Z-form extracellular DNA is a structural component of the bacterial biofilm matrix

Abstract

Biofilms are community architectures adopted by bacteria inclusive of a self-formed extracellular matrix that protects resident bacteria from diverse environmental stresses and, in many species, incorporates extracellular DNA (eDNA) and DNABII proteins for structural integrity throughout biofilm development. Here, we present evidence that this eDNA-based architecture relies on the rare Z-form. Z-form DNA accumulates as biofilms mature and, through stabilization by the DNABII proteins, confers structural integrity to the biofilm matrix. Indeed, substances known to drive B-DNA into Z-DNA promoted biofilm formation whereas those that drive Z-DNA into B-DNA disrupted extant biofilms. Importantly, we demonstrated that the universal bacterial DNABII family of proteins stabilizes both bacterial- and host-eDNA in the Z-form in situ. A model is proposed that incorporates the role of Z-DNA in biofilm pathogenesis, innate immune response, and immune evasion.

Keywords: DNABII proteins; DNase resistance; Z-DNA; biofilm matrix; extracellular DNA.

Figure 1.. Z-DNA was present in biofilms formed by multiple bacterial species in vitro and in vivo

(A) Both B and Z-DNA were present in biofilms. NTHI, UPEC, P. aeruginosa, S. mutans, and K. pneumoniae biofilms were established for 40h. Unfixed biofilms were incubated with rabbit monoclonal antibody against Z-DNA[Z22] [5 μg/ml] a murine monoclonal antibody against B-DNA[3519] [5 μg/ml], murine isotype IgG2a, and IgG purified from unimmunized rabbit serum [5μg/ml]. Then, biofilms were incubated with goat α-mouse IgG conjugated Alexa Fluor^® 405 [cyan] and goat α-rabbit IgG conjugated Alexa Fluor^® 488 [yellow], and counterstained with bacterial membrane stain FM^™4–64 [white]. NTHI, n=3; UPEC, n=3; Pa, n=4; S.mu, n=3; Kp, n=4. (B) NTHI biofilms could be readily established on human airway epithelial cells (HAEs) NTHI [pMDC-P1, a GFP reporter isolate, (fuchsia)] biofilms were established for 16h on primary HAEs [gray], which were incubated with murine IgG1, IgG purified from unimmunized rabbit serum as negative controls. Differential interference contrast (DIC) microscopy was used to visualize HAEs. (C) B and Z DNA could be visualized on HAEs but only when NTHI biofilms were present. No fluorescent signal was detected when HAEs only (no NTHI inoculated) were labeled with B- and Z-DNA specific monoclonal antibodies (as described in A) followed by goat-α-rabbit IgG conjugated Alexa Fluor^® [yellow], and goat-α-mouse IgG conjugated Alexa Fluor®594 [cyan]. (D) NTHI biofilms formed on HAEs immunolabeled for B-DNA and Z-DNA, as described above. Individual and merged fluorescent channel images shown. B-D, representative images shown, scale bars 5μM (n=6). (E) Representative images of immunohistochemical labeling of B-DNA with murine α-B-DNA[3519] and Z-DNA with rabbit αZ-DNA[Z22] in specimens collected during experimental and clinical diseases. Labeling was revealed with goat anti-rabbit IgG conjugated to Alexa Fluor® 647, goat anti-mouse IgG conjugated to Alexa Fluor® 488. Left: Sputum from an individual with cystic fibrosis (n=7). Right: NTHI biofilm formed for 14 days within the middle ear of a chinchilla with experimental otitis media (n=2). Insets, neg. control murine monoclonal antibody isotype IgG2a and IgG from unimmunized rabbit serum. Scale bar 10μm. (e.g., see also Figs. S2&S3)

Figure 2.. Z-DNA remained intact after DNase treatment

(A) UPEC, NTHI, and K. pneumoniae were allowed to form biofilms for 24h then incubated with DNase (Pulmozyme®; 40 U/ml in media) for 16h. Biofilms were then incubated with murine monoclonal raised against B-DNA[3519] [5 μg/ml] and rabbit monoclonal antibody raised against Z-DNA[Z22] [5 μg/ml] or IgG2a and IgG purified from unimmunized rabbit serum, then counterstained with FM^™4–64. (B) Changes in abundance of B-DNA or Z-DNA after DNase treatment were quantified as the ratio of fluorescence intensity (F.I.) of B-DNA or Z-DNA compared to untreated biofilms (- DNase) using ImageJ software. F.I. was normalized to total biomass (FM^™4–64 fluorescent signal). Error bars represent the SEM. UPEC, n=3; NTHI, n=3; Kp, n=6. Statistical significance compared to control (- DNase) was assessed by paired t-tests, **P<0.01; ****P<0.0001. (e.g., see also Figs. S1&S4).

