Characterization of Fractions Obtained from Two Industrial Softwood Kraft Lignins

With increasing interest in using lignin as an alternative material to petroleum-based chemicals (e.g., in the manufacture of carbon fibers or adhesives), it is becoming important to understand what properties of lignin are required to impart key features in the final product. Commercial lignins are complex, heterogeneous, macromolecular mixtures. To obtain maximum value, lignins will require classification and possibly fractionation or modification to improve properties and enable their utilization in high-value applications. To this end, the physicochemical properties of fractions derived from two industrial softwood Kraft lignins (New Bern Mill, Weyerhaeuser, U.S.A., and Backhammar Mill in Kristinehamn, Sweden) have been determined and compared to previously published data on commercially available Indulin AT lignin from MeadWestvaco., The fractions were obtained by successive extraction with organic solvents and analyzed using a range of techniques (e.g., DSC, ¹³C NMR, ³¹P NMR). The results showed that these industrial softwood Kraft lignins varied significantly in both the amounts of the various fractions and in the properties of the analogous fractions. These differences emphasize the issues industry faces in the utilization of industrial lignins for high-value applications where minor inconsistencies between lignin sources could pose major technical challenges

Broad-Spectrum Anti-biofilm Peptide That Targets a Cellular Stress Response

<div><p>Bacteria form multicellular communities known as biofilms that cause two thirds of all infections and demonstrate a 10 to 1000 fold increase in adaptive resistance to conventional antibiotics. Currently, there are no approved drugs that specifically target bacterial biofilms. Here we identified a potent anti-biofilm peptide 1018 that worked by blocking (p)ppGpp, an important signal in biofilm development. At concentrations that did not affect planktonic growth, peptide treatment completely prevented biofilm formation and led to the eradication of mature biofilms in representative strains of both Gram-negative and Gram-positive bacterial pathogens including <i>Pseudomonas aeruginosa</i>, <i>Escherichia coli</i>, <i>Acinetobacter baumannii</i>, <i>Klebsiella pneumoniae</i>, methicillin resistant <i>Staphylococcus aureus</i>, <i>Salmonella</i> Typhimurium and <i>Burkholderia cenocepacia</i>. Low levels of the peptide led to biofilm dispersal, while higher doses triggered biofilm cell death. We hypothesized that the peptide acted to inhibit a common stress response in target species, and that the stringent response, mediating (p)ppGpp synthesis through the enzymes RelA and SpoT, was targeted. Consistent with this, increasing (p)ppGpp synthesis by addition of serine hydroxamate or over-expression of <i>relA</i> led to reduced susceptibility to the peptide. Furthermore, <i>relA</i> and <i>spoT</i> mutations blocking production of (p)ppGpp replicated the effects of the peptide, leading to a reduction of biofilm formation in the four tested target species. Also, eliminating (p)ppGpp expression after two days of biofilm growth by removal of arabinose from a strain expressing <i>relA</i> behind an arabinose-inducible promoter, reciprocated the effect of peptide added at the same time, leading to loss of biofilm. NMR and chromatography studies showed that the peptide acted on cells to cause degradation of (p)ppGpp within 30 minutes, and <i>in vitro</i> directly interacted with ppGpp. We thus propose that 1018 targets (p)ppGpp and marks it for degradation in cells. Targeting (p)ppGpp represents a new approach against biofilm-related drug resistance.</p></div

Peptide 1018 exhibited potent broad-spectrum direct anti-biofilm activity but weak antibacterial activity for planktonic cells.

<p>Comparison of planktonic cell MIC to MBIC₅₀ and MBIC₁₀₀, which are the minimal biofilm inhibitory concentrations leading to 50% and 100% decrease in biofilm growth, respectively.</p

Genetic complementation of (p)ppGpp synthetase enzymes restored the ability to form biofilms.

<p>The biofilm deficiency of <i>Pseudomonas aeruginosa</i> PAO1 and <i>Staphylococcus aureus</i> HG001 (p)ppGpp mutants (Δ<i>relAspoT</i> and <i>rsh_Syn</i> respectively) was rescued by genetic complementation [Δ<i>relAspoT</i> + <i>relAspoT⁺</i> (+SR) as described <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004152#ppat.1004152-Nguyen1" target="_blank">[12]</a> and <i>rsh⁺</i><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004152#ppat.1004152-Geiger1" target="_blank">[30]</a>, respectively] leading to biofilm formation equivalent to WT shown in the left-most panels. After 3 days, bacteria were stained green with the all bacteria stain Syto-9 and red with the dead-bacteria stain propidium iodide (merge shows as yellow to red) prior to confocal imaging. Each panel shows reconstructions from the top in the large panel and sides in the right and bottom panels (xy, yz and xz dimensions).</p

Peptide 1018 prevented (p)ppGpp accumulation <i>in vivo</i> as revealed by thin layer chromatography separation of guanine nucleotides extracted from intact cells.

