Patent Application
20220064219
The present disclosure pertains to peptides with antimicrobial and antibiofilm properties.
The emergence of resistant bacteria coupled to the decline in antibiotic innovation has led to an urgent need for alternatives to standard-of-care antibiotics, which often have limited efficacy1,2. Chronic and recalcitrant infections are on the rise, such as those caused by highly resistant Gram-negative bacteria. Carbapenem-resistant Enterobacteriaceae (CRE), in particular, may also be resistant to acylureidopenicillins, third generation cephalosporins, and fluoroquinolones3. In addition to various antibiotic resistance mechanisms, CRE also deploy virulence factors, including the formation of biofilms4.
Bacterial biofilms15 are responsible for ˜80% of all nosocomial infections16. The communities of pathogenic microbes that form biofilms are associated with indwelling medical devices, including catheters, stents, and prosthetic implants. Bacterial biofilms represent a physiologically distinct growth state, with hundreds of genes changing expression compared to the expression profiles observed in bacteria grown under planktonic conditions17. These multicellular structures constitute an extracellular matrix, composed by extracellular polymeric substances (EPS) that protect the bacteria within the biofilm from exogenous agents such as antibiotics and constituents of the host immune system. EPS, a heterogeneous combination of polysaccharides, extracellular DNA (eDNA), proteins, and lipids held together by adhesins, forms an intricate network that immobilizes the microbes to the surface they colonize. Bacteria growing in biofilms cause chronic infections, which are extremely refractory towards even high concentrations of last resort antibiotics. These infections are, therefore, associated with high treatment costs and with high mortality and morbidity18. Unfortunately, no anti-biofilm drug has yet been approved for clinical use17,19-21 despite the importance of such drugs in combating biofilm-associated infections.
Several peptides have been described to date that are effective against biofilms18,22-24 and that can synergize with conventional antibiotics and antifungal agents against drug-resistant organisms such as the ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species)22-26. Yet, although these peptides have shown great promise as novel anti-biofilm agents for treating recalcitrant infections, significant hurdles have delayed their translation to the clinic.
Gram-negative bacteria such as CRE are among the most common pathogens found in biofilm-related infections. In fact, it is well established that carbapenamase-producing K. pneumoniae (KpC-Kpn) and multidrug resistant isolates of bacteria carrying the beta-lactamase encoding genes blaPER-1 and blaVIM-2 have a high propensity to form robust biofilms on medical implants, such as urinary catheters27,28. Furthermore, Gram-negative bacteria may escape killing because of the presence of lipopolysaccharide (LPS), which acts as a permeability barrier around the cell. Therefore, efforts are being made to design synthetic peptides that have enhanced activity against these hardy, highly resistant organisms.
Antimicrobial peptides (AMPs) potentially represent alternative strategies to combat the global health problem of antibiotic resistance. However, naturally occurring AMPs are generally not sufficiently active for use as antibiotics, are cytotoxic, or both. Accordingly, there exists an urgent need for strategies for identifying biocompatible AMPs or for developing modified AMPs that do not possess the aforementioned drawbacks that render them unsuitable for antimicrobial use among human and animal subjects.
Provided herein are antimicrobial peptides that comprises a peptide portion and an amino-terminal Cu(II) and Ni(II) binding motif that is conjugated to the N-terminus of the peptide portion, wherein the amino-terminal Cu(II) and Ni(II) binding motif is any one of SEQ ID NOS:31-59. The present disclosure also provides compositions comprising an antimicrobial peptide, and optionally an acceptable carrier.
Also provided are methods of treating a microbial infection in a subject comprising administering to the subject a therapeutically effective amount of an antimicrobial peptide according to the present disclosure.
Also disclosed are methods comprising contacting a biofilm with an effective amount of an antimicrobial peptide according to the present disclosure.
The present disclosure also pertains to a method of forming an antimicrobial peptide comprising conjugating an amino-terminal Cu(II) and Ni(II) binding motif comprising any one of SEQ ID NOS:31-59 to a peptide comprising any one of SEQ ID NOS:1-30 at the N-terminus of the peptide.
The file of this patent or application contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.
The presently disclosed inventive subject matter may be understood more readily by reference to the following detailed description taken in connection with the accompanying examples, which form a part of this disclosure. It is to be understood that these inventions are not limited to the specific formulations, methods, articles, or parameters described and/or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed inventions.
The entire disclosures of each patent, patent application, and publication cited or described in this document are hereby incorporated herein by reference.
As employed above and throughout the disclosure, the following terms and abbreviations, unless otherwise indicated, shall be understood to have the following meanings.
