Marcos Pires

The major constituent of bacterial cell walls is peptidoglycan, which, in its crosslinked form, is a polymer of considerable complexity that encases the entire bacterium. A functional cell wall is indispensable for survival of the organism. There are several dozen enzymes that assemble and disassemble the peptidoglycan dynamically within each bacterial generation. Understanding of the nature of these transformations is critical knowledge for these events. Octasaccharide peptidoglycans were prepared and studied with seven recombinant cell-wall-active enzymes (SltB1, MltB, RlpA, mutanolysin, AmpDh2, AmpDh3, and PBP5). With the use of highly sensitive mass spectrometry methods, we described the breadth of reactions that these enzymes catalyzed with peptidoglycan and shed light on the nature of the cell wall alteration performed by these enzymes. The enzymes exhibit broadly distinct preferences for their substrate peptidoglycans in the reactions that they catalyze.

Modifying cell walls: Homogeneous and heterogeneous octasaccharide peptidoglycans were synthesized for characterization of reactions of seven enzymes that modify the bacterial cell wall: SltB1, MltB, RlpA, mutanolysin, AmpDh2, AmpDh3, and PBP5. Three lytic transglycosylases and mutanolysin exhibited broadly distinct preferences for their substrate peptidoglycans in the reactions that they catalyze.

The surge in drug-resistant bacterial infections threatens to overburden healthcare systems worldwide. Bacterial cell walls are essential to bacteria, thus making them unique targets for the development of antibiotics. We describe a cellular reporter to directly monitor the phenotypic switch in drug-resistant bacteria with temporal resolution. Vancomycin-resistant enterococci (VRE) escape the bactericidal action of vancomycin by chemically modifying their cell-wall precursors. A synthetic cell-wall analogue was developed to hijack the biosynthetic rewiring of drug-resistant cells in response to antibiotics. Our study provides the first in vivo VanX reporter agent that responds to cell-wall alteration in drug-resistant bacteria. Cellular reporters that reveal mechanisms related to antibiotic resistance can potentially have a significant impact on the fundamental understanding of cellular adaption to antibiotics.

Can they resist? Drug-resistant bacteria pose a major threat. Vancomycin-resistant enterococci express VanX dipeptidases to remove drug-sensitive building blocks. A series of compounds were used to metabolically label bacterial cell walls of live VRE cells (see picture) to report on structural alterations linked with antibiotic resistance.

Quaternary ammonium compounds (QACs) are commonly used antiseptics that are now known to be subject to bacterial resistance. The prevalence and mechanisms of such resistance, however, remain underexplored. We investigated a variety of QACs, including those with multicationic structures (multiQACs), and the resistance displayed by a variety of Staphylococcus aureus strains with and without genes encoding efflux pumps, the purported main driver of bacterial resistance in MRSA. Through minimum inhibitory concentration (MIC)-, kinetic-, and efflux-based assays, we found that neither the qacR/qacA system present in S. aureus nor another efflux pump system is the main reason for bacterial resistance to QACs. Our findings suggest that membrane composition could be the predominant driver that allows CA-MRSA to withstand the assault of conventional QAC antiseptics.

Pièce de résistance: We describe herein our efforts to better understand the role that qac efflux genes play in biocide resistance through the implementation of our multiQAC chemical library. We identified that membrane composition, and not qac efflux, could be the predominant driver of resistance in community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA).

Pathogens frequently rely on lectins for adhesion and cellular entry into the host. Since these interactions typically result from multimeric binding of lectins to cell-surface glycans, novel therapeutic strategies are being developed with the use of glycomimetics as competitors of such interactions. Herein we study the benefit of nucleic acid based oligomeric assemblies with PNA–fucose conjugates. We demonstrate that the interactions of a lectin with epithelial cells can be inhibited with conjugates that do not form stable assemblies in solution but benefit from cooperativity between ligand–protein interactions and PNA hybridization to achieve high affinity. A dynamic dimeric assembly fully blocked the binding of the fucose-binding lectin BambL of Burkholderia ambifaria, a pathogenic bacterium, to epithelial cells with an efficiency of more than 700-fold compared to l-fucose.

Short and sweet: The interactions of lectins (blue) with cells can be inhibited with nucleic acid based oligomeric assemblies with PNA–fucose conjugates. These conjugates do not form stable assemblies in solution but benefit from cooperativity between ligand–protein interactions and PNA hybridization. A dynamic dimeric assembly fully blocked the binding of the fucose-binding lectin BambL of the pathogenic bacterium Burkholderia ambifaria to epithelial cells.

