Selective Recruitment of Antibodies to Cancer Cells and Immune Cell-mediated Killing via In Situ Click Chemistry.

Many current cancer immunotherapies function by redirecting immune system components to recognize cancer biomarkers and initiate a cytotoxic attack. The lack of a universal tumor biomarker limits the therapeutic potential of these approaches. However, one feature characteristic of nearly all solid tumors is extracellular acidity. This inherent acidity provides the basis for targeted drug delivery via the pH-low insertion peptide (pHLIP), which selectively accumulates in tumors in vivo due to a pH-dependent membrane insertion propensity. Previously, we established that we could selectively decorate cancer cells with antigen-pHLIP conjugates to facilitate antibody recruitment and subsequent killing by engineered effector cells via antibody-depended cellular cytotoxicity (ADCC). Here, we present a novel strategy for opsonizing antibodies on target cell surfaces using click chemistry. We utilize pHLIP to facilitate selective tetrazine - trans-cyclooctene ligation of human IgGs to the cancer cell surface and induce ADCC. We demonstrate that our approach activates the primary ADCC signaling pathway via CD16a (FcγRIIIa) receptors on effector cells and induces the killing of cancer cell targets by engineered NK cells.

A Chemical Approach to Assess the Impact of Post-translational Modification on MHC Peptide Binding and Effector Cell Engagement.

The human major histocompatibility complex (MHC) plays a pivotal role in the presentation of peptidic fragments from proteins, which can originate from self-proteins or from nonhuman antigens, such as those produced by viruses or bacteria. To prevent cytotoxicity against healthy cells, thymocytes expressing T cell receptors (TCRs) that recognize self-peptides are removed from circulation (negative selection), thus leaving T cells that recognize nonself-peptides. Current understanding suggests that post-translationally modified (PTM) proteins and the resulting peptide fragments they generate following proteolysis are largely excluded from negative selection; this feature means that PTMs can generate nonself-peptides that potentially contribute to the development of autoreactive T cells and subsequent autoimmune diseases. Although it is well-established that PTMs are prevalent in peptides present on MHCs, the precise mechanisms by which PTMs influence the antigen presentation machinery remain poorly understood. In the present work, we introduce chemical modifications mimicking PTMs on synthetic peptides. This is the first systematic study isolating the impact of PTMs on MHC binding and also their impact on TCR recognition. Our findings reveal various ways PTMs alter antigen presentation, which could have implications for tumor neoantigen presentation.

Identification of a family of peptidoglycan transpeptidases reveals that Clostridioides difficile requires noncanonical cross-links for viability.

Most bacteria are surrounded by a cell wall that contains peptidoglycan (PG), a large polymer composed of glycan strands held together by short peptide cross-links. There are two major types of cross-links, termed 4-3 and 3-3 based on the amino acids involved. 4-3 cross-links are created by penicillin-binding proteins, while 3-3 cross-links are created by L,D-transpeptidases (LDTs). In most bacteria, the predominant mode of cross-linking is 4-3, and these cross-links are essential for viability, while 3-3 cross-links comprise only a minor fraction and are not essential. However, in the opportunistic intestinal pathogen Clostridioides difficile, about 70% of the cross-links are 3-3. We show here that 3-3 cross-links and LDTs are essential for viability in C. difficile. We also show that C. difficile has five LDTs, three with a YkuD catalytic domain as in all previously known LDTs and two with a VanW catalytic domain, whose function was until now unknown. The five LDTs exhibit extensive functional redundancy. VanW domain proteins are found in many gram-positive bacteria but scarce in other lineages. We tested seven non-C. difficile VanW domain proteins and confirmed LDT activity in three cases. In summary, our findings uncover a previously unrecognized family of PG cross-linking enzymes, assign a catalytic function to VanW domains, and demonstrate that 3-3 cross-linking is essential for viability in C. difficile, the first time this has been shown in any bacterial species. The essentiality of LDTs in C. difficile makes them potential targets for antibiotics that kill C. difficile selectively.

Bioluminescence-Based Determination of Cytosolic Accumulation of Antibiotics in Escherichia coli.

Antibiotic resistance is an alarming public health concern that affects millions of individuals across the globe each year. A major challenge in the development of effective antibiotics lies in their limited ability to permeate cells, noting that numerous susceptible antibiotic targets reside within the bacterial cytosol. Consequently, improving the cellular permeability is often a key consideration during antibiotic development, underscoring the need for reliable methods to assess the permeability of molecules across cellular membranes. Currently, methods used to measure permeability often fail to discriminate between the arrival within the cytoplasm and the overall association of molecules with the cell. Additionally, these techniques typically possess throughput limitations. In this work, we describe a luciferase-based assay designed for assessing the permeability of molecules in the cytosolic compartment of Gram-negative bacteria. Our findings demonstrate a robust system that can elucidate the kinetics of intracellular antibiotic accumulation in live bacterial cells in real time.

