Antimicrobial effect of Tetraspanin CD9 Peptides on ...

The development of a new antimicrobial treatment with low resistance risk is the current goal to combat microbial resistance and chronic infections. P. aeruginosa is known to utilise many resistance mechanisms including intrinsic, acquired, and adaptive resistance mechanisms, to resist antibiotics.16 P. aeruginosa usually has low outer membrane permeability, efflux pump systems, antibiotic-inactivating enzymes, and biofilm formation to confine and expel antibiotics.16 To our knowledge, this is the first study to address the potential direct antimicrobial and antibiofilm inhibitory effects of tetraspanin peptides against P. aeruginosa, as there are currently no reports highlighting the direct effect of tetraspanin peptides against this bacterium.

In this study, DMSO was used to dissolve the CD9 peptides, a water-insoluble drug, according to the manufacturer’s protocol. DMSO is known to be widely used as a solvent for water-insoluble drugs.17 It is well-established that high concentrations of DMSO attenuate the virulence factors of P. aeruginosa.17 Interestingly, however, this study showed that DMSO did not affect the growth of either of the P. aeruginosa isolates. This finding thereby ruled out DMSO influence on the efficacy of the antibacterial activity of CD9 peptides to inhibit bacteria. In addition, even though pure DMSO was used to dissolve the peptide at early stages, the final concentration was usually kept lower than the stock, as it had been diluted.

This study showed that CD9 peptides have a small inhibitory effect on the standard strain of P. aeruginosa, as seen with the small zone of inhibition formed on the agar plate. There was no green pigment formed by P. aeruginosa colonies surrounding the CD9 peptide disc on the agar plate, which is known to be released by the production of pyocyanin (blue-green) and pyoverdins1. The zone of inhibition caused by CD9 peptides was not as clear as the zone of inhibition formed by the antibiotics, which may be due to the fluorescent pink colour of the peptide. In addition, the potency of the antibiotics was stronger than that of CD9 peptides in forming a clear zone of inhibition. The efficacy of antibiotics is well-known in healthcare settings and they have been well-regulated by the FDA. Unfortunately, this study showed that a higher concentration of CD9 peptides may be needed to fully inhibit the growth of both standard and MDR- P. aeruginosa strains. The results of the current study support the theory put forward by Ventress et al. and Green et al. that tetraspanin peptides and recombinant tetraspanin peptides do not affect bacteria when added before infection to the cells. However, because the inhibition of bacterial growth does not result in bacterial death, this method cannot distinguish between the bactericidal and bacteriostatic effects of CD9 peptides.

Based on the cut-off point, the MIC and MBC results showed that the effects of CD9 peptides were considered bactericidal against the standard strain of P. aeruginosa, as the ratio of MBC to MIC was less than 4 (0.5 mg/mL: 0.25 mg/mL).18 In addition, the CD9 peptides were considered bacteriostatic against MDR- P. aeruginosa, as the ratio of MBC to MIC was equal to 4 (1 mg/mL: 0.25 mg/mL).18 This is because this treatment did not fully kill the bacteria and only slightly inhibited bacterial growth. This phenomenon can be beneficial for humans, as this treatment has been shown previously to cause no toxicity to the cells, which in turn may help in strengthening immunity and subsequently eliminating the bacteria.13,14,19,20

It was reported by Benkova et al. that the diameter of the zone of inhibition is inversely proportional to the MIC value; that is, the smaller the zone of inhibition, the higher the MIC value, and thus, higher the concentration of antimicrobial needed.21 Our study shows that a higher concentration of CD9 peptides is needed to inhibit the growth of P. aeruginosa. With these high values, a higher dose of CD9 peptides could be used as an adjuvant in any antimicrobial drug, but still be safely administered, especially if the designed peptide was less than 100 amino acids and well-regulated under the Federal Food, Drug, and Cosmetic Act (FD&C) of the FDA.22 The results from the current study also suggest that CD9 peptides may only achieve significant therapeutic effects at the tissue level by inhibiting bacterial growth by neutralising the charge on the bacterial cell membrane and/or altering the permeability of bacterial cells.20,23

