Effects of Trp- and Arg-Containing Antimicrobial-Peptide Structure on Inhibition of Escherichia coli Planktonic Growth and Biofilm Formation

Biofilms are sessile microbial communities that cause serious chronic infections with high morbidity and mortality. In order to develop more effective approaches for biofilm control, a series of linear cationic antimicrobial peptides (AMPs) with various arginine (Arg or R) and tryptophan (Trp or W) repeats (RW)_n-NH₂, where n = 2, 3, or 4] were rigorously compared to correlate their structures with antimicrobial activities affecting the planktonic growth and biofilm formation of Escherichia coli. The chain length of AMPs appears to be important for inhibition of bacterial planktonic growth, since the hexameric and octameric peptides significantly inhibited E. coli growth, while tetrameric peptide did not cause noticeable inhibition. In addition, all AMPs except the tetrameric peptide significantly reduced E. coli biofilm surface coverage and the viability of biofilm cells, when added at inoculation. In addition to inhibition of biofilm formation, significant killing of biofilm cells was observed after a 3-hour treatment of preformed biofilms with hexameric peptide. Interestingly, treatment with the octameric peptide caused significant biofilm dispersion without apparent killing of biofilm cells that remained on the surface; e.g., the surface coverage was reduced by 91.5 ± 3.5% by 200 μM octameric peptide. The detached biofilm cells, however, were effectively killed by this peptide. Overall, these results suggest that hexameric and octameric peptides are potent inhibitors of both bacterial planktonic growth and biofilm formation, while the octameric peptide can also disperse existing biofilms and kill the detached cells. These results are helpful for designing novel biofilm inhibitors and developing more effective therapeutic methods.Antimicrobial peptides (AMPs) are promising alternatives to traditional antibiotics (5). Native AMPs are part of the host defense in organisms ranging from bacteria to insects, plants, and animals (14). They are capable of eliminating a broad spectrum of microorganisms, including viruses, bacteria, and fungi (4, 14). Compared with widespread antibiotic resistance (38), resistance to AMPs is rare, possibly because AMPs directly target cell membranes that are essential to microbes (14, 29). In addition, no cross-resistance has been observed in clinic due to the diversity of peptide sequences (42). Thus, native and synthetic AMPs offer potential alternatives to antibiotics for treating drug-resistant infections (3, 26, 27).In mammalian innate immune systems, some AMPs are produced constitutively, while others are inducible within hours after detection of invading microorganisms (4, 13). Although the detailed mechanism of AMPs'' activities remains elusive (5), AMPs are known to disrupt cell membranes of microbes, interfere with metabolism, and/or target cytoplasmic components (41). Most known AMPs are cationic and amphiphilic (29). It is hypothesized that the initial interaction occurs via an electrostatic attraction between the AMP molecule and microbial membrane. Cationic AMPs can cover bacterial membranes, disrupt the membrane potential, create pores across the membrane, and consequently cause the leak of cell contents and cell death (27, 41). AMPs are relatively selective in targeting microbes rather than mammalian cells, most likely because of the fundamental differences between microbial and host membranes (41), e.g., a higher abundance of negatively charged phospholipids and an absence of cholesterol in microbial membranes.Known AMPs vary dramatically in sequence, size (from 12 to 50 amino acids), and structure (α-helices or β-sheets) (23). However, most AMPs have two types of side chains with relatively conservative sequences: positively charged basic residues, containing arginine (R), lysine (K), and/or histidine (H), that presumably mediate the interaction with the negatively charged microbial membrane, and bulky hydrophobic residues, rich in tryptophan (W), proline (P), and/or phenylalanine (F), that facilitate permeabilization and membrane disruption (26).Although AMPs are promising agents for antimicrobial therapies (15), only a few have made it to clinical trials and applications, with varied success (15, 42). There are several issues that need further development. First, the MICs of AMPs are relatively high compared to those of conventional antibiotics. Recent studies suggest that the peptide/lipid (P/L) ratio needs to be higher than a threshold to allow the AMPs to be oriented perpendicular to the membrane so that pores can be created to kill bacteria (22, 30). Thus, an optimization of peptide structure and size may improve their antimicrobial activities. In addition to the high MICs, the wide application of AMPs is also hindered by their high manufacturing costs and the cytotoxicity of some AMPs.Given the limit of currently available AMPs, it is important to develop more effective AMPs with reduced manufacturing cost and enhanced activity (17, 26, 28, 39). Strøm et al. (39) chemically synthesized a series of short cationic AMPs containing repeating R and W residues in order to identify the minimal pharmacophore with high antimicrobial activities. The data suggest that tetrapeptides or capped tripeptides are effective and there is no correlation between the order of amino acids and antimicrobial activity. Liu et al. (26) analyzed the effects of chain length on the activities of AMPs with repeating pharmacophore sequences (RW)_n-NH₂ (n = 1, 2, 3, 4, or 5). The tests of antimicrobial activities and the hemolysis of red blood cells suggest that (RW)₃-NH₂ has the optimal chain length. Although longer chains are more potent antimicrobials, they can stimulate hemolysis.Most of the AMP studies to date are focused on planktonic bacteria. However, the majority of pathogenic bacteria tend to adhere to surfaces and form sessile microbial communities with highly hydrated structures of secreted polysaccharide matrix, collectively known as biofilms (9). Biofilms can tolerate up to 1,000 times more antibiotics and disinfectants than their planktonic counterparts (2, 7, 8). For example, Folkesson et al. (12) reported that biofilm formation of E. coli K-12 increases its tolerance to polymyxin E, a polypeptide antibiotic that kills Gram-negative bacteria by disrupting membranes (34, 40). Since biofilms are involved in 80% of human bacterial infections (1), it is necessary to study biofilm inhibition and dispersion by AMPs.In this study, a series of linear peptides (RW)_n-NH₂ (where n = 2, 3, or 4) were studied for the effects of their activities on planktonic cells and biofilms of E. coli to understand the structural effects on the antimicrobial activities of AMPs. We chose E. coli RP437 in this study because it is one of the model strains for biofilm research and allows us to compare the data with those of our previous studies (6, 16, 19, 20).