Massively parallel combination screen reveals small molecule sensitization of antibiotic-resistant Gram-negative ESKAPE pathogens.

Antibiotic resistance, especially in multidrug-resistant ESKAPE pathogens, remains a worldwide problem. Combination antimicrobial therapies may be an important strategy to overcome resistance and broaden the spectrum of existing antibiotics. However, this strategy is limited by the ability to efficiently screen large combinatorial chemical spaces. Here, we deployed a high-throughput combinatorial screening platform, DropArray, to evaluate the interactions of over 30,000 compounds with up to 22 antibiotics and 6 strains of Gram-negative ESKAPE pathogens, totaling to over 1.3 million unique strain-antibiotic-compound combinations. In this dataset, compounds more frequently exhibited synergy with known antibiotics than single-agent activity. We identified a compound, P2-56, and developed a more potent analog, P2-56-3, which potentiated rifampin (RIF) activity against Acinetobacter baumannii and Klebsiella pneumoniae. Using phenotypic assays, we showed P2-56-3 disrupts the outer membrane of A. baumannii. To identify pathways involved in the mechanism of synergy between P2-56-3 and RIF, we performed genetic screens in A. baumannii. CRISPRi-induced partial depletion of lipooligosaccharide transport genes (lptA-D, lptFG) resulted in hypersensitivity to P2-56-3/RIF treatment, demonstrating the genetic dependency of P2-56-3 activity and RIF sensitization on lpt genes in A. baumannii. Consistent with outer membrane homeostasis being an important determinant of P2-56-3/RIF tolerance, knockout of maintenance of lipid asymmetry complex genes and overexpression of certain resistance-nodulation-division efflux pumps - a phenotype associated with multidrug-resistance - resulted in hypersensitivity to P2-56-3. These findings demonstrate the immense scale of phenotypic antibiotic combination screens using DropArray and the potential for such approaches to discover new small molecule synergies against multidrug-resistant ESKAPE strains.

Genetic factors affecting storage and utilization of lipids during dormancy in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) can adopt a non-growing dormant state during infection that may be critical to both active and latent tuberculosis. During dormancy, Mtb is widely tolerant toward antibiotics, a significant obstacle in current anti-tubercular drug regimens, and retains the ability to persist in its environment. We aimed to identify novel mechanisms that permit Mtb to survive dormancy in an in vitro carbon starvation model using transposon insertion sequencing and gene expression analysis. We identified a previously uncharacterized component of the lipid transport machinery, omamC, which was upregulated and required for survival during carbon starvation. We show that OmamC plays a role both in increasing fatty acid stores during growth in rich media and enhancing fatty acid utilization during starvation. Besides its involvement in lipid metabolism, OmamC levels affected the expression of the anti-anti-sigma factor rv0516c and other genes to improve Mtb survival during carbon starvation and increase its tolerance toward rifampicin, a first-line drug effective against non-growing Mtb. Importantly, we show that Mtb can be eradicated during carbon starvation, in an OmamC-dependent manner, by inhibiting lipid metabolism with the lipase inhibitor tetrahydrolipstatin. This work casts new light into the survival processes of non-replicating, drug-tolerant Mtb by identifying new proteins involved in lipid metabolism required for the survival of dormant bacteria and exposing a potential vulnerability that could be exploited for antibiotic discovery.IMPORTANCETuberculosis is a global threat, with ~10 million yearly active cases. Many more people, however, live with "latent" infection, where Mycobacterium tuberculosis survives in a non-replicative form. When latent bacteria activate and regrow, they elicit immune responses and result in significant host damage. Replicating and non-growing bacilli can co-exist; however, non-growing bacteria are considerably less sensitive to antibiotics, thus complicating treatment by necessitating long treatment durations. Here, we sought to identify genes important for bacterial survival in this non-growing state using a carbon starvation model. We found that a previously uncharacterized gene, omamC, is involved in storing and utilizing fatty acids as bacteria transition between these two states. Importantly, inhibiting lipid metabolism using a lipase inhibitor eradicates non-growing bacteria. Thus, targeting lipid metabolism may be a viable strategy for treating the non-growing population in strategies to shorten treatment durations of tuberculosis.

