Synergistic Inhibition of Plantaricin E/F and Lactic Acid Against Aeromonas hydrophila LPL-1 Reveals the Novel Potential of Class IIb Bacteriocin

Abstract

Plantaricin E/F (PlnEF) is a pair of two-component class IIb bacteriocin produced by lactic acid bacteria. PlnEF commonly displays potent antimicrobial activity against certain Gram-positive organisms. In this study, we investigated the synergistic activity of PlnEF combined with lactic acid against Gram-negative food and aquaculture potential pathogen Aeromonas hydrophila LPL-1, which is naturally resistant to PlnEF. We applied SDS-PAGE, wavelength-scanning, laser confocal microscopy, flow cytometer, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and two-dimensional electrophoresis to investigate their synergistic inhibitory activities. The results showed that L-lactic acid drove the release of LPS from A. hydrophila, making it possible for PlnEF to contact the inner cell membrane of A. hydrophila. Besides, co-treatment of lactic acid and PlnEF caused severe morphological and intracellular changes of A. hydrophila, including blebs on the cell surface, abnormal cell elongation, inner membrane disruption, pore-forming through the outer and inner membrane, coagulation of the cytoplasm, and structural transformation of DNA. Protein profile analysis revealed that combined treatment of lactic acid and PlnEF inhibited the energy metabolism, protein synthesis, protein folding, and DNA replication in A. hydrophila. These findings proved that PlnEF combined with lactic acid was efficient against A. hydrophila and shed light on bacteriocin's potential and a new inhibition mechanism against A. hydrophila.

Importance: Bacteriocins and their producing strains are increasingly used to substitute artificial preservatives and antibiotics in the food and aquaculture industries. However, the bacteriocins produced by lactic acid bacteria are efficient to mainly Gram-positive bacteria. Our paper had demonstrated the antimicrobial activity of class IIb bacteriocin against potential Gram-negative pathogen, A. hydrophila LPL-1, when combined with lactic acid. The results could refresh our knowledge about the potential of class IIb bacteriocins produced by lactic acid bacteria.

Keywords: Gram-negative bacteria; bacteriocin; inhibition; lactic acid; lipopolysaccharide.

FIGURE 1

Effects of bacteriocin PlnEF and lactic acid co-treatment on inhibiting the growth and promoting lethality of A. hydrophila LPL-1. (A) Aeromonas hydrophila was cultured with lactic acid (10 mM) and different concentrations of PlnEF (0.5-25 μM) in 96-well cell culture plates for 12 h. The co-treatment of PlnEF and lactic acid significantly inhibited the growth of A. hydrophila. The results are representative of three independent experiments expressed as Means ± SD. (B) Aeromonas hyrophila cells were collected and treated with 10 mM lactic acid, 25 μM PlnEF and 10 mM lactic acid combined with 25 μM PlnEF at 30°C for 2, 4, 6, and 8 h. The co-treatment of PlnEF and lactic acid significantly promoted the proportion of dead cells. The results are representative of three independent experiments expressed as Means ± SD. Mean without a common letter indicated p < 0.05. Different letters of a–c indicated a significant difference between statistics of lactic acid and combined treatment at different time, ** indicated p < 0.01.

FIGURE 2

Interaction between bacteriocin PlnEF and LPS released from A. hydrophila LPL-1 (A) and effects of lactic acid on increasing LPS released from A. hydrophila LPL-1 (B). LPS released from A. hydrophila LPL-1 was treated with different concentrations of PlnEF (0.5, 2.5, 5, and 10 μM) showed a red shift in absorption peak. Aeromonas hyrophila LPL-1 cells were treated with different concentrations of lactic acid (5, 10, and 12 mM) for 0, 0.5, 1, 2.5, and 5 h. The release of LPS from A. hyrophila LPL-1 significantly increased by lactic acid in a dose-dependent and time-dependent manner.

FIGURE 3

Effects of lactic acid on promoting the accumulation of FITC labeled PlnEF in A. hydophila LPL-1. Aeromonas hyrophila LPL-1 cells were treated with FITC labeled PlnEF and lactic acid (25 μM) for 2, 4, and 8h. The co-treatment significantly increased the number of FITC labeled PlnEF in A. hyrophila LPL-1. (A) The FITC positive cells under a laser confocal microscope. (B) The proportions of FITC-positive cells in A. hyrophila LPL-1. The results are representative of three independent experiments expressed as Means ± SD. Means without a common letter are significantly different (p < 0.05).

