Antibacterial Fusion Proteins Enhance Moraxella catarrhalis Killing

Abstract

Moraxella catarrhalis is a human-specific commensal of the respiratory tract and an opportunistic pathogen. It is one of the leading cause of otitis media in children and of acute exacerbations in patients with chronic obstructive pulmonary disease, resulting in significant morbidity and economic burden. Vaccines and new immunotherapeutic strategies to treat this emerging pathogen are needed. Complement is a key component of innate immunity that mediates the detection, response, and subsequent elimination of invading pathogens. Many pathogens including M. catarrhalis have evolved complement evasion mechanisms, which include the binding of human complement inhibitors such as C4b-binding protein (C4BP) and Factor H (FH). Inhibiting C4BP and FH acquisition by M. catarrhalis may provide a novel therapeutic avenue to treat infections. To achieve this, we created two chimeric proteins that combined the Moraxella-binding domains of C4BP and FH fused to human immunoglobulin Fcs: C4BP domains 1 and 2 and FH domains 6 and 7 fused to IgM and IgG Fc, respectively. As expected, FH6-7/IgG displaced FH from the bacterial surface while simultaneously activating complement via Fc-C1q interactions, together increasing pathogen elimination. C4BP1-2/IgM also increased serum killing of the bacteria through enhanced complement deposition, but did not displace C4BP from the surface of M. catarrhalis. These Fc fusion proteins could act as anti-infective immunotherapies. Many microbes bind the complement inhibitors C4BP and FH through the same domains as M. catarrhalis, therefore these Fc fusion proteins may be promising candidates as adjunctive therapy against many different drug-resistant pathogens.

Keywords: Moraxella catarrhalis; antibacterial; complement; fusion proteins; pathogen.

FIGURE 1

FH 6-7/IgG and C4BP1-2/IgM bind to M. catarrhalis. (A,B) Schematic representation of fusion proteins FH6-7/IgG and C4BP1-2/IgM, respectively. (C) Binding of FH6-7/IgG and (D) C4BP1-2/IgM to M. catarrhalis RH4 [wild-type (WT) reference strain] assessed by flow cytometry. (E–H) Binding of RH4 WT and isogenic mutants lacking uspA1 and/or uspA2 to (E) FH6-7/IgG, (F) C4BP1-2/IgM, (G) factor H (FH), and (F) C4b-binding protein (C4BP). Mean (±SD) from three independent experiments are shown. Statistical significance of differences was calculated using one-way ANOVA with Dunnett’s post-test (E–H); **p < 0.01 and ****p < 0.0001.

FIGURE 2

FH6-7/IgG competes out serum FH, but C4BP1-2/IgM does not compete out serum C4BP. (A) M. catarrhalis were pre-incubated in increasing amounts of FH6-7/IgG, followed by the addition of OmCI-treated serum (C5-inhibitor). The binding of FH from NHS was assessed by flow cytometry using antibodies specific to domains present in serum derived FH but absent on the fusion proteins. (B) FH6-7/IgG and NHS were added simultaneously to the bacteria and binding of FH from NHS was assessed. (C) M. catarrhalis pre-incubated in increasing amounts of C4BP1-2/IgM, followed by OmCI-treated serum were tested for the binding of C4BP from NHS by flow cytometry using antibodies specific to domains present in serum derived C4BP but absent on the fusion proteins. (D) Alexa Fluor 488-labeled C4BP-IgM was added simultaneously with differing concentrations of OmCI-treated serum to bacteria to assess the binding of the fusion protein. Mean (±SD) from three independent experiments are shown. Statistical significance of differences was calculated using one-way ANOVA with Dunnett’s post-test; ***p < 0.001 and ****p < 0.0001.

FIGURE 3

In a whole blood model, M. catarrhalis is killed through MAC-mediated killing. CFSE-labeled bacteria were added to whole blood previously incubated with 1 μM OmCI, 10 μM cytochalasin D or both. (A,B) M. catarrhalis clinical isolate #473 was picked as more serum resistant than most clinical isolates and the reference strain RH4 (C,D) Staphylococcus aureus JE2 was used as an example of MAC-resistant bacteria (A,C) Survival of bacteria overtime was assessed by collecting a sample at t = 0, 30, 60, and 90 min post-infection, and plating bacteria on agar plates. The number of bacteria is represented as log of CFU/ml. (B,D) Phagocytosis was assessed by detection of CFSE+ cells among granulocytes and monocytes from whole blood. At 1 and 2 h post-infection, a sample was collected, red blood cells were lysed, cells were fixed and stained with anti-CD14, and cells were analyzed using flow cytometry. The percentage of CFSE+ cells was measured in granulocytes and monocytes (gated using Side Scatter and CD14 staining). Mean (±SD) from four (A) or three (B–D) independent experiments are shown. Statistical significance of differences was calculated using two-way ANOVA with Dunnett’s post-test; *p < 0.05; **p < 0.01; and ***p < 0.001.