In the present disclosure the singular forms “a,” “an,” and “the” include the plural reference, and reference to a particular numerical value includes at least that particular value, unless the context clearly indicates otherwise. Thus, for example, a reference to “an antibiotic” is a reference to one or more of such compounds and equivalents thereof known to those skilled in the art, and so forth. Furthermore, when indicating that a certain element “may be” X, Y, or Z, it is not intended by such usage to exclude in all instances other choices for the element.
When values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. As used herein, “about X” (where X is a numerical value) preferably refers to ±10% of the recited value, inclusive. For example, the phrase “about 8” can refer to a value of 7.2 to 8.8, inclusive. This value may include “exactly 8”. Where present, all ranges are inclusive and combinable. For example, when a range of “1 to 5” is recited, the recited range should be construed as optionally including ranges “1 to 4”, “1 to 3”, “1-2”, “1-2 & 4-5”, “1-3 & 5”, and the like. In addition, when a list of alternatives is positively provided, such a listing can also include embodiments where any of the alternatives may be excluded. For example, when a range of “1 to 5” is described, such a description can support situations whereby any of 1, 2, 3, 4, or 5 are excluded; thus, a recitation of “1 to 5” may support “1 and 3-5, but not 2”, or simply “wherein 2 is not included.”
In the present disclosure, superscripted numerals refer to the correspondingly numbered references that appear infra under the heading “References”.
As noted above, naturally occurring AMPs are generally not sufficiently active for use as antibiotics. Previous studies have described the concept of using catalytic metallodrugs as an effective strategy for improving the bioactivity of AMPs23,29-31. The amino terminal Cu(II) and Ni(II) (ATCUN) binding motif chelates metal ions and elicits the release of reactive oxygen species (ROS). The insertion of this motif at the N-terminal extremity of AMPs creates a molecular scaffold that catalyses the formation of ROS, which is absent in the original AMP32-37. The presence of the ATCUN motif in many naturally-occurring AMPs isolated from all branches of the phylogenetic tree indicates their potential as templates for the design of antimicrobial compounds (http://angeles-boza.chemistry.uconn.edu/atcun-amps/). The ATCUN motif shows high affinity for Cu(II) or Ni(II) ions (log K˜14-15), forming with these ions a stable square pyramidal coordination complex38. The high affinity of ATCUN motifs for labile copper ions in bacteria that flow in response to environmental stimuli39 suggests that the insertion of these motifs within an AMP or an anti-biofilm peptide (ABP) sequence would be likely to generate ROS in the surroundings of the bacterial cell membranes. The ATCUN-Cu(II) complex, in the presence of hydrogen peroxide and ascorbic acid, generates ROS via a Fenton-like reaction as the bound copper cycles between its +2 and +3 oxidation states40. This leads to ROS build-up at a high turnover rate, which can irreversibly degrade therapeutic targets such as nucleic acids and proteins, even when the AMP is present at sub-therapeutic doses32,41. The ATCUN-AMP molecular scaffold has been exploited to degrade extracellular DNA (eDNA), one of the main components of EPS42. Degrading eDNA, which is considered a major target of antibiofilm agents43, may cause biofilms to disintegrate.
The present inventors have engineered amino-terminal Cu(II) and Ni(II) (ATCUN) binding motifs, which can enhance biological function, into the native sequence of AMPs, including, for example, CM15 and citropin1.1. The incorporation of metal-binding motifs modulated the antimicrobial activity of synthetic peptides against a panel of carbapenem-resistant enterococci (CRE) bacteria, including carbapenem-resistant Klebsiella pneumoniae (KpC+) and Escherichia coli (KpC+). Activity modulation depended on the type of ATCUN variant utilized. Membrane permeability assays revealed that the inventive peptides increased bacterial cell death. Mass spectrometry, circular dichroism and molecular dynamics simulations indicated that coordinating ATCUN derivatives with Cu(II) ions did not increase the helical tendencies of the AMPs. In addition, when combined with meropenem, streptomycin, or chloramphenicol, the present antimicrobial peptides showed synergistic effects against biofilms, such as those comprising E. coli (KpC+1812446). Motif addition also reduced the hemolytic activity of the wild-type AMP and improved the survival rate of mice in a systemic infection model. The dependence of these bioactivities on the particular amino acids of the ATCUN motif highlights the use of size, net charge, and hydrophobicity to fine-tune AMP biological function. The obtained results demonstrated that incorporating metal-binding motifs into peptide sequences leads to synthetic variants with modified biological properties.
