# Roles of D-Amino Acids on the Bioactivity of Host Defense Peptides
## 5 Mechanisms of Bioactivities
### AntibioticIn Activity  
In the midst of environmental stress, bacteria tend to form multicellular communities known as biofilms in which the living bacterial cells are encased in a protective extracellular matrix consisting of polysaccharides and amyloid fibers. Biofilms render the bacterial cells highly resistant to antibiotics (i.e., up to 1000 folds when compared to the planktonic state [76]) and host immune response as well as being a significant obstacle in the treatment of a large variety of clinical infections [77]. Significant effort is being expended in the search for effective countermeasures and HDPs are amongst the many candidates that are being investigated for the ability to eliminate biofilms [78].  
HDPs are capable of disrupting biofilms through multiple mechanisms and at different stages as follows: (i) interfere with the adhesion of the bacteria to surfaces or to other cells in the initial stages of biofilm formation [79]; (ii) directly neutralizing bacterial cells inside the extracellular matrix [79] and (iii) disrupting metabolic pathways related to biofilm formation [80]. It has been suggested by [PERSON] et al. [78] that HDPs may have the potential to display all the \"ideal\" properties of an antibiofilm agent.  
As discussed in previous sections, the incorporation of d-AAs in HDPs enhances the therapeutic effectiveness through various mechanisms such as increased resistance to proteases and greater selectivity for pathogen membranes. In the use of d-AA containing HDPs against biofilms, such improvements to the activity is still valid [76]. In fact, a rather large number of studies have reported the use of d-AA incorporating HDPs to disrupt biofilms as a therapeutic route [76; 78]. However, instead of directly lysing pathogen cell membranes, the major focus of current antibiofilm HDP research is the disruption of metabolic pathways related to extracellular matrix formation. In fact, a number of potent antibiofilm HDPs (both all-l and d-AA containing) relied on mechanisms other than cell membrane disruption [80; 81; 82]. Notably, this included a peptide known as 1018, which was capable of completely preventing biofilm formation as well as eliminating established biofilms from both Gram-positive and Gram-negative bacterial pathogens and via synergistic reduction of the antibiotic concentration needed for complete biofilm inhibition by 64 folds [83].  
The study by [PERSON] [80] presented two synthetic all-d enantiomeric HDPs that were even more effective than the peptide 1018 as well as being found to protect the tested invertebrate animals from lethal _P. aeruginosa_ infection in vivo. This study also discussed in detail the mechanism of action of d-AA containing HDPs against the biofilm that was based on the inhibition of stringent intracellular response signal (p)ppGpp. The study observed that direct membrane lytic activity of both all-l and d-AA containing HDPs were not strictly correlated with that of the antibiofilm activity, which is unlike the antimicrobial activity that is directly dependent on membrane lyses. In terms of the underlying physicochemical properties of the peptide, the former is very sensitive to amino acid sequence changes while the latter is reliant on hydrophobicity and positive charge as discussed in the antimicrobial activity section.