A Novel Generation of Tailored Antimicrobial Drugs Based on Recombinant Multidomain Proteins

Abstract

Antibiotic resistance has exponentially increased during the last years. It is necessary to develop new antimicrobial drugs to prevent and treat infectious diseases caused by multidrug- or extensively-drug resistant (MDR/XDR)-bacteria. Host Defense Peptides (HDPs) have a versatile role, acting as antimicrobial peptides and regulators of several innate immunity functions. The results shown by previous studies using synthetic HDPs are only the tip of the iceberg, since the synergistic potential of HDPs and their production as recombinant proteins are fields practically unexplored. The present study aims to move a step forward through the development of a new generation of tailored antimicrobials, using a rational design of recombinant multidomain proteins based on HDPs. This strategy is based on a two-phase process, starting with the construction of the first generation molecules using single HDPs and further selecting those HDPs with higher bactericidal efficiencies to be combined in the second generation of broad-spectrum antimicrobials. As a proof of concept, we have designed three new antimicrobials, named D5L37βD3, D5L37D5L37 and D5LAL37βD3. After an in-depth exploration, we found D5L37D5L37 to be the most promising one, since it was equally effective against four relevant pathogens in healthcare-associated infections, such as methicillin-susceptible (MSSA) and methicillin-resistant (MRSA) Staphylococcus aureus, methicillin-resistant Staphylococcus epidermidis (MRSE) and MDR Pseudomonas aeruginosa, being MRSA, MRSE and P. aeruginosa MDR strains. The low MIC values and versatile activity against planktonic and biofilm forms reinforce the use of this platform to isolate and produce unlimited HDP combinations as new antimicrobial drugs by effective means.

Keywords: antimicrobial; host defense peptides; nosocomial infections; recombinant.

Figure 1

Scheme depicting the fabrication approach of enhanced broad-spectrum antimicrobials. The 1st generation of HDPs linked to a GFP carrier were evaluated in a triple assay, allowing the selection of the most promising ones to generate in phase 2, the 2nd generation of antimicrobials, devoid of a non-functional carrier, fully tunable and with enhanced antimicrobial features.

Figure 2

Schematic representation of both 1st and 2nd generations of antimicrobial proteins. (a) The 1st generation constructs are constituted from N- to C-terminal by a single HDP-based domain (LAP, HβD2, HβD3, HD5 or LL37) fused to the GFP gene. (b) The 2nd generation constructs are multidomain proteins combining HD5, LL37 and HβD3 domains (D5L37βD3), combining the last three with LAP (D5LAL37βD3) and using HD5 and LL37 tandem repetitions (D5L37D5L37). All constructs have an H6-tag at C-terminal for protein purification purposes.

Figure 3

Antimicrobial activity of 1st generation antimicrobials. Antimicrobial activity (reduction of log of cfu/mL) of the different 1st generation constructs against (a) MRSA, (b) MSSA, (c) MRSE and (d) P. aeruginosa. The constructs were tested against an initial concentration of 10⁵ cfu/mL (dark bars) or 10³ cfu/mL (white bars). Data shown are the mean of a triplicate ± SEM. Different letters depict statistically significant differences (p < 0.0001) examined by ANOVA and Tukey test analysis. Capital and lowercase letters refer to 10⁵ cfu/mL and 10³ cfu/mL, respectively.

Figure 4

Minimal inhibitory concentration of 1st generation antimicrobials. (a) Minimal inhibitory concentration (MIC) assay of proteins based on LAP, HβD2, HβD3 and HD5 against MRSA (filled squares), MSSA (filled circles), MRSE (filled triangles) and P. aeruginosa (filled inversed triangles). Each construct was tested at its maximum concentration and serial two-fold dilution to determine MIC against the four tested microorganisms. (b) Summary of MIC values.

Figure 5

Antibiofilm performance of 1st generation molecules. Antibiofilm activity of the different 1st generation constructs at 10, 5 and 1 µM, against pre-formed biofilm of MRSA. Complete survival of biofilm (100%) is indicated by dotted lines. Data shown are the mean of triplicate ± SEM. Different letters represent statistically significant differences (p < 0.0001) assessed by ANOVA and Tukey test analysis.

Figure 6

Analysis of the antimicrobial mechanisms using scanning electron microscopic study (FE-SEM). FESEM cell integrity images of P. aeruginosa and MRSA after (a) control, (b) HD5, (c) HβD3, (d) LL-37 1st generation construct treatment, (e) D5L37D5L37 and (f) D5L37βD3 multidomain proteins (2nd generation antimicrobial) treatment. All treatments were applied at 5 µM. Scale bars are indicated in each image. Red arrows point out relevant image areas.

Figure 7

Bactericidal activity of 2nd generation HDPs. Antimicrobial activity of D5L37D5L37, D5L37βD3 and D5LAL37βD3 multidomain constructs at 5 μM against (a) MRSA, (b) MSSA, (c) MRSE and (d) P. aeruginosa. All constructs were tested against an initial 10⁵ cfu/mL of each of the bacteria. Data shown are the mean of triplicate ± SEM. Different letters represent significant differences (p < 0.0001) assessed by ANOVA and Tukey test analysis.

Figure 8

Optimized minimal inhibitory concentration of multidomain antimicrobial proteins. (a) MIC of the 2nd generation of antimicrobial constructs D5L37βD3, D5L37D5L37 and D5LAL37βD3 against MRSA (filled squares), MSSA (filled circles), MRSE (filled triangles) and P. aeruginosa (filled inversed triangles). All constructs were evaluated at their maximum achieved concentration and a serial of two-fold dilution was performed to determine MIC against the examined microorganism. (b) Summary of MIC values of 1st and 2nd generation.

Figure 9

Protein toxicity assay. Percentage of propidium iodide positive PBMC as a measure of toxicity after 24 h treatment with several concentrations of protein D5L37D5L37.

Figure 10

Antibiofilm performance of 2nd generation molecules. Biofilm eradication capacity of the different multidomain constructs D5L37βD3, D5L37D5L37 and D5LAL37βD3 at 5 and 1 μM against MRSA pre-formed biofilms. Complete survival of biofilm (100%) is indicated by dotted lines. Plots are the mean of triplicate ± SEM. Different letters indicate statistically significant differences (p < 0.0001) assessed by ANOVA and Tukey test analysis.

Figure 11

Characterization of recombinant HDPs structuration. Size distribution plots of (a) 1st generation proteins based on HβD2, HβD3, LL37, LAP and HD5, and (b) 2nd generation proteins D5LAL37βD3, D5L37D5L37 and D5L37βD3. The mean size ± SEM and polydispersity index (PI) are indicated in brackets.