Systematic Survey of Serine Hydrolase Activity in Mycobacterium tuberculosis Defines Changes Associated with Persistence

Abstract

SUMMARY The transition from replication to non-replication underlies much of Mycobacterium tuberculosis (Mtb) pathogenesis, as non- or slowly replicating Mtb are responsible for persistence and poor treatment outcomes. Therapeutic targeting of non-replicating populations is a priority for tuberculosis treatment, but few drug targets in non-replicating Mtb are currently known. Here, we directly measured the activity of the highly diverse and druggable serine hydrolases (SHs) during active replication and non-replication using activity-based proteomics. We predict SH activity for 78 proteins, including 27 proteins with unknown function, and identify 37 SHs that remain active in the absence of replication, providing a set of candidate persistence targets. Non-replication was associated with major shifts in SH activity. These activity changes were largely independent of SH abundance, indicating extensive post-translational regulation of SHs. By probing a large cross-section of druggable Mtb enzyme space during replication and non-replication, we identify new SHs and suggest new persistence targets.

Design of Selective Substrates and Activity-Based Probes for Hydrolase Important for Pathogenesis 1 (HIP1) from Mycobacterium tuberculosis

“…8 The first site features a lysine in the P2 position, in agreement with the substrate preference we observed by MSP-MS and PS-SCL. The second cleavage appears to have many suboptimal amino acids in the P4–P4′ positions with the exception of glycine in the P1′ position.…”

“…M. tuberculosis Hip1 was produced as described previously, 8 and our initial studies revealed that it cleaved fluorogenic substrate WKLL-ACC with a k cat / K M value of 1.9 × 10 3 M –1 s –1 (Table 1). This substrate was designed to be cleaved by another cell envelope-associated Mtb protease, MarP, 10 and was unlikely to be optimal for Hip1.…”

“…A recent chemoproteomics study identified 78 putative Mtb serine hydrolases under replicating and nonreplicating growth conditions. 8 However, with the notable exception of inhibitors for Mtb caseinolytic protease clpP1P2, 18 there are no selective inhibitors or ABPs for any of these targets. Because many of these serine hydrolases have not been functionally characterized, 8 they are currently not accessible to target-based probe design using purified recombinant enzymes.…”

“…8 However, with the notable exception of inhibitors for Mtb caseinolytic protease clpP1P2, 18 there are no selective inhibitors or ABPs for any of these targets. Because many of these serine hydrolases have not been functionally characterized, 8 they are currently not accessible to target-based probe design using purified recombinant enzymes. In ongoing work in our laboratory to overcome this limitation, we are establishing a competitive activity-based protein profiling approach that would allow the direct screening of focused libraries to discover candidate inhibitors and probes for unidentified serine hydrolases in complex mycobacterial lysates.…”

“…5 Suitable chemical probes for these applications include activity-based probes (ABPs, i.e., irreversible inhibitors of a protease with a reporter tag 6 ) as well as protease-activated reporters such as quenched fluorescent substrates. 7 A recent chemoproteomic study revealed that Mtb expresses more than 70 predicted serine hydrolases (including 6 annotated serine proteases and 27 hypothetical hydrolases of unknown function) 8 that have the potential to be useful as imaging or drug targets. The prioritization of a suitable target for the design of new chemical probes for Mtb is based on a number of basic criteria.…”