C-reactive protein collaborates with plasma lectins to boost immune response against bacteria

Abstract

Although human C-reactive protein (CRP) becomes upregulated during septicemia, its role remains unclear, since purified CRP showed no binding to many common pathogens. Contrary to previous findings, we show that purified human CRP (hCRP) binds to Salmonella enterica, and that binding is enhanced in the presence of plasma factors. In the horseshoe crab, Carcinoscorpius rotundicauda, CRP is a major hemolymph protein. Incubation of hemolymph with a range of bacteria resulted in CRP binding to all the bacteria tested. Lipopolysaccharide-affinity chromatography of the hemolymph co-purified CRP, galactose-binding protein (GBP) and carcinolectin-5 (CL5). Yeast two-hybrid and pull-down assays suggested that these pattern recognition receptors (PRRs) form pathogen recognition complexes. We show the conservation of PRR crosstalk in humans, whereby hCRP interacts with ficolin (CL5 homologue). This interaction stabilizes CRP binding to bacteria and activates the lectin-mediated complement pathway. We propose that CRP does not act alone but collaborates with other plasma PRRs to form stable pathogen recognition complexes when targeting a wide range of bacteria for destruction.

Figure 1

Plasma factors enhance the binding of CRP onto bacteria. (A) Immunodetection of hCRP bound to S. enterica. S. enterica were incubated with (1) purified hCRP in human serum albumin (HSA, control), (2) purified hCRP in 10% plasma or (3) directly with 10% plasma. The experiment was performed in the presence of 1 mM CaCl₂. Proteins bound to bacteria were eluted (lanes 1–3) and analyzed by Western blot using anti-hCRP antibody. Purified hCRP (50 ng) and plasma (100 μg) were loaded as controls. (B) Immunodetection of hCRP that was bound to S. enterica (in VBS), with and without 2 mM EDTA, showed no difference in that both lacked hCRP binding, confirming that the purified hCRP and the plasma samples did not have any calcium that would have interfered with the study of hCRP binding in these experiments. (C) Immunodetection of hCRP bound to S. enterica during pretreatment, co-treatment or post-treatment with plasma and/or HSA in the indicated order of incubation. (D) SDS–PAGE of hemolymph proteins (e.g., 35, 52, 74 and 75 kDa) binding to bacteria and immunodetection of CrCRP (lower panels). Hemolymph was incubated with E. coli, P. aeruginosa, S. aureus and S. enterica. The bound proteins were eluted after various time periods of incubation with the bacteria. Lane H shows the relative abundance of hemocyanin and CRP in untreated hemolymph. Lane C shows proteins eluted from bacteria after incubation with buffer alone. (E) Immunodetection of CrCRP and GBP bound to S. enterica. Purified CrCRP, ‘hemolymph CrCRP' with an equal amount of CrCRP, or BSA alone (negative control) was incubated with S. enterica. In each treatment, either 1 mM CaCl₂, 2 mM EDTA or neither was included in the buffer. Proteins bound to the bacteria were eluted and analyzed by Western blot using anti-CrCRP and anti-GBP antibodies. Hemolymph, purified CrCRP and GBP were loaded as controls.

Figure 2

GBP and CL5 co-purified with CRP as an LPS-binding complex. (A) Schematic representation of LPS showing the O-polysaccharide and core polysaccharide and lipid A regions. Hexagons represent monosaccharides. KDO is shaded. The lipid A has acyl chains (wavy lines) attached to a disaccharide. Filled circles represent phosphate. The common substituents that are associated with the core region and lipid A moiety include phosphorylethanolamine (filled triangle) and 4-amino-4-dehydroarabinose (open triangles). The O-polysaccharide has a variable number (n) of the repeating units (in parentheses). ReLPS has only the lipid A region and two KDO residues. (B) SDS–PAGE of the plasma proteins purified with ReLPS-conjugated Sepharose or control Sepharose. Fractions from the EDTA wash and urea elution are shown. The proteins eluted from the ReLPS column (arrowed) are CRP, GBP and isoforms of CL5. Lanes ‘m' represent protein markers. (C) Peptide mass fingerprint (PMF) of trypsin-digested proteins from the p26–28 shows peaks corresponding to CRP (white box) and GBP (black box). The 1205 and 1432 peaks were common to CRP-1 and CRP-2. The 1350 and 1381 peaks were from CRP-1 and the 985 was from CRP-2. The m/z values of peaks that are unidentified are set in smaller font. A representative CrCRP sequence (CrCRP-1 hp1, GenBank accession no. AAV65022) is shown. Shaded regions represent parts of the sequences detected by peptide sequencing via ESI-Q-TOF. (D) PMF of the CL5 isoforms p35 and p40 and the peptide sequences obtained by MS sequencing of the peptide fragments show that they belong to CL5s. The CL5 peaks are set in bigger and bolder fonts. The unidentified peaks are set in smaller font. (E) PMF of p52 is similar to the fingerprint of the p26, which was confirmed to be GBP by peptide sequencing and cDNA cloning. (F) Alignment of the cloned C. rotundicauda GBP sequence against T. tridentatus GBP (TtGBP) sequence. Shaded sequences represent the fragments detectable by MS (via m/z peaks and/or peptide sequencing). In silico trypsin digestion of the CrGBP protein sequence yielded m/z values corresponding to the peaks ((C) and (E), black box) in p26–p28 and p52.

