Susceptibility of Pseudomonas aeruginosa Biofilm to Alpha-Helical Peptides: D-enantiomer of LL-37

Abstract

Pseudomonas aeruginosa is a highly versatile opportunistic pathogen and its ability to produce biofilms is a direct impediment to the healing of wounds and recovery from infection. Interest in anti-microbial peptides (AMPs) has grown due to their potential therapeutic applications and their possible use against antibiotic resistant bacteria. LL-37 is the only cathelicidin expressed by humans. In this study, we tested LL-37 and the effect of a protease-resistant LL-37 peptide mimetic, the peptide enantiomer D-LL-37, for anti-microbial and anti-biofilm activity against P. aeruginosa. Both forms of the peptide were equally effective as AMPs with similar killing kinetics. Circular dichroism spectra were obtained to demonstrate the chirality of D- and L-LL-37, and the trypsin resistance of D-LL-37 was confirmed. The helical cathelicidin from the cobra Naja atra (NA-CATH), and synthetic peptide variations (ATRA-1, ATRA-2, NA-CATH:ATRA1-ATRA1) were also tested. Although the cobra cathelicidin and related peptides had strong anti-microbial activity, those tested did not inhibit Pseudomonas biofilm formation, neither did control peptides. Both D- and L-LL-37 inhibited the attachment of Pseudomonas to a 96-well plate and decreased the amount of pre-formed (established) biofilm. D-LL-37 is able to promote Pseudomonas motility and decrease biofilm formation by altering the rate of twitching as well as by downregulating the expression of the biofilm-related genes, rhlA and rhlB, similar to L-LL-37. Both L- and D-LL-37 protected Galleria mellonella in vivo against Pseudomonas infection, while NA-CATH:ATRA1-ATRA1 peptide did not. This study demonstrates the ability and equivalence of D-LL-37 compared to L-LL-37 to promote bacterial twitching motility and inhibit biofilm formation, and protect against in vivo infection, and suggests that this peptide could be a critical advancement in the development of new treatments for P. aeruginosa infection.

The black soldier fly, Hermetia illucens – a promising source for sustainable production of proteins, lipids and bioactive substances

“…In recent years several AMPs were discovered for their ability to affect bacterial biofilms [60]. For example, the cathelicidin LL-37 produced by human neutrophils is very effective against P. aeruginosa biofilm formation and already existing biofilms of the multidrug-resistant S. aureus [61][62][63][64]. Despite these advantages, currently no commercial antibiofilm peptides are available to challenge biofilm infections specifically [60].…”

A Novel RNase 3/ECP Peptide for Pseudomonas aeruginosa Biofilm Eradication That Combines Antimicrobial, Lipopolysaccharide Binding, and Cell-Agglutinating Activities

“…First, from the cathelicidin family, we selected the antimicrobial peptide LL-37, which presents a high interaction for the negatively charged bacterial membranes and LPS molecules and has reported antimicrobial activity against planktonic P. aeruginosa (42). Furthermore, LL-37 is able to both inhibit P. aeruginosa biofilm formation (43,44) and remove preformed P. aeruginosa biofilms (43,45,46). Second, the antimicrobial synthetic fusion peptide cecropin A (1-7)-melittin (2-9), referred to here as CA-M, was chosen as it is a potent antimicrobial peptide against a wide variety of microorganisms.…”

Burkholderia Diffusible Signal Factor Signals to Francisella novicida To Disperse Biofilm and Increase Siderophore Production

“…Overnight cultures of bacteria were diluted 1:30 into 20 ml of TSBC, and 200 l was added to each well of a 96-well plate (Falcon 353072; BD). After 24 h at 37°C, the optical densities (ODs) at 600 nm of the wells were taken to normalize for growth, and then the liquid was removed by washing as previously described (23,24). The plates were then incubated at 70°C for 1 h and stained with 0.1% (wt/vol) crystal violet for 15 min.…”