Glycan-costumed virus-like particles promote type 1 anti-tumor immunity.

Cancer vaccine development is inhibited by a lack of strategies for directing dendritic cell (DC) induction of effective tumor-specific cellular immunity. Pathogen engagement of DC lectins and toll-like receptors (TLRs) shapes immunity by directing T cell function. Strategies to activate specific DC signaling pathways via targeted receptor engagement are crucial to unlocking type 1 cellular immunity. Here, we engineered a glycan-costumed virus-like particle (VLP) vaccine that delivers programmable peptide antigens to induce tumor-specific cellular immunity in vivo. VLPs encapsulating TLR7 agonists and decorated with a selective mannose-derived ligand for the lectin DC-SIGN induced robust DC activation and type 1 cellular immunity, whereas VLPs lacking this key DC-SIGN ligand failed to promote DC-mediated immunity. Vaccination with glycan-costumed VLPs generated tumor antigen-specific Th1 CD4+ and CD8+ T cells that infiltrated solid tumors, inhibiting tumor growth in a murine melanoma model. Thus, VLPs employing lectin-driven immune reprogramming provide a framework for advancing cancer immunotherapies.

Selective Glycan Labeling of Mannose-Containing Glycolipids in Mycobacteria.

Mycobacterium tuberculosis (Mtb) is one of history's most successful human pathogens. By subverting typical immune responses, Mtb can persist within a host until conditions become favorable for growth and proliferation. Virulence factors that enable mycobacteria to modulate host immune systems include a suite of mannose-containing glycolipids: phosphatidylinositol mannosides, lipomannan, and lipoarabinomannan (LAM). Despite their importance, tools for their covalent capture, modification, and imaging are limited. Here, we describe a chemical biology strategy to detect and visualize these glycans. Our approach, biosynthetic incorporation, is to synthesize a lipid-glycan precursor that can be incorporated at a late-stage step in glycolipid biosynthesis. We previously demonstrated selective mycobacterial arabinan modification by biosynthetic incorporation using an exogenous donor. This report reveals that biosynthetic labeling is general and selective: it allows for cell surface mannose-containing glycolipid modification without nonspecific labeling of mannosylated glycoproteins. Specifically, we employed azido-(Z,Z)-farnesyl phosphoryl-ß-d-mannose probes and took advantage of the strain-promoted azide-alkyne cycloaddition to label and directly visualize the localization and dynamics of mycobacterial mannose-containing glycolipids. Our studies highlight the generality and utility of biosynthetic incorporation as the probe structure directs the selective labeling of distinct glycans. The disclosed agents allowed for direct tracking of the target immunomodulatory glycolipid dynamics in cellulo. We anticipate that these probes will facilitate investigating the diverse biological roles of these glycans.

Assessing Squarates as Amine-Reactive Probes.

Probes that covalently label protein targets facilitate the identification of ligand-binding sites. Lysine residues are prevalent in the proteome, making them attractive substrates for covalent probes. However, identifying electrophiles that undergo amine-specific, regioselective reactions with binding site lysine residues is challenging. Squarates can engage in two sequential conjugate addition-elimination reactions with amines. Nitrogen donation reduces the second reaction rate, making the mono squaramide a mild electrophile. We postulated that this mild electrophilicity would demand a longer residence time near the amine, affording higher selectivity for binding site lysines. Therefore, we compared the kinetics of squarate and monosquaramide amine substitution to alternative amine bioconjugation handles. The data revealed that N-hydroxy succinimidyl esters react 4 orders of magnitude faster, consistent with their labeling promiscuity. Squarate reactivity can be tuned by a substitution pattern. Electron-withdrawing groups on the vinylogous ester or amide increase reaction rates. Dithionosquarates react more rapidly than squarates, while vinylogous thioester analogs, dithiosquarates, react more slowly. We assessed squarate selectively using the UDP-sugar processing enzyme GlfT2 from Mycobacterium tuberculosis, which possesses 21 surface-exposed lysines. The reaction predominately modified one lysine proximal to a binding site to afford covalent inhibition. These findings demonstrate the selectivity of squaric esters and squaramides, which is a critical feature for affinity-based chemoproteomic probes.

