Evaluation of the kinetics and mechanism of action of anti-integration host factor-mediated disruption of bacterial biofilms

Abstract

The extracellular polymeric substance produced by many human pathogens during biofilm formation often contains extracellular DNA (eDNA). Strands of bacterial eDNA within the biofilm matrix can occur in a lattice-like network wherein a member of the DNABII family of DNA-binding proteins is positioned at the vertex of each crossed strand. To date, treatment of all biofilms tested with antibodies directed against one DNABII protein, Integration Host Factor (IHF), results in significant disruption. Here, using non-typeable Haemophilus influenzae as a model organism, we report that this effect was rapid, IHF-specific and mediated by binding of transiently dissociated IHF by anti-IHF even when physically separated from the biofilm by a nucleopore membrane. Further, biofilm disruption fostered killing of resident bacteria by previously ineffective antibiotics. We propose the mechanism of action to be the sequestration of IHF upon dissociation from the biofilm eDNA, forcing an equilibrium shift and ultimately, collapse of the biofilm. Further, antibodies against a peptide positioned at the DNA-binding tips of IHF were as effective as antibodies directed against the native protein. As incorporating eDNA and associated DNABII proteins is a common strategy for biofilms formed by multiple human pathogens, this novel therapeutic approach is likely to have broad utility.

Fig. 1

Disruption of NTHI biofilms by anti-IHF_{E. coli}. (A) Representative images and (B) calculated mean biomass of NTHI biofilms established for the indicated times then treated for 16 hr with either a, medium; b, naive serum; or c, anti-IHF_{E. coli}. Sera were used at a 1:50 dilution for 16, 24 and 48 hr biofilms, and at a 1:10 dilution for older biofilms. At all timepoints tested NTHI biofilms of all ages were significantly disrupted by incubation with anti-IHF_{E. coli} as compared to naive serum or medium. Data are expressed as the mean ± SEM of three independent assays. *p<0.05; **p<0.01 compared to respective naive serum treatment, one way ANOVA.

Fig. 2

Kinetics of biofilm disruption by anti-IHF_{E. coli}. (A) Representative images and (B) calculated mean biomass of NTHI biofilms established for 24 hr then treated for indicated times with a, medium, or a 1:50 dilution of b, naive serum; or c, anti-IHF_{E. coli}. Reduction in biomass mediated by anti-IHF_{E. coli} was maximal at 6 hr and sustained for 24 hr. (C) 24 hr biofilm treated for 16 hr with a, medium, or a 1:5 dilution of b, naive serum or c, anti-IHF_{E. coli}. Treatment with this greater concentration of anti-IHF_{E. coli} eradicated the biofilm, leaving a monolayer of bacteria. Data are expressed as the mean ± SEM of three independent assays. *p<0.01 compared to respective naive serum treatment, one way ANOVA.

Fig. 3

Direct contact between anti-IHF_{E. coli} and biofilm was not required to mediate disruption. (A) Representative images of 24 hr biofilms treated with either sterile medium or a 1:50 dilution of serum added to the basolateral chamber. (B-C) Biofilms treated by placement of indicated amount of antibody coupled to agarose beads into the apical chamber of a transwell, (D) NTHI biofilms after naked beads were layered under antibody-coupled beads in the apical chamber, (E) Biofilms after mixing of naked and antibody-coupled beads. (F) Biomass values after incubation with: a, medium; b, naive serum; c, anti-IHF_{E. coli}; d, coupled IgG-enriched naive serum; e, coupled IgG-enriched anti-IHF_{E. coli}; f, naked beads layered under coupled IgG-enriched naive serum; g, naked beads layered under coupled IgG-enriched anti-IHF_{E. coli}; h, mixed naked and coupled IgG-enriched naive serum; i, mixed naked and coupled IgG-enriched anti-IHF_{E. coli}. Data are expressed as the mean ± SEM of three independent assays. *p<0.05 compared to respective naive serum or IgG-enriched naive serum conjugated to agarose beads treatment, one way ANOVA.