A modified BCG with depletion of enzymes associated with peptidoglycan amidation induces enhanced protection against tuberculosis in mice.

Mechanisms by which Mycobacterium tuberculosis (Mtb) evades pathogen recognition receptor activation during infection may offer insights for the development of improved tuberculosis (TB) vaccines. Whilst Mtb elicits NOD-2 activation through host recognition of its peptidoglycan-derived muramyl dipeptide (MDP), it masks the endogenous NOD-1 ligand through amidation of glutamate at the second position in peptidoglycan side-chains. As the current BCG vaccine is derived from pathogenic mycobacteria, a similar situation prevails. To alleviate this masking ability and to potentially improve efficacy of the BCG vaccine, we used CRISPRi to inhibit expression of the essential enzyme pair, MurT-GatD, implicated in amidation of peptidoglycan side-chains. We demonstrate that depletion of these enzymes results in reduced growth, cell wall defects, increased susceptibility to antibiotics, altered spatial localization of new peptidoglycan and increased NOD-1 expression in macrophages. In cell culture experiments, training of a human monocyte cell line with this recombinant BCG yielded improved control of Mtb growth. In the murine model of TB infection, we demonstrate that depletion of MurT-GatD in BCG, which is expected to unmask the D-glutamate diaminopimelate (iE-DAP) NOD-1 ligand, yields superior prevention of TB disease compared to the standard BCG vaccine. In vitro and in vivo experiments in this study demonstrate the feasibility of gene regulation platforms such as CRISPRi to alter antigen presentation in BCG in a bespoke manner that tunes immunity towards more effective protection against TB disease.

Tuberculosis is the leading cause of death from an infectious disease worldwide, partially due to a lack of access to drug treatments in certain countries where the disease is common. The only available tuberculosis vaccine ­ known as the BCG vaccine ­ is useful for preventing cases in young children, but is ineffective in teenagers and adults. So, there is a need to develop new vaccines that offer better, and longer lasting, durable protection in people of all ages. During an infection, our immune system recognizes markers known as PAMPs on the surface of bacteria, viruses or other disease-causing pathogens. The recognition of PAMPs by the immune system enables the body to distinguish foreign invading organisms from its own cells and tissues, thus triggering a response that fights the infection. If the body encounters the infectious agent again in the future, the immune system is able to quickly recognize and eliminate it before it can cause disease. Vaccines protect us by mimicking the appearance of the pathogen to trigger the first immune response without causing the illness. The BCG vaccine contains live bacteria that are closely related to the bacterium responsible for tuberculosis called Mycobacterium tuberculosis. Both M. tuberculosis and the live bacteria used in the BCG vaccine are able to hide an important PAMP, known as the NOD-1 ligand, from the immune system, making it harder for the body to detect them. The NOD-1 ligand forms part of the bacterial cell wall and modifying the BCG bacterium so it cannot disguise this PAMP may lead to a new, more effective vaccine. To investigate this possibility, Shaku et al. used a gene editing approach to develop a modified version of the BCG bacterium which is unable to hide its NOD-1 ligand when treated with a specific drug. Immune cells trained with the modified BCG vaccine were more effective at controlling the growth of M. tuberculosis than macrophages trained using the original vaccine. Furthermore, mice vaccinated with the modified BCG vaccine were better able to limit M. tuberculosis growth in their lungs than mice that had received the original vaccine. These findings offer a new candidate vaccine in the fight against tuberculosis. Further studies will be needed to modify the vaccine for use in humans. More broadly, this work demonstrates that gene editing can be used to expose a specific PAMP present in a live vaccine. This may help develop more effective vaccines for other diseases in the future.

Neisseria gonorrhoeae scavenges host sialic acid for Siglec-mediated, complement-independent suppression of neutrophil activation.

