Since all bile acid-based cationic polymers contain the same hydrophobic four fused rings in each repeating unit, the change in hydrophilicity is critical for the antimicrobial activities. The cholic acid-based polymer contains three QAC groups in each repeating unit, making it more hydrophilic with higher charge densities, whereas deoxycholic acid and lithocholic acid are less hydrophilic because of fewer charged groups.
Our results demonstrated that the higher charge densities of a polymer could lead to more significant interactions with bacterial membranes, similar to observations made by Yang and colleagues 46 , Though all polymers can inhibit bacterial growth, they again exhibited enhanced potency towards Gram-negative bacteria. This is significant as there are few antibiotics available for the treatment of infections by Gram-negative bacteria, in particular, pathogenic P.
We further studied the effect of methylene spacers in cholic acid-based polymers on antimicrobial activity Fig. We observed that polymers containing a longer spacer showed more potent killing efficacy compared to those with shorter spacers. According to the snorkeling effect in peptides 48 , 49 , 50 , a longer spacer unit could provide increased hydrophobicity, and the additional distance between the QAC groups and the hydrophobic multicyclic ring attached to the polymer backbone would facilitate a deeper insertion of the polymer chain into the bacterial membrane.
In contrast, a shorter spacer has less flexibility and room for extending the charge group through the membrane A longer spacer could not only facilitate the charge group easier to reach a target substrate here cell membrane , but provide a flexible anchoring on surfaces without requiring a configurational change of the bulky triterpene structure. Cholic acid-based cationic polymers with different spacers. Chemical structures of polymers and their illustration. In case of P. For E.
In case of Gram-positive bacteria, S. This could be explained by the potential trapping of higher molecular weight polymers in the dense, outmost peptidoglycan layer of S. This observation is consistent with the sieving effect, as also identified by Lienkamp et al This might be due to the increase of the density of local facial amphiphilicity from polycations than monomers, which was similarly observed by many other groups on different systems Antimicrobial activity was further investigated using a clinically isolated MDR strain of E.
These MIC values increased with polymers containing the shorter spacer unit. However, the MIC values are comparatively higher than those for regular strains of E. Bacteria were exposed multiple times to the polymer at a sub-MIC level, and the MIC was measured for every consecutive passage.
Detailed experimental procedures are provided in the supplementary information. After 10 passages, no significant changes in the MIC values were observed, as detailed in Fig. This important result demonstrated that developing resistance against cholic acid-based cationic polymers is inherently difficult for both P. The Data are collected from the three replicates and the error bars represent the s.
The toxicity of bile acid-derived cationic polymers was evaluated by measuring hemoglobin release from mouse red blood cells RBCs at various concentrations. As mentioned previously, the hydrophobic and hydrophilic balance of an antimicrobial polymer plays a critical role for the selective attachment to a bacterial cell membrane. It is well established that a polymer with higher hydrophobicity or lower hydrophilicity produces hemolysis to a greater extent, due to the strong interaction with the lipid portion of a mammalian cell membrane 25 , 47 , Bile acid derivatives are intrinsically hydrophobic due to the presence of a four fused-ring structure.
All cholic acid polymers contain three positive head groups in each repeat unit, which reduces hydrophobicity. In contrast, the deoxycholic acid-based polymer possesses only two positive charged head groups in each repeat unit, making it more hydrophobic with a substantial level of toxicity. The hemolysis activity of lithocholic acid-based cationic polymers was not determined due to poor solubility in water. We observed that HC 50 increased with the increase of molecular weight of cholic acid-based polymers. There are many parameters to influence the hemolytic activity, especially the balance of hydrophilicity and hydrophobicity.
The concentration of polymers is two times that of the MIC value. As shown in Fig. These findings revealed that the antimicrobial activity of bile acid-based cationic polymers occurred by the disruption of bacterial membrane, consistent with the membrane lytic mechanism of various synthetic antimicrobial polymers 28 , 39 , 46 , In case of S.
Bacteria E. Most bacterial cells were shown to be significantly fragmented. The significant physical damage of cell membranes was observed for S. The loss of original morphology with cell membrane damage was more apparent in the case of Gram-negative bacteria compared to that of Gram-positive bacteria.