Lectin-Seq: A method to profile lectin-microbe interactions in native communities.

Soluble human lectins are critical components of innate immunity. Genetic models suggest that lectins influence host-resident microbiota, but their specificity for commensal and mutualist species is understudied. Elucidating lectins' roles in regulating microbiota requires an understanding of which microbial species they bind within native communities. To profile human lectin recognition, we developed Lectin-Seq. We apply Lectin-Seq to human fecal microbiota using the soluble mannose-binding lectin (MBL) and intelectin-1 (hItln1). Although each lectin binds a substantial percentage of the samples (10 to 20%), the microbial interactomes of MBL and hItln1 differ markedly in composition and diversity. MBL binding is highly selective for a small subset of species commonly associated with humans. In contrast, hItln1's interaction profile encompasses a broad range of lower-abundance species. Our data uncover stark differences in the commensal recognition properties of human lectins.

Lectin Fingerprinting Distinguishes Antibody Neutralization in SARS-CoV-2.

Enveloped viruses co-opt host glycosylation pathways to decorate their surface proteins. As viruses evolve, emerging strains can modify their glycosylation patterns to influence host interactions and subvert immune recognition. Still, changes in viral glycosylation or their impact on antibody protection cannot be predicted from genomic sequences alone. Using the highly glycosylated SARS-CoV-2 Spike protein as a model system, we present a lectin fingerprinting method that rapidly reports on changes in variant glycosylation state, which are linked to antibody neutralization. In the presence of antibodies or convalescent and vaccinated patient sera, unique lectin fingerprints emerge that distinguish neutralizing versus non-neutralizing antibodies. This information could not be inferred from direct binding interactions between antibodies and the Spike receptor-binding domain (RBD) binding data alone. Comparative glycoproteomics of the Spike RBD of wild-type (Wuhan-Hu-1) and Delta (B.1.617.2) variants reveal O-glycosylation differences as a key determinant of immune recognition differences. These data underscore the interplay between viral glycosylation and immune recognition and reveal lectin fingerprinting to be a rapid, sensitive, and high-throughput assay to distinguish the neutralization potential of antibodies that target critical viral glycoproteins.

Human oral lectin ZG16B acts as a cell wall polysaccharide probe to decode host-microbe interactions with oral commensals.

The oral microbiome is critical to human health and disease, yet the role that host salivary proteins play in maintaining oral health is unclear. A highly expressed gene in human salivary glands encodes the lectin zymogen granule protein 16 homolog B (ZG16B). Despite the abundance of this protein, its interaction partners in the oral microbiome are unknown. ZG16B possesses a lectin fold, but whether it binds carbohydrates is unclear. We postulated that ZG16B would bind microbial glycans to mediate recognition of oral microbes. To this end, we developed a microbial glycan analysis probe (mGAP) strategy based on conjugating the recombinant protein to fluorescent or biotin reporter functionality. Applying the ZG16B-mGAP to dental plaque isolates revealed that ZG16B predominantly binds to a limited set of oral microbes, including Streptococcus mitis, Gemella haemolysans, and, most prominently, Streptococcus vestibularis. S. vestibularis is a commensal bacterium widely distributed in healthy individuals. ZG16B binds to S. vestibularis through the cell wall polysaccharides attached to the peptidoglycan, indicating that the protein is a lectin. ZG16B slows the growth of S. vestibularis with no cytotoxicity, suggesting that it regulates S. vestibularis abundance. The mGAP probes also revealed that ZG16B interacts with the salivary mucin MUC7. Analysis of S. vestibularis and MUC7 with ZG16B using super-resolution microscopy supports ternary complex formation that can promote microbe clustering. Together, our data suggest that ZG16B influences the compositional balance of the oral microbiome by capturing commensal microbes and regulating their growth using a mucin-assisted clearance mechanism.

