A Thermostable Salmonella Phage Endolysin,Lys68, with Broad Bactericidal Properties against Gram-Negative Pathogens in Presence of Weak Acids

Resistance rates are increasing among several problematic Gram-negative pathogens, a fact that has encouraged the development of new antimicrobial agents. This paper characterizes a Salmonella phage endolysin (Lys68) and demonstrates its potential antimicrobial effectiveness when combined with organic acids towards Gram-negative pathogens. Biochemical characterization reveals that Lys68 is more active at pH 7.0, maintaining 76.7% of its activity when stored at 4°C for two months. Thermostability tests showed that Lys68 is only completely inactivated upon exposure to 100°C for 30 min, and circular dichroism analysis demonstrated the ability to refold into its original conformation upon thermal denaturation. It was shown that Lys68 is able to lyse a wide panel of Gram-negative bacteria (13 different species) in combination with the outer membrane permeabilizers EDTA, citric and malic acid. While the EDTA/Lys68 combination only inactivated Pseudomonas strains, the use of citric or malic acid broadened Lys68 antibacterial effect to other Gram-negative pathogens (lytic activity against 9 and 11 species, respectively). Particularly against Salmonella Typhimurium LT2, the combinatory effect of malic or citric acid with Lys68 led to approximately 3 to 5 log reductions in bacterial load/CFUs after 2 hours, respectively, and was also able to reduce stationary-phase cells and bacterial biofilms by approximately 1 log. The broad killing capacity of malic/citric acid-Lys68 is explained by the destabilization and major disruptions of the cell outer membrane integrity due to the acidity caused by the organic acids and a relatively high muralytic activity of Lys68 at low pH. Lys68 demonstrates good (thermo)stability properties that combined with different outer membrane permeabilizers, could become useful to combat Gram-negative pathogens in agricultural, food and medical industry.     

The human antimicrobial peptide LL-37 and its fragments possess both antimicrobial and antibiofilm activities against multidrug-resistant Acinetobacter baumannii

Acinetobacter baumannii infections are difficult to treat due to multidrug resistance. Biofilm formation by A. baumannii is an additional factor in its ability to resist antimicrobial therapy. The antibacterial and antibiofilm activities of the human antimicrobial peptide LL-37 and its fragments KS-30, KR-20 and KR-12 against clinical isolates of multidrug-resistant (MDR) A. baumannii were evaluated. The minimal inhibitory concentration (MIC) of LL-37 against MDR A. baumannii isolates ranged from 16 to 32 μg/mL. The MIC of KS-30 fragment varied from 8.0 to16 μg/mL and the KR-20 fragment MIC ranged from 16 to 64 μg/mL. LL-37 and KS-30 fragment exhibited 100% bactericidal activity against five A. baumannii strains, including four MDR clinical isolates, within 30 min at concentrations of 0.25–1 μg/mL. By 0.5 h, the fragments KR-20 and KR-12 eliminated all tested strains at 8 and 64 μg/mL respectively. LL-37 and its fragments displayed anti-adherence activities between 32-128 μg/mL. A minimum biofilm eradication concentration (MBEC) biofilm assay demonstrated that LL-37 inhibited and dispersed A. baumannii biofilms at 32 μg/mL respectively. Truncated fragments of LL-37 inhibited biofilms at concentrations of 64–128 μg/mL. KS-30, the truncated variant of LL-37, effectively dispersed biofilms at 64 μg/mL. At 24 h, no detectable toxicity was observed at the efficacious doses when cytotoxicity assays were performed. Thus, LL-37, KS-30 and KR-20 exhibit significant antimicrobial activity against MDR A. baumannii. The prevention of biofilm formation in vitro by LL-37, KS-30 and KR-20 adds significance to their efficacy. These peptides can be potential therapeutics against MDR A. baumannii infections.     

