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Cell Wall Remodeling of Staphylococcus aureus in Live C. elegans Sean E Pidgeon, and Marcos M. Pires Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00363 • Publication Date (Web): 24 Jul 2017 Downloaded from http://pubs.acs.org on July 25, 2017

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Bioconjugate Chemistry

Cell Wall Remodeling of Staphylococcus aureus in Live C. elegans Sean E. Pidgeon, and Marcos M. Pires*

*Department of Chemistry Lehigh University Bethlehem, Pennsylvania 18015, United States

Corresponding e-mail: [email protected]

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Abstract

Peptidoglycan (PG) scaffolds are critical components of bacterial cell walls. They counter internal turgor pressure to prevent lysis and protect against external insults. It was recently discovered that various types of bacteria release large quantities of PG building blocks (D-amino acids) into their surrounding medium. Contrarily, cultured bacteria were also found to incorporate D-amino acids (both natural and synthetic) from the medium directly into their PG scaffold. These two processes may potentially function, in concert, to metabolically remodel PG in live host organisms. Yet, demonstration that bacteria can decorate their cell surfaces with exogenous D-amino acids was limited to in vitro culture conditions. We present the first evidence that bacteria remodel their PG with exogenous D-amino acids in a live host animal. A tetrazine click partner was conjugated onto the sidechain of a D-amino acid to capture incorporation into the bacterial PG scaffold using a complementary click-reactive fluorophore. Staphylococcus aureus infected Caenorhabditis elegans treated with exogenous D-amino acids readily revealed in vivo PG labeling. These results suggest that extracellular D-amino acids may provide pathogens with a mode of late-stage in vivo cell surface remodeling.


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Introduction

The adaptation of bacteria to their surrounding environment is vital to their survival. As a prominent example, bacteria must adequately respond to challenges encountered during colonization of their host organisms. There is increasing evidence that structural remodeling of bacterial cell surfaces is a powerful mode of promoting colonization.1,2 For example, Staphylococcus aureus (S. aureus) cells decorate their surfaces to establish host adhesion and evade immune response.3 Surface-bound sortases anchor full length proteins onto the peptidoglycan (PG) scaffold for their display to the host organism. Similarly, the PG structure can be chemically altered in response to environmental cues, including challenges with antibiotics and host immune system.4-12

Recently, it was discovered that diverse species of bacteria grown in culture release PG building blocks (D-amino acids) into the surrounding medium;13 in turn, released Damino acids modulated PG amount and strength (Figure 1). These findings were further extended to live host animals, which provided direct evidence that D-amino acid release may be integral to host-microbiome relationships.14 Conversely, bacteria (both Grampositive and -negative) incorporate extracellular D-amino acids within their own PG scaffolds.13,15



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Figure 1. Scheme showing the two processes (D-amino acid release and incorporation) by bacterial cells both in vitro and in vivo.

D-amino

acids are found within stem peptides from PG chains, which is a distinctive

characteristic of bacteria. Bacteria incorporate exogenous D-amino acids into their expanding PG scaffold during cellular growth via surface bound transpeptidases such as Penicillin Binding Proteins (PBPs) and L,D-transpeptidases.13,15,16 More specifically, exogenous D-amino acids supplemented in the surrounding medium replace D-alanine residues on PG stem peptides during crosslinking (Figure 2A). In addition to naturally produced D-amino acids, swapping of surface bound D-alanine was also established with entirely unnatural synthetic D-amino acids.17,18 A striking feature of cell wall 4 ACS Paragon Plus Environment

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remodeling with synthetic D-amino acids is the vast promiscuity displayed by surface anchored transpeptidases in tolerating entirely unnatural sidechains.

Figure 2. (A) Representation of PG remodeling in S. aureus. PBP transpeptidases catalyze the swapping of the 5th position amino acid on the stem peptide (D-Ala) with exogenous D-amino acids (D-A.A.). (B) Inverse electron demand-Diels Alder (IED-DA) results in the covalent ligation of a TCO and a tetrazine-modified fragment.



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Metabolic PG labeling with synthetic D-amino acids provides a unique mode of installing handles (e.g., fluorophores) within the growing cell wall. In turn, fluorescent D-amino acids have become invaluable PG probes and have underpinned important new findings related to cell wall biosynthesis.19-25 Similarly, our research group has leveraged relaxed substrate specificity by PG biosynthetic enzymes to graft unnatural handles onto bacterial cell surfaces.26-34 Incorporation and release (“catch and release”) of D-amino acids potentially provide mechanisms to modulate the composition of the surrounding environment and remodel PG scaffolds. Yet, incorporation of exogenous D-amino acids into bacterial PG scaffolds in live host animals has not been demonstrated. Here, we designed a D-amino acid reporter probe that captured PG incorporation of exogenous Damino acids in live Caenorhabditis elegans (C. elegans).

