Chitinases Are Negative Regulators of <i>Francisella novicida</i> Biofilms

<div><p>Biofilms, multicellular communities of bacteria, may be an environmental survival and transmission mechanism of <i>Francisella tularensis.</i> Chitinases of <i>F. tularensis</i> ssp. <i>novicida</i> (<i>Fn</i>) have been suggested to regulate biofilm formation on chitin surfaces. However, the underlying mechanisms of how chitinases may regulate biofilm formation are not fully determined. We hypothesized that <i>Fn</i> chitinase modulates bacterial surface properties resulting in the alteration of biofilm formation. We analyzed biofilm formation under diverse conditions using chitinase mutants and their counterpart parental strain. Substratum surface charges affected biofilm formation and initial attachments. Biophysical analysis of bacterial surfaces confirmed that the <i>chi</i> mutants had a net negative-charge. Lectin binding assays suggest that chitinase cleavage of its substrates could have exposed the concanavalin A-binding epitope. <i>Fn</i> biofilm was sensitive to chitinase, proteinase and DNase, suggesting that <i>Fn</i> biofilm contains exopolysaccharides, proteins and extracellular DNA. Exogenous chitinase increased the drug susceptibility of <i>Fn</i> biofilms to gentamicin while decreasing the amount of biofilm. In addition, chitinase modulated bacterial adhesion and invasion of A549 and J774A.1 cells as well as intracellular bacterial replication. Our results support a key role of the chitinase(s) in biofilm formation through modulation of the bacterial surface properties. Our findings position chitinase as a potential anti-biofilm enzyme in <i>Francisella</i> species.</p></div

<i>Fn</i> chitinase affects biofilm formation in different surface charged microplates.

<p>(<b>A</b>) Biofilm formation based on CV staining (CV570) of cells adherent to negatively (TC), positively (Amine), neutral (PS) and positively/negatively (Primaria) charged 96-well plates, normalized by bacterial growth (OD600) expressed as CV570/OD600. (<b>B</b>) Attachment was assessed by CV staining 1 h post-inoculation of stationary-phase cultures (OD = 1.0). Initial attachment of <i>Fn</i> WT was very low to the TC and Primaria plates, but high to the amine and PS. *P<0.01 (n = 6) and NS (not significant) by unpaired Student's t-test.</p

Effect of chitinase inhibitors SAN and DEQ on antibacterial and antibiofilm activity.

<p>(<b>A, B</b>) Susceptibility of <i>Fn</i> WT and <i>chi</i> mutants to SAN (<b>A</b>) and DEQ (<b>B</b>). Survival percentage of bacteria was calculated by OD₆₀₀ measurements after 24 h incubation with various concentrations of SAN and DEQ in TSBC. The EC₅₀s (μM) were determined by GraphPad software as indicated in the bottom table. (<b>C</b>) Effect of chitinase inhibitors SAN and DEQ on biofilm formation. Biofilm formation (CV570/OD600) was calculated by normalization with bacterial growth in each concentration of inhibitors. *P<0.05 compared to untreated (NT) control (n = 4).</p

Figure 4

<p>(<b>A</b>) EPS contents of the cells and (<b>B</b>) culture supernatants of the strains. EPS contents were determined by phenol extraction followed by phenol-sulfuric acid method for carbohydrates as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093119#s4" target="_blank">Materials and Methods</a>. (<b>C</b>) Lectin binding assay to biofilms. FITC-Con A and FITC-WGA lectins were used for biofilm binding. Lectin binding capacity to biofilms was measured by a fluorescence plate reader and calculated relative fold to WT binding. Fluorescence microscopic images of biofilms of WT, <i>chi</i>A and <i>chi</i>B grown in TC plate are shown in the top panel. Biofilms in the TC plate were shown by CV staining (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093119#pone.0093119.s001" target="_blank">Fig. S1C</a>). Scale bar, 100 μm.</p

Chitinase alters drug susceptibility of <i>Fn</i> biofilms.

<p>(<b>A, C</b>) Effect of chitinase on drug susceptibility of biofilms pre-formed in the TC plates to (<b>A</b>) gentamicin (Gm) and (<b>C</b>) ciprofloxacin (Cipro). (<b>B, D</b>) Effect of chitinase on drug susceptibility of biofilms pre-formed in the amine plates to (<b>B</b>) gentamicin and (<b>D</b>) ciprofloxacin. (<b>E</b>) Susceptibility of chitinase-pretreated biofilms to gentamicin. Biofilms were formed on Amine plates in the presence of chitinase (0, 0.2 and 2 μg/ml) for 24 h then Gm (2 μg/ml) was added to the biofilms for 24 h. The remaining bacteria were calculated by the relative bacteria to no Gm-treated control in each concentration of chitinase. *P<0.05 compared to no Gm-treated control (n = 3).</p

Abrogation of <i>Fn chi</i> genes enhances ability to adhere to, to invade to and to replicate in host cells.