Provided herein are antimicrobial peptides that comprises a peptide portion and an amino-terminal Cu(II) and Ni(II) binding motif that is conjugated to the N-terminus of the peptide portion, wherein the amino-terminal Cu(II) and Ni(II) binding motif is any one of SEQ ID NOS:31-59.
The peptide portion of the antimicrobial peptides may include a protein is a member of the general class of antimicrobial peptides (AMPs). Such proteins may be provided in wild-type form, or may include one or more desirable mutations. In some embodiments, the peptide portion is a cationic α-helical peptide. Peptide portions having a sequence of about 10-20 amino acids may be used. Such sequences have been found to be more readily synthesized, and are typically more potent and less toxic than larger sequences, although in instances where such limitations are not present, peptide portions that have an amino acid sequence of greater than about 20 amino acids may also be used. For example, the peptide portion may have a sequence that includes about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39. 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more amino acids. In certain embodiments, the peptide portion is any one of SEQ ID NOS:1-30:
*—NH2 indicates amidated C-terminal extremity.
For example, the peptide portion may comprise SEQ ID NO:11. In other embodiments, the peptide portion comprises SEQ ID NO:14.
The peptide portion may represent a sequence having a change to single amino acid of any one of SEQ ID NOS: 1-30, i.e., such that one amino acid from any one of SEQ ID NOS: 1-30 is replaced with a different amino acid. Other embodiments of the peptide portion may have a sequence that differs by more than one amino acid, e.g., by two, three, or four amino acids, from SEQ ID NOS: 1-30, respectively. Any such changes to the respective sequences of SEQ ID NOS: 1-30 should preferably not alter the α-helical structure of the peptide.
The amino-terminal Cu(II) and Ni(II) (“ATCUN”) binding motif may comprise any one of SEQ ID NOS: 31-59:
The ATCUN binding motif is conjugated to the N-terminus of the peptide portion, i.e., the N-terminus of the stand-alone peptide portion, before the ATCUN motif is conjugated to it. In certain embodiments, the ATCUN binding motif comprises SEQ ID NO:31. In other embodiments, the ATCUN binding motif comprises SEQ ID NO:32.
The present disclosure also pertains to methods of forming an antimicrobial peptide comprising conjugating an amino-terminal Cu(II) and Ni(II) binding motif comprising any one of SEQ ID NOS:31-59 to a peptide comprising any one of SEQ ID NOS:1-30 at the N-terminus of the peptide. The conjugation of the ATCUN motif to the peptide may be in accordance with procedures as disclosed herein in the illustrative examples, or using any other art-acceptable procedure, with which those of ordinary skill in the art will be familiar.
The present disclosure also provides compositions for treating a microbial infection comprising a therapeutically effective amount of an antimicrobial peptide according to any one of the embodiments described above. Also provided herein are methods of treating a microbial infection in a subject comprising administering to the subject a therapeutically effective amount of an antimicrobial peptide according to the present disclosure. As described above, the present inventors have discovered that the antimicrobial peptides disclosed herein possess enhanced antimicrobial characteristics relative to wild-type AMPs, with better biocompatibility, and therefore represent alternatives both to traditional antibiotic compounds to which microbial resistance has arisen or is likely to arise, and to naturally occurring AMPs that possess unacceptably high levels of toxicity to mammalian cells.
As used herein, the phrase “therapeutically effective amount” refers to the amount of active agent (here, the antimicrobial peptide) that elicits the biological or medicinal response that is being sought in a tissue, system, animal, individual or human by a researcher, veterinarian, medical doctor or other clinician, which includes one or more of the following:
(1) at least partially preventing the disease or condition or a symptom thereof; for example, preventing a disease, condition or disorder in an individual who may be predisposed to the disease, condition or disorder but does not yet experience or display the pathology or symptomatology of the disease;
(2) inhibiting the disease or condition; for example, inhibiting a disease, condition or disorder in an individual who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., including arresting further development of the pathology and/or symptomatology); and
(3) at least partially ameliorating the disease or condition; for example, ameliorating a disease, condition or disorder in an individual who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., including reversing the pathology and/or symptomatology).