Results and Discussion

Selectivity, minimal cytotoxicity, diverse chemistries, and compatibility with complex biomacromolecules make bioorthogonal reactions powerful chemical tools to study biology.35-37 Bertozzi and co-workers have previously shown that D-amino acids displaying copper-mediated (azide-alkyne) and copper-free (strained alkyne) click


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chemistry handles are tolerated by several Gram-positive bacteria.18,38,39 More recently, we demonstrated that alkene- and tetrazine-displaying D-amino acids are readily incorporated onto bacterial PG scaffolds.34 The tetrazine-trans-cyclooctene ligation is a rapid method for the assembly of biomolecular constructs (Figure 2B).40 With rate constants up to 106 M-1s-1, this inverse electron demand Diels-Alder (IED-DA) reaction provides exceptional versatility and compatibility with in vivo ligations.41,42

In our original design, we reasoned that either the alkene handle or tetrazine handle could be conjugated onto the sidechain of a D-amino acid. We were concerned that the large size of trans-cyclooctene (TCO) would dramatically lower labeling efficiency, having previously shown that the sidechain size can have an inverse relationship with incorporation efficiency.29 Therefore, the smaller norbornene was chosen in the place of TCO. Unfortunately, low ring strain in norbornene had a deleterious effect on conjugation efficiency with tetrazine-conjugated fluorophores. The reactive partners were swapped in a second set of D-amino acids, which were modified to display the tetrazine handle. A small tetrazine was assembled onto the sidechain of D-propionic acid. Although cell surface labeling was observed, the electron donating effect of the amino group dramatically reduced IED-DA efficiency.

The slow reaction rates from our previous alkene-tetrazine strategy, which required several hours of treatment for observable ligation, became a significant impediment in capturing PG remodeling in live organisms. We set out to improve on this design by using optimized handles that result in fast ligation without compromising incorporation



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efficiency. Towards this goal, we synthesized D-TetPG, a tetrazine derivative flanked by a phenyl substituent (Figure 3A). Its enantiomer was recently shown to display high reactivity and stability when incorporated into recombinant green fluorescent protein (GFP).43 Electron-withdrawing by the phenyl ring led to diene activation with minimal increase in structural size. Ligation with TCO-modified handles was exceedingly fast with product formation complete within ~2 min.


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Figure 3. (A) Chemical structures of D-TetPG and L-TetPG. (B) Schematic representation of PG labeling of S. aureus by D-TetPG and its ligation with a TCOfluorophore. (C) S. aureus cells were treated with D-TetPG (1 mM), L-TetPG (1 mM), or untreated media overnight followed by incubation with 50 µM Cy5-TCO (30 min) and analyzed using flow cytometry. Data are represented as mean + SD (n =3). (D) Fluorescence microscopy (top) and phase contrast (bottom) imaging of S. aureus treated with D-TetPG from (C). Scale bar represents 2 µm.



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Synthesis of D-TetPG was initiated by reacting Boc-4-cyano-D-Phenylalanine with anhydrous hydrazine and nickel triflate catalyst, followed by oxidation with sodium nitrite (Figure 3A). Conversion to the C-terminus carboxamide form was performed by using standard solid phase peptide coupling to Rink Amide resin, followed by global release and deprotection. We, and others, have found that C-terminus amidation improves incorporation and retention of exogenous D-amino acids in Gram-positive bacteria relative to its carboxylic acid counterpart.25,30 The final D-TetPG was purified by RPHPLC and characterized by NMR and HRMS. The control enantiomeric L-TetPG was synthesized using a similar route with the exception of the starting building block, which was the L-stereocenter.

The ability of D-TetPG to label bacterial PG was first assessed in cultured S. aureus (Figure 3B). S. aureus is an excellent model Gram-positive pathogen due to its high clinical burden and emerging drug-resistant strains. S. aureus cells were treated overnight with D-TetPG, formaldehyde fixed, incubated with the fluorophore conjugate Cy5-TCO, and labeling was quantified via flow cytometry (Figure 3C). A marked increase in fluorescence levels (>100-fold) was observed for cells treated with D-TetPG relative to untreated cells. In contrast, treatment of S. aureus with the enantiomeric LTetPG led to a 2-fold increase in fluorescence levels compared to untreated cells. These results are consistent with the requirement of D-stereocenters for incorporation into PG scaffolds. Confocal microscopy analysis of D-TetPG treated S. aureus revealed concentrated signals at the septal region, which is the primary site of new PG


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biosynthesis (Figure 3D). In addition, a labeling competition experiment was performed by co-incubation of D-TetPG with D-Ala, which resulted in a significant decrease in cellular fluorescence (Figure S1). These results clearly suggest that D-TetPG is readily accommodated by bacterial cells to reveal in vitro PG remodeling.

Next, the kinetics of surface-bound tetrazine-TCO ligation was evaluated using live bacterial cells. Ligation between phenyl-flanked tetrazine and TCO was previously found to be extremely fast using model proteins.43 However, tetrazine handles are imbedded within the PG matrix in D-TetPG labeled cells, which can potentially alter reaction kinetics. To test this concept, S. aureus cells were once again treated with DTetPG, incubated with Cy5-TCO, and samples were periodically analyzed for fluorescence levels for 180 min (Figure 4). By the first time point (5 min), fluorescence levels were nearly 100-fold over background and reached a plateau level within 15 min. From these results, it was evident that PG-anchored tetrazine handles are readily reacted with Cy5-TCO and the ligation is mostly complete within a few minutes.



