Mechanisms of Incorporation for D-Amino Acid Probes That Target Peptidoglycan Biosynthesis

Abstract

Bacteria exhibit a myriad of different morphologies, through the synthesis and modification of their essential peptidoglycan (PG) cell wall. Our discovery of a fluorescent D-amino acid (FDAA)-based PG labeling approach provided a powerful method for observing how these morphological changes occur. Given that PG is unique to bacterial cells and a common target for antibiotics, understanding the precise mechanism(s) for incorporation of (F)DAA-based probes is a crucial determinant in understanding the role of PG synthesis in bacterial cell biology and could provide a valuable tool in the development of new antimicrobials to treat drug-resistant antibacterial infections. Here, we systematically investigate the mechanisms of FDAA probe incorporation into PG using two model organisms Escherichia coli (Gram-negative) and Bacillus subtilis (Gram-positive). Our in vitro and in vivo data unequivocally demonstrate that these bacteria incorporate FDAAs using two extracytoplasmic pathways: through activity of their D,D-transpeptidases, and, if present, by their L,D-transpeptidases and not via cytoplasmic incorporation into a D-Ala-D-Ala dipeptide precursor. Our data also revealed the unprecedented finding that the DAA-drug, D-cycloserine, can be incorporated into peptide stems by each of these transpeptidases, in addition to its known inhibitory activity against D-alanine racemase and D-Ala-D-Ala ligase. These mechanistic findings enabled development of a new, FDAA-based, in vitro labeling approach that reports on subcellular distribution of muropeptides, an especially important attribute to enable the study of bacteria with poorly defined growth modes. An improved understanding of the incorporation mechanisms utilized by DAA-based probes is essential when interpreting results from high resolution experiments and highlights the antimicrobial potential of synthetic DAAs.

Figure 1

PG biosynthetic pathways are promiscuous and accept D-amino acid (DAA) based probes. (a) Simplified cartoon representation of the PG synthesis and modification pathways relevant to this work, along with representative PG synthesis inhibitors. (b) Representative DAA-based probes utilized in the experiments described in this manuscript.

Figure 2

B. subtilis cells do not incorporate (F)DAAs cytoplasmically, but through reactions mediated by  D,D-TPases, which can be inhibited by D-cycloserine. (a) A brief pretreatment of live B. subtilis cells with vancomycin, penicillin G (Pen G), and D-cycloserine (DCS), significantly inhibited FDAA incorporation. No inhibition of FDAA incorporation was obsereved with fosfomycin. (b) LdtA_Vc incorporated DCS into M4, comparable to other DAAs, e.g., D-Met, in vitro. (c) PBP4_Sa incorporated DCS into a synthetic N_α, N_ε-Diacetyl-L-Lys-D-Ala-D-Ala tripeptide (3P), in vitro. (d) Live B. subtilis wild-type and B. subtilis Δddl cells, grown in S7₅₀ minimal media supplemented with DA–DA, incorporated HADA comparably; HADA incorporation was significantly inhibited by ampicillin pretreatment. (e) DA–EDA was a poor substrate for MurF_Bs in vitro, but EDA–DA performed similarly well to the endogenous substrate, DA–DA. Column bar graphs represent mean relative signal ± SD quantified from at least N > 100 cells. Error bars are SEM.

Figure 3

E. coli cells incorporate (F)DAAs through reactions mediated by L,D- and D,D-TPases, and not cytoplasmically. (a) HADA incorporation in E. coli BW25113Δ6LDT cells (Δ6LDT) was ∼10-fold lower than that observed in E. coli wild-type cells. Unlike wild-type cells, which reveal extensive accumulation of FDAA signal around the entire cell, FDAA labeling in the Δ6LDT strain was predominantly limited to the sites of new growth. (b) Live E. coli wild-type cells, but not Δ6LDT cells, incorporated FDAAs in a growth-independent manner. (c) Live E. coli Δ6LDTΔdacA cells accumulated significantly more HADA signal compared to Δ6LDT. (d) Live Δ6LDT and E. coli Δ6LDTΔddlAB cells, grown in M9 minimal media supplemented with DA–DA, incorporated HADA comparably; HADA incorporation was inhibited by ampicillin pretreatment. (e) Compared to D-Ala, EDA was a poor substrate for DdlB_E. coliin vitro. Micrographs are adjusted for qualitative comparison only. Values in column bar graphs represent mean relative signal quantified from at least N > 100 cells. Scale bars, 2 μm.

Figure 4

FDAAs are incorporated by periplasmic transpeptidases and DAADs cytoplasmically. (a) MurF_Pa incorporated DAADs as well as the native substrate (DA–DA), in vitro. (b) Live Δ6LDT cells incorporated a greater EDA—DA (1 mM for 1 h) signal than E. coli wild-type cells. Values in column bar graphs represent the mean relative signal quantified from at least N > 150 cells. (c) Cartoon representation depicting that in E. coli DAAs, including FDAAs and DCS, are substrates for periplasmic L,D-TPases, D,D-TPases, or D,D-CPases and that DAADs are substrates for cytoplasmic MurF.

Figure 5

FDAAs are efficiently incorporated into PG precursors. (a) LdtA_Vc incorporated HADA into the soluble muropeptide disaccharide tetrapeptide (M4) comparable to other DAAs, e.g., D-Met, in vitro. (b) Different high-molecular-weight D,D-TPases from diverse bacteria incorporated HADA and NADA during the in vitro synthesis of nascent PG from lipid-II without significantly changing their total D,D-TPase and dD,D-CPase product distribution. The values are the mean ± SD of three independent experiments.

Figure 6

FDAAs report on the abundance and subcellular distribution of muropeptides in ethanol fixed and permeabilized bacterial cells in vitro. (a) Ethanol fixed V. cholerae cells are substrates for LdtA_Vc and FDAAs, e.g., HADA, in vitro. (b) Ethanol fixed S. aureus cells are substrates for PBP4_Sa and FDAAs in vitro. (c–d) Ethanol fixed E. coli cells are substrates for LdtA_Vc or PBP4_Sa and FDAAs in vitro. (c) FDAAs and LdtA_Vc can report on PG tetrapeptide abundance of the E. coli Δ6LDT and ΔdacA strains in vitro. (d) FDAAs and PBP4_Sa can report on PG pentapeptide abundance of the E. coli Δ6LDT and ΔdacA strains in vitro. (e) Sequential PBP4_Sa (with HADA) and LdtA_Vc (with BADA) labeling can report on differential subcellular muropeptide distribution of a strain, e.g., in ethanol fixed A. tumefaciens cells, red arrows. Column bar graphs represent mean relative signal quantified from at least N > 100 cells. Error bars are SEM. Scale bars, 2 μm.