The antimicrobial peptides according to the present disclosure may be provided in a composition that is formulated for any type of administration. For example, the compositions may be formulated for administration orally, topically, parenterally, enterally, or by inhalation (e.g., intranasally). The active agent may be formulated for neat administration, or in combination with conventional pharmaceutical carriers, diluents, or excipients, which may be liquid or solid. The applicable solid carrier, diluent, or excipient may function as, among other things, a binder, disintegrant, filler, lubricant, glidant, compression aid, processing aid, color, sweetener, preservative, suspensing/dispersing agent, tablet-disintegrating agent, encapsulating material, film former or coating, flavoring agent, or printing ink. Any material used in preparing any dosage unit form is preferably pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active agent may be incorporated into sustained-release preparations and formulations. Administration in this respect includes administration by, inter alia, the following routes: intravenous, intramuscular, subcutaneous, intraocular, intrasynovial, transepithelial including transdermal, ophthalmic, sublingual and buccal; topically including ophthalmic, dermal, ocular, rectal and nasal inhalation via insufflation, aerosol, and rectal systemic.
In powders, the carrier, diluent, or excipient may be a finely divided solid that is in admixture with the finely divided active ingredient. In tablets, the active ingredient is mixed with a carrier, diluent or excipient having the necessary compression properties in suitable proportions and compacted in the shape and size desired. For oral therapeutic administration, the active compound may be incorporated with the carrier, diluent, or excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. The amount of active agent(s) in such therapeutically useful compositions is preferably such that a suitable dosage will be obtained.
Liquid carriers, diluents, or excipients may be used in preparing solutions, suspensions, emulsions, syrups, elixirs, and the like. The active ingredient of this invention can be dissolved or suspended in a pharmaceutically acceptable liquid such as water, an organic solvent, a mixture of both, or pharmaceutically acceptable oils or fat. The liquid carrier, excipient, or diluent can contain other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, colors, viscosity regulators, stabilizers, or osmo-regulators.
Suitable solid carriers, diluents, and excipients may include, for example, calcium phosphate, silicon dioxide, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, methyl cellulose, ethylcellulose, sodium carboxymethyl cellulose, microcrystalline cellulose, polyvinylpyrrolidine, low melting waxes, ion exchange resins, croscarmellose carbon, acacia, pregelatinized starch, crospovidone, HPMC, povidone, titanium dioxide, polycrystalline cellulose, aluminum methahydroxide, agar-agar, tragacanth, or mixtures thereof.
Suitable examples of liquid carriers, diluents and excipients, for example, for oral, topical, or parenteral administration, include water (particularly containing additives as above, e.g. cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g. glycols) and their derivatives, and oils (e.g. fractionated coconut oil and arachis oil), or mixtures thereof.
For parenteral administration, the carrier, diluent, or excipient can also be an oily ester such as ethyl oleate and isopropyl myristate. Also contemplated are sterile liquid carriers, diluents, or excipients, which are used in sterile liquid form compositions for parenteral administration. Solutions of the active agents can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. A dispersion can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.
The pharmaceutical forms suitable for injectable use include, for example, sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form is preferably sterile and fluid to provide easy syringability. It is preferably stable under the conditions of manufacture and storage and is preferably preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier, diluent, or excipient may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of a dispersion, and by the use of surfactants. The prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In some instances, the antimicrobial peptides themselves may be sufficient to prevent contamination by microorganisms. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions may be achieved by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions may be prepared by incorporating the active agent in the pharmaceutically appropriate amounts, in the appropriate solvent, with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions may be prepared by incorporating the sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation may include vacuum drying and freeze drying techniques that yield a powder of the active ingredient or ingredients, plus any additional desired ingredient from the previously sterile-filtered solution thereof.
Thus, an antimicrobial peptide may be in the present compositions and methods in an effective amount by any of the conventional techniques well-established in the medical field. For example, the administration may be in the amount of about 0.1 mg/day to about 500 mg per day. In some embodiments, the administration may be in the amount of about 250 mg/kg/day. Thus, administration may be in the amount of about 0.1 mg/day, about 0.5 mg/day, about 1.0 mg/day, about 5 mg/day, about 10 mg/day, about 20 mg/day, about 50 mg/day, about 100 mg/day, about 200 mg/day, about 250 mg/day, about 300 mg/day, or about 500 mg/day.
Also disclosed are methods comprising contacting a biofilm with an effective amount of an antimicrobial peptide according to the present disclosure. Such methods may be effective to remove or reduce the presence of an unwanted biofilm, such as in hospitals or other medical settings, in sewer and filtration systems, in industrial settings, on equipment involved in food preparation or manufacture, in aquaculture or hydroponics, or in any other context that is prone to unwanted biofilm formation.
