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Streptococcus mutans is commonly associated with dental caries and the ability to form biofilms is essential for its pathogenicity. We recently identified the Pgf glycosylation machinery of S. mutans, responsible for the post-translational modification of the surface-associated adhesins Cnm and WapA. Since the four-gene pgf operon (pgfS-pgfM1-pgfE-pgfM2) is part of the S. mutans core genome, we hypothesized that the scope of the Pgf system goes beyond Cnm and WapA glycosylation. In silico analyses and tunicamycin sensitivity assays suggested a functional overlap between the Pgf machinery and the rhamnose-glucose polysaccharide synthesis pathway. Phenotypic characterization of pgf mutants (ΔpgfS, ΔpgfE, ΔpgfM1, ΔpgfM2, and Δpgf) revealed that the Pgf system is important for biofilm formation, surface charge, membrane stability, and survival in human saliva. Moreover, deletion of the entire pgf operon (Δpgf strain) resulted in significantly impaired colonization in a rat oral colonization model. Using Cnm as a model, we showed that Cnm is heavily modified with N-acetyl hexosamines but it becomes heavily phosphorylated with the inactivation of the PgfS glycosyltransferase, suggesting a crosstalk between these two post-translational modification mechanisms. Our results revealed that the Pgf machinery contributes to multiple aspects of S. mutans pathobiology that may go beyond Cnm and WapA glycosylation., (© 2023 John Wiley & Sons Ltd.)

An optimum hydrophilic/hydrophobic balance has been recognized as a crucial parameter in designing cationic polymers that mimic antimicrobial peptides (AMPs). To date, this balance was achieved either by hydrophilicity variation through altering the nature and the number of cationic charges or by hydrophobicity modulation through incorporation of alkyl groups of different chain lengths. However, how the hydrophobicity variation through AMPs' building blocks-amino acids-influences the antibacterial efficacy of AMP-mimicking cationic polymers has rarely been explored. Toward this goal, herein we report a class of amino acid conjugated polymers (ACPs) with tunable antibacterial activity through a simple post-polymer-functionalization strategy. Our polymeric design comprised a permanent cationic charge in every repeating unit, whereby the hydrophobicity was tuned through incorporation of different amino acids. Our results revealed that the amino acid alteration has a strong influence on antibacterial efficacy. Upon increasing the amino acid side-chain hydrophobicity, both the antibacterial activity (against broad spectrum of bacteria) and toxicity increased. However, the distinct feature of this class of polymers was their good activity against Acinetobacter baumannii -the top most critical pathogen according to WHO, which has created an alarming situation worldwide, causing the majority of infections in humans. A nontoxic (no hemolysis even at 1000 μg/mL) ACP including a glycine residue (ACP- 1 (Gly)) showed very good activity (MIC = 8-16 μg/mL) against both drug-sensitive and drug-resistant strains of A. baumannii , including clinical isolates. This polymer not only was rapidly bactericidal against growing planktonic A. baumannii but also killed nondividing stationary-phase cells instantaneously (<2 min). Moreover, it eradicated the established biofilm formed by drug-resistant A. baumannii clinical isolates. No propensity for bacterial resistance development against this polymer was seen even after 14 continuous passages. Taken together, the results highlight that hydrophobicity modulation through incorporation of amino acids in cationic polymers will provide a significant opportunity in designing new ACPs with potent antibacterial activity and minimum toxicity toward mammalian cells. More importantly, the excellent anti- A. baumannii efficacy of the optimized lead polymer indicates its immense potential for being developed as therapeutic agent.

l-Lysines were conjugated to lipidated biphenyls using simple synthetic chemistry to obtain selective membrane-active antibacterial agents that inhibit cell-wall biosynthesis. The most selective compound bore promising activity against biofilm-related infections and intracellular bacteria, and also suppressed the stimulation of TNF-α induced by lipoteichoic acid. Belligerent to resistance development, it was active in a murine model of MRSA infection.

