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.

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.

Protected N-Acetyl Muramic Acid Probes Improve Bacterial Peptidoglycan Incorporation via Metabolic Labeling.

Metabolic glycan probes have emerged as an excellent tool to investigate vital questions in biology. Recently, methodology to incorporate metabolic bacterial glycan probes into the cell wall of a variety of bacterial species has been developed. In order to improve this method, a scalable synthesis of the peptidoglycan precursors is developed here, allowing for access to essential peptidoglycan immunological fragments and cell wall building blocks. The question was asked if masking polar groups of the glycan probe would increase overall incorporation, a common strategy exploited in mammalian glycobiology. Here, we show, through cellular assays, that E. coli do not utilize peracetylated peptidoglycan substrates but do employ methyl esters. The 10-fold improvement of probe utilization indicates that (i) masking the carboxylic acid is favorable for transport and (ii) bacterial esterases are capable of removing the methyl ester for use in peptidoglycan biosynthesis. This investigation advances bacterial cell wall biology, offering a prescription on how to best deliver and utilize bacterial metabolic glycan probes.

Chemical Biology Tools for Examining the Bacterial Cell Wall.

Bacteria surround themselves with cell walls to maintain cell rigidity and protect against environmental insults. Here we review chemical and biochemical techniques employed to study bacterial cell wall biogenesis. Recent advances including the ability to isolate critical intermediates, metabolic approaches for probe incorporation, and isotopic labeling techniques have provided critical insight into the biochemistry of cell walls. Fundamental manuscripts that have used these techniques to discover cell wall-interacting proteins, flippases, and cell wall stoichiometry are discussed in detail. The review highlights that these powerful methods and techniques have exciting potential to identify and characterize new targets for antibiotic development.

Target-specific cytotoxic activity of recombinant immunotoxin scFv(MUC1)-ETA on breast carcinoma cells and primary breast tumors.

MUC1 is a mucin family protein, overexpressed in more than 90% of breast cancers in an underglycosylated form, exposing the core peptides of the extracellular domain that act as a potential target for antibody-mediated therapy. We have developed an anti-MUC1 scFv antibody from a phage library of mice immunized with synthetic peptide MUC1-variable number of tandem repeats. MUC1 binding phages were affinity selected through biopanning using a biotin-streptavidin pull-down method. The selected phage clones showed target-specific binding to MUC1-expressing cells. Fusion of truncated Pseudomonas aeruginosa exotoxin A (ETA) to a high binder, phage-derived scFv clone and bacterial expression and purification of recombinant scFv(MUC1)-ETA immunotoxin were done with good yield and purity. In vitro target-specific cytotoxic activity and target-specific binding of immunotoxin were shown on MUC1-expressing cells and primary breast tumor samples. A truncated ETA fusion protein expressed from the same vector but lacking scFv did not show cytotoxic effects, confirming target specificity. Our results suggest that the scFv(MUC1)-ETA immunotoxin has therapeutic potential and deserves further development and characterization for MUC1-specific breast cancers treatment.

From the Battlefield to the Bedside: Supporting Warfighter and Civilian Health With the "ART" of Whole Genome Sequencing for Antibiotic Resistance and Outbreak Investigations.

Awareness, responsiveness, and throughput characterize an approach for enhancing the clinical impact of whole genome sequencing for austere environments and for large geographically dispersed health systems. This Department of Defense approach is informing interagency efforts linking antibiograms of multidrug-resistant organisms to their genome sequences in a public database.

Generation and functional characterization of intracellular antibodies interacting with the kinase domain of human EGF receptor.