In accordance with the methods of treating a microbial infection in a subject or the methods comprising contacting a biofilm according to the present disclosure, microbes against which the present antimicrobial peptides are effective may be, for example, any unicellular organism, such as gram-negative bacteria, gram-positive bacteria, protozoa, viruses, bacteriophages, and archaea. The present peptides can have an antimicrobial effect with respect to any such microbe. Examples of bacteria against which the present compounds are effective to cause reduction in numbers include gram positive bacteria and gram negative bacteria, for example, Salmonella enterica, Listeria monocytogenes, Escherichia coli, Clostridium botulinum, Clostridium difficile, Campylobacter, Bacillus cereus, Vibrio parahaemolyticus, Vibrio cholerae, Vibrio vulnificus, Staphylococcus aureus, Yersinia enterocolitica, Shigella, Moraxella spp., Helicobacter, Stenotrophomonas, Bdellovibrio, Legionella spp. (e.g., pneumophila), Neisseria gonorrhoeae, Neisseria meningitidis, Haemophilus influenzae, Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis, Enterobacter cloacae, Serratia marcescens, Helicobacter pylori, Salmonella enteritidis, Salmonella typhi, and combinations thereof. Examples of Salmonella enterica serovars that can be reduced using the compounds of the disclosure include, for example, Salmonella enteriditis, Salmonella typhimurium, Salmonella poona, Salmonella heidelberg, and Salmonella anatum. Exemplary viruses against which the present peptides are effective to cause reduction in numbers include coronaviruses, rhinoviruses, and influenza viruses.
Hereinafter, the present disclosure will be described in more detail through Examples, which are intended to be illustrative to the present disclosure, although present disclosure is not limited to the Examples.
In silico selection of AMPs. Fourteen cationic α-helical AMPs 10-20 amino acid residues long (corresponding to SEQ ID NOS:1-14) with broad-spectrum antimicrobial activity were selected by using the search tool from the Antimicrobial Peptide Database (APD) (http://aps.unmc.edu/AP/database/antiA.php).
The N-terminus of each AMP was modified by adding the tripeptide motifs Gly-Gly-His (GGH) or Val-Ile-His (VIH), also known as the ATCUN motifs. A total of 28 ATCUN-variants was generated and subjected to a 3-step selection process. Each variant was submitted to two prediction modules, available at http://www.camp.bicnirrh.res.in/predict/44 and http://www.biomedicine.org.ge/dbaasp/(DBAASP—Database of Antimicrobial Activity and Structure of Peptides), to determine whether antimicrobial activity would be retained. Peptides were ranked based on their likelihood of having antimicrobial activity, determined by whether the predictor returned a probability of <0.9 or “non-AMP”45, as shown in Table 1, below:
Moreover, molecular models of ATCUN-variants were initially built on the I-TASSER server46 using a hierarchical approach for peptide structure prediction (as reliable template structures could not be determined for the comparative modeling of all peptides) to identify peptides whose α-helical structures were likely to be disrupted upon the insertion of an ATCUN motif. Those not having this structure were ranked last (Table 1). Finally, the HeliQuest server (http://heliquest.ipmc.cnrs.fr/)47 was used to generate helical wheel projections for all variants to study the properties of the hydrophobic and hydrophilic portions such as the polar angle and mean hydrophobic moment (
CM15 and citropin1.1 were modified by using two well-known ROS-generating ATCUN motifs: Gly-Gly-His (GGH) and Val-Ile-His (VIH). These motifs, which had been reported by Libardo et al.64, do not occur naturally but were rationally designed according to the general NH2-XXH ATCUN motif sequence, where X is any canonical amino acid except proline, and H is histidine, which must always be in the third position of the N-terminal extremity. ROS generation confers cytotoxic activity to ATCUN motifs in the presence of Cu(II) ions via Fenton-like reactions65. CM15, citropin1.1, and their ATCUN variants were synthesised by solid-phase peptide synthesis and a fluorenylmethyloxycarbonyl strategy by AminoTech (São Paulo, Brazil). Model molecules and the motifs GGH and VIH were used as controls.
Mass spectrometry (MS) analysis of copper coordination. To determine whether the ATCUN-variants of CM15 coordinated to Cu(II), peptides were analyzed using electrospray ionization mass spectrometry on a 4,000 Q-Trap mass spectrometer at room temperature and with a cone voltage of 5 kV. The peptides were diluted in a solution containing 47.5% H2O, 47.5% acetonitrile, 5% dimethylsulfoxide, and 0.1% formic acid to a concentration of 50 μM. The Cu(II)-containing samples were incubated with 0.9× CuCl2 for 30 min prior to injection in the mass spectrometer.