More than 80% of the bacterial infections are associated with biofilm formation. To combat infections, amphiphilic small molecules have been developed as promising antibiofilm agents. However, cytotoxicity of such molecules still remains a major problem. Herein we demonstrate a concept in which antibacterial versus cytotoxic activities of cationic small molecules are tuned by spatial positioning of hydrophobic moieties while keeping positive charges constant. Compared to the molecules with more pendent hydrophobicity from positive centers (MIC = 1-4 μg/mL and HC 50 = 60-65 μg/mL), molecules with more confined hydrophobicity between two centers show similar antibacterial activity but significantly less toxicity toward human erythrocytes (MIC = 1-4 μg/mL and HC 50 = 805-1242 μg/mL). Notably, the optimized molecule is shown to be nontoxic toward human cells (HEK 293) at a concentration at which it eradicates established bacterial biofilms. The molecule is also shown to eradicate preformed bacterial biofilm in vivo in a murine model of superficial skin infection.

Bacterial biofilms represent the root-cause of chronic or persistent infections in humans. Gram-negative bacterial infections due to nosocomial and opportunistic pathogens such as Acinetobacter baumannii are more difficult to treat because of their inherent and rapidly acquiring resistance to antibiotics. Due to biofilm formation, A. baumannii has been noted for its apparent ability to survive on artificial surfaces for an extended period of time, therefore allowing it to persist in the hospital environment. Here we report, maleic anhydride based novel cationic polymers appended with amide side chains that disrupt surface established multi-drug resistant A. baumannii biofilms. More importantly, these polymers significantly (p < 0.0001) decrease the bacterial burden in mice with chronic A. baumannii burn wound infection. The polymers also show potent antibacterial efficacy against methicillin resistant Staphylococcus aureus (MRSA), vancomycin resistant Enterococci (VRE) and multi-drug resistant clinical isolates of A. baumannii with minimal toxicity to mammalian cells. We observe that optimal hydrophobicity dependent on the side chain chemical structure of these polymers dictate the selective toxicity to bacteria. Polymers interact with the bacterial cell membranes by causing membrane depolarization, permeabilization and energy depletion. Bacteria develop rapid resistance to erythromycin and colistin whereas no detectable development of resistance occurs against these polymers even after several passages. These results suggest the potential use of these polymeric biomaterials in disinfecting biomedical device surfaces after the infection has become established and also for the topical treatment of chronic bacterial infections., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The emergence of bacterial resistance and biofilm associated infections has created a challenging situation in global health. In this present state of affairs where conventional antibiotics are falling short of being able to provide a solution to these problems, development of novel antibacterial compounds possessing the twin prowess of antibacterial and antibiofilm efficacy is imperative. Herein, we report a library of amino acid tunable lipidated norspermidine conjugates that were prepared by conjugating both amino acids and fatty acids with the amine functionalities of norspermidine through amide bond formation. These lipidated conjugates displayed potent antibacterial activity against various planktonic Gram-positive and Gram-negative bacteria including drug-resistant superbugs such as methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecium, and β-lactam-resistant Klebsiella pneumoniae. This class of nontoxic and fast-acting antibacterial molecules (capable of killing bacteria within 15 min) did not allow bacteria to develop resistance against them after several passages. Most importantly, an optimized compound in the series was also capable of killing metabolically inactive persisters and stationary phase bacteria. Additionally, this compound was capable of disrupting the preformed biofilms of S. aureus and E. coli. Therefore, this class of antibacterial conjugates have potential in tackling the challenging situation posed by both bacterial resistance as well as drug tolerance due to biofilm formation.

Rationally designed amphiphilic small molecules selectively kill drug-sensitive and drug-resistant bacteria over mammalian cells. The small molecules disperse preformed biofilms and reduce viable bacterial count in the biofilms. Moreover, this class of membrane-active molecules disarms the development of bacterial resistance.

Treating bacterial biofilms with conventional antibiotics is limited due to ineffectiveness of the drugs and higher propensity to develop bacterial resistance. Development of new classes of antibacterial therapeutics with alternative mechanisms of action has become imperative. Herein, we report the design, synthesis, and biological evaluations of novel membrane-active small molecules featuring two positive charges, four nonpeptidic amide groups, and variable hydrophobic/hydrophilic (amphiphilic) character. The biocides synthesized via a facile methodology not only displayed good antibacterial activity against wild-type bacteria but also showed high activity against various drug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecium (VRE), and β-lactam-resistant Klebsiella pneumoniae. Further, these biocides not only inhibited the formation of biofilms but also disrupted the established S. aureus and E. coli biofilms. The membrane-active biocides hindered the propensity to develop bacterial resistance. Moreover, the biocides showed negligible toxicity against mammalian cells and thus bear potential to be used as therapeutic agents.