The endolysin of the Acinetobacter baumannii phage vB_AbaP_D2 shows broad antibacterial activity

The emergence and rapid spread of multidrug-resistant bacteria has induced intense research for novel therapeutic approaches. In this study, the Acinetobacter baumannii bacteriophage D2 (vB_AbaP_D2) was isolated, characterized and sequenced. The endolysin of bacteriophage D2, namely Abtn-4, contains an amphipathic helix and was found to have activity against multidrug-resistant Gram-negative strains. By more than 3 log units, A. baumannii were killed by Abtn-4 (5 µM) in 2 h. In absence of outer membrane permeabilizers, Abtn-4 exhibited broad antimicrobial activity against several Gram-positive and Gram-negative bacteria, such as Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumonia, Enterococcus and Salmonella. Furthermore, Abtn-4 had the ability to reduce biofilm formation. Interestingly, Abtn-4 showed antimicrobial activity against phage-resistant bacterial mutants. Based on these results, endolysin Abtn-4 may be a promising candidate therapeutic agent for multidrug-resistant bacterial infections.     

Fighting Acinetobacter baumannii infections with the acylase PvdQ

Acinetobacter baumannii is an opportunistic Gram-negative bacterial pathogen that poses a threat for frail patients worldwide. The high ability to withstand environmental stresses as well as its resistance towards a broad range of antibiotics make A. baumannii an effective hard-to-eradicate pathogen. One of the key mechanisms mediating tolerance against antibiotic treatment is the formation of biofilms, a process that is controlled by a multitude of different regulatory mechanisms. A key factor with major impact on biofilm formation is cell-to-cell communication by quorum-sensing, which in A. baumannii is mediated by acyl homoserine lactone signaling molecules. Here we show that the Ntn-Hydrolase PvdQ from Pseudomonas aeruginosa can reduce biofilm formation by the A. baumannii ATCC 17978 type strain and several clinical isolates on abiotic surfaces. Further, our study shows that a combination treatment of PvdQ-mediated quorum-quenching with the antibiotic gentamicin has a synergistic effect on the clearance of A. baumannii biofilms and possible biofilm dispersal. Moreover, we demonstrate in a Galleria mellonella larval infection model that PvdQ administration significantly prolongs survival of the larvae. Altogether, we conclude that the acylase-mediated irreversible cleavage of quorum-sensing signaling molecules as exemplified with PvdQ can set a profound limit to the progression of A. baumannii infections.     

Antibacterial activity of <Emphasis Type="Italic">Acinetobacter baumannii</Emphasis> phage ϕAB2 endolysin (LysAB2) against both Gram-positive and Gram-negative bacteria

To investigate the nature and origin of the antibacterial activity of the lytic phage ϕAB2 toward Acinetobacter baumannii, we successfully isolated and characterized a novel phage lysozyme (endolysin) from ϕAB2 and named it LysAB2. To analyze antibacterial activity of LysAB2, the complete LysAB2 and two deletion derivatives were constructed, purified and characterized. Zymographic assays showed that only the intact LysAB2 could lyse the peptidoglycan of A. baumannii and the Staphylococcus aureus cell wall. Antibacterial analysis also showed that only the intact LysAB2 retained the complete bactericidal activity. When applied exogenously, LysAB2 exhibited a broad bacteriolytic activity against a number of Gram-negative and Gram-positive bacteria. Thermostability assays indicated that LysAB2 was stable at 20∼40°C. Its optimal pH was 6.0, and it was active from pH 4 to 8. Scanning electron microscopy revealed that exposure to 500 μg ml⁻¹ LysAB2 for up to 60 min caused a remarkable modification of the cell shape of the bacteria. Treating bacteria with LysAB2 clearly enhanced permeation of the bacterial cytoplasmic membrane. These results indicate that LysAB2 is an effective lysozyme against bacteria, and they suggest that it is a good candidate for a therapeutic/disinfectant agent to control nosocomial infections caused by multiple drug-resistant bacteria.     