Thus, as initial selection criteria, the length, cationicity, helicity, and spectrum of activity were used to narrow down a list of potential AMPs. CM15 and citropin1.1, two known pore-forming AMPs62,63, were selected for further studies based on 1) prediction algorithms considering the effect of the insertion of ATCUN motifs on the helical tendency, 2) analyses of the helical wheel projections for ATCUN-AMP variants, and 3) molecular modelling of the three-dimensional structures (
In vitro growth inhibition assays. The following strains were used in antimicrobial susceptibility assays: E. coli (ATCC 25922), E. coli MG1655, E. coli (KpC+ 1812446), E. coli (KpC+2101123), K. pneumoniae (KpC+1825971), K. pneumoniae (ATCC 13883), P. aeruginosa (ATCC 27853), methicillin-resistant S. aureus (3730529), and S. aureus (ATCC 25923). Antimicrobial susceptibility tests were performed using the broth microdilution method38. Minimum inhibitory concentration (MIC) was defined as the minimal 100% inhibitory concentration of peptide after 12 h of incubation at 37° C. To study the influence of Cu(II) ions on the antimicrobial activity of ATCUN-peptides, Mueller Hinton (MH) broth was supplemented with CuSO4.5H2O to achieve a final concentration of 0.25 mM of Cu(II) ions, below the toxic level (7.8 mM) against E. coli (ATCC 25922) and S. aureus (ATCC 25923) determined in our laboratory. As controls, the cell-impermeable Cu(II) chelator triethylenetetramine (TETA) and the cell-permeable Cu(II) chelator tetrathiolmolybdate (TTM) were used to determine the importance of Cu(II) in antimicrobial activity. The chelators at a concentration of 200 μM were incubated with E. coli MG1655 for 10 min prior to exposure to the peptide solutions at room temperature. All bacterial cell cultures used for these experiments were in exponential growth phase.
Table 2 summarizes the activity of peptides CM15 and citropin1.1 with and without the incorporation of the ATCUN motif and with and without exposure to Cu(II) ions.
E. coli
E. coli
E. coli
K. pneumoniae
K. pneumoniae
P. aeruginosa
S. aureus
E. coli
E. coli
K. pneumoniae
S. aureus
MIC is defined as the lowest concentration required to inhibit visible growth of bacteria, confirmed by optical density at 600 nm (OD600). Data correspond to the mean of three independent experiments.
These data indicate that, for some bacterial strains, the antimicrobial activity of CM15 was modulated by incorporation of the ATCUN motif: the potency of the GGH and VIH variants of CM15 against carbapenem-resistant K. pneumoniae (KpC+ 1825971), for example, was 4-fold and 8-fold higher, respectively, than the potency of the original peptide. The antibacterial activity of CM15 against the carbapenem-resistant strains E. coli (KpC +1812446), E. coli (KpC +2101123), E. coli (ATCC 25922), and P. aeruginosa (ATCC27853) was marginally improved by the presence of the ATCUN motif (Tables 3 and 4).
K.
K.
E. coli
E. coli
pneumoniae
pneumoniae
K.
K.
E. coli
E. coli
pneumoniae
pneumoniae
This small improvement is nevertheless significant considering that pulmonary infections caused by carbapenem-resistant K. pneumoniae (KpC+ 1825971) have a mortality rate on the order of 40%67. However, ATCUN modification lowered the antimicrobial activity of citropin1.1, resulting in as much as a 3-fold decrease in antimicrobial activity against the Gram-negative and Gram-positive bacteria tested (Table 2). As ATCUN motifs require Cu(II) ions for their catalytic activity, we supplemented the growth medium with 0.25 μM Cu(II) solution. Antimicrobial activities obtained after the addition of Cu(II) ions were not changed when compared to those assayed in MH broth only (Table 2). The lack of enhanced activity upon the addition of Cu(II) underscores the ability of ATCUN-AMPs to scavenge labile Cu(II) ions from the media or the bacteria themselves64.
ATCUN-CM15 peptides enhance their antimicrobial activity in the presence of copper ions. MIC values were also obtained in the presence of Cu(II) chelators to determine the importance of the concentrations of trace Cu(II) ions found in the growth medium used to evaluate the antimicrobial activity of the peptide CM15 and its ATCUN variants. Table 5 shows that the activity of the ATCUN peptides decreased 2-fold in the presence of the cell-impermeable Cu(II) chelator triethylenetetramine (TETA), indicating that GGH-CM15 and VIH-CM15 require Cu(II) for their observed activity; whereas the activity of the wild-type peptide, which remained unchanged, is independent of the presence of Cu(II) ions. In contrast, addition of the cell-permeable Cu(II) chelator tetrathiolmolybdate (TTM) led to smaller MIC values for all three peptides, likely the result of ionic interactions between the negatively charged chelator and the cationic peptides, decreasing the overall availability of the peptides. However, the GGH-CM15 and VIH-CM15 variants were more strongly affected than CM15, showing that the competition for Cu(II) ion chelation by TTM influences the antimicrobial activity of the ATCUN motif-containing peptides.
