Salmonella Typhimurium biofilm disruption by a human antibody that binds a pan-amyloid epitope on curli

Abstract

Bacterial biofilms, especially those associated with implanted medical devices, are difficult to eradicate. Curli amyloid fibers are important components of the biofilms formed by the Enterobacteriaceae family. Here, we show that a human monoclonal antibody with pan-amyloid-binding activity (mAb 3H3) can disrupt biofilms formed by Salmonella enterica serovar Typhimurium in vitro and in vivo. The antibody disrupts the biofilm structure, enhancing biofilm eradication by antibiotics and immune cells. In mice, 3H3 injections allow antibiotic-mediated clearance of catheter-associated S. Typhimurium biofilms. Thus, monoclonal antibodies that bind a pan-amyloid epitope have potential to prevent or eradicate bacterial biofilms.

Fig. 1. Incubation of S. Typhimurium biofilms with anti-amyloid mAbs reduces biofilm thickness and curli content.

a S. Typhimurium was cultured in the absence of mAb (untreated) or in the presence of 0.5 mg/ml control antibody 6A or 0.5 mg/ml 4A6, 4G1, 2C10, or 3H3. Isogenic mutant S. Typhimurium csgBA was included as a negative control. After 72 h, biofilms were stained with the bacterial stain Syto9 (green) and amyloid stain Congo Red (red), washed extensively, and imaged using a Leica TCS confocal microscopy at ×63. ImageJ was used to create 3D reconstructions of z-stacks using the 3D projection application. Scale bars represent 25 μm. b Biofilm thickness (μm) was determined from z-stacks using Leica TCS software. c Mean relative fluorescent units (RFU) of the red channel calculated from z-stacks using ImageJ. d Biofilms were grown in the absence (untreated) of antibody or in the presence of 0.5 mg/ml 6A, 4A6, 4G1, 2C10, or 3H3. csgBA was included as a negative control. After 72 h, biofilms were stained with crystal violet, and the optical density at 570 nm was determined. Representative images of crystal violet staining are shown below the graph. Mean and SE were calculated from results from at least two independent experiments. *p < 0.05, **p < 0.01 as determined by Student’s t-test.

Fig. 2. Incubation with 3H3 alters the biofilm architecture.

a S. Typhimurium biofilms were formed in the absence of mAb (untreated) or in the presence of 0.5 mg/ml 6A, 0.5 mg/ml 3H3, or anti-CsgA serum. After 72 h, biofilms were stained with Syto9 (green) and visualized using Leica TCS confocal microscopy at ×63. 3D surface plots were created in ImageJ and all Syto9 particles of the biofilms appear as green. Particles are colored black at the bottom of the z-plane and are increased in green intensity from 0 to 220 μm on the z-plane. b Particles above the mean biofilm mass of the untreated sample appear in white using the 3D surface plot application of ImageJ. c Number of particles above the mean biofilm mass (white particles) enumerated using ImageJ. d S. Typhimurium biofilms formed in the absence of mAb (untreated) or in the presence of 0.5 mg/ml 6A, 0.5 mg/ml 3H3, or anti-CsgA. After 72 h, biofilms were stained with Syto9 (green) and incubated with 10 μl Crimson FluoSpheres 1 μm red-fluorescent glyoxylate beads (red). Biofilms were imaged using confocal microscopy and biofilm projections of z-stacks were created using ImageJ (3D projections application). Scale bars represent 25 μm. Fluorescently labeled beads (red) were visualized by removing the Syto9 green channel. ImageJ was used to create 3D reconstructions of z-stacks. White arrows indicate location of beads in the untreated sample. e Number of red Crimson FluoSpheres within biofilms enumerated using ImageJ. Mean and SE were calculated from results of at least three independent experiments. *p < 0.05, **p < 0.01 as determined by Student’s t-test.

Fig. 3. 3H3 alters the architecture of pre-established S. Typhimurium biofilms.

a Experimental schematic and representative images of biofilms established for 24 and 48 h and then incubated for an additional 24 h without or with 0.5 mg/ml 3H3. Biofilms were stained with Syto9 (green) and visualized using Leica TCS confocal microscopy at ×63. Scale bars represent 25 μm. b 3D surface plots of biofilms depicted in a created in ImageJ. All biofilm particles are colored green. c 3D surface plots in which particles above the mean biofilm mass appear in white as determined by the 3D surface plot application in ImageJ. d Number of particles above the mean biofilm mass enumerated using ImageJ. Mean and SE were calculated from results from at least three independent experiments. *p < 0.05, **p < 0.01 as determined by Student’s t-test.

Fig. 4. Synergistic effect of 3H3 and antibiotic reduces S. Typhimurium biofilm.

a S. Typhimurium biofilms were untreated or were incubated in the presence of 0.5 mg/ml control antibody 6A or 0.5 mg/ml 3H3. After 48 h, biofilms were subjected to 30 μg/ml ampicillin or not for an additional 24 h. Biofilms were stained with Syto9 (green), washed extensively, and visualized using Leica TCS confocal microscopy at ×63. Scale bars represent 25 μm. b Biofilm thickness (μm) was determined from z-stacks using Leica TCS software. Mean and SE were calculated from results from at least three independent experiments. *p < 0.05, **p < 0.01 as determined by Student’s t-test.

Fig. 5. Combination treatment of 3H3 and antibiotic leads to biofilm eradication.

a Biofilms (dashed white arrow) were established in the presence (untreated) or absence of 0.5 mg/ml 3H3 on medical grade catheters in vitro. After 72 h biofilms were stained with Syto9 (green) and amyloid curli stain Congo Red (red) and imaged using Leica TCS confocal microscopy at ×63. Scale bars represent 25 μm. Inserts are ×3 zoom images. Catheters exhibit green autofluorescence (solid white arrow). b Biofilms were established on medical grade catheters in vitro 24 h prior to insertion of catheters into flanks of mice. At 24 and 48 h post insertion, 100 μg 3H3 was injected in vivo percutaneously into the catheter lumen. Where applicable, drinking water was supplemented with 1 mg/ml ampicillin beginning 24 h prior to catheter insertion. At 72 h after catheter insertion mice were euthanized and catheters were removed, stained with Syto9 (green) and Congo Red (red), and imaged using a Leica TCS confocal microscopy at ×63. Scale bars represent 25 μm. Insets are ×3 images. Catheters exhibit green autofluorescence.

Fig. 6. Treatment with monoclonal antibody 3H3 enhances uptake of bacteria by macrophages.

a Wild-type bone marrow derived macrophages (BMDMs) were treated with 100 μl supernatants from biofilms grown in the absence (untreated) or presence of 0.5 mg/ml 3H3. After 1 h, gentamicin was added, cells were lysed, and bacteria were enumerated as colony forming units. b Wild-type BMDMs were treated with supernatants from biofilms grown in the absence (untreated) or presence of 0.5 mg/ml 3H3 adjusted to an optical density of 0.5 at 600. After 1 h, gentamicin was added, cells were lysed, and bacteria were enumerated as colony forming units. c 1 × 10⁶ wild-type immortalized macrophages were stimulated with 5 μg of untreated curli (CR Curli) or 3H3 incubated curli (3H3 CR Curli) for 1 h at 37 C and 5% CO_2. Cells were washed 3× with PBS to remove excess curli and macrophages were stained with DAPI (blue). Internalization of the curli (red) was imaged using a Leica TCS confocal microscopy at ×63. Scale bars represent 25 μm. Mean and SE were calculated from results from at least three independent experiments. *p < 0.05, **p < 0.01 as determined by Student’s t-test.