Identification of aryl 2-aminoimidazoles as biofilm inhibitors in Gram-negative bacteria

The synthesis and biofilm inhibitory activity of a 30-member aryl amide 2-aminoimidazole library against the three biofilm forming Gram-negative bacteria Escherichia coli, Psuedomonas aeruginosa, and Acinetobacter baumannii is presented. The most active compound identified inhibits the formation of E. coli biofilms with an IC₅₀ of 5.2 μM and was observed to be non-toxic to planktonic growth, demonstrating that analogues based on an aryl framework are viable options as biofilm inhibitors within the 2-aminoimidazole family.     

New trifluoromethyl quinolone derivatives: Synthesis and investigation of antimicrobial properties

A series of quinolone derivatives, containing different heterocyclic amines were prepared. Synthesized compounds were evaluated for their in vitro antimicrobial activities against two Gram-positive bacteria, three Gram-negative bacteria as well as four fungi. All the derivatives showed good activity towards Gram-positive bacteria and less activity towards Gram-negative bacteria. They also showed moderate to comparable activity against Aspergillus niger and Candida albicans and low to moderate antifungal activity against Aspergillus fumigatus and Aspergillus flavus.     

Synergistic Antimicrobial Activity of Camellia sinensis and Juglans regia against Multidrug-Resistant Bacteria

Synergistic combinations of antimicrobial agents with different mechanisms of action have been introduced as more successful strategies to combat infections involving multidrug resistant (MDR) bacteria. In this study, we investigated synergistic antimicrobial activity of Camellia sinensis and Juglans regia which are commonly used plants with different antimicrobial agents. Antimicrobial susceptibility of 350 Gram-positive and Gram-negative strains belonging to 10 different bacterial species, was tested against Camellia sinensis and Juglans regia extracts. Minimum inhibitory concentrations (MICs) were determined by agar dilution and microbroth dilution assays. Plant extracts were tested for synergistic antimicrobial activity with different antimicrobial agents by checkerboard titration, Etest/agar incorporation assays, and time kill kinetics. Extract treated and untreated bacteria were subjected to transmission electron microscopy to see the effect on bacterial cell morphology. Camellia sinensis extract showed higher antibacterial activity against MDR S. Typhi, alone and in combination with nalidixic acid, than to susceptible isolates.” We further explore anti-staphylococcal activity of Juglans regia that lead to the changes in bacterial cell morphology indicating the cell wall of Gram-positive bacteria as possible target of action. The synergistic combination of Juglans regia and oxacillin reverted oxacillin resistance of methicillin resistant Staphylococcus aureus (MRSA) strains in vitro. This study provides novel information about antimicrobial and synergistic activity of Camellia sinensis and Juglans regia against MDR pathogens     

Structure-activity relationship-based screening of antibiotics against Gram-negative Acinetobacter baumannii

To discover potent antibiotics against the Gram-negative bacteria, we performed a structure-activity relationship (SAR) study of YKsa-6, which was the most potent inhibitor of Staphylococcus aureus β-ketoacyl acyl carrier protein III in our previous study. We identified and selected 11 candidates, and finally screened two active compounds, YKab-4 (4-[(3-chloro-4-methylphenyl)aminoiminomethyl]benzene-1,3-diol) and YKab-6 (4-[[3-(trifluoromethyl)phenyl]aminoiminomethyl]phenol) as inhibitors of Acinetobacter baumannii KAS III (abKAS III). They showed potent antimicrobial activities at 2 or 8 μg/mL, specifically against Acinetobacter baumannii and a strong binding affinity for abKAS III. From the homology modeling, we defined the three-dimensional (3D) structure of abKAS III for the first time and found that it had an extra loop region compared with common Gram-negative bacteria derived KAS IIIs. The docking study revealed that the hydroxyl groups of inhibitors formed extensive hydrogen bonds and the complicated hydrophobic and cation-stacking interactions are important to binding with abKAS III. We confirmed that the hydrophobicity of these compounds might be the essential factor for their antimicrobial activities against Gram-negative bacteria as well as their structural rigidity, a cooperative feature for retaining the hydrophobic interactions between abKAS III and its inhibitors. This study may provide an insight developing strategies for potent antibiotics against A. baumannii.     

