Fluorescent Probes for Imaging Bacteria

Progress and prospects for small-molecule probes of bacterial imaging

Fluorescence microscopy is an essential tool for the exploration of cell growth, division, transcription and translation in eukaryotes and prokaryotes alike. While bacteria do not have the distinct internal compartments seen in eukaryotes, these unicellular organisms possess highly organized cellular structures that are essential to orchestrating cell growth, division, motility, DNA transcription, protein translation and secretion in a well-defined synchrony. Visualization of interactions between the constituents of this organization is critical to our understanding of bacterial growth and pathogenesis, and although great progress has been made in these areas in the past decade, many questions cannot be addressed using existing techniques. Imaging-based methods are and will continue to be essential for the study of the biomolecules involved in cellular and physiological processes in their native environment, with fluorescence microscopy being particularly valuable for examination of the subcellular structures of bacteria.

Until the application of fluorescent tagging techniques in bacteria, these cells were considered amorphous bags in which chromosomes and proteins were randomly distributed. However, work over the last several decades has revealed that bacteria are well-organized multicomponent systems and that individual proteins, such as the cytoskeletal proteins MreB and FtsZ and the chemoreceptors, localize to specific sites of the cell in a dynamic fashion. Fluorescent protein fusion (for example, with green fluorescent protein, GFP) and epitope tagging (for example, with FLAG or c-Myc) are the most widely used strategies for monitoring protein expression and localization in bacteria. Small protein and peptide tags have also been used to facilitate labeling, whereby a fluorescent tag is conjugated to the protein of interest by association with a tetracysteine motif (CCXXCC; FlAsH or ReAsH) or through the action of enzymes such as biotin ligase or sortase. Alternatively, the protein target is fused to an enzyme that can be specifically labeled with a small dye-containing substrate (such as HaloTag or SNAP-tag)9.

Antibiotic-inspired chemical probes

PG, also called murein, is a major component of the bacterial cell wall, which maintains the organism’s shape and protects it from turgor pressure. PG is a polymeric structure composed of alternating β-(1,4)-linked N-acetylglucosamine (GlcNAc) and N-acetylmuramic (MurNAc) acid glycan chains that are crosslinked by pentapeptides. Although most bacteria have PG, the length of the glycan chains and the amino acid composition of the stem peptides vary between organisms. This structure is unique to bacteria, and inhibition of its biosynthesis by antibacterial agents leads to cell lysis. A particularly fruitful strategy for probe design has been to harness the power of known antibiotic scaffolds that interfere with cell wall construction.

Peptidoglycan-inspired chemical probes

Imaging of the bacterial cell wall has also been accomplished through the incorporation of unnatural biosynthetic precursors or substrate mimics. For example, fluorophore-tagged UDP-MurNAc pentapeptide was synthesized and tested for incorporation into bacterial cells. Fluorescence microscopy images show that this UDP-MurNAc analog is readily incorporated by Gram-positive bacteria. Pretreatment of Gram-negative bacteria with EDTA weakened the thick liposaccharide layer and enabled incorporation of the probe in these cells. In another study, a derivative of the PG tripeptide, L-alanyl-γ-D-glutamyl-L-lysine, was generated with N-7-nitro-2,1,3-benzoxadiazol-4-yl in the lysine position for in situ detection of nascent PG synthesis in E. coli.

Reference:

Budin I.; et al. Membrane assembly driven by a biomimetic coupling reaction. Journal of the American Chemical Society. 2012,134 (2): 751–3.

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