Targeting Iron - Respiratory Reciprocity Promotes Bacterial Death.

Discovering new bacterial signaling pathways offers unique antibiotic strategies. Here, through an unbiased resistance screen of 3,884 gene knockout strains, we uncovered a previously unknown non-lytic bactericidal mechanism that sequentially couples three transporters and downstream transcription to lethally suppress respiration of the highly virulent P. aeruginosa strain PA14 - one of three species on the WHO's 'Priority 1: Critical' list. By targeting outer membrane YaiW, cationic lacritin peptide 'N-104' translocates into the periplasm where it ligates outer loops 4 and 2 of the inner membrane transporters FeoB and PotH, respectively, to suppress both ferrous iron and polyamine uptake. This broadly shuts down transcription of many biofilm-associated genes, including ferrous iron-dependent TauD and ExbB1. The mechanism is innate to the surface of the eye and is enhanced by synergistic coupling with thrombin peptide GKY20. This is the first example of an inhibitor of multiple bacterial transporters.

Measurement of Accumulation of Antibiotics to Staphylococcus aureus in Phagosomes of Live Macrophages.

Staphylococcus aureus (S. aureus) has evolved the ability to persist after uptake into host immune cells. This intracellular niche enables S. aureus to potentially escape host immune responses and survive the lethal actions of antibiotics. While the elevated tolerance of S. aureus to small-molecule antibiotics is likely to be multifactorial, we pose that there may be contributions related to permeation of antibiotics into phagocytic vacuoles, which would require translocation across two mammalian bilayers. To empirically test this, we adapted our recently developed permeability assay to determine the accumulation of FDA-approved antibiotics into phagocytic vacuoles of live macrophages. Bioorthogonal reactive handles were metabolically anchored within the surface of S. aureus, and complementary tags were chemically added to antibiotics. Following phagocytosis of tagged S. aureus cells, we were able to specifically analyze the arrival of antibiotics within the phagosomes of infected macrophages. Our findings enabled the determination of permeability differences between extra- and intracellular S. aureus, thus providing a roadmap to dissect the contribution of antibiotic permeability to intracellular pathogens.

Measurement of Accumulation of Antibiotics to Staphylococcus aureus in Phagosomes of Live Macrophages.

Staphylococcus aureus ( S. aureus ) has evolved the ability to persist after uptake into host immune cells. This intracellular niche enables S. aureus to potentially escape host immune responses and survive the lethal actions of antibiotics. While the elevated tolerance of S. aureus to small-molecule antibiotics is likely to be multifactorial, we pose that there may be contributions related to permeation of antibiotics into phagocytic vacuoles, which would require translocation across two mammalian bilayers. To empirically test this, we adapted our recently developed permeability assay to determine the accumulation of FDA-approved antibiotics into phagocytic vacuoles of live macrophages. Bioorthogonal reactive handles were metabolically anchored within the surface of S. aureus, and complementary tags were chemically added to antibiotics. Following phagocytosis of tagged S. aureus cells, we were able to specifically analyze the arrival of antibiotics within the phagosomes of infected macrophages. Our findings enabled the determination of permeability differences between extra- and intracellular S. aureus , thus providing a roadmap to dissect the contribution of antibiotic permeability to intracellular pathogens.

Measurement of Small Molecule Accumulation into Diderm Bacteria.

Some of the most dangerous bacterial pathogens (Gram-negative and mycobacterial) deploy a formidable secondary membrane barrier to reduce the influx of exogenous molecules. For Gram-negative bacteria, this second exterior membrane is known as the outer membrane (OM), while for the Gram-indeterminate Mycobacteria, it is known as the "myco" membrane. Although different in composition, both the OM and mycomembrane are key structures that restrict the passive permeation of small molecules into bacterial cells. Although it is well-appreciated that such structures are principal determinants of small molecule permeation, it has proven to be challenging to assess this feature in a robust and quantitative way or in complex, infection-relevant settings. Herein, we describe the development of the bacterial chloro-alkane penetration assay (BaCAPA), which employs the use of a genetically encoded protein called HaloTag, to measure the uptake and accumulation of molecules into model Gram-negative and mycobacterial species, Escherichia coli and Mycobacterium smegmatis, respectively, and into the human pathogen Mycobacterium tuberculosis. The HaloTag protein can be directed to either the cytoplasm or the periplasm of bacteria. This offers the possibility of compartmental analysis of permeation across individual cell membranes. Significantly, we also showed that BaCAPA can be used to analyze the permeation of molecules into host cell-internalized E. coli and M. tuberculosis, a critical capability for analyzing intracellular pathogens. Together, our results show that BaCAPA affords facile measurement of permeability across four barriers: the host plasma and phagosomal membranes and the diderm bacterial cell envelope.