Antimicrobial and cytotoxic activities of the crude extracts of Dietes bicolor leaves,flowers and rhizomes

The crude extracts of Dietes bicolor leaves, flowers and rhizomes were subjected to comparative antimicrobial screening against two Gram-positive, two Gram-negative bacteria and four fungal strains using the agar well diffusion method. The minimum inhibitory concentrations (MIC) of the tested extracts were also determined. Furthermore, the cytotoxic activity was evaluated. D. bicolor extracts generally demonstrated notable broad spectrum antimicrobial activities (MIC values ≤ 500 μg/mL) against all tested pathogens. D. bicolor leaf extract showed potent broad spectrum antimicrobial activity with MIC values ranging between 0.24 and 31.25 μg/mL against all tested pathogens. Moreover, the flowers extract exhibited promising antimicrobial activities, displaying MIC values ranging between 1.95 and 125 μg/mL against the tested bacteria and fungi. However, the rhizomes extract showed moderate antimicrobial activity with MIC values ranging between 31.25 and 500 μg/mL. Despite the potent antimicrobial activity of D. bicolor extracts, they were ineffective as cytotoxic agents against nine tested cancer cell lines, displaying 50% inhibitory concentration (IC₅₀) values above 100 μg/mL. The reported potent antimicrobial activity along with the lack of measurable cytotoxic activity indicated that the antimicrobial activity of D. bicolor crude extracts is mediated through a mechanism other than cytotoxicity. These results suggest that D. bicolor can act as a potential source for natural antibacterial and antifungal agents with a good safety profile at a preliminary level.     

Screening of Indonesian peat soil bacteria producing antimicrobial compounds

The development and world-wide spread of multidrug-resistant (MDR) bacteria have a high concern in the medicine, especially the extended-spectrum of beta-lactamase (ESBL) producing Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA). There are currently very limited effective antibiotics to treat infections caused by MDR bacteria. Peat-soil is a unique environment in which bacteria have to compete each other to survive, for instance, by producing antimicrobial substances. This study aimed to isolate bacteria from peat soils from South Kalimantan Indonesia, which capable of inhibiting the growth of Gram-positive and Gram-negative bacteria. Isolates from peat soil were grown and identified phenotypically. The cell-free supernatant was obtained from broth culture by centrifugation and was tested by agar well-diffusion technique against non ESBL-producing E. coli ATCC 25922, ESBL-producing E. coli ATCC 35218, methicillin susceptible Staphylococcus aureus (MSSA) ATCC 29,213 and MRSA ATCC 43300. Putative antimicrobial compounds were separated using SDS-PAGE electrophoresis and purified using electroelution method. Antimicrobial properties of the purified compounds were confirmed by measuring the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). In total 28 isolated colonies were recovered; three (25PS, 26PS, and 27PS) isolates produced proteins with strong antimicrobial activities against both reference strains. The substance of proteins from three isolates exerted strong antimicrobial activity against ESBL-producing E. coli ATCC 35,218 (MIC = 2,80 µg/mL (25PS), 3,76 µg/mL (26PS), and 2,41 µg/mL (27PS), and MRSA ATCC 43,300 (MIC = 4,20 µg/mL (25PS), 5,65 µg/mL (26PS), and 3,62 µg/mL (27PS), and also had the ability bactericidal properties against the reference strains. There were isolates from Indonesian peat which were potentials sources of new antimicrobials.     

