Plant defensin antibacterial mode of action against Pseudomonas species

Abstract

Background: Though many plant defensins exhibit antibacterial activity, little is known about their antibacterial mode of action (MOA). Antimicrobial peptides with a characterized MOA induce the expression of multiple bacterial outer membrane modifications, which are required for resistance to these membrane-targeting peptides. Mini-Tn5lux mutant strains of Pseudomonas aeruginosa with Tn insertions disrupting outer membrane protective modifications were assessed for sensitivity against plant defensin peptides. These transcriptional lux reporter strains were also evaluated for lux gene expression in response to sublethal plant defensin exposure. Also, a plant pathogen, Pseudomonas syringae pv. syringae was modified through transposon mutagenesis to create mutants that are resistant to in vitro MtDef4 treatments. Results: Plant defensins displayed specific and potent antibacterial activity against strains of P. aeruginosa. A defensin from Medicago truncatula, MtDef4, induced dose-dependent gene expression of the aminoarabinose modification of LPS and surface polycation spermidine production operons. The ability for MtDef4 to damage bacterial outer membranes was also verified visually through fluorescent microscopy. Another defensin from M. truncatula, MtDef5, failed to induce lux gene expression and limited outer membrane damage was detected with fluorescent microscopy. The transposon insertion site on MtDef4 resistant P. syringae pv. syringae mutants was sequenced, and modifications of ribosomal genes were identified to contribute to enhanced resistance to plant defensin treatments. Conclusions: MtDef4 damages the outer membrane similar to polymyxin B, which stimulates antimicrobial peptide resistance mechanisms to plant defensins. MtDef5, appears to have a different antibacterial MOA. Additionally, the MtDef4 antibacterial mode of action may also involve inhibition of translation.

Proteomic response of Escherichia coli to a membrane lytic and iron chelating truncated Amaranthus tricolor defensin

“…Three of these (denoted Atr-DEF1–3) included a highly basic γ-core motif similar to known active synthetic analogs (So-D2, BcDEF, Solyc07g007760, and MtDef4) (Fig. 1 b) [ 8 , 17 , 18 ]. Atr-DEF2 displayed the most positive charge and thus was used to design a synthetic analog spanning the γ-core motif yielding Atr-DEF2(G39-C54) (Fig.…”

“…Lipid A aminoarabinose modification has been previously shown to impact the antibacterial activity of γ-core motif analogs of plant defensins Mtdef4 and So-D2 against Gram-negative bacteria [ 8 ]. Gram-negative P. aeruginosa strains with inhibited aminoarabinose modification pathways were more susceptible to Mtdef4/So-D2 γ-core motif analogs than the wild type strain [ 8 ]. So-D2, Mtdef4, and Atr-DEF2 all have similar highly charged γ-core motifs including a “RRR” repeat, (Fig.…”

“…The significant length and complexity of defensins can hinder synthetic approaches to obtain sufficient quantities of defensins for extensive biological characterization. To circumvent this limitation and expedite the screening processes, previous studies have designed truncated defensins which are smaller (~ 1.2 kDa), less structurally complex (no disulfide bonds) and more synthetically tractable [ 8 , 15 , 17 – 20 ]. These analogs include the γ-core motif (GXCX 3-9 C, where X n is the number of residues between cysteines) of target full length defensins [ 15 , 17 – 20 ] (Fig.…”

“…( b ) Alignment of Atr-DEF2 (predicted mature sequence) with other plant defensins including known sequence of Atr-DEF1 [ 16 ] and predicted mature sequence of Atr-DEF3. Underlined regions have been synthesized as γ-core motif analogs [ 8 , 17 , 18 ]. Cysteines forming disulfide bonds are green, basic residues are red, and acidic residues are blue.…”

The Xanthomonas RaxH-RaxR Two-Component Regulatory System Is Orthologous to the Zinc-Responsive Pseudomonas ColS-ColR System

“…Further variation comes from using PP-EtN vs P-EtN ( Figure 3 B vs. Figure 3 D) or sugars other than Ara4N [ 79 , 80 ]. Plants also synthesize cationic antimicrobial peptides, and at least one of these induces ArnT synthesis in P. aeruginosa [ 91 ].…”