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.

Minimalist Tetrazine N-Acetyl Muramic Acid Probes for Rapid and Efficient Labeling of Commensal and Pathogenic Peptidoglycans in Living Bacterial Culture and During Macrophage Invasion.

N-Acetyl muramic acid (NAM) probes containing alkyne or azide groups are commonly used to investigate aspects of cell wall synthesis because of their small size and ability to incorporate into bacterial peptidoglycan (PG). However, copper-catalyzed alkyne-azide cycloaddition (CuAAC) reactions are not compatible with live cells, and strain-promoted alkyne-azide cycloaddition (SPAAC) reaction rates are modest and, therefore, not as desirable for tracking the temporal alterations of bacterial cell growth, remodeling, and division. Alternatively, the tetrazine-trans-cyclooctene ligation (Tz-TCO), which is the fastest known bioorthogonal reaction and not cytotoxic, allows for rapid live-cell labeling of PG at biologically relevant time scales and concentrations. Previous work to increase reaction kinetics on the PG surface by using tetrazine probes was limited because of low incorporation of the probe. Described here are new approaches to construct a minimalist tetrazine (Tz)-NAM probe utilizing recent advancements in asymmetric tetrazine synthesis. This minimalist Tz-NAM probe was successfully incorporated into pathogenic and commensal bacterial PG where fixed and rapid live-cell, no-wash labeling was successful in both free bacterial cultures and in coculture with human macrophages. Overall, this probe allows for expeditious labeling of bacterial PG, thereby making it an exceptional tool for monitoring PG biosynthesis for the development of new antibiotic screens. The versatility and selectivity of this probe will allow for real-time interrogation of the interactions of bacterial pathogens in a human host and will serve a broader utility for studying glycans in multiple complex biological systems.

Purification and Characterization of a Stable, Membrane-Associated Peptidoglycan Responsive Adenylate Cyclase LRR Domain from Human Commensal Candida albicans.

The evolutionarily conserved leucine rich repeat (LRR) protein domain is a unique structural motif found in many viral, bacterial, archaeal, and eukaryotic proteins. The LRR domain serves many roles, including being a signaling domain and a pathogen recognition receptor. In the human innate immune system, it serves an essential role by recognizing fragments of bacterial cell walls. Interestingly, the human fungal pathogen Candida albicans also uses an LRR domain-containing protein, Cyrp1, to sense bacterial cell wall fragments. However, the dynamics of signaling and detection of bacterial peptidoglycan fragments by the LRR of Cyr1p remains poorly characterized. Here we develop optimal recombinant expression workflows and provide characterization of the entire region of the LRR domain of Cyr1p as a peripheral membrane protein. Using a newly designed peptidoglycan enrichment bead assay, we demonstrate that this domain can bind bacterial peptidoglycan fragments under native conditions. The new membrane-associated Cyr1p-LRR construct sets the stage for the development of antifungal agents via high-throughput campaigns to inhibit cell wall-Cyr1p interactions.

Staphylococcus aureus resistance to albocycline can be achieved by mutations that alter cellular NAD/PH pools.

Small molecule target identification is a critical step in modern antibacterial drug discovery, particularly against multi-drug resistant pathogens. Albocycline (ALB) is a macrolactone natural product with potent activity against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant S. aureus (VRSA) whose mechanism of action has been elusive to date. Herein, we report biochemical and genomic studies that reveal ALB does not target bacterial peptidoglycan biosynthesis or the ribosome; rather, it appears to modulate NADPH ratios and upregulate redox sensing in the cell consistent with previous studies at Upjohn. Owing to the complexity inherent in biological pathways, further genomic assays are needed to identify the true molecular target(s) of albocycline.

Differential Peptidoglycan Recognition Assay Using Varied Surface Presentations.

Bacterial peptidoglycan (PG) is recognized by the human innate immune system to generate an appropriate response. To gain an appreciation of how this essential polymer is sensed, a surface plasmon resonance (SPR) assay using varied PG surface presentation was developed. PG derivatives were synthesized and immobilized on the surface at different positions on the molecule to assess effects of ligand orientation on the binding affinities of NOD-like receptors (NLRs). NLRP1 and NOD2 are cytosolic innate immune proteins known to generate an immune response to PG. Both possess conserved leucine rich repeat domains (LRR) as proposed sites of molecular recognition, though limited biochemical evidence exists regarding the mechanisms of PG recognition. Here direct biochemical evidence for the association of PG fragments to NOD2 and NLRP1 with nanomolar affinity is shown. The orientations in which the fragments were presented on the SPR surface influenced the strength of PG recognition by both NLRs. This assay displays fundamental differences in binding preferences for PG by innate immune receptors and reveals unique recognition mechanisms between the LRRs. Each receptor uses specific ligand structural features to achieve optimal binding, which will be critical information to manipulate these responses and combat diseases.