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Figure 4. S. aureus cells were treated with D-TetPG (1 mM) or L-TetPG (1 mM) overnight followed by incubation with 50 µM Cy5-TCO and analyzed at various time points using flow cytometry. Data are represented as mean + SD (n =3).

With a synthetic D-amino acid in hand operating in a time-scale compatible with in vivo studies, we turned our attention to capturing PG remodeling in live host organisms. The nematode C. elegans has proven to be a highly valuable animal model for studying host-pathogen interactions.44-46 We had anticipated that direct monitoring of PG remodeling with fluorescently-labeled D-amino acids would suffer from two severe drawbacks: (1) low incorporation efficiency due to the large sidechain and (2) high 12 ACS Paragon Plus Environment

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background fluorescence levels. The high background fluorescence was expected due to the high concentrations required for robust PG labeling by unnatural D-amino acids – a product of the inherent high KM of PBPs for its substrates. Nonetheless, we tested the possibility of directly capturing PG remodeling in C. elegans with FITC-modified DLysine, D-Lys(FITC). S. aureus-infected C. elegans were treated with D-Lys(FITC) and analyzed by fluorescence microscopy. mCherry-expressing S. aureus were observed in various compartments of C. elegans (Figure S2). However, green fluorescence signal from D-Lys(FITC) was observed indiscriminately throughout the inner cavity of C. elegans. The high fluorescence background highlights the need for a two-step ligation that optimizes incorporation and signal-to-noise levels.

We next evaluated the compatibility of tetrazine-TCO ligations in live C. elegans by prelabeling bacterial cells with the PG probe D-TetPG (ex vivo labeling, Figure S3). GFPexpressing S. aureus cells were used to visualize co-localization of bacteria and DTetPG labels on bacterial PG. After overnight treatment with D-TetPG, S. aureus cells were washed and transferred to a liquid culture of C. elegans (infection period). After a 4 h incubation period, non-colonized bacteria were removed, followed by treatment with Cy5-TCO for 20 min (Figure 5A). Confocal microscopy imaging showed prominent PG labeling co-localized with GFP-expressing S. aureus (Figure 5A). Bacterial treatment with the control L-TetPG led to no discernible Cy5 fluorescence signals (Figure S4). These results establish the constructive ligation of tetrazine-TCO handles within PG scaffolds in the host organism C. elegans. In the future, we plan to explore this facile



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mode of grafting unnatural handles onto bacterial cell surfaces with the goal of revealing key aspects of host-pathogen interactions.

Figure 5. (A) S. aureus cells were treated overnight with D-TetPG (1 mM), transferred to a liquid culture of C. elegans (infection period) for 4 h, washed to remove non-colonized bacteria, and incubated with Cy5-TCO for 20 min. (B) S. aureus were introduced into C. 14 ACS Paragon Plus Environment

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elegans, washed to remove uncolonized bacteria, treated with D-TetPG (1 mM) for 3 h, and Cy5-TCO for 20 min. For (A) and (B), C. elegans were anesthetized, mounted on a bed of agarose, and imaged using confocal microscopy. Scale bar presents 10 µm.

Finally, we evaluated PG remodeling by exogenous D-amino acids in S. aureus infected C. elegans (in vivo labeling, Figure S3). Untreated bacterial cells were first introduced into C. elegans to initiate colonization. Consistent with previous reports, C. elegans infected with S. aureus cells showed distention of the lumen, suggestive of S. aureus pathogenicity (Figure 5B).47 After which point, C. elegans were treated with D-TetPG for 3 h to induce PG remodeling and ligation was performed with Cy5-TCO in live hosts. Remarkably, clear co-localization of Cy5-fluorescence signals and GFP-expressing S. aureus was observed, which is indicative of in vivo PG remodeling. To our knowledge, this represents the first example of exogenous D-amino acids incorporation by bacteria in a live host organism.

In conclusion, we have demonstrated that a D-amino acid conjugated with an optimized tetrazine led to PG remodeling of bacterial cells. We showed that a complimentary click partner led to rapid ligation onto PG-anchored tetrazine handles in cultured bacteria and in S. aureus infected C. elegans. Our results close the loop on the D-amino acid “catch & release” phenomenon observed for bacteria both in vitro and in vivo. Going forward, we will investigate the functional consequence of PG remodeling by both pathogenic and commensal bacteria in C. elegans.



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Acknowledgements This study was supported by Lehigh University (M.P.). We would like to thank Dr. Horswill (University of Iowa) for kindly sharing the mCherry and GFP expressing S. aureus strains.

The Supporting Information (protocols, additional figures, and characterization) is available free of charge on the ACS Publications website at DOI:


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Cell Wall Remodeling of Staphylococcus aureus in ... - ACS Publications

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