Furthermore, mass and CD spectroscopy data coupled to MD simulations confirmed that the addition of the ATCUN motifs to the original peptide sequence as well as the formation of the Cu(II) ion-ATCUN motifs complex did not interfere with the helical content of the AMPs compared to the template peptide CM15. Changes in the helical content were calculated using CD spectrometry assays, which revealed small variations on helical tendency compared to the wild-type peptide CM15 (>5%).
Cell permeability assays. Confocal fluorescence microscopy was used to observe the permeabilizing effect of CM15 and its ATCUN-derivatives on E. coli membranes. Briefly, E. coli MG1655 was treated with the peptides at their MICs for 1 h at 37° C. in a shaking incubator. The cell membranes were stained for 30 min with FM4-64 and SYTOX green, a cell-impermeable dye. The bacteria were then spotted onto agarose pads on glass microscope slides and covered with a glass coverslip. Cells were imaged using a Nikon A1R spectral confocal microscope with a 60× oil immersion lens in order to observe possible morphological damage in E. coli MG1655 treated with the peptides. Images were analysed using ImageJ 1.8.0.
As noted, the cells were first treated for one hour with one of the three peptides (CM15 and the GGH-CM15 and VIH-CM15 variants), followed by staining with FM4-64 to label the bacterial membranes, and SYTOX Green, a cell-impermeable dye.
Inhibition of biofilm formation by the peptides was assessed against susceptible and carbapenem-resistant E. coli and K. pneumoniae in BM2 minimal medium [62 mM potassium phosphate buffer, pH 7.0, 7 mM (NH4)2SO4, 2 mM MgSO4, 10 mM FeSO4, 0.5% glucose] using methods described by Wiegand et al.49. Planktonic cell growth was determined by measuring absorbance at 600 nm at the end of the incubation period in the presence of peptide that was added at the beginning of the experiment, and biofilm formation was assessed using the crystal violet (CV) assay for biofilm biomass quantification. CV binds to negatively charged bacteria and to polysaccharides of the EPS. The amount of CV adsorbed is directly proportional to the biofilm biomass50. Biofilms were stained with 0.1% CV solution (100 μL per well) for 20 min at room temperature. The plates were washed three times by flooding with double distilled water and thoroughly dried by tapping onto paper towels several times followed by air drying. The bound CV was solubilized with 95% ethanol (110 μL per well) for 10 min and the absorbance of extracted CV was measured at 595 nm51.
A sought-after feature of AMPs and ABPs is their potential ability to enhance the antimicrobial and antibiofilm activity of conventional antibiotics, which for some peptides has been demonstrated previously. AMPs might enhance the sensitivity of resistant bacteria to the conventional antibiotic or decrease cross-resistance to both AMPs and antibiotics68. The peptides CM15 and its GGH- or VIH-variants were tested to determine if they changed the activity of several antibiotics: meropenem, ampicillin, trimethropim, streptomycin, and chloramphenicol. Results of the checkerboard assays for the inhibition of biofilm formation and the growth of planktonic cells for each combination are presented in Table 6. Results for each peptide and antibiotic combination are provided in Table 3, and Tables 7 and 8.
495
0.5
132
495
0.33
4
0.27
32
0.185
16
The data indicated that the ATCUN motifs had influence on the antibiofilm activity of antibiotics used in the checkerboard assays in a manner highly dependent on the type of ATCUN motif (GGH or VIH), the growth phase of the bacteria (whether in biofilm or planktonic phase), and the mechanism of action of the antibiotic. Biofilm formation by carbapenem-resistant E. coli (KpC+ 1812446) was completely prevented with a combination of meropenem and either the parental peptide CM15, GGH-CM15, or VIH-CM15 (Table 7). The same combinations, however, were ineffective at inhibiting carbapenem-resistant E. coli (KpC+ 1812446) planktonic cells (Table 8). Complete inhibition of the proliferation of carbapenem-resistant E. coli (KpC+ 1812446) in the planktonic phase was observed only when the peptides were combined with chloramphenicol or streptomycin (Table 3). This variance in effect among several conventional antibiotics in combination with the same peptide highlights possible differences in the mechanisms that inhibit biofilm formation or prevent the growth of planktonic cells. There may also be differences in defense mechanisms deployed by E. coli (KpC+ 1812446). Planktonic cell growth was inhibited by streptomycin only in combination with VIH-CM15, clearly pointing to the importance of the ATCUN motif to the outcome of combinations. The synergies with FICI of 0.33, 0.27 and 0.185, for the combination of meropenem with CM15, GGH-CM15, and VIH-CM15, respectively (Table 3), translate into a 32-fold (meropenem and GGH-CM15) and 16-fold (meropenem and VIH-CM15) decrease in the concentration of meropenem required to completely inhibit E. coli (KpC+ 1812446) biofilm formation; compared to a 4-fold reduction for meropenen and CM15 wild-type (Table 6).