Biofilm is more resistant to physical and chemical changes by different antibacterial agents up to 1000 times more than bacteria in their planktonic growth.5,6 Biofilms are a complex matrix of microbial cells composed of proteins, extracellular DNA, and exopolysaccharides, which in P. aeruginosa include alginate, Psl, and Pel, and which help the bacterial cells evade antimicrobial agents.23 Although CD9 peptides showed no visible inhibitory effect against the growth of the MDR- P. aeruginosa isolate on the agar plate, they successfully inhibited 70% of the biofilm formation of both the standard strain P. aeruginosa and the MDR- P. aeruginosa in the microtiter plate. This study showed that the untreated P. aeruginosa isolates formed a thick biofilm layer on the bottom surface of the well in the absence of any effector or inhibitor other than the broth medium and the presence of water. This condition provided an aqueous and moist surface for the bacteria to develop a biofilm attached to the bottom surface of the plate. Since DMSO did not show any inhibition in the bacterial growth in the previous section, it is suggested that it may not interfere with the efficacy of CD9 peptides as an anti-biofilm agent.

Biofilm formation is the typical characteristic of P. aeruginosa, which is often present on contaminated and moist surfaces such as bottles, sinks, and medical devices such as urine catheters.4 In addition, biofilm formation can also be favoured by the changes in the pH of the environment, which may be due to secreted bacterial metabolites.24,25 This study confirmed the inhibitory effect of CD9 peptides on P. aeruginosa biofilm formation. The effect of CD9 peptides could be due to the cross-reaction of CD9 peptides with P. aeruginosa, which may limit the ability of bacteria to form a biofilm under static conditions and may inhibit the adhesion of biofilm to host surfaces, thus reducing the potential for pathogenesis at a later stage. This inhibitory mechanism may be due to the ability of CD9 peptides to destabilise the biofilm on the surface of the wells, making the bacteria less susceptible to cell adhesion.23,26 Moreover, CD9 peptides can interfere with acidic environments during biofilm formation, as demonstrated by the reduction in the stained crystal violet.24,27 In general, biofilm formation is associated with the chronicity of bacterial infectious diseases because biofilms act as a barrier that limits the penetration of antimicrobial agents into bacteria.28 We concluded that the CD9 peptides can reduce chronicity and recurrent P. aeruginosa infections by inhibiting biofilm formation, with a lower risk of resistance development. However, crystal violet staining has the disadvantage of staining both viable and non-viable cells adhering to the surface.

This study successfully produced additional insights into the antimicrobial action of CD9 peptides against P. aeruginosa, in addition to the known anti-adherence properties. The findings in this study were consistent with our recent work which reported the antimicrobial effect of CD9 peptides against Gram-positive bacteria causing axillary malodor.29 In addition, this mode of action may be similar to D-mannose against uropathogenic Escherichia coli, which exploits strong anti-adherence properties and does not exert “antibiotic-like” activity such as destroying and killing the bacteria.30 Furthermore, this study proved that the tetraspanin CD9 peptides have an indirect inhibitory effect on bacterial attachment to host cells due to their direct inhibitory effects on P. aeruginosa and reducing the bacterial load on host cells. It is reported that the antimicrobial activity of antimicrobial peptides is influenced by a few physio-chemical properties including the length, charge, hydrophobicity, amphipathicity and hydrophobic or hydrophilic angle of the peptides.26 Positively charged amino acids of CD9 peptides such as lysine and glutamine enable the binding to the negatively charged bacterial surfaces, inducing electrostatic forces, that may cause the penetration of CD9 peptides to the cytoplasmic membrane of P. aeruginosa, without causing membrane damage and rupture.20,26

Although these results suggest that tetraspanin-based treatments cause little direct inhibition of P. aeruginosa, they prove that tetraspanins on tetraspanin enriched microdomain facilitate P. aeruginosa binding to host cells, recruit cell surface proteins, and provide bacterial adhesion interactions.9,31 Moreover, these results reflect the role of tetraspanins as indirect receptors for bacterial pathogenesis, perhaps because of their properties not only as receptors but as mediators of the adhesion platform for bacterial pathogenesis.8,10 The treatment studied here, derived from the EC2 domain of CD9, will mainly disturb the TEMs, altering the microdomain and simultaneously disrupting the interaction of tetraspanins with partner proteins on the surface of host cells.9,14 However, this study has some limitations as it was the first attempt to investigate the direct antimicrobial activity of tetraspanin CD9 against bacterial isolates. Further experiments should include other bacterial species to fully demonstrate the antimicrobial effects of tetraspanin CD9 peptides, and should further investigate the related virulence factors of P. aeruginosa, to thoroughly understand the mode of action of CD9 peptides. Due to the high cost of peptides, further research also needs to include another tetraspanins to explore the antimicrobial properties of the tetraspanin family; those shown in this study may not reflect the entire tetraspanins family.