We previously reported an efficient peptide synthesis method, AJIPHASE®, that comprises repeated reactions and isolations by precipitation. This method utilizes an anchor molecule with long-chain alkyl groups as a protecting group for the C-terminus. To further improve this method, we developed a one-pot synthesis of a peptide sequence wherein the synthetic intermediates were isolated by solvent extraction instead of precipitation. A branched-chain anchor molecule was used in the new process, significantly enhancing the solubility of long peptides and the operational efficiency compared with the previous method, which employed precipitation for isolation and a straight-chain aliphatic group. Another prerequisite for this solvent-extraction-based strategy was the use of thiomalic acid and DBU for Fmoc deprotection, which facilitates the removal of byproducts, such as the fulvene adduct.

Extracted, not precipitated: AJIPHASE® is a new method for one-pot peptide synthesis that makes use of solvent extraction during peptide elongation and does not require any isolation steps. This efficient approach leads to peptides of high purity and benefits from significantly reduced solvent consumption.

Penetratin (RQIKIWFQNRRMKWKK) enters cells by different mechanisms, including membrane translocation, thus implying that the peptide interacts with the lipid bilayer. Penetratin also crosses the membrane of artificial vesicles, depending on their phospholipid content. To evaluate the phospholipid preference of penetratin, as the first step of translocation, we exploited the benzophenone triplet kinetics of hydrogen abstraction, which is slower for secondary than for allylic hydrogen atoms. By using multilamellar vesicles of varying phospholipid content, we identified and characterized the cross-linked products by MALDI-TOF mass spectrometry. Penetratin showed a preference for negatively charged (vs. zwitterionic) polar heads, and for unsaturated (vs. saturated) and short (vs. long) saturated phospholipids. Our study highlights the potential of using benzophenone to probe the environment and insertion depth of membranotropic peptides in membranes.

Fathoming the depths: Evidence for the direct interaction of the cell-penetrating peptide penetratin with specific phospholipids was obtained by a photolabeling strategy (see picture). The method exploits the benzophenone triplet kinetics of hydrogen abstraction, which is faster for allylic hydrogen atoms. Benzophenone-modified penetratin analogues were found to interact preferentially with phospholipids in disordered membrane domains.

Antimicrobial resistance (AMR), the ability of a bacterial species to resist the action of an antimicrobial drug, has been on the rise due to the widespread use of antimicrobial agents. Per the World Health Organization, AMR has an estimated annual cost of USD 34 billion in the US and is predicted to be the number one cause of death worldwide by 2050. One way AMR bacteria can spread, and by which individuals can contract AMR infections, is through contaminated water. Monitoring AMR bacteria in the environment currently requires that samples be transported to a central laboratory for slow and labor intensive tests. We have developed an inexpensive assay using paper-based analytical devices (PADs) that can test for the presence of β-lactamase-mediated resistance. To demonstrate viability, the PAD was used to detect β-lactam resistance in wastewater and sewage and identified resistance in individual bacterial species isolated from environmental water sources.

Resistance is futile: Antimicrobial resistance (AMR), the ability of a bacterial species to resist the action of an antimicrobial drug, has been on the rise because of the widespread use of antimicrobial agents. An inexpensive, fast assay using a paper-based analytical device (PAD) has been developed to monitor water sources for the presence of β-lactamase-mediated resistance.

Salt bridges are very common in proteins. But what drives the formation of protein salt bridges is not clear. In this work, we determined the strength of four salt bridges in the protein GB3 by measuring the ΔpK_a values of the basic residues that constitute the salt bridges with a highly accurate NMR titration method at different temperatures. The results show that the ΔpK_a values increase with temperature, thus indicating that the salt bridges are stronger at higher temperatures. Fitting of ΔpK_a values to the van't Hoff equation yields positive ΔH and ΔS values, thus indicating that entropy drives salt-bridge formation. Molecular dynamics simulations show that the protein and solvent make opposite contributions to ΔH and ΔS. Specifically, the enthalpic gain contributed from the protein is more than offset by the enthalpic loss contributed from the solvent, whereas the entropic gain originates from the desolvation effect.

Out of disorder: The strengths of four salt bridges in the protein GB3 were determined by measuring the ΔpK_a values of the basic residues that constitute the salt bridges with a highly accurate NMR titration method at different temperatures. Fitting of the ΔpK_a values to the van't Hoff equation showed that entropy drives the formation of protein salt bridges, while the enthalpic contribution disfavors salt-bridge formation.