<p>(<b>A</b>) Anti-biofilm peptide 1018 at 5 µg/ml directly prevented (p)ppGpp accumulation. (<b>B</b>) Treatment with peptide 1018 led to (p)ppGpp elimination within 30 min in <i>P. aeruginosa</i> PAO1 cells containing pre-accumulated (p)ppGpp due to SHX treatment. In panel <b>A</b>, bacteria were grown overnight in modified MOPS minimal medium containing 0.4% glucose, 2 mM phosphate (KH₂PO₄), and 0.2% CAA. For experiments evaluating the ability of the peptide to directly degrade (p)ppGpp in panel <b>B</b>, the cells were grown as described previously, induced with SHX and allowed to synthesize (p)ppGpp for 3 h prior to peptide treatment. After growth for both A and B, the cells were then diluted 1∶20 in the same MOPS minimal medium except containing 0.4 mM phosphate (KH₂PO₄) and 500 µM serine hydroxamate (SHX) to induce (p)ppGpp synthesis, in the presence or absence of peptide 1018 and cells were labelled with 10 µCi/ml ³²P for 3 h. Samples were then extracted with frozen 13 M formic acid by three cycles of freeze-thaw. Aliquots of the supernatants were applied to 20×20 cm PEI cellulose TLC plates, resolved with 1.5 M KH₂PO₄, pH 3.4 for 4 h. After chromatography, nucleotides were visualized by autoradiography and quantified with a MolecularImager FX PhosphorImager and Quantity One software (Bio-Rad). Controls were performed to demonstrate that the Δ<i>relAspoT</i> mutation also prevented (p)ppGpp formation.</p

Modulation of <i>relA</i> expression impacts on biofilm development.

<p>(<b>A</b>) <b><i>relA</i></b><b> expression modulated biofilm formation and disassembly.</b> The <i>relA</i> gene under the control of an arabinose-inducible promoter was introduced into a <i>P. aeruginosa</i> PAO1 Δ<i>relAspoT</i> background. Induction of <i>relA</i> led to biofilm formation in flow cells after 3 days. On the other hand, induction of <i>relA</i> for 2 days followed by 24 h of non-induction led to biofilm dispersal. Biofilm biovolume was calculated using Imaris software from Bitplane AG. Experiments were performed at least in triplicate. Student's <i>t</i> test was used (****, P<0.0001). (<b>B</b>) Repression of <i>relA</i> expression (after 2 days of induction) led to biofilm dispersal in a <i>P. aeruginosa</i> PAO1 Δ<i>relAspoT</i> strain, while continuous induction of <i>relA</i> expression during the 3 days of the experiment resulted in significantly fewer cells dispersed from biofilms. Dispersed cells from 2-day old biofilms were collected and viable cell counts performed 3 and 6 h after induction of <i>relA</i> expression was either stopped or continued. Statistical significance was determined using Student's <i>t</i> test (*, P<0.05).</p

Peptide 1018 bound to ppGpp <i>in vitro</i> and led to degradation of (p)ppGpp <i>in vivo</i>.

