Chapter Three - Fluorescent stem peptide mimics: In situ probes for peptidoglycan crosslinking
Introduction
With few exceptions, all bacteria are surrounded by a peptidoglycan cell wall—a mesh-like polymer that confers protection and shape to prokaryotic organisms (Otten, Brilli, Vollmer, Viollier, & Salje, 2018). Its ability to withstand severe environmental stresses, such as turgor pressures up to 140 kPa (Auer & Weibel, 2017), and yet undergo the intricate processes of cell growth and division has long fascinated biologists. Meanwhile, its indispensability for cell survival makes it an attractive therapeutic target for physicians treating bacterial infections. Given the spread of drug-resistant pathogens, which are projected to claim 10 million lives per year by 2050 (O'Neill, 2016), and the slowed pace of antimicrobial development in recent decades (Brown & Wright, 2016), it is imperative to elucidate the mechanisms of peptidoglycan synthesis in order to accelerate discovery of novel antibiotics.
Structurally, peptidoglycan consists of long glycan strands crosslinked together by peptide bridges (Pazos & Peters, 2019), The enzymes responsible for crosslink formation are penicillin-binding proteins (PBPs), the target of several classes of antibiotics including beta-lactams and glycopeptides (Fig. 1A) (Frère & Page, 2014; Pazos, Peters, & Vollmer, 2017; Sauvage, Kerff, Terrak, Ayala, & Charlier, 2008). The past decade has witnessed explosive progress in PBP biology, in part due to the emergence of novel chemical probes for studying PBP function in live bacteria (Gautam, Gniadek, Kim, & Spiegel, 2013; Radkov, Hsu, Booher, & VanNieuwenhze, 2018; Taguchi, Kahne, & Walker, 2019). These probes include the fluorescent stem peptide mimics (FSPMs) developed in our laboratory (Fig. 1B) (Gautam, Kim, Shoda, et al., 2015; Gautam, Kim, & Spiegel, 2015), fluorescent d-amino acids (FDAAs) introduced by VanNieuwenhze and colleagues (Kuru et al., 2012; Radkov et al., 2018), and other agents as recently reviewed (Radkov et al., 2018; Taguchi et al., 2019).
FSPMs consist of a fluorophore conjugated to a short synthetic peptide that mimics the endogenous substrate of PBPs—the stem peptide. Covalent attachment of FSPMs to the cell wall by PBPs proceeds as described in Fig. 1B, resulting in installation of a fluorophore precisely at the site of PBP activity. Thus, FSPMs enable evaluation of crosslinking in live bacteria. This in situ labeling technique represents an important advance over conventional methods for studying crosslinking, which require lysis, enzymatic digestion, and biochemical analysis of the resulting muropeptides (Desmarais, De Pedro, Cava, & Huang, 2013).
Using the Gram-positive pathogen Staphylococcus aureus as a model organism, we have shown that FSPMs are exclusively incorporated by a single isoform—PBP4 (Gautam, Kim, Shoda, et al., 2015). This property enables monitoring of PBP4-specific crosslinking activity in vivo. Utilizing FSPMs we have demonstrated the PBP4 activity is restricted to the septum during division by cell surface polysaccharides called wall teichoic acids (Gautam, Kim, Shoda, et al., 2015), and occurs globally throughout the cell well between divisions (Gautam, Kim, & Spiegel, 2015). We have since extended the scope of FSPM labeling to several additional Gram-positive organisms including the rod-shaped Bacillus subtilis (unpublished results).
Here we provide protocols for FSPM synthesis, bacterial labeling, quantification of crosslinking by flow cytometry, and direct visualization in situ using super-resolution microscopy.
Section snippets
Peptide synthesis
Two peptides must be synthesized: a functional stem peptide mimic containing the PBP recognition motif, (l-Lys)–(d-Ala)–(d-Ala); and a diastereomeric control containing (l-Lys)–(l-Ala)–(l-Ala), which indicates non-enzymatic labeling (i.e., non-specific binding to the cell wall). Both contain a short peptide linker, (l-Lys)–(Gly)–(Gly), which allows conjugation of a fluorophore to the epsilon amine of the N-terminal lysine. The full sequences are thus (l-Lys)–(Gly)–(Gly)–(l-Lys)–(d-Ala)–(d-Ala)
Maintenance of S. aureus
The model staphylococcal strain, Newman, is commonly used in our laboratory, but any strain expressing PBP4 can be utilized to study FSPM incorporation, including methicillin-resistant S. aureus. Luria-Bertani (LB) agar plates are streaked weekly from 20% glycerol stocks stored at − 80 °C.
FSPM labeling
Liquid cultures of LB broth (3 mL) are inoculated with single colonies and grown overnight at 37 °C with agitation at 200 RPM to stationary phase. Cultures are then diluted 1:100 and grown 2 h to reach logarithmic
Flow cytometry analysis
To assess the magnitude of FSPM labeling, we use an Accuri C6 flow cytometer (Becton Dickinson) on medium speed fluidics with a minimum threshold of 40,000 FSC-H. Maximum FSC and SSC gates are set to exclude multi-cell aggregates (e.g., 50,000 FSC-A and 50,000 SSC-A for S. aureus). 10,000 Events per sample are analyzed. Traces are processed using FlowJo software. Representative data indicating inhibition of FSPM labeling with the PBP4-specific beta-lactam antibiotic, cefoxitin, are shown in
Image acquisition
While any fluorescent microscope can be used to image FSPM incorporation, the small size of bacteria (~ 1 μm in diameter) often requires super-resolution imaging techniques for accurate subcellular localization of probe labeling. Here we describe our laboratory's methodology using three-dimensional structured illumination microscopy (3D-SIM). Images are acquired using a UPLANAPO 60X/1.42 PSF, oil immersion objective lens (Olympus) and CoolSNAP HQ2 CCD cameras with a pixel size of 0.080 μm
Concluding remarks
It is important to contrast the FSPMs described here with the other available probes for peptidoglycan synthesis—particularly FDAAs, a powerful class of reagents that label active sites of cell wall synthesis in a wide array of bacterial species (Kuru et al., 2012; Radkov et al., 2018). FDAAs are covalently incorporated into the PG stem peptide via an exchange reaction mediated by transpeptidases, wherein FDAAs serve as surrogate acceptor strands in the transpeptidation reaction (Fig. 1) (Kuru
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New approaches and techniques for bacterial cell wall analysis
2021, Current Opinion in MicrobiologyCitation Excerpt :Besides their importance in investigating PG synthesis [51•,52•,53•,54•], DAAs-based probes (including radiolabelled DAAs) have been applied to studies of the mammalian gut microbiota [55•], as well as in monitoring bacterial infections [56•,57•]. Finally, new alternatives to DAAs-based probes have emerged for PG labelling: for example, fluorescent synthetic peptides that mimic the peptide part of the PG precursor and allow the study of PG crosslinking [58•,59•] and synthetic muramic acid derivatives, which enable labelling of the PG sugar backbone via click chemistry [60•] (Figure 3). Despite the cell wall is one of the most studied bacterial components, our understanding of the PG biosynthesis and dynamics remains limited.
β-Lactams against the Fortress of the Gram-Positive Staphylococcus aureus Bacterium
2021, Chemical Reviews