The drastic reduction in MICs and MBICs have far reaching implications for the treatment of infections caused by carbapenem-resistant E. coli (KpC+ 1812446) and are clearly dependent on the type of ATCUN motif conjugated to CM15 (Tables 3 and 4). It is also interesting to note that, although some combinations were not synergistic, the presence of the ATCUN motifs nevertheless enhanced the activity of the antibiotic, resulting in a marginal reduction in the concentration required to prevent biofilm formation. This was the case when CM15 or its variants was combined with streptomycin or trimethoprim, which reduced the concentration of antibiotic needed to inhibit biofilm formation by 4-fold (FICI>0.5) (Table 6). Similarly, the combination of trimethoprim with CM15, GGH-CM15, or VIH-CM15, inhibited the growth of planktonic cells by 8-fold, 4-fold, and 2-fold, respectively, compared to the activity of the antibiotic alone (Table 6). The wild-type CM15 and VIH motif appear to enhance antibiotic action most in the biofilm phase, whereas the influence of the GGH motif in antibiotic synergy is more pronounced in the planktonic phase (Table 6).
Hemolytic assays were conducted to assess the suitability of peptides for in vivo trials and selectivity towards bacterial membranes. Hemolysis was measured for red blood cells (RBCs) from mice (approved by the Ethics Committee of Universidade Católica Dom Bosco number 019/2016). Fresh blood was collected into EDTA-coated vacutainers and washed three times with sterile PBS. A small aliquot of washed cells was resuspended in fresh PBS to make a 0.8% (v/v) solution of RBCs. Then a 75 μL aliquot of RBCs was mixed with a 75 mL aliquot of a two-fold serial dilution series of the peptides, and the mixture was incubated at 37° C. for 1 h in polypropylene PCR tubes. Triton X-100 and PBS were used as positive and negative controls, respectively. After incubation, the tubes were spun down at 4,400 rpm at 4° C. for 10 min, and 100 mL of the supernatant was transferred into a clear 96-well plate. The absorbance at 414 nm was measured and normalized against the absorbance of the positive and negative controls. Data were obtained from four independent trials and presented as the mean±standard deviation.
The hemolytic activity of the ATCUN-variants was reduced as compared with that of the parent peptides at the MIC value for E. coli KpC+ (Table 9, below). This reduced hemolysis suggests that AMPs that have incorporated an ATCUN motif may be less harmful to mammalian cells than AMPs lacking the motif
Male Balb/C (18-20 g) mice from the Biotério Central do Campus da USP in Ribeirão Preto, São Paulo, were kept in groups of 5 per cage at 22° C. with normal cycles of light and free access to food and water. The care and use of the animals were approved by the Ethics Committee of Universidade Católica Dom Bosco number 019/2016. We assessed the ability of the peptides to protect the mice from lethal systemic infections induced with E. coli KpC+ 1812446. The mice (n=40) were randomly placed into eight groups of five mice each, and each mouse was injected intraperitoneally with 200 μL of saline solution containing 2×107 CFU of E. coli KpC+ 1812446. Intraperitoneal treatment with 5 or 10 mg·Kg−1 of each peptide was initiated after one hour of infection and repeated at 24 h intervals for seven days61. Gentamicin at 10 mg·Kg−1 of body weight and normal saline were used as positive and negative controls, respectively.
Survival at 24 h intervals for one week is presented in
Additional information concerning the disclosed subject matter can also be found in Agbale C M, et al., Biochemistry. 2019 Sep. 10; 58(36):3802-3812, the entire contents of which are incorporated herein by reference.
The following publications may also be relevant to the present disclosure:
This application claims the benefit of priority to U.S. Provisional Application No. 63/070,644, filed Aug. 26, 2020, the entire contents of which are incorporated by reference herein.
| Number | Date | Country | |
|---|---|---|---|
| 63070644 | Aug 2020 | US |