Synthesis of Bacterial-Derived Peptidoglycan Cross-Linked Fragments.

Peptidoglycan (PG) is the core structural motif of the bacterial cell wall. Fragments released from the PG serve as fundamental recognition elements for the immune system. The structure of the PG, however, encompasses a variety of chemical modifications among different bacterial species. Here, the applicability of organic synthetic methods to address this chemical diversity is explored, and the synthesis of cross-linked PG fragments, carrying biologically relevant amino acid modifications and peptide cross-linkages, is presented using solution and solid phase approaches.

Tools for probing host-bacteria interactions in the gut microenvironment: From molecular to cellular levels.

Healthy function of the gut microenvironment is dependent on complex interactions between the bacteria of the microbiome, epithelial and immune (host) cells, and the surrounding tissue. Misregulation of these interactions is implicated in disease. A range of tools have been developed to study these interactions, from mechanistic studies to therapeutic evaluation. In this Digest, we highlight select tools at the cellular and molecular level for probing specific cell-microenvironment interactions. Approaches are overviewed for controlling and probing cell-cell interactions, from transwell and microfluidic devices to engineered bacterial peptidoglycan fragments, and cell-matrix interactions, from three-dimensional scaffolds to chemical handles for in situ modifications.

Synthesis and Application of Methyl N,O-Hydroxylamine Muramyl Peptides.

The innate immune system's interaction with bacterial cells plays a pivotal role in a variety of human diseases. Carbohydrate units derived from a component of bacterial cell wall, peptidoglycan (PG), are known to stimulate an immune response. Nonetheless, access to modified late-stage peptidoglycan intermediates is limited due to their synthetic complexity. A method to rapidly functionalize PG fragments is needed to better understand the natural host-PG interactions. Here methyl N,O-hydroxylamine linkers are incorporated onto a synthetic PG derivative, muramyl dipeptide (MDP). The modification of MDP maintained the ability to stimulate a nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) immune response dependent on the expression of nucleotide-binding oligomerization domain-containing protein 2 (Nod2). Intrigued by this modification's maintenance of biological activity, several applications were explored. Methyl N,O-hydroxylamine MDP was amendable to N-hydroxylsuccinimide (NHS) chemistry for bioconjugation to fluorophores as well as a self-assembled monolayer for Nod2 surface plasmon resonance analysis. Finally, linker incorporation was applicable to larger PG fragments, both enzymatically generated from Escherichia coli or chemically synthesized. This methodology provides rapid access to PG probes in one step and allows for the installation of a variety of chemical handles to advance the molecular understanding of PG and the innate immune system.

Modulation of the NOD-like receptors NOD1 and NOD2: A chemist's perspective.

The innate immune system is the body's first defense against invading microorganisms, relying on the recognition of bacterial-derived small molecules by host protein receptors. This recognition event and downstream immune response rely heavily on the specific chemical features of both the innate immune receptors and their bacterial derived ligands. This review presents a chemist's perspective on some of the most crucial and complex components of two receptors (NOD1 and NOD2): starting from the structural and chemical characteristics of bacterial-derived small molecules, to the specific proposed models of molecular recognition of these molecules by immune receptors, to the subsequent post-translational modifications that ultimately dictate downstream immune signaling. Recent advances in the field are discussed, as well as the potential for the development of targeted therapeutics.

Synthesis of Functionalized N-Acetyl Muramic Acids To Probe Bacterial Cell Wall Recycling and Biosynthesis.

