The antibiotic trimethoprim (TMP) is used to treat a variety of Escherichia coli infections, but its efficacy is limited by the rapid emergence of TMP-resistant bacteria. Previous laboratory evolution experiments have identified resistance-conferring mutations in the gene encoding the TMP target, bacterial dihydrofolate reductase (DHFR), in particular mutation L28R. Here, we show that 4’-desmethyltrimethoprim (4’-DTMP) inhibits both DHFR and its L28R variant, and selects against the emergence of TMP-resistant bacteria that carry the L28R mutation in laboratory experiments. Furthermore, antibiotic-sensitive E. coli populations acquire antibiotic resistance at a substantially slower rate when grown in the presence of 4’-DTMP than in the presence of TMP. We find that 4’-DTMP impedes evolution of resistance by selecting against resistant genotypes with the L28R mutation and diverting genetic trajectories to other resistance-conferring DHFR mutations with catalytic deficiencies. Our results demonstrate how a detailed characterization of resistance-conferring mutations in a target enzyme can help identify potential drugs against antibiotic-resistant bacteria, which may ultimately increase long-term efficacy of antimicrobial therapies by modulating evolutionary trajectories that lead to resistance.

Significance: Light-chain amyloidosis (AL) is a devastating human disease involving the clonal expansion of a plasma cell and the secretion of destabilized, amyloidogenic immunoglobulin light chains (LCs). Secreted amyloidogenic LCs aggregate extracellularly, leading to proteotoxicity on distal tissues. Available therapeutic strategies to treat AL specifically target the cancerous plasma cell population. While this approach is effective in ∼70% of patients, patients who present with substantial LC-related organ proteotoxicity are generally too sick to tolerate standard chemotherapeutics. Here, we show that stress-independent activation of unfolded protein response-associated transcription factors selectively reduces secretion of amyloidogenic LCs and decreases extracellular soluble LC aggregates associated with proteotoxicity in AL. These results identify a promising therapeutic strategy to treat AL patients unserved by current treatments.

Mitochondrial dysfunction, proteasome inhibition, and α‐synuclein aggregation are thought to play important roles in the pathogenesis of Parkinson’s disease (PD). Rare cases of early‐onset PD have been linked to mutations in the gene encoding DJ‐1, a protein with antioxidant and chaperone functions. In this study, we examined whether DJ‐1 protects against various stresses involved in PD, and we investigated the underlying mechanisms. Expression of wild‐type DJ‐1 rescued primary dopaminergic neurons from toxicity elicited by rotenone, proteasome inhibitors, and mutant α‐synuclein. Neurons with reduced levels of endogenous DJ‐1 were sensitized to each of these insults, and DJ‐1 mutants involved in familial PD exhibited decreased neuroprotective activity. DJ‐1 alleviated rotenone toxicity by up‐regulating total intracellular glutathione. In contrast, inhibition of α‐synuclein toxicity by DJ‐1 correlated with up‐regulation of the stress‐inducible form of Hsp70. RNA interference studies revealed that this increase in Hsp70 levels was necessary for DJ‐1‐mediated suppression of α‐synuclein aggregation, but not toxicity. Our findings suggest that DJ‐1 acts as a versatile pro‐survival factor in dopaminergic neurons, activating different protective mechanisms in response to a diverse range of PD‐related insults.

Accumulation of amyloid β-peptide (Aβ) and tau aggregates, possibly linked to age-associated deficiencies in protein homeostasis, appear to cause Alzheimer's disease. Schiff-base formation between Aβ and the aldehyde-bearing cholesterol oxidation product 3-β-hydroxy-5-oxo-5,6- seco cholestan-6-al is known to increase Aβ amyloidogenicity. Here, we synthesized Aβ variants site-specifically modified with the cholesterol aldehyde at Asp-1, Lys-16, or Lys-28, rather than studying mixtures. These distinct modifications have a similar effect on the thermodynamic propensity for aggregation, enabling aggregation at low concentrations. In contrast, the modification site differentially influences the aggregation kinetics; Lys-16-modified Aβ formed amorphous aggregates fastest and at the lowest concentration (within 2 h at a concentration of 20 nM), followed by the Lys-28 and Asp-1 conjugates. Also, the aggregates resulting from Aβ Lys-16 cholesterol aldehyde conjugation were more toxic to primary rat cortical neurons than treatment with unmodified Aβ under identical conditions and at the same concentration. Our results show that Aβ modification by cholesterol derivatives, especially at Lys-16, renders it kinetically and thermodynamically competent to form neurotoxic aggregates at concentrations approaching the physiologic concentration of Aβ.