Determination of synergistic effects of antibiotics and Zno NPs against isolated E. Coli and A. Baumannii bacterial strains from clinical samples

The mortality rates has been increased globally due to multidrug resistant (MDR) E.coli and A.baumanii bacterial strains and also there is an emerging resistance of the Enterobacteriaceae family of bacteria to Carbapenem antibiotics (CRE) in Saudi Arabia. The main aim of our research study is to isolate E.coli and A. baumannii bacterial species from various collected clinical samples and to evaluate the MIC and FICI of Colistin, Ciprofloxacin, Meropenem and ZnO NPs and in combination of Colistin, Ciprofloxacin, Meropenem with ZnO NPs.The clinical isolated strains of A. baumannii (MRO-17-13) and A. baumannii (MRO-17–25) was found to be sensitive towards colistin with 0.5 μg/mL concentration, whereas, all the isolated A. baumannii strains showed similar MIC value 2 mg/mL when tested with ZnO NPs, the MIC value for the ZnO NPs was found to be similar for all the E.coli strains 0.25 mg/mL. The effects of all Ciprofloxacin concentrations used in the study were bacteriostatic against E. coli (01UR19006568-01) strain but 1 mg/mL concentration of ZnO NPs alone is showed bactericidal activity, ZnO NPs effect was found to be concentration dependent, as highest concentration of ZnO NPs showed strongest antibacterial effect. In conclusion, more investigation is required to evaluate the acceptable concentration of Zno NPs and antibiotics selected to avoid toxicity and must be tested against more clinically isolated gram-negative bacterial strains.     

Analogues of the frog skin peptide alyteserin‐2a with enhanced antimicrobial activities against Gram‐negative bacteria

The emergence of strains of multidrug‐resistant Gram‐negative bacteria mandates a search for new types of antimicrobial agents. Alyteserin‐2a (ILGKLLSTAAGLLSNL.NH₂) is a cationic, α‐helical peptide, first isolated from skin secretions of the midwife toad, Alytes obstetricans, which displays relatively weak antimicrobial and haemolytic activities. Increasing the cationicity of alyteserin‐2a while maintaining amphipathicity by the substitution Gly¹¹→ Lys enhanced the potency against both Gram‐negative and Gram‐positive bacteria by between fourfold and 16‐fold but concomitantly increased cytotoxic activity against human erythrocytes by sixfold (mean concentration of peptide producing 50% cell death; LC₅₀ = 24 µm ). Antimicrobial potency was increased further by the additional substitution Ser⁷→Lys, but the resulting analogue remained cytotoxic to erythrocytes (LC₅₀ = 38 µm ). However, the peptide containing d ‐lysine at positions 7 and 11 showed high potency against a range of Gram‐negative bacteria, including multidrug‐resistant strains of Acinetobacter baumannii and Stenotrophomonas maltophilia (minimum inhibitory concentration = 8 µm ) but appreciably lower haemolytic activity (LC₅₀ = 185 µm ) and cytotoxicity against A549 human alveolar basal epithelial cells (LC₅₀ = 65 µm ). The analogue shows potential for treatment of nosocomial pulmonary infections caused by bacteria that have developed resistance to commonly used antibiotics. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis and biological evaluation of a new series of N-acyldiamines as potential antibacterial and antifungal agents

In continuation of our efforts to find new antimicrobial compounds, series of fatty N-acyldiamines were prepared from fatty methyl esters and 1,2-ethylenediamine, 1,3-propanediamine or 1,4-butanediamine. The synthesized compounds were screened for their antibacterial activity against Gram-positive bacteria (Staphylococcus aureus, Staphylococcus epidermidis), Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa) and for their antifungal activity against four species of Candida (C. albicans, C. tropicalis, C. glabrata and C. parapsilosis). Compounds 5a (N-(2-aminoethyl)dodecanamide), 5b (N-(2-aminoethyl)tetracanamide) and 6d (N-(3-aminopropyl)oleamide) were the most active against Gram-positive bacteria, with MIC values ranging from 1 to 16 μg/mL and were evaluated for their activity against 21 clinical isolates of methicillin-resistant S. aureus. All the compounds exhibited good to moderate antifungal activity. Compared to chloramphenicol, compound 6b displayed a similar activity (MIC₅₀ = 16 μg/mL). A positive correlation could be established between lipophilicity and biological activity.     