Identification of D-arabinan-degrading enzymes in mycobacteria.

Bacterial cell growth and division require the coordinated action of enzymes that synthesize and degrade cell wall polymers. Here, we identify enzymes that cleave the D-arabinan core of arabinogalactan, an unusual component of the cell wall of Mycobacterium tuberculosis and other mycobacteria. We screened 14 human gut-derived Bacteroidetes for arabinogalactan-degrading activities and identified four families of glycoside hydrolases with activity against the D-arabinan or D-galactan components of arabinogalactan. Using one of these isolates with exo-D-galactofuranosidase activity, we generated enriched D-arabinan and used it to identify a strain of Dysgonomonas gadei as a D-arabinan degrader. This enabled the discovery of endo- and exo-acting enzymes that cleave D-arabinan, including members of the DUF2961 family (GH172) and a family of glycoside hydrolases (DUF4185/GH183) that display endo-D-arabinofuranase activity and are conserved in mycobacteria and other microbes. Mycobacterial genomes encode two conserved endo-D-arabinanases with different preferences for the D-arabinan-containing cell wall components arabinogalactan and lipoarabinomannan, suggesting they are important for cell wall modification and/or degradation. The discovery of these enzymes will support future studies into the structure and function of the mycobacterial cell wall.

Dynamic Changes in Heparan Sulfate Nanostructure in Human Pluripotent Stem Cell Differentiation.

Heparan sulfate (HS) is a heterogeneous, cell-surface polysaccharide critical for transducing signals essential for mammalian development. Imaging of signaling proteins has revealed how their localization influences their information transfer. In contrast, the contribution of the spatial distribution and nanostructure of information-rich, signaling polysaccharides like HS is not known. Using expansion microscopy (ExM), we found striking changes in HS nanostructure occur as human pluripotent stem (hPS) cells differentiate, and these changes correlate with growth factor signaling. Our imaging studies show that undifferentiated hPS cells are densely coated with HS displayed as hair-like protrusions. This ultrastructure can recruit fibroblast growth factor for signaling. When the hPS cells differentiate into the ectoderm lineage, HS is localized into dispersed puncta. This striking change in HS distribution coincides with a decrease in fibroblast growth factor binding to neural cells. While developmental variations in HS sequence were thought to be the primary driver of alterations in HS-mediated growth factor signaling, our high-resolution images indicate a role for the HS nanostructure. Our study highlights the utility of high-resolution glycan imaging using ExM. In the case of HS, we found that changes in how the polysaccharide is displayed link to profound differences in growth factor binding.

Rapid Single-Shot Synthesis of the 214 Amino Acid-Long N-Terminal Domain of Pyocin S2.

The impermeable outer membrane of Pseudomonas aeruginosa is bypassed by antibacterial proteins known as S-type pyocins. Because of their properties, pyocins are investigated as a potential new class of antimicrobials against Pseudomonas infections. Their production and modification, however, remain challenging. To address this limitation, we employed automated fast-flow peptide synthesis for the rapid production of a pyocin S2 import domain. The N-terminal domain sequence (PyS2NTD) was synthesized in under 10 h and purified to yield milligram quantities of the desired product. To our knowledge, the 214 amino acid sequence of PyS2NTD is among the longest peptides produced from a "single-shot" synthesis, i.e., made in a single stepwise route without the use of ligation techniques. Biophysical characterization of the PyS2NTD with circular dichroism was consistent with the literature reports. Fluorescently labeled PyS2NTD binds to P. aeruginosa expressing the cognate ferripyoverdine receptor and is taken up into the periplasm. This selective uptake was validated with confocal and super resolution microscopy, flow cytometry, and fluorescence recovery after photobleaching. These modified, synthetic S-type pyocin domains can be used to probe import mechanisms of P. aeruginosa and leveraged to develop selective antimicrobial agents that bypass the outer membrane.

