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.

A Metabolic-Tag-Based Method for Assessing the Permeation of Small Molecules Across the Mycomembrane in Live Mycobacteria.

The general lack of permeability of small molecules observed for Mycobacterium tuberculosis (Mtb) is most ascribed to its unique cell envelope. More specifically, the outer mycomembrane is hypothesized to be the principal determinant for access of antibiotics to their molecular targets. We describe a novel assay that combines metabolic tagging of the peptidoglycan, which sits directly beneath the mycomembrane, click chemistry of test molecules, and a fluorescent labeling chase step, to measure the permeation of small molecules. We showed that the assay workflow was robust and compatible with high-throughput analysis in mycobacteria by testing a small panel of azide-tagged molecules. The general trend is similar across the two types of mycobacteria with some notable exceptions. We anticipate that this assay platform will lay the foundation for medicinal chemistry efforts to understand and improve uptake of both existing drugs and newly-discovered compounds into mycobacteria.

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.

Immunotargeting of Gram-Positive Pathogens via a Cell Wall Binding Tick Antifreeze Protein.

Immunological agents that supplement or modulate the host immune response have proven to have powerful therapeutic potential, although this modality is less explored against bacterial pathogens. We describe the application of a bacterial binding protein to re-engage the immune system toward pathogenic bacteria. More specifically, a hapten was conjugated to a protein expressed by Ixodes scapularis ticks, called I. scapularis antifreeze glycoprotein (IAFGP), that has high affinity for the d-alanine residue on the bacterial peptidoglycan. We showed that a fragment of this protein retained high surface binding affinity. Moreover, conjugation of a hapten to this peptide led to the display of haptens on the cell surface of vancomycin-resistant Enterococcus faecalis. Hapten display then induced the recruitment of antibodies and promoted uptake of bacterial pathogens by immune cells. These results demonstrate the feasibility in using cell wall binding agents as the basis of a class of bacterial immunotherapies.

Real-time non-invasive fluorescence imaging of gut commensal bacteria to detect dynamic changes in the microbiome of live mice.

In mammals, gut commensal microbiota interact extensively with the host, and the same interactions can be dysregulated in diseased states. Animal imaging is a powerful technique that is widely used to diagnose, measure, and track biological changes in model organisms such as laboratory mice. Several imaging techniques have been discovered and adopted by the research community that provide dynamic, non-invasive assessment of live animals, but these gains have not been universal across all fields of biology. Herein, we describe a method to non-invasively image commensal bacteria based on the specific metabolic labeling of bacterial cell walls to illuminate the gut bacteria of live mice. This tagging strategy may additionally provide unprecedented insight into cell wall turnover of gut commensals, which has implications for bacterial cellular growth and division, in a live animal.

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.

SaccuFlow: A High-Throughput Analysis Platform to Investigate Bacterial Cell Wall Interactions.

Bacterial cell walls are formidable barriers that protect bacterial cells against external insults and oppose internal turgor pressure. While cell wall composition is variable across species, peptidoglycan is the principal component of all cell walls. Peptidoglycan is a mesh-like scaffold composed of cross-linked strands that can be heavily decorated with anchored proteins. The biosynthesis and remodeling of peptidoglycan must be tightly regulated by cells because disruption to this biomacromolecule is lethal. This essentiality is exploited by the human innate immune system in resisting colonization and by a number of clinically relevant antibiotics that target peptidoglycan biosynthesis. Evaluation of molecules or proteins that interact with peptidoglycan can be a complicated and, typically, qualitative effort. We have developed a novel assay platform (SaccuFlow) that preserves the native structure of bacterial peptidoglycan and is compatible with high-throughput flow cytometry analysis. We show that the assay is facile and versatile as demonstrated by its compatibility with sacculi from Gram-positive bacteria, Gram-negative bacteria, and mycobacteria. Finally, we highlight the utility of this assay to assess the activity of sortase A from Staphylococcus aureus against potential antivirulence agents.

Induction of Endogenous Antibody Recruitment to the Surface of the Pathogen Enterococcus faecium.

For the foreseeable future, conventional small molecule antibiotics will continue to be the predominant treatment option due to wide patient coverage and low costs. Today, however, there is already a significant portion of patients that fail to respond to small molecule antibiotics and, according to the Centers for Disease Control and Prevention, this number is poised to increase in the coming years. Therefore, this rise in drug resistant bacteria must be countered with the development of nontraditional therapies. We propose a measure based on the re-engagement of the immune system toward pathogenic bacteria by grafting bacterial cell surfaces with immunogenic agents. Herein, we describe a class of cell wall analogues that selectively graft bacterial cell surfaces with epitopes that promote their opsonization. More specifically, synthetic analogues of peptidoglycan conjugated to haptens were designed to be incorporated by the cell wall biosynthetic machinery into live Enterococcus faecium. E. faecium is a formidable human pathogen that poses a considerable burden to healthcare and often results in fatalities. We showed that treatment of E. faecium and vancomycin-resistant strains with the cell wall analogues led to the display of haptens on the cell surface, which induced the recruitment of antibodies existing in the serum of humans. These results demonstrate the feasibility in using cell wall analogues as the basis of a class of bacterial immunotherapies against dangerous pathogens.