3-Aryl-4-acyloxyethoxyfuran-2(5H)-ones as inhibitors of tyrosyl-tRNA synthetase: Synthesis,molecular docking and antibacterial evaluation

Thirty-eight 3-aryl-4-acyloxyethoxyfuran-2(5H)-ones were designed, prepared and tested for antibacterial activities. Some of them showed significant antibacterial activity against Gram-positive organism, Gram-negative organism and fungus. Out of these compounds, 4-(2-(3-chlorophenylformyloxy)ethoxy)-3-(4-chlorophenyl)furan-2(5H)-one (d40) showed the widest spectrum of activity with MIC₅₀ of 2.0 μg/mL against Staphylococcus aureus, 4.3 μg/mL against Escherichia coli, 1.5 μg/mL against Pseudomonas aeruginosa and 1.2 μg/mL against Candida albicans. Our data disclosed that MIC₅₀ values against whole cell bacteria are positive correlation with MIC₅₀ values against tyrosyl-tRNA synthetase. Meanwhile, molecular docking of d40 into S. aureus tyrosyl-tRNA synthetase active site was also performed, and the inhibitor tightly fitting the active site might be an important reason why it has high antimicrobial activity.     

Novel 3-arylfuran-2(5H)-one-fluoroquinolone hybrid: Design,synthesis and evaluation as antibacterial agent

3-Arylfuran-2(5H)-one, a novel antibacterial pharmacophore targeting tyrosyl-tRNA synthetase (TyrRS), was hybridized with the clinically used fluoroquinolones to give a series of novel multi-target antimicrobial agents. Thus, twenty seven 3-arylfuran-2(5H)-one-fluoroquinolone hybrids were synthesized and evaluated for their antimicrobial activities. Some of the hybrids exhibited merits from both parents, displaying a broad spectrum of activity against resistant strains including both Gram-negative and Gram-positive bacteria. The most potent compound (11) in antibacterial assay shows MIC₅₀ of 0.11 μg/mL against Multiple drug resistant Escherichia coli, being about 51-fold more potent than ciprofloxacin. The enzyme assays reveal that 11 is a potent multi-target inhibitor with IC₅₀ of 1.15 ± 0.07 μM against DNA gyrase and 0.12 ± 0.04 μM against TyrRS, respectively. Its excellent inhibitory activities against isolated enzymes and intact cells strongly suggest that 11 deserves to further research as a novel antibiotic.     

Novel antimicrobial peptides from the venom of the eusocial bee Halictus sexcinctus (Hymenoptera: Halictidae) and their analogs

Two novel antimicrobial peptides, named halictines, were isolated from the venom of the eusocial bee Halictus sexcinctus. Their primary sequences were established by ESI-QTOF mass spectrometry, Edman degradation and enzymatic digestion as Gly-Met-Trp-Ser-Lys-Ile-Leu-Gly-His-Leu-Ile-Arg-NH₂ (HAL-1), and Gly-Lys-Trp-Met-Ser-Leu-Leu-Lys–His-Ile-Leu-Lys-NH₂ (HAL-2). Both peptides exhibited potent antimicrobial activity against Gram-positive and Gram-negative bacteria but also noticeable hemolytic activity. The CD spectra of HAL-1 and HAL-2 measured in the presence of trifluoroethanol or SDS showed ability to form an amphipathic α-helical secondary structure in an anisotropic environment such as bacterial cell membrane. NMR spectra of HAL-1 and HAL-2 measured in trifluoroethanol/water confirmed formation of helical conformation in both peptides with a slightly higher helical propensity in HAL-1. Altogether, we prepared 51 of HAL-1 and HAL-2 analogs to study the effect of such structural parameters as cationicity, hydrophobicity, α-helicity, amphipathicity, and truncation on antimicrobial and hemolytic activities. The potentially most promising analogs in both series are those with increased net positive charge, in which the suitable amino acid residues were replaced by Lys. This improvement basically relates to the increase of antimicrobial activity against pathogenic Pseudomonas aeruginosa and to the mitigation of hemolytic activity.     