Structure Activity Relationship of the Stem Peptide in Sortase A Mediated Ligation from Staphylococcus aureus.

The surfaces of most Gram-positive bacterial cells, including that of Staphylococcus aureus (S. aureus), are heavily decorated with proteins that coordinate cellular interactions with the extracellular space. In S. aureus, sortase A is the principal enzyme responsible for covalently anchoring proteins, which display the sorting signal LPXTG, onto the peptidoglycan (PG) matrix. Considerable efforts have been made to understand the role of this signal peptide in the sortase-mediated reaction. In contrast, much less is known about how the primary structure of the other substrate involved in the reaction (PG stem peptide) could impact sortase activity. To assess the sortase activity, a library of synthetic analogs of the stem peptide that mimic naturally existing variations found in the S. aureus PG primary sequence were evaluated. Using a combination of two unique assays, we showed that there is broad tolerability of substrate variations that are effectively processed by sortase A. While some of these stem peptide derivatives are naturally found in mature PG, they are not known to be present in the PG precursor, lipid II. These results suggest that sortase A could process both lipid II and mature PG as acyl-acceptor strands that might reside near the membrane, which has not been previously described.

Targeting of Pseudomonas aeruginosa cell surface via GP12, an Escherichia coli specific bacteriophage protein.

Bacteriophages are highly abundant molecular machines that have evolved proteins to target the surface of host bacterial cells. Given the ubiquity of lipopolysaccharides (LPS) on the outer membrane of Gram-negative bacteria, we reasoned that targeting proteins from bacteriophages could be leveraged to target the surface of Gram-negative pathogens for biotechnological applications. To this end, a short tail fiber (GP12) from the T4 bacteriophage, which infects Escherichia coli (E. coli), was isolated and tested for the ability to adhere to whole bacterial cells. We found that, surprisingly, GP12 effectively bound the surface of Pseudomonas aeruginosa cells despite the established preferred host of T4 for E. coli. In efforts to elucidate why this binding pattern was observed, it was determined that the absence of the O-antigen region of LPS on E. coli improved cell surface tagging. This indicated that O-antigens play a significant role in controlling cell adhesion by T4. Probing GP12 and LPS interactions further using deletions of the enzymes involved in the biosynthetic pathway of LPS revealed the inner core oligosaccharide as a possible main target of GP12. Finally, we demonstrated the potential utility of GP12 for biomedical applications by showing that GP12-modified agarose beads resulted in the depletion of pathogenic bacteria from solution.

Broadening Activity of Polymyxin by Quaternary Ammonium Grafting.

Bacterial pathogens continue to impose a tremendous health burden across the globe. Here, we describe a novel series of polymyxin-based agents grafted with membrane-active quaternary ammonium warheads to combine two important classes of Gram-negative antimicrobial scaffolds. The goal was to deliver a targeted quaternary ammonium warhead onto the surface of bacterial pathogens using the outer membrane homing properties of polymyxin. The most potent agents resulted in new scaffolds that retained the ability to target Gram-negative bacteria and had limited toxicity toward mammalian cells. We showed, using a molecular dynamics approach, that the new agents retained their ability to engage in specific interactions with lipopolysaccharide molecules. Significantly, the combination of quaternary ammonium and polymyxin widens the activity to the pathogen Staphylococcus aureus. Our results serve as an example of how two membrane-active agents can be combined to produce a class of novel scaffolds with potent biological activity.