Bovine Lactoferrin and Lactoferrin-Derived Peptides Inhibit the Growth of Vibrio cholerae and Other Vibrio species

Abstract

Vibrio is a genus of Gram-negative bacteria, some of which can cause serious infectious diseases. Vibrio infections are associated with the consumption of contaminated food and classified in Vibrio cholera infections and non-cholera Vibrio infections. In the present study, we investigate whether bovine lactoferrin (bLF) and several synthetic peptides corresponding to bLF sequences, are able to inhibit the growth or have bactericidal effect against V. cholerae and other Vibrio species. The antibacterial activity of LF and LF-peptides was assessed by kinetics of growth or determination of colony forming unit in bacteria treated with the peptides and antibiotics. To get insight in the mode of action, the interaction between bLF and bLF-peptides (coupled to FITC) and V. cholera was evaluated. The damage of effector-induced bacterial membrane permeability was measured by inclusion of the fluorescent dye propidium iodide using flow cytometry, whereas the bacterial ultrastructural damage in bacteria treated was observed by transmission electron microscopy. The results showed that bLF and LFchimera inhibited the growth of the V. cholerae strains; LFchimera permeabilized the bacteria which membranes were seriously damaged. Assays with a multidrug-resistant strain of Vibrio species indicated that combination of sub-lethal doses of LFchimera with ampicillin or tetracycline strongly reduced the concentration of the antibiotics to reach 95% growth inhibition. Furthermore, LFchimera were effective to inhibit the V. cholerae counts and damage due to this bacterium in a model mice. These data suggest that LFchimera and bLF are potential candidates to combat the V. cholerae and other multidrug resistant Vibrio species.

Keywords: LFchimera; Vibrio cholerae; bactericide; lactoferrin; lactoferrin peptides.

Figure 1

Bactericidal effect of bLF and LF-peptides on Vibrio cholerae O1 and non-O1. Approximately 1 × 10⁷ CFU/ml of V. cholerae O1 and non-O1 strains were incubated with bLF and LFpeptides solutions at final concentrations of 40 μM bLF or 20 μM of LFcin17-30, LFampin265-284, respectively; and 5 μM of LFchimera at 37°C with constant agitation for 1, 2, 4, or 6 h. Bacteria grown in LB broth were used as a control for optimal growth and 100 μM of Gentamicin was used as a control for growth inhibition. Bacterial growth was followed by measuring the OD660 nm of cultures. Percentage of viable cells was determined in relation to cultures Gentamicin without peptides or antibiotics (A). All Experiments were repeated at least twice in triplicates. V. cholerae O1 (B) and non-O1 (C) strains were incubated with bLF and LFpeptides solutions at final concentrations of 40 μM bLF and 20 μM LFcin17-30, LFampin265-284, and 5 μM LFchimera; respectively. Viability was monitored by enumerating colony forming units CFU/ml (viable cells) obtained from serial 10-fold dilutions plated onto MH agar (B,C). Percentage of viable cells was calculated relative to viable bacteria untreated grown in MH agar. Experiments were performed in triplicate; mean and standard deviation are indicated. Statistical significance was determined using a Student's t-test for P-values < 0.05, and ANOVA (with Bonferroni correction).

Figure 2

Determination of membrane permeabilization in Vibrio cholerae O1 treated with bLF and LF peptides. V. cholerae O1 was incubated with 40 μM LF, or with 20 μM LFcin17-30, or LFampin265-284, respectively; or 5 μM LFchimera, at 37°C with constant agitation for 2 h. Then, samples were processed and stained with the fluorescent dye Propidium iodide. Untreated bacteria were used as control of membrane integrity and 0.5% Triton X-100 treated bacteria were used as control of permeabilized membranes. Experiments were performed at least twice in duplicate. Samples were processed to be analyzed by Flow Cytometry.

Figure 3

LF and LFpeptides cause ultrastructural damage to Vibrio cholerae O1 cells. V. cholerae O1 cells (1.0 × 10⁸ cells/ml) were incubated in LB alone (negative control for damage) or with 0.5% SDS (positive control for damage), or with 40 μM of bLF, 20 μM LFcin 17-30 or LFampin265-284 respectively, or 5 μM LFchimera for 1.5 h at 37°C. Cells were harvested, resuspended in PBS and fixed with 4% para-formaldehyde plus 0.5% glutaraldehyde. Next, bacterial samples were placed on 200-mesh Formvar-coated copper grids (3%), post-stained with phosphotungstic acid and examined with a JEOL electron microscope JEM1400 at 40 kv.

Figure 4

LF and LFpeptides cause ultrastructural damage to Vibrio cholerae non-O1 cells. V. cholerae non-O1 cells (1.0 × 10⁸ cells/ml) were incubated in LB alone (negative control for damage) or with 0.5% SDS (positive control for damage) or with 40 μM of bLF, or 20 μM LFcin 17-30 and LFampin265-284 respectively, or 5 μM LFchimera, for 1.5 h at 37°C. Cells were harvested, resuspended in PBS and fixed with 4% para-formaldehyde plus 0.5% glutaraldehyde. Next, bacterial samples were placed on 200-mesh Formvar-coated copper grids (3%), post-stained with phosphotungstic acid and examined with a JEOL electron microscope JEM1400 at 40 kv.

Figure 5

Interaction of Lactoferrin derived peptides with Vibrio cholerae O1 and non-O1 strains. Vibrio cholerae O1 cells (10⁷ CFU/ml) were incubated with 2 μM FITC-labeled peptides for 30 min. Bacteria were centrifuged (5 min, 10,000 × g), resuspended and incubated with 2 μM of FITC-bLF or FITC-labeled peptides for 30 min (A), or fixed (4% paraformaldehyde, pH 7.4 during 30 min at 37°C) (B), washed and then incubated with 2 μM bLF and LFpeptides as before was described. In both cases samples were washed twice with PBS mounted on slides and processed. All samples were analyzed under confocal microscopy by using a confocal laser-scanning microscope (Leica, Heidelberg, Germany). Bar 20 nm.

Figure 6

Alterations in gut morphology of mice. Representative macroscopic images for the gross morphological alterations of the small intestine and caecum from mice at 24 h of treatment. (A) Small intestine Black arrows indicate normal small intestine in the uninfected group (blue arrowhead) indicate normal caecum. (B) White arrows point to injury in small intestine in the infected and untreated group (blue arrow) shows caecum is swelling. (C) Black arrows indicate injury in small intestine (blue arrows) caecum is swelling and enlarged in the tetracycline group. (D) Black arrows indicate normal small intestine as in A (blue arrowhead) indicates normal caecum. (E) Black arrows indicate normal small intestine in the LFchimera group as in D (blue arrow) indicate caecum.

Figure 7

LF chimera and bLF diminish Vibrio cholerae O1 counts in (A) feces and (B) intestine. Recovery feces and intestines from mice infected and treated were homogenized and serial dilutions were prepared and plated onto LB agar plates with 100 mg/ml streptomycin for 24 h at 37°C. For confirmation, developed colonies were counted and then plated onto CHROMagar™ Vibrio (CHROMagar; Paris, France). On the other hand, the intestines were dissected, homogenized in PBS and serial dilutions were prepared in PBS and plated in LB agar plates with 100 mg/ml streptomycin. Finally, colonies were counted and plated onto CHROMagar™ Vibrio.