Intracellular expression of single-chain antibodies (scFvs) represents a promising approach for selective interference with cellular proto-oncogenes such as the epidermal growth factor receptor (EGFR). Previously, we have shown that intrabodies targeted to the lumen of the endoplasmic reticulum prevent the transit of EGFR or the related ErbB2 molecule to the cell surface, thereby inactivating their transforming potential. While intramolecular disulfide bridges important for antibody stability are correctly formed during expression in the secretory pathway, scFvs expressed in the reducing environment of the cytosol are often inactive. To overcome this problem and to generate antibody fragments that interact with the intracellular domain of human EGFR in the cytoplasm, here we have chosen a two-step approach combining classical selection of scFvs by phage display with subsequent expression in yeast. After enrichment of EGFR-specific antibody fragments from a combinatorial library by biopanning, a yeast two-hybrid screen was performed using the intracellular domain of EGFR as bait. Screening of 1.5 x 10(5) preselected scFv plasmids under highly stringent conditions yielded 223 colonies that represented at least five independent scFv clones functional in the intracellular milieu of eukaryotic cells. Interaction of selected antibody fragments with the intracellular domain of EGFR was confirmed in GST pull-down and coimmunoprecipitation experiments. Upon cytoplasmic expression in human tumor cells, scFvs colocalized with EGFR at the plasma membrane demonstrating their functionality in vivo.

A universal strategy for stable intracellular antibodies.

The expression of intracellular antibodies (intrabodies) in mammalian cells has provided a powerful tool to manipulate microbial and cellular signalling pathways in a highly precise manner. However, several technical hurdles have thus far restricted their more widespread use. In particular, single-chain antibodies (scFvs) have been reported to fold poorly in the reducing environment of the cytoplasm and as such there has been a reluctance to use scFv-phage libraries as a source of intrabodies unless a preselection step was applied to identify these rare scFvs that could fold properly in the absence of disulfide bonds. Recently, we reported that scFvs can be efficiently expressed within the cytoplasm of bacteria when fused at the C-terminus of the Escherichia coli maltose-binding protein (MBP). Here, we demonstrate that such MBP-scFvs are similarly stabilized when expressed in the mammalian cell cytoplasm as well as other compartments. This was demonstrated by comparing MBP-scFv fusions to the corresponding unfused scFvs that activate a defective beta-galactosidase enzyme, others that neutralize the wild-type beta-galactosidase enzyme, and an antibody that blocks the epidermal growth factor receptor. In all cases, the MBP-scFvs significantly outperformed their unfused counterparts. Our results suggest that fusion of scFvs to MBP, and possibly to other "chaperones in the context of a fusion protein", may provide a universal approach for efficient expression of intrabodies in the mammalian cell cytoplasm. This strategy should allow investigators to bypass much of the in vitro scFv characterization that is often not predictive of in vivo intrabody function and provide a more efficient use of large native and synthetic scFv-phage libraries already in existence to identify intrabodies that will be active in vivo.

Cortical gene expression in the vitamin E-deficient rat: possible mechanisms for the electrophysiological abnormalities of visual and neural function.

In mammals, severe and chronic deficiency of vitamin E (alpha-tocopherol) is associated with a characteristic neurological syndrome. Previously, we have shown that this syndrome is accompanied by electrophysiological abnormalities of neural and visual function. To investigate the molecular basis of the observed abnormalities, we used microarrays to monitor the expression of approximately 14,000 genes in the cerebral cortex from rats which had received diets containing 0, 1.25 and 5.0 mg/kg diet of all-rac-alpha-tocopheryl acetate for 14 months. Compared to the groups receiving 1.25 and 5.0 mg/kg alpha-tocopheryl acetate, a total of 11 genes were statistically significantly upregulated (> or =1.3-fold) and 34 downregulated (< or =1.3-fold) in the vitamin E-deficient group. Increased expression was observed for the genes encoding the antioxidant enzyme catalase and the axon guidance molecule tenascin-R, while decreased expression was detected for genes encoding protein components of myelin and determinants of neuronal signal propagation. Thus our observations suggest that vitamin E deficiency results in transcriptional alterations in the cerebral cortex of the rat which are consistent with the observed neurological and electrophysiological alterations.