Fluorescent stem peptide mimics: In situ probes for peptidoglycan crosslinking

Abstract

Understanding the mechanisms of bacterial cell wall synthesis is essential for microbiology and medicine alike. A key step in this process is peptidoglycan crosslinking, which confers mechanical strength to the cell wall and represents a target for numerous classes of antibiotics. However, the biology of crosslinking remains poorly understood due to a lack of tools for studying the reaction in vivo. Recently, we developed a class of synthetic probes called fluorescent stem peptide mimics (FSPMs) that meet this need, allowing quantification and localization of crosslinking activity in live bacteria. We have utilized FSPMs to describe novel aspects of peptidoglycan synthesis in the human pathogen, Staphylococcus aureus. To enable wider use of our methodology, we provide detailed protocols herein for the synthesis of FSPMs, labeling of live bacteria, and evaluation of crosslinking by flow cytometry and super-resolution microscopy. We believe that FSPMs, together with complementary biosynthetic probes and traditional bacteriologic methods, will help to advance our understanding of peptidoglycan biology and accelerate the search for new antibiotics.

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.