Fig. 4

Adsorption of anti-IHF-specific antibody. (A) Representative images of 24 hr biofilms after incubation for 16 hr with a, naive serum or b, anti-IHF_{E. coli} at a 1:50 dilution or anti-IHF_{E. coli} (4.4 μg) adsorbed with: c, 0 μg IHF _{E. coli}; d, 2.2 μg IHF _{E. coli}; e, 4.4 μg IHF _{E. coli}; or f, 4.4 μg rsPilA. (B) Changes in biofilm biomass and mean thickness.. Adsorption of IHF-specific antibody counteracted biofilm disruption capability of anti-IHF_{E. coli}. Data are expressed as the mean ± SEM of three independent assays. *p<0.05 compared to naive serum, one way ANOVA.

Fig. 5

Anti-IHF_{E. coli} acted synergistically with antibiotics. (A-D) Representative images of 24 hr biofilms treated for 16 hr with a 1:50 dilution of antiserum in the presence of medium or antibiotic at the MIC₉₀ for planktonically grown NTHI strain 86-028NP. (E) Biomass and mean thickness of treated biofilms. a, medium; b, naive serum; c, anti-IHF_{E. coli}. Incubation of NTHI biofilms with anti-IHF_{E. coli} plus antibiotic markedly altered biofilm architecture, significantly reduced biofilm biomass and mean thickness and negatively impacted viability (note yellow color of biofilms which indicates that bacteria are dying). Data are expressed as the mean ± SEM of three independent assays. Bars indicate p<0.05, one way ANOVA.

Fig. 6

Bacteria newly released from the biofilm by treatment with anti-IHF_{E. coli} showed enhanced sensitivity to antibiotics. 24 hr biofilms were incubated with ampicillin (A&D), amoxicillin-clavulanate (B&E), or cefdinir (C&F) for 16 hr in the absence or presence of a 1:50 dilution of anti-IHF_{E. coli} or naive serum. (A-C) CFU of NTHI adherent within the biofilms. (DF) Sum of the planktonic and adherent NTHI. a, medium alone; b, naive serum; c, anti-IHF_{E. coli}. Data are expressed as the mean ± SEM of three independent assays. Bars indicate p< 0.05, one way ANOVA.

Fig. 7

Epitope mapping IHF_NTHI and design of a minimal IHF_NTHI-targeted peptide. 3-D model depicting reactivity of (A) chinchilla anti-IHF_{E. coli} and (B) chinchilla anti-IHF _{E. coli} – complexed to DNA to synthetic peptides representing IHF_NTHI. Regions with reactivity are indicated in fuchsia, nonreactive regions in blue. (C) 3-D model of DNA bound to IHF to show occlusion of tip-binding regions. (D) Localization of IhfA-3_NTHI (yellow) and IhfA-5 _NTHI (green) within IHF model. (E) Representative images and (F) mean biomass of 24 hr biofilms after incubation with a 1:50 dilutions of chinchilla sera for 16 hr. Data are expressed as the mean ± SEM of three independent assays. Bars indicate p< 0.01, one way ANOVA. a, naive serum; b, anti-IHF_{E. coli}; c, anti-IHF_{E. coli} complexed to DNA; d, anti-IhfA-3_NTHI; e, anti-IhfA-5_NTHI.

Fig. 8

Model of proposed mechanism by which antibodies directed against DNABII protein(s) disrupt a bacterial biofilm. In an established biofilm, IHF is bound to bacterial eDNA, thus stabilizing the 3-dimensional structure of the biofilm matrix. As such, bacteria within the biofilm are protected. 2. Antibodies directed against IHF bind to free IHF as it transiently dissociates from the eDNA matrix. 3. As the equilibrium shifts, loss of IHF from the matrix induces structural collapse of the biofilm with release of resident bacteria. 4. Newly released bacteria are highly susceptible to therapeutics, including antibodies and antibiotics.