Activity-Based Protein Profiling Reveals That Cephalosporins Selectively Active on Non-replicating Mycobacterium tuberculosis Bind Multiple Protein Families and Spare Peptidoglycan Transpeptidases

Abstract

As β-lactams are reconsidered for the treatment of tuberculosis (TB), their targets are assumed to be peptidoglycan transpeptidases, as verified by adduct formation and kinetic inhibition of Mycobacterium tuberculosis (Mtb) transpeptidases by carbapenems active against replicating Mtb. Here, we investigated the targets of recently described cephalosporins that are selectively active against non-replicating (NR) Mtb. NR-active cephalosporins failed to inhibit recombinant Mtb transpeptidases. Accordingly, we used alkyne analogs of NR-active cephalosporins to pull down potential targets through unbiased activity-based protein profiling and identified over 30 protein binders. None was a transpeptidase. Several of the target candidates are plausibly related to Mtb's survival in an NR state. However, biochemical tests and studies of loss of function mutants did not identify a unique target that accounts for the bactericidal activity of these beta-lactams against NR Mtb. Instead, NR-active cephalosporins appear to kill Mtb by collective action on multiple targets. These results highlight the ability of these β-lactams to target diverse classes of proteins.

Nonredundant functions of Mycobacterium tuberculosis chaperones promote survival under stress

“…We show that Mtb ΔclpB cells are sensitive to oxidants, especially diamide, while ΔhtpG cells are not. While htpG, like clpB, is not essential in Mtb (Lopez Quezada et al, 2020;Vaubourgeix et al, 2015), we find that cells lacking both nonessential chaperones are hypersensitive to host-like stresses that induce a nonreplicating state. Finally, a small molecule probe that targets HtpG mimics the effect of htpG disruption in ClpB-deficient Mtb.…”

“…While GA treatment demonstrated off-target effects in cells treated under nonreplicating conditions, a wealth of available GA analogs can be tested for cellular HtpG selectivity (Hadden et al, 2006;Ueda et al, 2019). We recently reported that Mtb ΔhtpG shows fourfold more cidality than wild type in the presence of select cephalosporins under nonreplicating conditions (Lopez Quezada et al, 2020). Perhaps this family of beta-lactams will synergize with GA analogs to kill Mtb.…”

“…Recombinant tagless HtpG (~1.4 mg) was purified as previously described (Lopez Quezada et al, 2020) and used to immunize rabbits without Freund's incomplete adjuvant, instead using TiterMax (Pierce). HtpG antiserum was further purified using purified HtpG with the AminoLink Plus Immobilization Kit (Thermo Scientific) following the manufacturer's protocols.…”

“…Tagless recombinant HtpG was purified from 4 L of culture (strain 4, Table S8) using a previously described method (Lopez Quezada et al, 2020). Nucleotide free Mtb HtpG (1.6 ml) was obtained by following a previously described 4-day dialysis protocol (Rauch and Gestwicki, 2014) using 1 L each of the following buffers: (1) 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 150 mM NaCl, 5 mM EDTA (pH 8), (2) 25 mM HEPES, 150 mM NaCl, 1 mM EDTA (pH 8), (3) 10 mM KCl, 5 mM MgCl 2 (pH 8), and 4…”

Click and Release Chemistry for Activity‐Based Purification of β‐Lactam Targets

“…These combinations differ both between and within bacterial species following the acquisition of the so-called "low-affinity" PBPs responsible for β-lactam resistance. In addition, the complexity of these combinations includes evolutionary unrelated enzymes, [5,[41][42][43] including unidentified ones, [44] in particular in M. tuberculosis. [42,45] This context prompted us to develop here a new strategy for affinity purification of β-lactam targets.…”