OmpA Binding Mediates the Effect of Antimicrobial Peptide LL-37 on Acinetobacter baumannii

Multidrug-resistant Acinetobacter baumannii has recently emerged as an important pathogen in nosocomial infection; thus, effective antimicrobial regimens are urgently needed. Human antimicrobial peptides (AMPs) exhibit multiple functions and antimicrobial activities against bacteria and fungi and are proposed to be potential adjuvant therapeutic agents. This study examined the effect of the human cathelicidin-derived AMP LL-37 on A. baumannii and revealed the underlying mode of action. We found that LL-37 killed A. baumannii efficiently and reduced cell motility and adhesion. The bacteria-killing effect of LL-37 on A. baumannii was more efficient compared to other AMPs, including human ß–defensin 3 (hBD3) and histatin 5 (Hst5). Both flow cytometric analysis and immunofluorescence staining showed that LL-37 bound to A. baumannii cells. Moreover, far-western analysis demonstrated that LL-37 could bind to the A. baumannii OmpA (AbOmpA) protein. An ELISA assay indicated that biotin-labelled LL-37 (BA-LL37) bound to the AbOmpA_74-84 peptide in a dose-dependent manner. Using BA-LL37 as a probe, the ~38 kDa OmpA signal was detected in the wild type but the ompA deletion strain did not show the protein, thereby validating the interaction. Finally, we found that the ompA deletion mutant was more sensitive to LL-37 and decreased cell adhesion by 32% compared to the wild type. However, ompA deletion mutant showed a greatly reduced adhesion defect after LL-37 treatment compared to the wild strain. Taken together, this study provides evidence that LL-37 affects A. baumannii through OmpA binding.     

Antimicrobial activity and mechanism of action of a novel cationic α-helical dodecapeptide,a partial sequence of cyanate lyase from rice

CL(14-25), a dodecapeptide, that is a partial region near N-terminus of cyanate lyase (CL, EC 4.3.99.1) from rice (Oryza sativa L. japonica), contains three arginine and two lysine residues. It was a novel cationic α-helical antimicrobial peptide. The antimicrobial activity of CL(14-25) against Porphyromonas gingivalis, a periodontal pathogen, was quantitatively evaluated by a chemiluminescence method that measures ATP derived from viable cells. The 50% growth-inhibitory concentration of CL(14-25) against P. gingivalis cells was 145 μM. CL(14-25), even at a concentration of 800 μM, had no hemolytic activity. When giant unilamellar vesicles (GUVs) that mimic the membrane composition of Gram-negative bacteria were used, microscopy image analysis suggested that CL(14-25) disrupted GUVs in a detergent-like manner. Therefore, CL(14-25) appears to exhibit antimicrobial activity through membrane disruption. To investigate the contribution of cationic amino acid residues in CL(14-25) to its antimicrobial activity, we synthesized four truncated CL analogs, in which one or two cationic amino acid residues were deleted from the N- and C- termini of CL(14-25). The degrees of calcein leakage from large unilamellar vesicles (LUVs) and 3,3′-dipropylthiadicarbocyanine iodide (diSC₃-5) release from P. gingivalis cells induced by truncated CL analogs were closely related to their antimicrobial activities. CL analogs, which were truncated by removing an arginine residue from the N-terminus and a lysine residue from the C-terminus maintained their antimicrobial activity. However, CL analogs, which were further truncated by removing two arginine residues from the N-terminus, and an arginine and a lysine residue from the C-terminus, rarely exhibited antimicrobial activity.