Bacterial Cell Walls and Surfaces
Bacteria concentrations were 1. Bile acid derivatives mostly small molecules have been developed as antimicrobial agents. Moore et al. Diamond et al. Savage and co-workers claimed that membrane-active facial amphiphilic cationic molecules, such as bile acid derivatives, could disrupt bacterial membranes 58 , Cholic acid-derived cationic surfactants can form micellar structures that exhibit antimicrobial activity against Gram-positive and Gram-negative bacteria However, higher susceptibility to the resistance of these small molecules remains a significant issue.
In the current study, we developed a class of antimicrobial polymers from bile acids, which possess macromolecular conformations critical for interactions with bacterial membranes. We observed that cholic acid-based cationic polymers are more effective against Gram-negative bacteria, especially P. Different from Gram-positive bacteria using peptidoglycan as the major periphery enveloping their cell membranes, Gram-negative bacteria possess double membranes with the outer membrane made up of zwitterionic phosphatidylethanolamine PE and other anionic phospholipids as their periphery for self-defense.
Therefore, in Gram-negative bacteria it is more challenging for antimicrobial agents to balance their hydrophobicity and cationic charges as well as to adopt a conformation that is favorable for interactions with the outer membrane. The hydrophobic multicyclic structure and three oriented cationic charges in the modified cholic acid provide true facial amphiphilicity in contact with bacterial cell membranes. Initially, three cationic charges on each cholic acid unit localize onto the outer membrane as a result of electrostatic interactions Fig.
Since each of this unique moiety is attached to a flexible macromolecular chain, collectively tens of or even hundreds of these local facial amphiphilic structures would facilitate each other and promote the entire macromolecule to penetrate through the membrane Fig. Such a concerted penetration of macromolecular chains across the cell membrane would cause its destabilization and fragmentation, ultimately leading to cell death.
With this design, there is no need for an entire macromolecule to adopt a globally entropy-unfavorable facial amphiphilic conformation. Conversely, Gram-positive bacteria, like S. Bulky cholic acid-based polymers could be more easily trapped in this layer and thus less effective in disrupting these cell membranes. A proposed mechanism of action of cholic acid-based polymers on the bacterial cell membrane: 1 diffusion, 2 surface binding, 3 membrane insertion and 4 membrane disruption.
The illustrated cholic acid can be replaced by other multicyclic compounds that are modified with facial amphiphilicity. In summary, we reported a design of antimicrobial polymers with repeat units possessing local facial amphiphilicity, which could promote effective interactions of an entire macromolecule with bacterial cell membranes, circumventing the adoption of an energetically unfavorable global facial amphiphilicity. Specifically, we derivatized three different multicyclic natural products.
Among them, cholic acid polymers were shown to be more efficient than their deoxycholic and lithocholic acid counterparts, regarding both antimicrobial activity and selectivity. This is ascribed to the true facial amphiphilic structure from cholic acid derivatives, which have the hydrophobic multicyclic structure as one face and three oriented hydrophilic cationic charges as the other face.
It is worth noting that a lot of multicyclic natural and synthetic compounds could be used as the key building block. This macromolecular structure and conformation may open an avenue toward next-generation antimicrobial agents to treat multidrug-resistant Gram-negative bacteria. Initially, CA 5. After placing the reaction mixture in an ice bath, HEMA 1. The reaction mixture was filtered and evaporated. After drying the organic layer over anhydrous MgSO 4 , the solvent was removed by rotary evaporation.
Methacrylate monomers were polymerized using a typical RAFT polymerization technique After a certain period of time, the polymerization was quenched by exposure to air and cooled under an ice water bath. The reaction mixture was precipitated twice into a mixture of hexane and DCM and finally dissolved in THF and precipitated into hexane.