<p>(<b>A</b>) Comparison of the adhesive properties of <i>Fn</i> WT and <i>chiA</i> mutants to A549 cells. *P<0.001 compared to WT (n = 6). (<b>B</b>) Bacterial invasion to A549 cells assayed by gentamicin protection method. *P<0.01 compared to WT (n = 6). (<b>C</b>) Effect of exogenous chitinase on bacterial adhesion. The same number of chitinase-treated bacteria as untreated bacteria were subjected to adhesion assays. *P<0.05 compared to untreated control of each strain (n = 3). (<b>D</b>) Bacterial adhesion assays using planktonic and biofilm cultures. Values are expressed as fold-increase adhesion relative to the planktonic counterparts. *P<0.05 and **P<0.01 compared to WT (n = 3). (<b>E</b>) Intracellular replication of the bacteria in host cells. A549 cells were infected with either WT or <i>chi</i> mutants at 100:1 MOI. Colony-forming units (CFUs) were determined after recovering intracellular bacteria from A549 cell lysates at 0 h or 18 h after gentamicin treatment to infected cells. (<b>F</b>) CFUs recovered from A549 cells lysed at 18 h post infection were compared with CFUs recovered at 0 h time point to calculate fold replication rate (change in CFU/hr). *P<0.05 compared to WT. (<b>G</b>) Bacterial invasion to J774A.1 cells assayed by gentamicin protection method. *P<0.01 compared to WT (n = 3). (<b>H</b>) Intracellular replication of the bacteria in J774A.1 cells. *P<0.05 and **P<0.01 compared to WT (n = 3).</p

<i>Fn</i> chitinase affects the biophysical properties of the biofilm.

<p><i>Fn</i> was grown to mid-log phase prior to the analyses. (<b>A</b>) The relative hydrophobicity of WT and <i>chi</i> mutants assayed by phenyl-sepharose column chromatography (HIC) and microbial adhesion to the nonpolar solvent hexadecane. *P<0.05, **P<0.01, and ***P<0.001 compared to WT (n = 6). (<b>B</b>) Autoaggregation of WT and <i>chi</i> mutants in PBS assayed at 24 and 48 h. *P<0.05, and **P<0.01 compared to WT (n = 6). (<b>C</b>) Size distribution for planktonic cultures of the strains in PBS measured by qNano analysis. (<b>D</b>) Particle translocation time (fwhm). The <i>chi</i> mutants had a larger fwhm duration than that observed for WT, indicating that the lower charge <i>chi</i> mutants took longer to traverse the pore. Mean presented in dots was calculated from every 100 data points.</p

InhA contributes to increased permeability of infected HBMEC monolayers.

<p>(A, B) Real-time TEER of HBMECs (about 6×10⁴ cells/well) treated with <i>ΔinhA</i> of <i>B. anthracis</i> Ames 35 (<i>ΔinhA</i>, red line in panel A) and InhA-expressing <i>B. subtilis</i> (<i>inhA+</i>, red line in panel B) at MOI of 10. Signals corresponding to parental strains (WT) and culture medium without bacteria are shown as blue and black lines, respectively. Arrows indicate time points when bacteria were added to the cells. (C) Transwell permeability assays of HBMECs infected with <i>B. anthracis</i> (<i>Ba</i>) and <i>B. subtilis</i> (<i>Bs</i>) strains as indicated above for 4 h. Leakage of FITC-dextran added to the monolayers for 2 h was determined by measuring fluorescence in the bottom chamber at 485/538 nm. *<i>P</i><0.01.</p

InhA contributes to BBB breakdown during <i>B. anthracis</i> infection in mice.

<p>Histopathology of representative H&E-stained brain sections from mice challenge as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017921#pone-0017921-g006" target="_blank">Figure 6</a>. (A, B) Control mice; (C, D) Moribund mice challenged with <i>B. anthracis</i> Ames 35. Meningeal thickening is shown by double arrows. (E, F) Moribund mice infected with <i>B. anthracis ΔinhA</i>. Boxes in C, E show the areas enlarged in D, F, respectively.</p

Western blot of TJ proteins in HBMECs after treatment with InhA.

<p>(A) HBMECs cells were treated with increasing concentrations of InhA (0.1, 0.3, 1, 3 µg/ml from left to right) for 24 h at 37°C. Western blots were probed with antibodies to ZO-1 (225 kDa), occludin (56 kDa), claudin-1 (22 KDa), and JAM-1 (39 kDa). (B) Densitometry of ZO-1 protein bands from (A). All samples analyzed (<i>n</i> = 4) were normalized to the intensity of corresponding β-actin bands. (C) Western blot analysis of time-dependent degradation of ZO-1 in HBMECs. Black arrows indicate two isoforms for ZO-1. <i>C</i>, control; <i>N</i>, Npr599; and <i>I</i>, InhA; α+ and α-, two splice variants of α domain. (D) Immunofluorescence of ZO-1 in HBMECs treated with or without 0.25 µg/ml of cytochalasin D for 1 h prior to incubation with varying concentrations of InhA at 37°C for 12 h. Panels show HBMECs treated as follows: untreated (a); InhA-treated (b); cytochalasin D-treated (c); InhA-treated after cytochalasin D treatment (d). Scale bar: 20 µm. (E) Western blot of His₆-tagged rZO-1. Degradation of purified rZO-1 after treatment with 0.1 µg/ml of InhA for indicated time at 37°C. Putative cleavage sites within the rZO-1fragment after treatment with InhA were deduced from immunoblot using the molecular masses of the cleavage products. SH3, Src homology 3; GK, guanylate kinase homolog; α, 80-amino-acid splice variant; -, acidic domain; PRK, proline-rich domain. β-Actin band intensity was used as a loading control for all Western blots. The results shown in (A)-(D) are representative of 4 independent experiments.</p