Structure-activity relationship study using peptide arrays to optimize Api137 for an increased antimicrobial activity against Pseudomonas aeruginosa

Bluhm, Martina E.C.; Knappe, Daniel; Hoffmann, Ralf

doi:10.1016/j.ejmech.2015.09.022

Cited by 26 publications

(29 citation statements)

References 42 publications

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“…aeruginosa infections represent an urgent and worldwide health problem that needs to be addressed through the development of new therapeutic agents. PR-AMPs such as Bac7(1-35) and optimized apidaecins have been proved to be active against several strains of this opportunistic pathogen (19,25). Here we showed that it is active also against P. aeruginosa strains isolated from CF patients, and it acts through a complex mechanism that involves membranolytic activity.…”

Section: Discussionmentioning

confidence: 78%

The Mechanism of Killing by the Proline-Rich Peptide Bac7(1–35) against Clinical Strains of Pseudomonas aeruginosa Differs from That against Other Gram-Negative Bacteria

Runti

Benincasa

Giuffrida

et al. 2017

Antimicrob Agents Chemother

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Pseudomonas aeruginosa infections represent a serious threat to worldwide health. Proline-rich antimicrobial peptides (PR-AMPs), a particular group of peptide antibiotics, have demonstrated in vitro activity against P. aeruginosa strains. Here we show that the mammalian PR-AMP Bac7(1-35) is active against some multidrug-resistant cystic fibrosis isolates of P. aeruginosa. By confocal microscopy and cytometric analyses, we investigated the mechanism of killing against P. aeruginosa strain PAO1 and three selected isolates, and we observed that the peptide inactivated the target cells by disrupting their cellular membranes. This effect is deeply different from that previously described for PR-AMPs in Escherichia coli and Salmonella enterica serovar Typhimurium, where these peptides act intracellularly after having been internalized by means of the transporter SbmA without membranolytic effects. The heterologous expression of SbmA in PAO1 cells enhanced the internalization of Bac7(1-35) into the cytoplasm, making the bacteria more susceptible to the peptide but at the same time more resistant to the membrane lysis, similarly to what occurs in E. coli. The results evidenced a new mechanism of action for PRAMPs and indicate that Bac7 has multiple and variable modes of action that depend on the characteristics of the different target species and the possibility to be internalized by bacterial transporters. This feature broadens the spectrum of activity of the peptide and makes the development of peptide-resistant bacteria a more difficult process.

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Section: Discussionmentioning

confidence: 78%

The Mechanism of Killing by the Proline-Rich Peptide Bac7(1–35) against Clinical Strains of Pseudomonas aeruginosa Differs from That against Other Gram-Negative Bacteria

Runti

Benincasa

Giuffrida

et al. 2017

Antimicrob Agents Chemother

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“…A recent SAR study provided analogs of Api137 that were more active against P. aeruginosa due to an increased positive net charge and hydrophobicity, such as Api755 and Api760 that were 16 times more active in 50% MHB than Api137 (Bluhm et al, 2015 ). In order to increase the net charge and the hydrophobicity, we elongated the N-terminal sequence by repeating the Ile-Orn- and Trp-Orn-motifs two or three times, respectively.…”

Section: Resultsmentioning

confidence: 99%

“…Recently, this lead-peptide was optimized to enhance its activity against P. aeruginosa in full strength media using a structure-activity relationship (SAR) study (Bluhm et al, 2015 ). Among several interesting peptide analogs, Api755 (gu-OIORPVYOPRPRPPHPRL-OH) and Api760 (gu-OWORPVYOPRPRPPHPRL-OH) were particularly active.…”

Section: Introductionmentioning

confidence: 99%

N-Terminal Ile-Orn- and Trp-Orn-Motif Repeats Enhance Membrane Interaction and Increase the Antimicrobial Activity of Apidaecins against Pseudomonas aeruginosa

Bluhm

Schneider

Schäfer

et al. 2016

Front. Cell Dev. Biol.