Gonorrhea, caused by the bacterium Neisseria gonorrhoeae (Gc), is characterized by neutrophilic influx to infection sites. Gc has developed mechanisms to resist killing by neutrophils that include modifications to its surface lipooligosaccharide (LOS). One such LOS modification is sialylation: Gc sialylates its terminal LOS sugars with cytidine-5'-monophosphate-N-acetylneuraminic acid, which is scavenged from the host using LOS sialyltransferase (Lst) since Gc cannot make its sialic acid. Sialylation enables sensitive strains of Gc to resist complement-mediated killing in a serum-dependent manner. However, little is known about the contribution of sialylation to complement-independent, direct Gc-neutrophil interactions. In the absence of complement, we found sialylated Gc expressing opacity-associated (Opa) proteins decreased the oxidative burst and granule exocytosis from primary human neutrophils. In addition, sialylated Opa+ Gc survived better than vehicle treated or Δlst Gc when challenged with neutrophils. However, Gc sialylation did not significantly affect Opa-dependent association with or internalization of Gc by neutrophils. Previous studies have implicated sialic acid-binding immunoglobulin-type lectins (Siglecs) in modulating neutrophil interactions with sialylated Gc. Blocking neutrophil Siglecs with antibodies that bind to their extracellular domains eliminated the ability of sialylated Opa+ Gc to suppress the oxidative burst and resist neutrophil killing. These findings highlight a new role for sialylation in Gc evasion of human innate immunity, with implications for the development of vaccines and therapeutics for gonorrhea. IMPORTANCE: Neisseria gonorrhoeae, the bacterium that causes gonorrhea, is an urgent global health concern due to increasing infection rates, widespread antibiotic resistance, and its ability to thwart protective immune responses. The mechanisms by which Gc subverts protective immune responses remain poorly characterized. One way N. gonorrhoeae evades human immunity is by adding sialic acid that is scavenged from the host onto its lipooligosaccharide, using the sialyltransferase Lst. Here, we found that sialylation enhances N. gonorrhoeae survival from neutrophil assault and inhibits neutrophil activation, independently of the complement system. Our results implicate bacterial binding of sialic acid-binding lectins (Siglecs) on the neutrophil surface, which dampens neutrophil antimicrobial responses. This work identifies a new role for sialylation in protecting N. gonorrhoeae from cellular innate immunity, which can be targeted to enhance the human immune response in gonorrhea.

Identification of a new family of peptidoglycan transpeptidases reveals atypical crosslinking is essential for viability in Clostridioides difficile.

Clostridioides difficile, the leading cause of antibiotic-associated diarrhea, relies primarily on 3-3 crosslinks created by L,D-transpeptidases (LDTs) to fortify its peptidoglycan (PG) cell wall. This is unusual, as in most bacteria the vast majority of PG crosslinks are 4-3 crosslinks, which are created by penicillin-binding proteins (PBPs). Here we report the unprecedented observation that 3-3 crosslinking is essential for viability in C. difficile. We also report the discovery of a new family of LDTs that use a VanW domain to catalyze 3-3 crosslinking rather than a YkuD domain as in all previously known LDTs. Bioinformatic analyses indicate VanW domain LDTs are less common than YkuD domain LDTs and are largely restricted to Gram-positive bacteria. Our findings suggest that LDTs might be exploited as targets for antibiotics that kill C. difficile without disrupting the intestinal microbiota that is important for keeping C. difficile in check.

Noninvasive Analysis of Peptidoglycan from Living Animals.

The role of the intestinal microbiota in host health is increasingly revealed in its contributions to disease states. The host-microbiome interaction is multifactorial and dynamic. One of the factors that has recently been strongly associated with host physiological responses is peptidoglycan from bacterial cell walls. Peptidoglycan from gut commensal bacteria activates peptidoglycan sensors in human cells, including the nucleotide-binding oligomerization domain-containing protein 2. When present in the gastrointestinal tract, both the polymeric form (sacculi) and depolymerized fragments can modulate host physiology, including checkpoint anticancer therapy efficacy, body temperature and appetite, and postnatal growth. To utilize this growing area of biology toward therapeutic prescriptions, it will be critical to directly analyze a key feature of the host-microbiome interaction from living hosts in a reproducible and noninvasive way. Here we show that metabolically labeled peptidoglycan/sacculi can be readily isolated from fecal samples collected from both mice and humans. Analysis of fecal samples provided a noninvasive route to probe the gut commensal community including the metabolic synchronicity with the host circadian clock. Together, these results pave the way for noninvasive diagnostic tools to interrogate the causal nature of peptidoglycan in host health and disease.

Targeted acidosis mediated delivery of antigenic MHC-binding peptides.