Uridine diphosphate N-acetyl muramic acid (UDP NAM) is a critical intermediate in bacterial peptidoglycan (PG) biosynthesis. As the primary source of muramic acid that shapes the PG backbone, modifications installed at the UDP NAM intermediate can be used to selectively tag and manipulate this polymer via metabolic incorporation. However, synthetic and purification strategies to access large quantities of these PG building blocks, as well as their derivatives, are challenging. A robust chemoenzymatic synthesis was developed using an expanded NAM library to produce a variety of 2 -N-functionalized UDP NAMs. In addition, a synthetic strategy to access bio-orthogonal 3-lactic acid NAM derivatives was developed. The chemoenzymatic UDP synthesis revealed that the bacterial cell wall recycling enzymes MurNAc/GlcNAc anomeric kinase (AmgK) and NAM α-1 phosphate uridylyl transferase (MurU) were permissive to permutations at the two and three positions of the sugar donor. We further explored the utility of these derivatives in the fluorescent labeling of both Gram (-) and Gram (+) PG in whole cells using a variety of bio-orthogonal chemistries including the tetrazine ligation. This report allows for rapid and scalable access to a variety of functionalized NAMs and UDP NAMs, which now can be used in tandem with other complementary bio-orthogonal labeling strategies to address fundamental questions surrounding PG's role in immunology and microbiology.

Elucidating the inhibition of peptidoglycan biosynthesis in Staphylococcus aureus by albocycline, a macrolactone isolated from Streptomyces maizeus.

Antibiotic resistance is a serious threat to global public health, and methicillin-resistant Staphylococcus aureus (MRSA) is a poignant example. The macrolactone natural product albocycline, derived from various Streptomyces strains, was recently identified as a promising antibiotic candidate for the treatment of both MRSA and vancomycin-resistant S. aureus (VRSA), which is another clinically relevant and antibiotic resistant strain. Moreover, it was hypothesized that albocycline's antimicrobial activity was derived from the inhibition of peptidoglycan (i.e., bacterial cell wall) biosynthesis. Herein, preliminary mechanistic studies are performed to test the hypothesis that albocycline inhibits MurA, the enzyme that catalyzes the first step of peptidoglycan biosynthesis, using a combination of biological assays alongside molecular modeling and simulation studies. Computational modeling suggests albocycline exists as two conformations in solution, and computational docking of these conformations to an ensemble of simulated receptor structures correctly predicted preferential binding to S. aureus MurA-the enzyme that catalyzes the first step of peptidoglycan biosynthesis-over Escherichia coli (E. coli) MurA. Albocycline isolated from the producing organism (Streptomyces maizeus) weakly inhibited S. aureus MurA (IC50 of 480 µM) but did not inhibit E. coli MurA. The antimicrobial activity of albocycline against resistant S. aureus strains was superior to that of vancomycin, preferentially inhibiting Gram-positive organisms. Albocycline was not toxic to human HepG2 cells in MTT assays. While these studies demonstrate that albocycline is a promising lead candidate against resistant S. aureus, taken together they suggest that MurA is not the primary target, and further work is necessary to identify the major biological target.

Crohn's Disease Variants of Nod2 Are Stabilized by the Critical Contact Region of Hsp70.

Nod2 is a cytosolic, innate immune receptor responsible for binding to bacterial cell wall fragments such as muramyl dipeptide (MDP). Upon binding, subsequent downstream activation of the NF-κB pathway leads to an immune response. Nod2 mutations are correlated with an increased susceptibility to Crohn's disease (CD) and ultimately result in a misregulated immune response. Previous work had demonstrated that Nod2 interacts with and is stabilized by the molecular chaperone Hsp70. In this work, it is shown using purified protein and in vitro biochemical assays that the critical Nod2 CD mutations (G908R, R702W, and 1007fs) preserve the ability to bind bacterial ligands. A limited proteolysis assay and luciferase reporter assay reveal regions of Hsp70 that are capable of stabilizing Nod2 and rescuing CD mutant activity. A minimal 71-amino acid subset of Hsp70 that stabilizes the CD-associated variants of Nod2 and restores a proper immune response upon activation with MDP was identified. This work suggests that CD-associated Nod2 variants could be stabilized in vivo with a molecular chaperone.

Postsynthetic Modification of Bacterial Peptidoglycan Using Bioorthogonal N-Acetylcysteamine Analogs and Peptidoglycan O-Acetyltransferase B.