Mycobacterial nucleoid-associated protein Lsr2 is required for productive mycobacteriophage infection.

Mycobacteriophages are a diverse group of viruses infecting Mycobacterium with substantial therapeutic potential. However, as this potential becomes realized, the molecular details of phage infection and mechanisms of resistance remain ill-defined. Here we use live-cell fluorescence microscopy to visualize the spatiotemporal dynamics of mycobacteriophage infection in single cells and populations, showing that infection is dependent on the host nucleoid-associated Lsr2 protein. Mycobacteriophages preferentially adsorb at Mycobacterium smegmatis sites of new cell wall synthesis and following DNA injection, Lsr2 reorganizes away from host replication foci to establish zones of phage DNA replication (ZOPR). Cells lacking Lsr2 proceed through to cell lysis when infected but fail to generate consecutive phage bursts that trigger epidemic spread of phage particles to neighbouring cells. Many mycobacteriophages code for their own Lsr2-related proteins, and although their roles are unknown, they do not rescue the loss of host Lsr2.

Surface Presentation of Hyaluronic Acid Modulates Nanoparticle-Cell Association.

Nanoparticle (NP) drug carriers have revolutionized medicine and increased patient quality of life. Clinically approved formulations typically succeed because of reduced off-target toxicity of the cargo. However, increasing carrier accumulation at disease sites through precise targeting remains one of the biggest challenges in the field. Novel multivalent ligand presentations and self-assembled constructs can enhance cell association, but an inability to draw direct comparisons across formulations has hindered progress. Furthermore, how nanoparticle structure influences function often is unclear. In this report, we leverage the well-characterized hyaluronic acid (HA)-CD44 binding pair to investigate how the surface architecture of modified NPs impacts their association with ovarian cancer cells that overexpress CD44. We functionalized anionic liposomes with 5 kDa HA by either covalent conjugation via surface coupling or electrostatic self-assembly using the layer-by-layer (LbL) adsorption method. Comparing these two methods, we observed a consistent enhancement of NP-cell association with the self-assembly LbL technique, particularly with higher molecular weight (≥10 kDa) HA. To further optimize association, we increased the surface-available HA. We synthesized a bottlebrush glycopolymer composed of a polynorbornene backbone and pendant 5 kDa HA and layered this macromolecule onto NPs. Flow cytometry revealed that the LbL HA bottlebrush NP outperformed the LbL linear display of HA. Cellular visualization by deconvolution optical microscopy corroborated results from all three constructs. Using exogenous HA to block NP-CD44 interactions, we found the LbL HA bottlebrush NP had a 4-fold higher binding avidity than the best-performing LbL linear HA NP. We further observed that decreasing the density of HA bottlebrush side chains to 75% had minimal impact on LbL NP stability or cell association, though we did see a reduction in binding avidity with this side-chain-modified NP. Our studies indicate that LbL surfaces are highly effective for multivalent displays, and the mode in which they present a targeting ligand can be optimized for NP cell targeting.

Virtual screening for small-molecule pathway regulators by image-profile matching.

Identifying the chemical regulators of biological pathways is a time-consuming bottleneck in developing therapeutics and research compounds. Typically, thousands to millions of candidate small molecules are tested in target-based biochemical screens or phenotypic cell-based screens, both expensive experiments customized to each disease. Here, our uncustomized, virtual, profile-based screening approach instead identifies compounds that match to pathways based on the phenotypic information in public cell image data, created using the Cell Painting assay. Our straightforward correlation-based computational strategy retrospectively uncovered the expected, known small-molecule regulators for 32% of positive-control gene queries. In prospective, discovery mode, we efficiently identified new compounds related to three query genes and validated them in subsequent gene-relevant assays, including compounds that phenocopy or pheno-oppose YAP1 overexpression and kill a Yap1-dependent sarcoma cell line. This image-profile-based approach could replace many customized labor- and resource-intensive screens and accelerate the discovery of biologically and therapeutically useful compounds.