Figure 3.. B/Z eDNA ratio modulates the physical properties of biofilms

NTHI biofilms formed for 24h were incubated with CeCl₃ (500μM) for 16h, then incubated with unimmunized rabbit IgG (5 μg/ml) or rabbit α-Z-DNA[Z22] [5 μg/ml], then with goat α-rabbit IgG conjugated with Alexa Fluor^® 488 and imaged by CLSM. (A) Representative image of an NTHI biofilm with 500μM CeCl₃. (B&C) ImageJ quantifications of Z-DNA (Alexa Fluor^® 488) and bacterial cells (FM^™4–64) signal intensities. Statistical significance compared to control was assessed by paired t-tests, *P<0.05, **P<0.01, n=7. To determine the mechanical properties of biofilms, NTHI biofilms were formed for 24h on glass fluorodishes, then incubated with media or 500μM CeCl₃ for 16h at 37°C. Additionally, 24h biofilms incubated with media or CeCl₃ for 16h, then treated with Pulmozyme® (50μg/ml) for 1h.(D) Stress-strain curves of NTHI biofilms were determined from axial mechanical indentation analysis. (E) Young’s Modulus was determined from the lower linear region of the stress-strain curve depicted in (D). Statistical significance determined by One-way ANOVA. * p <0.05, ns; not significant compared to media only control. NTHI biofilms formed for 24h were incubated with chloroquine (5μM) for 16h then incubated with unimmunized rabbit IgG (5 μg/ml) or rabbit α-Z-DNA[Z22] (5 μg/ml) and then with goat α-rabbit IgG conjugated with Alexa Fluor^® 488 and imaged by CLSM. (F) Representative image of an NTHI biofilm with 5μM chloroquine. (G&H) ImageJ quantifications of Z-DNA (Alexa Fluor^® 488) and bacterial cells (FM^™4–64) in the presence of chloroquine. Statistical significance compared to control was assessed by paired t-tests, *P<0.05, **P<0.01, n=3. Mechanical properties of 40h NTHI biofilms formed on glass fluorodishes and incubated with media or chloroquine (5μM) for 1h at 37°C. (I) Stress-strain curves of NTHI biofilms treated with chloroquine were determined as described previously (D). (J) Young’s Modulus was determined as described in (E), from stress-strain curve in (G). Statistical significance determined by One-way ANOVA or unpaired t-test * p <0.05, ns; not significant compared to media only control, n=3 [2 biofilms for each replicate was analyzed for a total of 6 biofilms per group] (K, L & M) Modulation of B/Z eDNA increased the susceptibility of biofilms to DNase after treatment with chloroquine. (K) NTHI biofilms were formed for 40h and incubated with chloroquine, DNase, or both, then stained with LIVE/DEAD®, fixed, visualized via CLSM, and images analyzed by COMSTAT. Average thickness as a percent of media control, n=7. (L) Images of the sputum solids disruption assay (n=1) where clinical sputum exudates were incubated with DPBS (control), chloroquine (100μM), DNase (30U), or in combination for 1h at 37°C, then analyzed for change in OD₆₀₀ over time, n=4. (M) Fold change in OD₆₀₀ relative to t=0. Higher OD₆₀₀ relative to t=0 indicates biofilm disruption. Scale bar 10μm. Error bars represent the SEM. Statistical significance compared to control was assessed by unpaired t-tests, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, n=4. (e.g., see also Figs. S5, S6 & Table S1).