Cytotoxic T lymphocytes are the primary effector immune cells responsible for protection against cancer, as they target peptide neoantigens presented through the major histocompatibility complex (MHC) on cancer cells, leading to cell death. Targeting peptide-MHC (pMHC) complex offers a promising strategy for immunotherapy due to their specificity and effectiveness against cancer. In this work, we exploit the acidic tumor micro-environment to selectively deliver antigenic peptides to cancer using pH(low) insertion peptides (pHLIP). We demonstrated the delivery of MHC binding peptides directly to the cytoplasm of melanoma cells resulted in the presentation of antigenic peptides on MHC, and activation of T cells. This work highlights the potential of pHLIP as a vehicle for the targeted delivery of antigenic peptides and its presentation via MHC-bound complexes on cancer cell surface for activation of T cells with implications for enhancing anti-cancer immunotherapy.

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.

Neisseria gonorrhoeae scavenges host sialic acid for Siglec-mediated, complement-independent suppression of neutrophil activation.

Gonorrhea, caused by the bacterium Neisseria gonorrhoeae (Gc), is characterized by neutrophil influx to infection sites. Gc has developed mechanisms to resist killing by neutrophils that include modifications to its surface lipooligosaccharide (LOS). One such LOS modification is sialylation: Gc sialylates its terminal LOS sugars with cytidine-5'-monophosphate-N-acetylneuraminic acid (CMP-NANA) scavenged from the host using LOS sialyltransferase (Lst), since Gc cannot make its own sialic acid. Sialylation enables sensitive strains of Gc to resist complement-mediated killing in a serum-dependent manner. However, little is known about the contribution of sialylation to complement-independent, direct Gc-neutrophil interactions. In the absence of complement, we found sialylated Gc expressing opacity-associated (Opa) proteins decreased the oxidative burst and granule exocytosis from primary human neutrophils. In addition, sialylated Opa+ Gc survived better than vehicle treated or Δlst Gc when challenged with neutrophils. However, Gc sialylation did not significantly affect Opa-dependent association with or internalization of Gc by neutrophils. Previous studies have implicated sialic acid-binding immunoglobulin-type lectins (Siglecs) in modulating neutrophil interactions with sialylated Gc. Blocking neutrophil Siglecs with antibodies that bind to their extracellular domains eliminated the ability of sialylated Opa+ Gc to suppress oxidative burst and resist neutrophil killing. These findings highlight a new role for sialylation in Gc evasion of human innate immunity, with implications for the development of vaccines and therapeutics for gonorrhea.

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.

Bioluminescence-Based Determination of Cytosolic Accumulation of Antibiotics in Escherichia coli.

Antibiotic resistance is an alarming public health concern that affects millions of individuals across the globe each year. A major challenge in the development of effective antibiotics lies in their limited ability to permeate into cells, noting that numerous susceptible antibiotic targets reside within the bacterial cytosol. Consequently, improving cellular permeability is often a key consideration during antibiotic development, underscoring the need for reliable methods to assess the permeability of molecules across cellular membranes. Currently, methods used to measure permeability often fail to discriminate between arrival within the cytoplasm and the overall association of molecules with the cell. Additionally, these techniques typically possess throughput limitations. In this work, we describe a luciferase-based assay designed for assessing the permeability of molecules into the cytosolic compartment of Gram-negative bacteria. Our findings demonstrate a robust system that can elucidate the kinetics of intracellular antibiotics accumulation in live bacterial cells in real time.

Targeted Acidosis Mediated Delivery of Antigenic MHC-Binding Peptides.

Cytotoxic T lymphocytes are the primary effector immune cells responsible for protection against cancer, as they target peptide neoantigens presented through the major histocompatibility complex (MHC) on cancer cells, leading to cell death. Targeting peptide-MHC (pMHC) complexes offers a promising strategy for immunotherapy due to its specificity and effectiveness against cancer. In this work, we exploit the acidic tumor micro-environment to selectively deliver antigenic peptides to cancer cells using pH(low) insertion peptides (pHLIP). We demonstrated that the delivery of MHC binding peptides directly to the cytoplasm of melanoma cells resulted in the presentation of antigenic peptides on MHC, and subsequent activation of T cells. This work highlights the potential of pHLIP as a vehicle for targeted delivery of antigenic peptides and their presentation via MHC-bound complexes on cancer cell surfaces for activation of T cells with implications for enhancing anti-cancer immunotherapy.

Amidation of glutamate residues in mycobacterial peptidoglycan is essential for cell wall cross-linking.

