Plant-expressed pyocins for control of Pseudomonas aeruginosa.

The emergence, persistence and spread of antibiotic-resistant human pathogenic bacteria heralds a growing global health crisis. Drug-resistant strains of gram-negative bacteria, such as Pseudomonas aeruginosa, are especially dangerous and the medical and economic burden they impose underscore the critical need for finding new antimicrobials. Recent studies have demonstrated that plant-expressed bacteriocins of the colicins family can be efficient antibacterials against all major enteropathogenic strains of E. coli. We extended our studies of colicin-like bacteriocins to pyocins, which are produced by strains of P. aeruginosa for ecological advantage against other strains of the same species. Using a plant-based transient expression system, we expressed six different pyocins, namely S5, PaeM, L1, L2, L3 and one new pyocin, PaeM4, and purified them to homogeneity. Among these pyocins, PaeM4 demonstrated the broadest spectrum of activity by controlling 53 of 100 tested clinical isolates of P. aeruginosa. The activity of plant-made pyocins was confirmed in the agar drop, liquid culture susceptibility and biofilm assays, and in the Galleria mellonella animal infection model

Chimeric bacteriocin S5-PmnH engineered by domain swapping efficiently controls Pseudomonas aeruginosa infection in murine keratitis and lung models

Rampant rise of multidrug resistant strains among Gram-negative bacteria has necessitated investigation of alternative antimicrobial agents with novel modes of action including antimicrobial proteins such as bacteriocins. The main hurdle in the clinical development of bacteriocin biologics is their narrow specificity and limited strain activity spectrum. Genome mining of bacteria for broadly active bacteriocins have identified a number of promising candidates but attempts to improve these natural multidomain proteins further, for example by combining domains of different origin, have so far met with limited success. We have found that domain swapping of Pseudomonas bacteriocins of porin type, when carried out between phylogenetically related molecules with similar mechanism of activity, allows the generation of highly active molecules with broader spectrum of activity, for example by abolishing strain resistance due to the presence of immunity proteins. The most broadly active chimera engineered in this study, S5-PmnH, exhibits excellent control of Pseudomonas aeruginosa infection in validated murine keratitis and lung infection models

Survival of <i>Galleria melonella</i> larvae after injection with <i>P</i>. <i>aeruginosa</i> PAO1 or A19.

<p>Fifth-instar <i>G</i>. <i>mellonella</i> larvae were injected in the left hind proleg with different amounts of <i>P</i>. <i>aeruginosa</i> PAO1 and A19 bacteria. The image was captured 18 hours post infection. Healthy larvae are cream colored, and darker pigmentation indicates infection. Dead larvae can be recognized from their dark brown-black color.</p

Agar drop plate assay on different <i>P</i>. <i>aeruginosa</i> isolates with plant-produced pyocins.

<p>Aliquots of 10 μl (10 μg) of purified pyocins were spotted on <i>P</i>. <i>aeruginosa</i> agar lawn and incubated overnight. <b>A–</b><i>P</i>. <i>aeruginosa</i> agar lawn grown on CAA medium, <b>B–</b><i>P</i>. <i>aeruginosa</i> agar lawn grown on LB medium.</p

Survival of larvae pre-infected with <i>P</i>. <i>aeruginosa</i> PAO1 and A19 and treated therapeutically with pyocins.

<p><i>G</i>. <i>melonella</i> larvae were infected with 500 CFU of <i>P</i>. <i>aeruginosa</i> A19 or PAO1 and treated with pyocins 3 hours after infection. <b>A–</b>larvae infected with A19 strain and treated with 10 μg of the indicated pyocin. <b>B–</b>larvae infected with PAO1 strain and treated with 10 μg of the indicated pyocin. <b>C–</b>larvae infected with A19 strain and treated with 1 μg of the indicated pyocin. Killing curves were plotted and estimation of differences in survival analyzed by the Kaplan-Meier method using XLSTAT software.</p

Sensitivity of PAO1 and PAO1 (FiuA) to pyocins.

<p>Plant-produced pyocins S5, L1, PaeM4, PaeM_JJ692 and PaeM _NCTC10332 were applied (5 μg each) on <i>P</i>. <i>aeruginosa</i> PAO1, PW1861 (PAO1 (FiuA)) and PW1862 (PAO1 (FiuA)) lawn and the plates were incubated overnight at 37°C.</p

Sensitivity of clinical isolates to three plant-expressed pyocins.

<p>Venn diagram describing pyocin susceptibilities of clinical <i>Pseudomonas aeruginosa</i> isolates. Left–susceptibility of all 100 tested isolates, right–susceptibility of 21 antibiotic-resistant isolates.</p

Pyocin expression in plants.

<p><b>A–schematic presentation of T-DNA region with pyocin expression cassette.</b> RB–right T-DNA border, AtAct23Prom–<i>A</i>. <i>thaliana</i> actin promoter, RdRp–RNA-dependent RNA polymerase, MP–truncated TMV movement protein, LB–left T-DNA border. <b>B–expression of pyocins in <i>N</i>. <i>benthamiana</i> leaves.</b> Plant material (50 mg) was harvested at 5 or 7 days post spraying (dps) (pooled samples of three leaves—pyocins S5, PaeM, PaeM4, L1, L2 at 5 dps, L3 at 7 dps), ground in liquid nitrogen, extracted with 50 mM Tris-HCl, 150 mM NaCl (pH 7.0) (S5, PaeM and PaeM4) or 50 mM HEPES, 10 mM CH₃COOK, 5 mM Mg(CH₃COO)₂, 2 mM DTT, 1 mM EDTA, pH 5.0 (L1, L2, L3) and denatured at 98°C for 10 min. Solutions containing 5 μg of protein were resolved in 12% polyacrylamide gel for Coomassie staining. Mw–PageRuler Prestained protein ladder (ThermoFisher Scientific Baltics), Wt–crude extract of non—sprayed <i>N</i>. <i>benthamiana</i> leaves, S5, M, M4, L1, L2, L3 –extracts of <i>N</i>. <i>benthamiana</i> leaves, sprayed with pyocin expression constructs (pyocin S5, PaeM, PaeM4 and lectin–like pyocins L1, L2, L3). Bands corresponding to recombinant pyocins are marked by arrows. <b>C–purified pyocins</b>. Pyocins were purified by two-step chromatography as described in Purification section of Methods, and resolved in 12% polyacrylamide gel for Coomassie staining.</p

Pyocin activity against biofilms.

<p>One day-old <i>P</i>. <i>aeruginosa</i> biofilms grown in CAA medium were treated with pyocins. <b>A–</b>A19 strain treated with 10 μg mL^-1 of pyocins, when each pyocin is used separately, and 2.5 μg of each pyocin used as a mixture. <b>B</b>–PW2389 (PAO1(mucB)) treated with 100 μg mL^-1 of pyocins. <b>C</b>–PAO1 and PW2389 treated with 10 μg mL^-1 of pyocins. Data are the mean ± SD of at least three separate independent experiments. * Denotes statistical significance (p≤0.001) for comparison of treatment with antimicrobials versus control by a one-way ANOVA test with Bonferroni correction applied. ** no statistical significance (p>0.001).</p

Clustal W alignment of <i>E</i>. <i>coli</i> ColM, <i>P</i>. <i>aeruginosa</i> PaeM, syringacin M and PaeM4 amino acid sequences.

<p>Residues essential for activity of ColM [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185782#pone.0185782.ref019" target="_blank">19</a>] are marked by asterisks; residues essential for activity of PaeM [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185782#pone.0185782.ref020" target="_blank">20</a>] are marked by black asterisks.</p