L,D-Transpeptidase Specific Probe Reveals Spatial Activity of Peptidoglycan Cross-Linking

Abstract

Peptidoglycan (PG) is a cross-linked, meshlike scaffold endowed with the strength to withstand the internal pressure of bacteria. Bacteria are known to heavily remodel their peptidoglycan stem peptides, yet little is known about the physiological impact of these chemical variations on peptidoglycan cross-linking. Furthermore, there are limited tools to study these structural variations, which can also have important implications on cell wall integrity and host immunity. Cross-linking of peptide chains within PG is an essential process, and its disruption thereof underpins the potency of several classes of antibiotics. Two primary cross-linking modes have been identified that are carried out by D,D-transpeptidases and L,D-transpeptidases (Ldts). The nascent PG from each enzymatic class is structurally unique, which results in different cross-linking configurations. Recent advances in PG cellular probes have been powerful in advancing the understanding of D,D-transpeptidation by Penicillin Binding Proteins (PBPs). In contrast, no cellular probes have been previously described to directly interrogate Ldt function in live cells. Herein, we describe a new class of Ldt-specific probes composed of structural analogs of nascent PG, which are metabolically incorporated into the PG scaffold by Ldts. With a panel of tetrapeptide PG stem mimics, we demonstrated that subtle modifications such as amidation of iso-Glu can control PG cross-linking. Ldt probes were applied to quantify and track the localization of Ldt activity in Enterococcus faecium, Mycobacterium smegmatis, and Mycobacterium tuberculosis. These results confirm that our Ldt probes are specific and suggest that the primary sequence of the stem peptide can control Ldt cross-linking levels. We anticipate that unraveling the interplay between Ldts and other cross-linking modalities may reveal the organization of the PG structure in relation to the spatial localization of cross-linking machineries.

Figure 1

(A) PG cross-linking modes associated with Ltds and PBPs. X represents the third position amino acid (either m-DAP or L-lysine-based amino acids). (B) A synthetic mimic of the stem peptide modified with a fluorescent handle (green hexagon) is covalently incorporated within growing PG scaffold. First, a terminal D-Ala residue is removed by Ltd, leading to a covalent intermediate. Second, this acyl-donor is captured by the third position amino acid within existing PG thus leading to its cross-linking with PG and generating a measurable fluorescent signal.

Figure 2

(A) Schematic diagram delineating incorporation of synthesized fluorescent Ldt substrate and incorporation into bacterial PG. (B) Chemical structure of fluorescein-modified tetrapeptide (TetraFl) and pentapeptide (PentaFl) PG stem mimics. (C) Flow cytometry analysis of E. faecium (WT and drug resistant strain) treated overnight with 100 μM TetraFl or PentaFL. Data are represented as mean + SD (n = 3). (D) Mass spectrum and XIC of TetraFL-PG with 3-3 cross-link with observed [M + H]⁺²m/z of 818.3503.

Figure 3

Flow cytometry analysis of E. faecium (drug resistant) treated overnight with 100 μM of tetrapeptide (A) or pentapeptide (B) with variations. Data are represented as mean + SD (n = 3). Chemical series of tetrapeptides and pentapeptides with variations at the C-terminus (acid/amide), terminal residue(s) (D-Ala/L-Ala), second position (iso-Gln/iso-Glu), and third position (L-Lys/acetylated L-Lys).

Figure 4

(A) Flow cytometry analysis of E. faecium (M9) treated overnight with 100 μM TetraFl or PentaFl with or without ampicillin/Meropenem. Data are represented as mean + SD (n = 3). (B) E. faecium (M9) treated with 100 μM TetraFl with 16 μg/mL ampicillin, 8 μg/mL meropenum, or DMSO (vehicle control) at early log phase. Cells were collected at various time points and analyzed by flow cytometry. Data are represented as mean + SD (n = 3). (C) Flow cytometry analysis of E. faecium (M9) treated overnight with 100 μM TetraFl (blue bars) or PentaFl (orange bars) and increasing concentrations of ampicillin, amoxicillin, vancomycin, or erythromycin. Data are represented as mean + SD (n = 3).

Figure 5

(A) Confocal microscopy image of E. faecium (WT) treated with 5 min pulse of 500 μM TetraRh, 500 μM PentaFl, and 5 mM DADA (scale bar: 1 μm). (B) In vivo labeling of E. faecium in model host. C. elegans were infected with E. faecium for 4 h, washed to remove noncolonized bacteria, and incubated with 50 μM TetraRh for 2 h. The C. elegans were washed, anesthetized, mounted on a bed of agarose, and imaged using confocal microscopy (scale bar: 10 μm).

Figure 6

(A) Flow cytometry analysis of M. smegmatis (WT) treated overnight with 100 μM tetrapeptide or pentapeptide with variations (see Figure 3). Data are represented as mean + SD (n = 3). (B) Flow cytometry analysis of M. smegmatis (WT) and Ldt knockout mutants treated overnight with 100 μM TetraFl, TetraFl-2, or TetraFl-5. Data are represented as mean + SD (n = 3). (C) Confocal microscopy image of M. smegmatis (WT) treated with 30 min pulse of 500 μM TetraRh, 500 μM PentaFl, and 5 mM DADA (scale bar: 2 μm). (D) Flow cytometry analysis of M. smegmatis (WT) treated overnight with 100 μM TetraFl or PentaFl with increasing concentrations of ampicillin or meropenum. Data are represented as mean + SD (n = 3).

Figure 7

Confocal microscopy image of M. tuberculosis treated with 50 μM TetraRh for 30 min and 3 h (scale bar: 5 μm).