A genetic strategy to identify targets for the development of drugs that prevent bacterial persistence

Abstract

Antibacterial drug development suffers from a paucity of targets whose inhibition kills replicating and nonreplicating bacteria. The latter include phenotypically dormant cells, known as persisters, which are tolerant to many antibiotics and often contribute to failure in the treatment of chronic infections. This is nowhere more apparent than in tuberculosis caused by Mycobacterium tuberculosis, a pathogen that tolerates many antibiotics once it ceases to replicate. We developed a strategy to identify proteins that Mycobacterium tuberculosis requires to both grow and persist and whose inhibition has the potential to prevent drug tolerance and persister formation. This strategy is based on a tunable dualcontrol genetic switch that provides a regulatory range spanning three orders of magnitude, quickly depletes proteins in both replicating and nonreplicating mycobacteria, and exhibits increased robustness to phenotypic reversion. Using this switch, we demonstrated that depletion of the nicotinamide adenine dinucleotide synthetase (NadE) rapidly killed Mycobacterium tuberculosis under conditions of standard growth and nonreplicative persistence induced by oxygen and nutrient limitation as well as during the acute and chronic phases of infection in mice. These findings establish the dual-control switch as a robust tool with which to probe the essentiality of Mycobacterium tuberculosis proteins under different conditions, including those that induce antibiotic tolerance, and NadE as a target with the potential to shorten current tuberculosis chemotherapies.

Therapeutic Potential of the Mycobacterium tuberculosis Mycolic Acid Transporter, MmpL3

“…pGMCH-T38S38-P750-mmpL3-DAS ϩ 4 achieves transcriptional regulation of DAS-tagged MmpL3 as mmpL3-DAS is transcribed using a promoter (P750), which contains a tet operator (tetO4C5G) that is recognized by TetR38. TetR38 is a reverse TetR that binds tetO4C5G in the presence of ATc (20) and thus transcriptionally represses mmpL3-DAS with ATc. pGMCH-T38S38-P750-mmpL3-DAS ϩ 4 causes hygromycin resistance and integrates into the L5 attachment site.…”

“…To investigate whether MmpL3 is required for the growth and persistence of M. tuberculosis in vitro and in vivo, we generated M. tuberculosis knockdown strains in which MmpL3 levels are regulated either exclusively at the transcriptional level or by a dual control (DUC) switch so that the chemical compound, ATc or doxycycline, triggers transcriptional repression of the mmpL3 gene and simultaneous degradation of the MmpL3 protein (20). Conditional knockdowns in the virulent (ATCC 25618) and avirulent (mc 2 6206) M. tuberculosis H37Rv backgrounds were generated as follows.…”

“…1), MmpL3-DUC knockdowns were generated by transforming the merodiploid H37Rv strains with plasmid pGMCgSq19-TSC10M1-sspB, allowing for the expression of the E. coli adaptor protein SspB under the control of an ATc-inducible (TET-ON) promoter. The presence of a DAS ϩ 4 tag on MmpL3 is expected to target the cytosolic C-terminal domain of MmpL3 for proteolytic degradation by the ClpX protease upon coexpression of sspB, which allows the tagged proteins to be delivered to the protease (20,27). Because sspB is expressed under the control of a TET-ON promoter and mmpL3-DAS ϩ 4 under the control of a TET-OFF promoter in MmpL3-DUC, the addition of ATc to the culture medium is expected to simultaneously silence mmpL3-DAS ϩ 4 expression and turn on the expression of sspB, resulting in the C-terminal proteolytic degradation of MmpL3-DAS ϩ 4.…”

Phenotypic Heterogeneity, a Phenomenon That May Explain Why Quorum Sensing Does Not Always Result in Truly Homogenous Cell Behavior

“…Recently, the hipAB system of Shewanella oneidensis was characterized, and a ternary HipAB-DNA complex different from the one from E. coli was observed (46). Since persister cells are highly problematic with respect to antibiotic treatments of infectious diseases, much emphasis has been placed on the development of drugs that suppress the development and survival of persister cells (47)(48)(49)(50)(51)(52)(53).…”

Resuscitation-Promoting Factors Are Cell Wall-Lytic Enzymes with Important Roles in the Germination and Growth of Streptomyces coelicolor

“…Other bacteria have evolved less extreme dormant states and instead adopt cell states with low metabolic activity such that these cells are better able to tolerate stresses such as antibiotic exposure or immune system assault (6). In addition to low metabolic activity, dormant cells of all types often have a cell wall architecture that differs from that of their vegetative counterparts.…”