Introduction: Mycobacteria assemble a complex cell wall with cross-linked peptidoglycan (PG) which plays an essential role in maintenance of cell wall integrity and tolerance to osmotic pressure. We previously demonstrated that various hydrolytic enzymes are required to remodel PG during essential processes such as cell elongation and septal hydrolysis. Here, we explore the chemistry associated with PG cross-linking, specifically the requirement for amidation of the D-glutamate residue found in PG precursors. Methods: Synthetic fluorescent probes were used to assess PG remodelling dynamics in live bacteria. Fluorescence microscopy was used to assess protein localization in live bacteria and CRISPR-interference was used to construct targeted gene knockdown strains. Time-lapse microscopy was used to assess bacterial growth. Western blotting was used to assess protein phosphorylation. Results and discussion: In Mycobacterium smegmatis, we confirmed the essentiality for D-glutamate amidation in PG biosynthesis by labelling cells with synthetic fluorescent PG probes carrying amidation modifications. We also used CRISPRi targeted knockdown of genes encoding the MurT-GatD complex, previously implicated in D-glutamate amidation, and demonstrated that these genes are essential for mycobacterial growth. We show that MurT-rseGFP co-localizes with mRFP-GatD at the cell poles and septum, which are the sites of cell wall synthesis in mycobacteria. Furthermore, time-lapse microscopic analysis of MurT-rseGFP localization, in fluorescent D-amino acid (FDAA)-labelled mycobacterial cells during growth, demonstrated co-localization with maturing PG, suggestive of a role for PG amidation during PG remodelling and repair. Depletion of MurT and GatD caused reduced PG cross-linking and increased sensitivity to lysozyme and ß-lactam antibiotics. Cell growth inhibition was found to be the result of a shutdown of PG biosynthesis mediated by the serine/threonine protein kinase B (PknB) which senses uncross-linked PG. Collectively, these data demonstrate the essentiality of D-glutamate amidation in mycobacterial PG precursors and highlight the MurT-GatD complex as a novel drug target.

Non-invasive Analysis of Peptidoglycan from Living Animals.

The role of the intestinal microbiota in host health is increasingly revealed in its contributions to disease states. The host-microbiome interaction is multifactorial and dynamic. One of the factors that has recently been strongly associated with host physiological responses is peptidoglycan from bacterial cell walls. Peptidoglycan from gut commensal bacteria activate peptidoglycan sensors in human cells, including the Nucleotide-binding oligomerization domain containing protein 2 (NOD2). When present in the gastrointestinal tract, both the polymeric form (sacculi) and de-polymerized fragments can modulate host physiology, including checkpoint anticancer therapy efficacy, body temperature and appetite, and postnatal growth. To leverage this growing area of biology towards therapeutic prescriptions, it will be critical to directly analyze a key feature of the host-microbiome interaction from living hosts in a reproducible and non-invasive way. Here we show that metabolically labeled peptidoglycan/sacculi can be readily isolated from fecal samples collected from both mice and humans. Analysis of fecal samples provided a non-invasive route to probe the gut commensal community including the metabolic synchronicity with the host circadian clock. Together, these results pave the way for non-invasive diagnostic tools to interrogate the causal nature of peptidoglycan in host health and disease.

A modified BCG with depletion of enzymes associated with peptidoglycan amidation induces enhanced protection against tuberculosis in mice.

Mechanisms by which Mycobacterium tuberculosis (Mtb) evades pathogen recognition receptor activation during infection may offer insights for the development of improved tuberculosis (TB) vaccines. Whilst Mtb elicits NOD-2 activation through host recognition of its peptidoglycan-derived muramyl dipeptide (MDP), it masks the endogenous NOD-1 ligand through amidation of glutamate at the second position in peptidoglycan sidechains. As the current BCG vaccine is derived from pathogenic mycobacteria, a similar situation prevails. To alleviate this masking ability and to potentially improve efficacy of the BCG vaccine, we used CRISPRi to inhibit expression of the essential enzyme pair, MurT-GatD, implicated in amidation of peptidoglycan sidechains. We demonstrate that depletion of these enzymes results in reduced growth, cell wall defects, increased susceptibility to antibiotics and altered spatial localization of new peptidoglycan. In cell culture experiments, training of monocytes with this recombinant BCG yielded improved control of Mtb growth. In the murine model of TB infection, we demonstrate that depletion of MurT-GatD in BCG, resulting in unmasking of the D-glutamate diaminopimelate (iE-DAP) NOD-1 ligand, yields superior prevention of TB disease compared to the standard BCG vaccine. This work demonstrates the feasibility of gene regulation platforms such as CRISPRi to alter antigen presentation in BCG in a bespoke manner that tunes immunity towards more effective protection against TB disease.

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.

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.