Bacteria have the natural ability to install protective postsynthetic modifications onto its bacterial peptidoglycan (PG), the coat woven into bacterial cell wall. Peptidoglycan O-acetyltransferase B (PatB) catalyzes the O-acetylation of PG in Gram (-) bacteria, which aids in bacterial survival, as it prevents autolysins such as lysozyme from cleaving the PG. We explored the mechanistic details of PatB's acetylation function and determined that PatB has substrate specificity for bioorthgonal short N-acetyl cysteamine (SNAc) donors. A variety of functionality including azides and alkynes were installed on tri-N-acetylglucosamine (NAG)3, a PG mimic, as well as PG isolated from various Gram (+) and Gram (-) bacterial species. The bioorthogonal modifications protect the isolated PG against lysozyme degradation in vitro. We further demonstrate that this postsynthetic modification of PG can be extended to use click chemistry to fluorescently label the mature PG in whole bacterial cells of Bacillus subtilis. Modifying PG postsynthetically can aid in the development of antibiotics and immune modulators by expanding the understanding of how PG is processed by lytic enzymes.

Application of bioanalytical and computational methods in decoding the roles of glycans in host-pathogen interactions.

Host-pathogen interactions (HPIs) are complex processes that require tight regulation. A common regulatory mechanism of HPIs is through glycans of either host cells or pathogens. Due to their diverse sequences, complex structures, and conformations, studies of glycans require highly sensitive and powerful tools. Recent improvements in technology have enabled the application of many bioanalytical techniques and modeling methods to investigate glycans and their mechanisms in HPIs. This mini-review highlights how these advances have been used to understand the role glycans play in HPIs in the past 2 years.

Synthesis and validation of click-modified NOD1/2 agonists.

NOD1 and NOD2 sense small bacterial peptidoglycan fragments, often called muropeptides, that access the cytosol. These muropeptides include iE-DAP and MDP, the minimal agonists for NOD1 and NOD2, respectively. Here, we synthesized and validated alkyne-modified muropeptides, iE-DAP-Alk and MDP-Alk, for use in click-chemistry reactions. While it has long been known that many cell types respond to extracellular exposure to muropeptides, it is unclear how these innate immune activators access their cytosolic innate immune receptors, NOD1 and NOD2. The subcellular trafficking and transport mechanisms by which muropeptides access these cytosolic innate immune receptors are a major gap in our understanding of these critical host responses. The click-chemistry-enabled agonists developed here will be particularly powerful to decipher the underlying cell biology and biochemistry of NOD1 and NOD2 innate immune sensing.

Multiscale Invasion Assay for Probing Macrophage Response to Gram-Negative Bacteria.

The immune system is a complex network of various cellular components that must differentiate between pathogenic bacteria and the commensal bacteria of the human microbiome, where misrecognition is linked to inflammatory disorders. Fragments of bacterial cell wall peptidoglycan bind to pattern recognition receptors within macrophages, leading to immune activation. To study this complex process, a methodology to remodel and label the bacterial cell wall of two different species of bacteria was established using copper (I) catalyzed azide-alkyne cycloaddition (CuAAC) and strain-promoted azide-alkyne cycloaddition (SPAAC). Additionally, an approach for three-dimensional (3D) culture of human macrophages and their invasion with relevant bacteria in a well-defined hydrogel-based synthetic matrix inspired by the microenvironment of the gut was established. Workflows were developed for human monocyte encapsulation and differentiation into macrophages in 3D culture with high viability. Bacteria invaded into macrophages permitted in situ peptidoglycan labeling. Macrophages exhibited biologically-relevant cytokine release in response to bacteria. This molecularly engineered, multi-dimensional bacteria-macrophage co-culture system will prove useful in future studies to observe immunostimulatory, bacterial fragment production and localization in the cell at the carbohydrate level for insights into how the immune system properly senses bacteria.

Investigating Peptidoglycan Recycling Pathways in Tannerella forsythia with N-Acetylmuramic Acid Bioorthogonal Probes.

The human oral microbiome is the second largest microbial community in humans, harboring over 700 bacterial species, which aid in digestion and protect from growth of disease-causing pathogens. One such oral pathogen, Tannerella forsythia, along with other species, contributes to the pathogenesis of periodontitis. T. forsythia is unable to produce its own N-acetylmuramic acid (NAM) sugar, essential for peptidoglycan biosynthesis and therefore must scavenge NAM from other species with which it cohabitates. Here, we explore the recycling potential of T. forsythia for NAM uptake with a bioorthogonal modification into its peptidoglycan, allowing for click-chemistry-based visualization of the cell wall structure. Additionally, we identified NAM recycling enzyme homologues in T. forsythia that are similar to the enzymes found in Pseudomonas putida. These homologues were then genetically transformed into a laboratory safe Escherichia coli strain, resulting in the efficient incorporation of unnatural NAM analogues into the peptidoglycan backbone and its visualization, alone or in the presence of human macrophages. This strain will be useful in further studies to probe NAM recycling and peptidoglycan scavenging pathways of T. forsythia and other cohabiting bacteria.