The polymer was dried under vacuum. After cooling and concentrating the reaction mixture, the resulting solution was precipitated in THF and centrifuged to collect the product. The product was washed with THF and dried under vacuum. The solution in dialysis bag was collected and freeze-dried to obtain a white product. MAECA 0. Then, 6-bromohexanoyl chloride 2. The organic phase was dried over magnesium sulfate and concentrated, then precipitated in hexane twice to remove unreacted 6-bromohexanoyl chloride. The yellow product was dried under vacuum.
The product was further washed with THF and dried under vacuum. All the data supporting the findings of this study are available within the Article and its Supplementary Information file. Bush, K. Tackling antibiotic resistance. Taubes, G. The bacteria fight back. Science , — Lam, S. Combating multidrug-resistant Gram-negative bacteria with structurally nanoengineered antimicrobial peptide polymers.
Chemistry And Biological Activities Of Bacterial Surface Amphiphiles
Chin, W. A macromolecular approach to eradicate multidrug resistant bacterial infections while mitigating drug resistance onset. Antimicrobial polymeric nanoparticles. Nederberg, F. Biodegradable nanostructures with selective lysis of microbial membranes. Brogden, K. Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria?
Kohanski, M. How antibiotics kill bacteria: from targets to networks. Ling, L. A new antibiotic kills pathogens without detectable resistance. Nature , — Shi, Y. Radzishevsky, I. Improved antimicrobial peptides based on acyl-lysine oligomers. Takahashi, H. Synthetic random copolymers as a molecular platform to mimic host-defense antimicrobial peptides. Xiong, M. Zasloff, M.
Antimicrobial peptides of multicellular organisms. Mowery, B. Lienkamp, K. Antimicrobial polymers prepared by ROMP with unprecedented selectivity: a molecular construction kit approach. Ganewatta, M. Controlling macromolecular structures towards effective antimicrobial polymers. Polymer 63 , A1—A29 Ong, Z. Strategies employed in the design and optimization of synthetic antimicrobial peptide amphiphiles with enhanced therapeutic potentials. Drug Deliv. Porter, E. Nature , Chongsiriwatana, N.
Peptoids that mimic the structure, function, and mechanism of helical antimicrobial peptides. Natl Acad. USA , — Reuther, J. A versatile approach to noncanonical, dynamic covalent single- and multi-loop peptide macrocycles for enhancing antimicrobial activity. Ilker, M. Tuning the hemolytic and antibacterial activities of amphiphilic polynorbornene derivatives. Geng, Z. Thiabicyclononane-based antimicrobial polycations. Ergene, C.
Biomimetic antimicrobial polymers: recent advances in molecular design. Chen, Y. Amphipathic antibacterial agents using cationic methacrylic polymers with natural rosin as pendant group. RSC Adv. Bio-inspired resin acid-derived materials as anti-bacterial resistance agents with unexpected activities. Antibacterial and biofilm-disrupting coatings from resin acid-derived materials.
Biomacromolecules 16 , — Zhang, J. Antimicrobial metallopolymers and their bioconjugates with conventional antibiotics against multidrug-resistant bacteria. Yang, P. Trio act of boronolectin with antibiotic-metal complexed macromolecules toward broad-spectrum antimicrobial efficacy. ACS Infect. Yan, Y. Metal-containing and related polymers for biomedical applications. Pageni, P. Recyclable magnetic nanoparticles grafted with antimicrobial metallopolymer-antibiotic bioconjugates. Biomaterials , — Zhu, T.
Chemical Composition and Architecture Combat Bacterial Membranes
Metallo-polyelectrolytes as a class of ionic macromolecules for functional materials. Facially amphiphilic polyionene biocidal polymers derived from lithocholic acid. Usually 6 FA are found. All FA in Lipid A are saturated. Some FA are attached directly to the NAG dimer and others are esterified to the 3-hydroxy fatty acids that are characteristically present.
The structure of Lipid A is highly conserved among Gram-negative bacteria. Among Enterobacteriaceae Lipid A is virtually constant. The primary structure of Lipid A has been elucidated and Lipid A has been chemically synthesized. Its biological activity appears to depend on a peculiar conformation that is determined by the glucosamine disaccharide, the PO 4 groups, the acyl chains, and also the KDO-containing inner core.