FIGURE 4

Effects of bacteriocin PlnEF and lactic acid co-treatment on changing cell morphology of A. hyrophila LPL-1. Aeromonas hyrophila LPL-1 cells were treated with lactic acid (10 mM) and/or PlnEF (25 μM) at 4°C for 2, 4, and 8 h. Images were observed using a scanning electron microscope (SEM). The scale bar indicates length 1 μm, HV = 15-20 kV, direct mag (20,000-50,000). (A–C) the control cells, (D–F) the PlnEF treated cells, (G–I) lactic acid treated cells, (J–L) PlnEF combined lactic acid treated cells. Lactic acid treatment induced outer membrane damage (green arrows), deformation and shrinkage (blue arrows), and apical surface protrusion (cyan arrow). The co-treatment of PlnEF and lactic acid induced extra small vesicles (yellow arrows), multiple splitting points (pink arrows), holes (red arrows), increase in length (purple arrows) and fragmentation of cracking bacteria (orange arrows).

FIGURE 5

Effects of bacteriocin PlnEF and lactic acid co-treatment on affecting internal structural deformation of A. hydrophila. Aeromonas hyrophila LPL-1 cells were treated lactic acid (10 mM) and/or PlnEF (25 μM) at 4°C for 8 h. Images were observed using a Hitachi H-7650B transmission electron microscope (TEM). The scale bar indicates length 1 μm, HV = 80 kV, direct mag (20,000-1,00,000). (A1–A4) the control cells, (B1–B4) the PlnEF treated cells, (C1–C4) lactic acid treated cells, (D1–D8) PlnEF and lactic acid combined treated cells. The binary fission (BF), outer membranes (OM), cytoplasmic membrane (CM), periplasmic space (PS), supercoiled DNA (SCDNA) are visible. Lactic acid treatment induced protrusion (cyan arrows), en (purple arrows), outer membrane damage (red solid line arrows), cell inner membranes damaged (red dotted arrows), the outer and inner membrane separation (white arrows), and reduced electron density region (black dotted arrows). Co-treatment of PlnEF and lactic acid induced extra dark granules (black solid line arrows), small vesicles (yellow arrows), deepening (brown arrows), protruding part (cyan box), sag (blue arrows).

FIGURE 6

Effects of bacteriocin PlnEF and lactic acid co-treatment on the proteomic profile of A. hydrophila. (A–D) A two-dimensional electrophoresis (2-DE) separation of total proteins from A. hydrophila. The cells of A. hydrophila LPL-1 (1–3 × 10⁹ CFU/ml) were cultured in LB broth (control), LB broth with 25 μM PlnEF; 10 mM L-lactic acid; and 10 mM L-lactic acid + 25μM PlnEF aerobically at 30°C for 8 h. The first dimension comprised an 17-cm non-linear pH 4–7 immobilized pH gradient (IPG) subjected to isoelectric focusing. The second dimension was a 21-cm 12% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gel. Proteins were detected by Coomassie Brilliant Blue G-250 staining. The non-linear pH range of the first-dimension IPG strip is indicated along the top of the gel, acidic pH to the left. The Mr (relative molecular mass) scale can estimate the molecular weights of the separated proteins. (E) Two-dimensional polyacrylamide gel electrophoresis (PAGE) reference map of the whole proteins from A. hydrophila LPL-1. Using the PDQuest software (Bio-Rad), the average gel of each population was compared with the reference map gel to identify the differentially expressed protein spots (green cross). (F) The relative mRNA levels of several genes in A. hydrophila LPL-1 treated by la-10mM lactic acid, PlnEF-25 μM PlnEF, la + EF-10 mM lactic acid + 25 μM PlnEF for 8 h revealed by Quantitative RT-PCR. acnB: bifunctional aconitate hydratase 2/2-methylisocitrate; sdhA: succinate dehydrogenase flavoprotein subunit; pckA: phosphoenolpyruvate carboxykinase; prpD: 2-methylcitrate dehydratase; gap: glyceraldehyde-3-phosphate dehydrogenase (GAPDH); glpk: glycerol kinase (GK); purA: adenylosuccinate synthetase (ADSS); rspA: 30S ribosomal subunit protein S1; turf1, turf2: elongation factor Tu; aromatic amino acid aminotransferase (AAA-ATs) gyrB: DNA gyrase subunit B; ligA. NAD-dependent DNA ligase; tyrB: threonyl-tRNA synthetase; pnp: polynucleotide phosphorylase/polyadenylase; hptG: heat shock protein 90.

FIGURE 7

Schematic mechanism of inhibition of synergistic inhibition of PlnEF and lactic acid on A. hydrophila. The LPS works as a barrier against PlnEF. After the release of LPS induced by lactic acid, PlnEF insert into the inner membrane of A. hydrophila, causing the collapse of membrane potential. In the center of the cell, reduction of energy metabolism, down-regulation of Hsp90 but up-regulation of DNA gyrase GraB, suggesting abnormal protein folding and DNA status, which was in accordance with the outer membrane vesicles and reduced DNA electron density proved in SEM and TEM.