A two-track model for the spatiotemporal coordination of bacterial septal cell wall synthesis revealed by single-molecule imaging of FtsW.

Synthesis of septal peptidoglycan (sPG) is crucial for bacterial cell division. FtsW, an indispensable component of the cell division machinery in all walled bacterial species, was recently identified in vitro as a peptidoglycan glycosyltransferase (PGTase). Despite its importance, the septal PGTase activity of FtsW has not been demonstrated in vivo. How its activity is spatiotemporally regulated in vivo has also remained elusive. Here, we confirmed FtsW as an essential septum-specific PGTase in vivo using an N-acetylmuramic acid analogue incorporation assay. Next, using single-molecule tracking coupled with genetic manipulations, we identified two populations of processively moving FtsW molecules: a fast-moving population correlated with the treadmilling dynamics of the essential cytoskeletal FtsZ protein and a slow-moving population dependent on active sPG synthesis. We further identified that FtsN, a potential sPG synthesis activator, plays an important role in promoting the slow-moving population. Our results suggest a two-track model, in which inactive sPG synthases follow the 'Z-track' to be distributed along the septum and FtsN promotes their release from the Z-track to become active in sPG synthesis on the slow 'sPG-track'. This model provides a mechanistic framework for the spatiotemporal coordination of sPG synthesis in bacterial cell division.

Localizing Peptidoglycan Synthesis in Helicobacter pylori using Clickable Metabolic Probes.

The bacterial cell wall, composed of peptidoglycan (PG), provides structural integrity for the cell and is responsible for cell shape in most bacteria. Here we present tools to study the cell wall using a clickable PG-specific sugar, 2-alkyne muramic acid (MurNAc-alk), as a metabolic probe. Here we present a new reaction pathway for generating MurNAc-alk. We also include protocols for labeling PG synthesis in Helicobacter pylori, determining the identity of the labeled muropeptides using LC-MS/MS, sample preparation of cells labeled for a short fraction of the doubling time, and visualization using 3D structured illumination microscopy. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Alternative synthesis of MurNAc-alk (direct coupling) Support Protocol 1: Growing Helicobacter pylori in liquid culture Support Protocol 2: Fosfomycin rescue assay Basic Protocol 2: Mass spectrometry (MS) analysis to determine incorporation of MurNAc-alk within the peptidoglycan of H. pylori Support Protocol 3: Hayashi test to determine if SDS is present in the supernatant of peptidoglycan preparations Support Protocol 4: Creating custom cytocentrifuge units for use in a swinging-bucket tabletop centrifuge Basic Protocol 3: Labeling H. pylori with MurNAc-alk or D-Ala-alk Basic Protocol 4: Structured illumination microscopy (SIM) imaging on the DeltaVision OMX.

Bacterial Peptidoglycan Fragments Differentially Regulate Innate Immune Signaling.

The human innate immune system responds to both pathogen and commensal bacteria at the molecular level using bacterial peptidoglycan (PG) recognition elements. Traditionally, synthetic and commercially accessible PG monosaccharide units known as muramyl dipeptide (MDP) and N-glycolyl MDP (ng-MDP) have been used to probe the mechanism of innate immune activation of pattern recognition receptors, such as NOD-like receptors. However, bacterial PG is a dynamic and complex structure, with various chemical modifications and trimming mechanisms that result in the production of disaccharide-containing elements. These molecules pose as attractive targets for immunostimulatory screening; however, studies are limited because of their synthetic accessibility. Inspired by disaccharide-containing compounds produced from the gut microbe Lactobacillus acidophilus, a robust and scalable chemical synthesis of PG-based disaccharide ligands was implemented. Together with a monosaccharide PG library, compounds were screened for their ability to stimulate proinflammatory genes in bone-marrow-derived macrophages. The data reveal distinct gene induction patterns for monosaccharide and disaccharide PG units, suggesting that PG innate immune signaling is more complex than a one activator-one pathway program, as biologically relevant fragments induce transcriptional programs to different degrees. These disaccharide molecules will serve as critical immunostimulatory tools to more precisely define specialized innate immune regulatory mechanisms that distinguish between commensal and pathogenic bacteria residing in the microbiome.