAC deposition (Y-axis) was calculated as mentioned in the methodology section and the MAC deposition in the positive control was represented as 100% (denoted by the black dotted line). Data are presented as deposited MAC ± SEM calculated from 3 biological replicates. Statistical significance was determined by Student’s t -test in GraphPad Prism 9. * Represents p ≤ 0.05, ** represents p ≤ 0.01, and ns represents not significant.

Article Snippet: The test proteins, human complement serum (HCS; Innovative Research, Inc., Novi, MI, USA) were diluted to 10% in the pull-down buffer (6.5 mM sodium phosphate (pH 7.4), 140 mM NaCl, 0.02% Tween-20), added to the bead–bait complex, and incubated on a roller for 2 h at RT.

Techniques: Membrane, Incubation, Negative Control, Positive Control

Journal: Pathogens

Article Title: Recombinant Ixodes scapularis Calreticulin Binds Complement Proteins but Does Not Protect Borrelia burgdorferi from Complement Killing

doi: 10.3390/pathogens13070560

Figure Lengend Snippet: r Ixs CRT does not protect B. burgdorferi from complement killing. Normal human serum (NHS) was pre-incubated with serial dilutions of r Ixs CRT (1, 2, and 4 µM) or phosphate-buffered saline (PBS) at 37 °C for 30 min prior to the addition of 85 µL of 10 6 cells/mL of B. burgdorferi B314/pBBE22luc (complement-sensitive strain) and incubated in a bio-shaker at 32 °C and 100 rpm. NHS incubated with B. burgdorferi B314/pPCD100 (complement-resistant strain) was used as a positive control. Survival rates of B. burgdorferi were assessed at 3 h post-incubation. Data represent mean ± SEM of 3 biological replicates. Statistical significance was evaluated using t -test in GraphPad Prism 9 (ns: no significance, * represents p -value ≤ 0.05).

Article Snippet: The test proteins, human complement serum (HCS; Innovative Research, Inc., Novi, MI, USA) were diluted to 10% in the pull-down buffer (6.5 mM sodium phosphate (pH 7.4), 140 mM NaCl, 0.02% Tween-20), added to the bead–bait complex, and incubated on a roller for 2 h at RT.

Techniques: Incubation, Saline, Positive Control