<p>(<b>A</b>) Binding of peptide 1018 to various nucleotides based on co-precipitation. Peptide 1018 (0.25 mM) was separately mixed with increasing amounts of ppGpp, GTP, ATP, GDP and ADP in buffer (50 mM Tris, pH 7.4) and the extent of co-precipitation was assessed by measuring the increase in absorbance at 620 nm. The amount of co-precipitation induced by 1018 appeared to correlate with an increased negative charge on the nucleotides. A separate sample containing NaH₂PO₄ revealed that phosphate ions did not induce precipitation of 1018 in the concentration range tested. (<b>B</b>) Anti-biofilm peptide 1018 preferentially bound to ppGpp compared to GTP as revealed by ³¹P-NMR spectroscopy. In the absence of 1018 (top panel), a mixture of 0.5 mM ppGpp and 0.5 mM GTP revealed unique signals corresponding to the phosphorous atoms in ppGpp and GTP (indicated by arrows). Upon the addition of 1 mM 1018 (bottom panel), the peak intensity from the ppGpp signals was almost completely abolished, while the signals from GTP were reduced but to a lesser degree. (<b>C</b>) Samples containing an equimolar mixture of ppGpp and GTP at intermediate concentrations of 1018 were used to further evaluate the preferential binding of 1018 to ppGpp. Examination of specific spectral regions unique to ³¹P signals from either ppGpp (∼−4.2 ppm) or GTP (∼−20 ppm) showed that the ppGpp peak intensity decreased more readily than those from GTP (peptide concentrations, in mM, are indicated above each trace). The preferential precipitation of ppGpp by 1018 suggests that the peptide had a higher affinity for ppGpp over GTP under these conditions (See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004152#ppat.1004152.s005" target="_blank">Fig. S5B</a>). (<b>D</b>) The ppGpp levels in nucleotide extracts from <i>P. aeruginosa</i> PAO1 cultures induced with SHX and treated with 1018 were also measured using ³¹P-NMR spectroscopy. In these spectra, the ppGpp phosphorous signals were shifted because of the presence of 6.5 M formic acid used to extract the nucleotides from the PAO1 cells. The chemical shifts of GTP and ppGpp in 6.5 M formic acid were determined separately using samples of pure nucleotide (See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004152#ppat.1004152.s006" target="_blank">Fig. S6</a>). Only the region from 3 ppm to −0.5 ppm is shown as this contained a unique ppGpp phosphorous peak at 0.6 ppm (for comparison, the spectra of 0.5 mM ppGpp in 6.5 M formic acid is shown as the top trace). In the ³¹P spectrum of nucleotide extracts from PAO1 induced with SHX, the ppGpp peak at 0.6 ppm appeared as a shoulder on the large phosphate peak at 1.5 ppm (middle grey trace). This shoulder was absent in samples taken from PAO1 cells grown without SHX (lowest grey trace). When PAO1 induced with SHX was treated with 20 µg/ml 1018, the ppGpp peak was essentially lost (black trace) demonstrating that the addition of 1018 to bacteria leads to the degradation of ppGpp <i>in vivo</i>.</p

Low levels of 1018 led to biofilm dispersion while higher levels triggered biofilm cell death.

<p>Dispersed cells from mature <i>P. aeruginosa</i> flow cell biofilms were collected and viable cell counts performed after 0, 3 and 23 h of treatment with different concentrations of the peptide (0.8 and 10 µg/ml). Representative confocal images of the remaining cells present in the flow cell chambers after peptide treatment are shown for each condition. Statistical significance comparing peptide-treated groups to untreated was determined using Student's <i>t</i> test (ns, P>0.05; *, P<0.05; **, P<0.01; ****, P<0.0001).</p

Peptide 1018 potently inhibited bacterial biofilms at concentrations that did not affect planktonic cell growth.

<p>Sub-inhibitory concentrations of peptide 1018 prevented biofilm development and eradicated or reduced existing biofilms of Gram-negative and Gram-positive bacteria. Concentrations of peptide 1018 used were 10 µg/ml for <i>Pseudomonas aeruginosa</i> (labelled as strains PA14 and PAO1) <i>Escherichia coli</i> 0157, <i>Acinetobacter baumannii</i> and <i>Burkholderia cenocepacia</i>, 20 µg/ml for <i>Salmonella enterica</i> serovar Typhimurium 14028S experiments, 2 µg/ml for <i>Klebsiella pneumoniae</i> experiments, and 2.5 µg/ml for methicillin resistant <i>Staphylococcus aureus</i> (MRSA) experiments. Inhibition of biofilm development was tested by immediately adding 1018 into the flow-through medium of the flow cell apparatus and then monitoring biofilm formation for 3 days. Eradication conditions involved waiting two days before addition of 1018 into the flow-through medium. After 3 days, bacteria were stained green with the all bacteria stain Syto-9 and red with the dead-bacteria stain propidium iodide (merge shows as yellow to red) prior to confocal imaging. Each panel shows reconstructions from the top in the large panel and sides in the right and bottom panels (xy, yz and xz dimensions).</p

Enhanced (p)ppGpp production leads to altered susceptibility of biofilms to peptides.

<p>(<b>A</b>) Addition of SHX, which leads to overproduction of (p)ppGpp, resulted in the resistance of biofilm development to 20 µg/ml of peptide 1018; (<b>B</b>) (p)ppGpp overproduction through <i>relA</i> overexpression led to anti-biofilm peptide resistance. (<b>A–B</b>) Inhibition of biofilm development was tested by immediately adding 20 µg/ml 1018 (± SHX or IPTG) into the flow-through medium of the flow cell apparatus and then monitoring biofilm formation for 3 days. After 3 days, bacteria were stained green with the all bacteria stain Syto-9 prior to confocal imaging. Each panel shows reconstructions from the top in the large panel and sides in the right and bottom panels (xy, yz and xz dimensions).</p