FIGURE 4

FH6-7/IgG and C4BP1-2/IgM increase serum killing of M. catarrhalis. (A) Bacteria were pre-incubated with PBS (control), 50 μg/ml of FH6-7/IgG or 50 μg/ml of C4BP1-2/IgM for 30 min before addition of 10% NHS. Bacteria were counted at t = 0 and t = 30 min post-addition of serum and survival percentage was calculated. (B) Fusion proteins and serum were added simultaneously to the bacteria, and bacteria survival was calculated. (C) Increasing amounts of FH6-7/IgG were added simultaneously with NHS to bacteria and survival was calculated. Heat-inactivated NHS was used as a negative control of bacteria killing. Mean (±SD) from three (A,C) or four (B) independent experiments are shown. Statistical significance of differences was calculated using one-way ANOVA with Dunnett’s post-test; *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001.

FIGURE 5

Fusion proteins increase complement deposition on the bacterial surface. Flow cytometry was used to measure C3d, iC3b, and MAC binding at the surface of M. catarrhalis. (A–C) FH6-7/IgG and serum were added simultaneously to bacteria. Bacteria were incubated with 5% OmCI-treated serum for 30 min for C3 deposition, or 10% NHS for 20 min for MAC deposition. Heat-inactivated serum was used as a negative control of complement activation. (D–F) C4BP1-2/IgM and serum were added simultaneously to bacteria. (G–I) Bacteria were pre-incubated with C4BP1-2/IgM before addition of serum. Mean (±SD) from three (A–F) or four (G–I) independent experiments are shown. Statistical significance of differences was calculated using one-way ANOVA with Dunnett’s post-test; *p < 0.05; **p < 0.01.

FIGURE 6

FH6-7/IgG binds to and increases serum killing of a large majority of M. catarrhalis clinical isolates. (A) 21 clinical isolates and the laboratory strain RH4 were tested for FH6-7/IgG binding by flow cytometry. In the absence of FH6-7/IgG, bacteria incubated with detection antibody had a similar gMFI for all isolates (average 151, standard deviation 45). (B–D) Twelve clinical isolates were treated with 50 μg/ml of FH6-7/IgG or PBS, at the same time as 15% NHS, and their survival after 30 min was assessed by plating bacteria and counting remaining colony forming-units (CFU). The remaining nine isolates were not included because their survival in serum in the absence of fusion protein was less than 5%. (B) UspA2 NTER2A strains, (C) UspA2 NTER2B strains, (D) UspA2H strains. Mean (±SD) from three (A–C) or four (D) independent experiments are shown. Statistical significance of differences was calculated using two-way ANOVA with Holm–Sidak’s post-test; *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 7

C4BP1-2/IgM binds to and increases serum killing of many of the M. catarrhalis clinical isolates tested. (A) 21 clinical isolates and the lab strain RH4 were tested for C4BP1-2/IgM binding by flow cytometry. The gMFI for control without C4BP1-2/IgM was similar in all isolates (average 152, SD 75). (B) Clinical isolates were tested for C4BP binding from NHS by flow cytometry. Moraxella isolates were treated with 5% NHS + OmCI for 30 min. The gMFI for control without NHS was similar in all isolates (average 207, SD 132). (C–E) Twelve clinical isolates were treated with 50 μg/ml of C4BP1-2/IgM or PBS, before addition of as 15% NHS, and their survival after 30 min was assessed by plating bacteria and counting remaining CFU. The remaining nine isolates were not included because their survival in serum in the absence of fusion protein was less than 5%. (C) UspA2 NTER2A strains, (D) UspA2 NTER2B strains, (E) UspA2H strains. Mean (±SD) from three (A) or four (B–E) independent experiments are shown. Statistical significance of differences was calculated using two-way ANOVA with Holm–Sidak’s post-test; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.