Biosynthetic incorporation for visualizing bacterial glycans.

Cell-surface glycans are central to many biological processes, yet methods for their site-selective modification are limited. Strategies for interrogating the structure and function of proteins have been enabled by chemoselective reactions of sidechain functionality for covalent modification, capture, or imaging. However, unlike protein sidechains, glycan building blocks lack distinguishing reactivity. Moreover, glycans are not primary gene products, so encoding glycan variants through genetic manipulation is challenging. Reactive functional groups can be introduced into glycans through metabolic engineering, which involves the generation of modified nucleotide-sugar building blocks. Lipid-linked building blocks, which are also used in glycan biosynthesis, have the advantage that they can be delivered directly to glycosyltransferases to function as surrogate substrates. This process, termed "biosynthetic incorporation," takes advantage of the properties of bacterial glycosyltransferase: they are selective for the products they generate yet promiscuous in their donor preferences. We describe how this strategy can be implemented to label arabinofuranose-containing glycans on the surface of mycobacterial cells. We anticipate that this platform can be expanded to develop chemoselective labeling agents for other important bacterial monosaccharides.

Bifunctional Peptide that Anneals to Damaged Collagen and Clusters TGF-ß Receptors Enhances Wound Healing.

Transforming growth factor-ß (TGF-ß) plays important roles in wound healing. The activity of TGF-ß is initiated upon the binding of the growth factor to the extracellular domains of its receptors. We sought to facilitate the activation by clustering these extracellular domains. To do so, we used a known peptide that binds to TGF-ß receptors without diminishing their affinity for TGF-ß. We conjugated this peptide to a collagen-mimetic peptide that can anneal to the damaged collagen in a wound bed. We find that the conjugate enhances collagen deposition and wound closure in mice in a manner consistent with the clustering of TGF-ß receptors. This strategy provides a means to upregulate the TGF-ß signaling pathway without adding exogenous TGF-ß and could inspire means to treat severe wounds.

Synthetic Glycomacromolecules of Defined Valency, Absolute Configuration, and Topology Distinguish between Human Lectins.

Carbohydrate-binding proteins (lectins) play vital roles in cell recognition and signaling, including pathogen binding and innate immunity. Thus, targeting lectins, especially those on the surface of immune cells, could advance immunology and drug discovery. Lectins are typically oligomeric; therefore, many of the most potent ligands are multivalent. An effective strategy for lectin targeting is to display multiple copies of a single glycan epitope on a polymer backbone; however, a drawback to such multivalent ligands is they cannot distinguish between lectins that share monosaccharide binding selectivity (e.g., mannose-binding lectins) as they often lack molecular precision. Here, we describe the development of an iterative exponential growth (IEG) synthetic strategy that enables facile access to synthetic glycomacromolecules with precisely defined and tunable sizes up to 22.5 kDa, compositions, topologies, and absolute configurations. Twelve discrete mannosylated "glyco-IEGmers" are synthesized and screened for binding to a panel of mannoside-binding immune lectins (DC-SIGN, DC-SIGNR, MBL, SP-D, langerin, dectin-2, mincle, and DEC-205). In many cases, the glyco-IEGmers had distinct length, stereochemistry, and topology-dependent lectin-binding preferences. To understand these differences, we used molecular dynamics and density functional theory simulations of octameric glyco-IEGmers, which revealed dramatic effects of glyco-IEGmer stereochemistry and topology on solution structure and reveal an interplay between conformational diversity and chiral recognition in selective lectin binding. Ligand function also could be controlled by chemical substitution: by tuning the side chains of glyco-IEGmers that bind DC-SIGN, we could alter their cellular trafficking through alteration of their aggregation state. These results highlight the power of precision synthetic oligomer/polymer synthesis for selective biological targeting, motivating the development of next-generation glycomacromolecules tailored for specific immunological or other therapeutic applications.