Two unusual sugars, heptose and 2-ketodeoxyoctonoic acid KDO , are usually present, in the core polysaccharide. With minor variations, the core polysaccharide is common to all members of a bacterial genus e. Salmonella , but it is structurally distinct in other genera of Gram-negative bacteria. Salmonella , Shigella and Escherichia have similar but not identical cores. Region III. Somatic O antigen or O polysaccharide is attached to the core polysaccharide. It consists of repeating oligosaccharide subunits made up of 3 - 5 sugars.
The individual chains vary in length ranging up to 40 repeat units. The O polysaccharide is much longer than the core polysaccharide, and it maintains the hydrophilic domain of the LPS molecule. A major antigenic determinant antibody-combining site of the Gram-negative cell wall resides in the O polysaccharide.
Great variation occurs in the composition of the sugars in the O side chain between species and even strains of Gram-negative bacteria.
At least 20 different sugars are known to occur and many of these sugars are characteristically unique dideoxyhexoses, which occur in nature only in Gram-negative cell walls. Variations in sugar content of the O polysaccharide contribute to the wide variety of antigenic types of Salmonella and E. Particular sugars in the structure, especially the terminal ones, confer immunological specificity of the O antigen, in addition to "smoothness" colony morphology of the strain. Loss of the O specific region by mutation results in the strain becoming a "rough" colony morphology or R strain.
The elucidation of the structure of LPS Figure 3 relied heavily on the availability of mutants each blocked at a particular step in LPS synthesis. The biosynthesis of LPS is strictly sequential. The core sugars are added sequentially to Lipid A by successive additions, and the O side chain is added last, one preassembled subunit at a time. The properties of mutants producing incomplete LPS molecules suggests the nature and biological functions performed by various parts of the LPS molecule. It is known that such "rough" mutants are more susceptible to phagocytosis and serum bactericidal reactions.
Loss of the more proximal parts of the core, as in "deep rough" mutants i. This area contains a large number of charged groups and is thought to be important in maintaining the permeability properties of the outer membrane.
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Mutants in the assembly of Lipid A cannot be isolated except as conditional lethal mutants and this region must therefore be essential for cell viability. The innermost region of LPS, consisting of Lipid A and three residues of KDO, appears to be essential for viability, presumably for assembling the outer membrane. Both Lipid A the toxic component of LPS and the polysaccharide side chains the nontoxic but immunogenic portion of LPS act as determinants of virulence in Gram-negative bacteria.
How are the polysaccharide side chains involved in the expression of virulence?
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There are a number of possibilities:. O-specific antigens could allow organisms to adhere specifically to certain tissues, especially epithelial tissues. Smooth antigens probably allow resistance to phagocytes , since rough mutants are more readily engulfed and destroyed by phagocytes. The hydrophilic O polysaccharides could act as water-solubilizing carriers for toxic Lipid A.
It is known that the exact structure of the polysaccharide can greatly influence water binding capacity at the cell surface. The O antigens could provide protection from damaging reactions with antibody and complement. Rough strains of Gram-negative bacteria derived from virulent strains are generally non virulent. Smooth strains have polysaccharide "whiskers" which bear O antigens projecting from the cell surface. The O antigens are the key targets for the action of host antibody and complement, but when the reaction takes place at the tips of the polysaccharide chains, a significant distance external to the general bacterial cell surface, complement fails to have its normal lytic effect.
Such bacteria are virulent because of this resistance to immune forces of the host. If the projecting polysaccharide chains are shortened or removed, antibody reacts with antigens on the general bacterial surface, or very close to it, and complement can lyse the bacteria. This contributes to the loss of virulence in "rough" colonial strains. The O-polysaccharide or O antigen is the basis of antigenic variation among many important Gram-negative pathogens including E. Antigenic variation guarantees the existence of multiple serotypes of the bacterium, so that it is afforded multiple opportunities to infect its host if it can bypass the immune response against a different serotype.
Furthermore, even though the O polysaccharides are strong antigens, they seldom elicit immune responses which give full protection to the host against secondary challenge with specific endotoxin. These, in turn, stimulate production of prostaglandins and leukotrienes.