Figure 4.. Resolution of HJs prevent, whereas DNABII proteins facilitate Z-DNA content within the biofilm

NTHI biofilms were initiated in the presence or absence of RusA (10 μg/ml) for 16h at 37°C, then washed and incubated with rabbit monoclonal antibody against Z-DNA[Z22] [5 μg/ml] and a murine monoclonal antibody against B-DNA α-B-DNA[3519] [5 μg/ml], murine isotype IgG2a (Invitrogen, 02-6200), or IgG purified from unimmunized rabbit [5μg/ml], then with goat α-rabbit IgG conjugated Alexa Fluor® 405 (yellow) and goat α-mouse IgG conjugated Alexa Fluor® 488 (cyan). (A) Representative image of RusA prevention of Z-DNA. Signal intensities were quantified by ImageJ: (B) biomass (FM^™4–64 fluorescent signal), (C) B-DNA (Alexa Fluor® 594) or (D) Z-DNA intensity (Alexa Fluor® 488), n=3. (E) Representative image of NTHI biofilms that were initiated and maintained in the presence or absence of exogenous HU_NTHI (2 μg/ml) for 40h at 37°C. Biofilms were then washed and incubated with rabbit monoclonal antibody against Z-DNA[Z22] [5μg/ml] or IgG purified from unimmunized rabbit [5μg/ml], then with goat α-rabbit IgG conjugated Alexa Fluor® 488 (yellow). (F) Z-DNA intensity (Alexa Fluor® 488) and (G) changes in biomass (FM^™4–64) were quantified by ImageJ, n=3. To determine the mechanical properties of biofilms, NTHI biofilms were formed on fluorodishes and incubated with RusA or HU_NTHI as described above. Control experiments were performed, where HU_NTHI was added as described above, but was treated with Pulmozyme® (50μg/ml) or RusA (10μg/ml) + Pulmozyme® (50μg/ml) for 1h. Additionally, NTHI biofilms were grown for 24h, incubated with CeCl3 (500μM) for 16h, then treated with RusA (10μg/ml) for 1h at 37°C (H) Stress-strain curves were determined from axial mechanical indentation analysis. (I) Young’s Modulus was determined from the lower linear region of the stress-strain curve(H), n=3 [2 biofilms for each replicate were analyzed for a total of 6 biofilms per group]. Statistical significance determined by One-way ANOVA and Dunnett’s multiple comparison test. * p <0.05, a p-value ≤ 0.01 is indicated by**, a p-value ≤ 0.001 is indicated by ***, and a p-value <0.0001 is indicated by ****, ns; not significant compared to control. Error bars represent the SEM.

Figure 5.. DNABII proteins inactivated NET function through the conversion of B-form NET eDNA to Z-form DNA

Neutrophils (1×10⁵) were allowed to attach to an 8 well glass chamberslide and induced to NET with 200nM PMA (phorbol-12-myristate-13-acetate) in the presence or absence of HU_NTHI (500nM) for 3.5h at 37°C. NETs were then fixed, washed, blocked with 10% goat serum followed by incubation with rabbit polyclonal antibody against human neutrophil elastase (1:100), rabbit monoclonal antibody against Z-DNA[Z22] (5μg/ml), and a murine monoclonal antibody against B-DNA (5μg/ml). Then NETs were incubated with goat α-rabbit IgG conjugated Alexa Fluor^® 594 and goat α-mouse IgG conjugated Alexa Fluor^® 488, wheat germ agglutinin (WGA 350), and SYTOX^™ nucleic acid stain. NETs were imaged by CLSM. (A) Representative images of NETs stained with the traditional NET markers, SYTOX^™ (nucleic acid stain, green), neutrophil elastase (active NETs, red), and WGA (membrane stain, blue), n=3. (B) Representative images of NETs formed in the presence or absence of HU_NTHI demonstrated an increased Z-DNA signal only when exogenous DNABII proteins were added. Note: Merged Z-DNA (yellow) and B-DNA (cyan) co-localize and appear white, n=3. (C) NETs killing is inactivated by DNABII proteins. NTHI biofilms were challenged with human neutrophils (B+N) and treated with 10 units/ml of Pulmozyme^® (B+N+Pulmo), 1μM HU (B+N+HU) or 1 μM CbpA (B+N+CbpA) for 4h, NTHI biofilm without neutrophils was used as a control (B). The bacteria challenged with neutrophils (B+N) [mean= 40.3%] and the group treated with CbpA (B+N+CbpA) [mean=36.2%] showed a reduction in the relative % killing as compared to the biofilm control group (B). The bacteria treated with Pulmozyme^® (B+N+Pulmo) [mean= 5.64%] or with HU (B+N+HU) [mean= 12.2%] did not show differences in the relative % killing of bacteria by PMNs. The results suggest that Pulmozyme^® or HU treatment inactivated the NET function of bacterial killing. The graph plot replicates were derived from 4 healthy donors ± SEM. The statistical analysis was performed with One-way ANOVA and Dunett’s multiple comparison test. (**=p<0.01, ***=p<0.001). (e.g., see also Fig. S7)