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The Gram-negative bacterium Pseudomonas aeruginosa is a life-threatening nosocomial pathogen due to its generally low susceptibility toward antibiotics. Furthermore, many strains have acquired resistance mechanisms requiring new antimicrobials with novel mechanisms to enhance treatment options. Proline-rich antimicrobial peptides, such as the apidaecin analog Api137, are highly efficient against various Enterobacteriaceae infections in mice, but less active against P. aeruginosa in vitro. Here, we extended our recent work by optimizing lead peptides Api755 (gu-OIORPVYOPRPRPPHPRL-OH; gu = N,N,N′,N′-tetramethylguanidino, O = L-ornithine) and Api760 (gu-OWORPVYOPRPRPPHPRL-OH) by incorporation of Ile-Orn- and Trp-Orn-motifs, respectively. Api795 (gu-O(IO)2RPVYOPRPRPPHPRL-OH) and Api794 (gu-O(WO)3RPVYOPRPRPPHPRL-OH) were highly active against P. aeruginosa with minimal inhibitory concentrations of 8–16 and 8–32 μg/mL against Escherichia coli and Klebsiella pneumoniae. Assessed using a quartz crystal microbalance, these peptides inserted into a membrane layer and the surface activity increased gradually from Api137, over Api795, to Api794. This mode of action was confirmed by transmission electron microscopy indicating some membrane damage only at the high peptide concentrations. Api794 and Api795 were highly stable against serum proteases (half-life times >5 h) and non-hemolytic to human erythrocytes at peptide concentrations of 0.6 g/L. At this concentration, Api795 reduced the cell viability of HeLa cells only slightly, whereas the IC50 of Api794 was 0.23 ± 0.09 g/L. Confocal fluorescence microscopy revealed no colocalization of 5(6)-carboxyfluorescein-labeled Api794 or Api795 with the mitochondria, excluding interactions with the mitochondrial membrane. Interestingly, Api795 was localized in endosomes, whereas Api794 was present in endosomes and the cytosol. This was verified using flow cytometry showing a 50% higher uptake of Api794 in HeLa cells compared with Api795. The uptake was reduced for both peptides by 50 and 80%, respectively, after inhibiting endocytotic uptake with dynasore. In summary, Api794 and Api795 were highly active against P. aeruginosa in vitro. Both peptides passed across the bacterial membrane efficiently, most likely then disturbing the ribosome assembly, and resulting in further intracellular damage. Api795 with its IOIO-motif, which was particularly active and only slightly toxic in vitro, appears to represent a promising third generation lead compound for the development of novel antibiotics against P. aeruginosa.

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“…Generally, increased hydrophobicity of the non-polar face of an amphipathic α-helical peptide could improves antimicrobial activities (Dathe et al 1997). As reported by Mattei, et al, hydrophobicity is positively correlated with the membrane disrupting power of the peptides to a certain extent (Bluhm et al 2015).…”

Section: Discussionmentioning

confidence: 90%

LHH1, a Novel Antimicrobial Peptide with Anti-Cancer Activity Identified from Lactobacillus casei HZ1

Jin

Luo

et al. 2020

Preprint

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Antimicrobial peptides have been attracting increasing attention for their multiple beneficial effects. In present study, a novel AMP with a molecular weight of 1875.25 Da, was identified from the genome of Lactobacillus casei HZ1. The peptide, which was named as LHH1 was comprised of 16 amino acid residues, and its α-helix content was 95.34% when dissolved in 30 mM SDS. LHH1 exhibited a broad range of antimicrobial activities against gram-positive bacteria and fungus. It could effectively inhibit staphylococcus aureus with a minimum inhibitory concentration of 3.5 μM and showed a low hemolytic activity. The scanning electron microscope, confocal laser scanning microscope and flow cytometry results showed that LHH1 exerted its antibacterial activity by damaging the cell membrane of staphylococcus aureus. Meanwhile, LHH1 also exhibited anti-cancer activities against several cancer cells including MGC803, HCT116 and C666-1 cells, and one major action of its anti-cancer mechanism was breaking the cell membrane.

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Structure-activity relationship study using peptide arrays to optimize Api137 for an increased antimicrobial activity against Pseudomonas aeruginosa

Cited by 26 publications

References 42 publications

The Mechanism of Killing by the Proline-Rich Peptide Bac7(1–35) against Clinical Strains of Pseudomonas aeruginosa Differs from That against Other Gram-Negative Bacteria

The Mechanism of Killing by the Proline-Rich Peptide Bac7(1–35) against Clinical Strains of Pseudomonas aeruginosa Differs from That against Other Gram-Negative Bacteria

N-Terminal Ile-Orn- and Trp-Orn-Motif Repeats Enhance Membrane Interaction and Increase the Antimicrobial Activity of Apidaecins against Pseudomonas aeruginosa

LHH1, a Novel Antimicrobial Peptide with Anti-Cancer Activity Identified from Lactobacillus casei HZ1

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