Figure 3

CRP interacts with GBP, which interacts with CL5. (A) Yeast two-hybrid analysis shows that CRP1 interacts with GBP, which in turn interacts with CL5. As the dominant isoform on the p35 spectrum from ReLPS-affinity chromatography, CL5c, was used for analysis (see Figure 2B). Growth on SC-Trp-Leu (Trp- and Leu-dropout) agar indicates the presence of both plasmids. Growth on QDO (quadruple dropout lacking Trp, Leu, His and Ade) agar indicates interaction. Empty denotes AH109 yeast strain co-transformed with either pGBKT7 or pGADT7-Rec vector without cDNA inserts. (B) One- and two-dimensional SDS–PAGE analyses show that CRP and CL5 co-purified with GBP from hemolymph. Western analysis using anti-CrCRP antibody showed that co-purification of CrCRP and GBP occurred only with 6 hpi hemolymph. The p74 and p75 (not identified) are probably hemocyanin, which, due to its sheer abundance in the hemolymph, commonly associates non-specifically onto Sepharose. (C) A first-dimensional native PAGE followed by second-dimensional denaturing SDS–PAGE of the complex co-purified with GBP from naïve hemolymph. (D) SDS–PAGE of proteins co-immunoprecipitated by anti-CRP antibody from naïve hemolymph, 6 hpi hemolymph or buffer alone (negative control). Control using an unrelated antibody (anti-IgG) for co-immunoprecipitation showed that the 74 kDa protein was nonspecific. The untreated hemolymph (naive) used for co-immunoprecipitation is shown. The 26 kDa proteins (P1 and P2) were excised for MS. (E) Matrix-assisted laser desorption ionization time of flight (MALDI-TOF) spectra of P1 and P2 show CRP (white boxes) and GBP (black boxes) peaks.

Figure 4

Interaction of hCRP with L-ficolin or myc-tagged M-ficolin FBG domain. (A) Relative binding of L-ficolin or (B) M-ficolin FBG domain to immobilized hCRP. L-ficolin or M-ficolin FBG domain bound to hCRP, acetylated BSA (AcBSA) or L. polyphemus CRP (LpCRP) that was coated on wells, was detected by anti-L-ficolin or anti-myc antibody, respectively. The positive control was directly coated L-ficolin or M-ficolin FBG domain. (C) Binding curve of M-ficolin FBG domain to immobilized hCRP. Addition of an increasing amount of M-ficolin FBG domain results in a saturable increase in its binding. The k_D of 4 × 10⁻⁸ M was determined from non-linear regression analysis of the binding curve using Sigma plot (version 8.0). (D) Relative binding of hCRP to immobilized ficolins. M-ficolin FBG domain (FBG) or L-ficolin was coated onto wells. hCRP binding was detected by goat anti-CRP antibody. Direct coating of hCRP was used as the positive control for 100% binding. For panels A, B and D, 0.5 μg of proteins was used for coating and adding of interaction partner. Readings were subtracted off negative controls (BSA-blocked wells with corresponding treatment) and expressed as a percentage of the corresponding positive control. The means±s.e.m. of three independent experiments are plotted.

Figure 5

hCRP–ficolin crosstalk enhances pathogen recognition and activates the lectin-mediated complement pathway. (A) S. enterica incubated with CRP followed by M-ficolin FBG domain (FBG), in the reverse order or concurrently were analyzed by Western blot using anti-hCRP antibody. Addition of M-ficolin FBG domain (with myc tag) either before or together (C+F) with CRP enhanced the amount of pathogen-bound CRP. (B) CRP and L-ficolin (FL) collaboration triggered MASP-2 and C4b deposition. hCRP or BSA was coated onto the 96-well plate and incubated with L-ficolin/MASP-2 complex, followed by C4. C4b deposition was detected by anti-C4c antibody. Results are the means of triplicates. ^* indicates a significant difference of P<0.05 relative to the three controls.

Figure 6

Interaction between two previously unlinked PRRs, CRP and ficolin suggests cross-activation of the classical and lectin-mediated complement pathways. The model shows that pathogen-bound CRP activates the classical pathway and cross-activates the lectin pathway via interaction with ficolin. Conversely, pathogen-bound ficolin, which activates the lectin pathway, may potentially activate the classical pathway via interaction with CRP. This ensures full activation of the complement armaments. The evolutionarily ancient status of CRP and CL5 (ficolin homologue) suggests that CRP:ficolin crosstalk represents part of an ancient complement activation pathway that is entrenched in the immune system of organisms that predated the divergence of the protostome and the deuterostome.