Figure 6.. Tiled CSLM images of an immunolabeled biofilm recovered from the middle ear of a chinchilla 11 days after challenge with NTHI

[lu – lumen of the middle ear; PMNs – dense extensive PMN-rich region above the bacterial biofilm; b – NTHI biofilm adherent to the middle ear mucosa; e – epithelium that lines the middle ear space]. The cryosection was labeled with: (A) isotype control rabbit serum and isotype control mouse serum + Alexa Fluor® 594-conjugated goat α-rabbit serum and Alexa Fluor® 488-conjugated goat α-mouse serum respectively; (B) DAPI to identify ds-DNA; (C) rabbit α-Z-DNA + Alexa Fluor® 594-conjugated goat α-rabbit serum (pseudocolored yellow); (D) murine anti-B-DNA + Alexa Fluor® 488-conjugated goat α-mouse serum (pseudocolored cyan); (E) composite image of overlayed panels B-D. Insets F-I are higher magnification 3D reconstructions of z-stack images taken from the portions of the biomass as indicated by the boxed regions in Panel E. Inset F: the uppermost portion of the biofilm closest to the lumen of the middle ear where the most recently migrated PMNs are located; Inset G: top third of the PMN-rich region of this cryosection; Inset H: bottom third of the PMN-rich region; Inset I: bacterial biofilm. Note that ds-DNA is present throughout this cryosection as evidenced by DAPI labeling in Panel B, however it is only in B-form in the PMN-rich region and not within the bacterial biofilm, as evidence by Panel D. Conversely, DNA in the bacterial biofilm is in Z-form as evidenced by labeling visible in Panel C but absent in Panel D. Labeling within insets provide additional evidence for the B-form DNA that predominates in the NETs formed by the most recently migrated PMNs (F); strands of DNA labeled for both B- and Z-form DNA in PMN-rich regions that are ‘older’ (e.g. within the middle region of the cryosection) (G); exclusively Z-form DNA labeling of NETs formed by PMNs closest to the bacterial biofilm (H); and exclusively Z-form of the eDNA within the bacterial biofilm that is adherent to the middle ear mucosa (I). Scale bar in panels A-E = 200 μm; in panels F-I = 5 μm. [Note: to best resolve deep blue DAPI labeling against the black background in panels A & B, the brightness of these two panels only was increased].

Figure 7.. Models of the B/Z-DNA equilibrium and the HJ constrained eDNA of the biofilm

Top panel: (A) In the absence of DNABII proteins, there is no eDNA lattice structure. (B) In the presence of DNABII proteins, native biofilm the equilibrium shifts between B-form and Z-form eDNA. (C) Antibodies directed against DNABII proteins collapse bacterial biofilms as this favors B-form eDNA. (D) CeCl₃ favors Z-form eDNA, which stabilizes the biofilm matrix and promotes biofilm residence. (E) Chloroquine favors B-form eDNA, which destabilizes the biofilm matrix and releases bacteria from biofilm residence. (F) Exogenous DNABII proteins shift the equilibrium to favor Z-DNA via Holliday Junction-like stabilization. Bottom panel: (A) B-form predominates when linear ds-DNA has free ends, and the DNABII proteins are in equilibrium between HJ-bound and linear DNA-bound. (B) DNABII proteins migrate to stabilize HJs and a closed loop is formed. (C) The ends of DNA could be locked in a high energy state [>3 HJs]. The transition from (B) to (C) is inhibited by Chloroquine, whereas the CeCl₃ stimulates the transition from (C) to (D) [e.g., supercoiling “flips” to Z-form. (D) Z-DNA is constrained. (E) Z-DNA is stabilized within the ds-DNA matrix [4HJs].