Lawrence Mulcahy

The majority of cystic fibrosis (CF) patients succumb to a chronic infection of the airway with Pseudomonas aeruginosa. Paradoxically, pathogenic strains are often susceptible to antibiotics, but the infection cannot be eradicated with antimicrobial therapy. We find that in a majority of patients with airway infections, late isolates of P. aeruginosa produce increased levels of drug-tolerant persister cells. The genomes of a clonal pair of early/late isolates from a single patient have been previously sequenced, and the late isolate (obtained at age 96 months) showed a 100-fold increase in persister levels. The 96-month isolate carries a large number of mutations, including a mutation in mutS that confers a hypermutator phenotype. There is also a mutation in the mexZ repressor controlling the expression of the MexXY-OprM multidrug pump, which results in a moderate increase in the ofloxacin, carbenicillin, and tobramycin MICs. Knocking out the mexXY locus restored the resistance to that of the parent strain but did not affect the high levels of persisters formed by the 96-month isolate. This suggests that the late isolate is a high-persister (hip) mutant. Increased persister formation was observed in exponential phase, stationary phase, and biofilm populations of the 96-month isolate. Analysis of late isolates from 14 additional patients indicated that 10 of them are hip mutants. Most of these hip mutants did not have higher drug resistance. Increased persister formation appears to be their sole mechanism for surviving chemotherapy. Taken together, these findings suggest a link between persisters and recalcitrance of CF infection and identify an overlooked culprit-high-persister mutants producing elevated levels of drug-tolerant cells. Persisters may play a similarly critical role in the recalcitrance of other chronic infections.

Antibiotic Mechanisms Revisited Several recent studies have suggested that bactericidal antibiotics kill cells by a common mechanism involving reactive oxygen species (ROS). Two groups tested this hypothesis using diverse experiments, with both finding that quinolone, lactam, and aminoglycoside antibiotics had similar efficacy for killing in the presence or absence of oxygen (or nitrate). Liu et al. (p. 1210 ) saw no increase in hydrogen peroxide production in antibiotic-exposed cells and found no association between antibiotic exposure and the expected symptoms of oxidative damage, such as the breakdown of iron-sulfur clusters in enzymes or of hydroxyl radical injuries to DNA. Similarly, Keren et al. (p. 1213 ) found no correlation between the production of ROS, inferred from hydroxyphenyl fluorescein dye measurements, and bacterial survival, nor was there any significant protective effect engendered by thiourea. The results do not support a common mode of action for bactericidal antibiotics mediated by ROS.

Cpe(fat/fat) mice are obese, diabetic, and infertile. They have a mutation in carboxypeptidase E (CPE), an enzyme that converts prohormone intermediates to bioactive peptides. The Cpe(fat) mutation leads to rapid degradation of the enzyme. To test whether pro-thyrotropin-releasing hormone (TRH) conversion to TRH involves CPE, processing was examined in the Cpe(fat/fat) mouse. Hypothalamic TRH is depressed by at least 75% compared with wild-type controls. Concentrations of pro-TRH forms are increased in homozygotes. TRH-[Gly(4)-Lys(5)-Arg(6)] and TRH-[Gly(4)-Lys(5)] represent approximately 45% of the total TRH-like immunoreactivity in Cpe(fat/fat) mice; they constitute approximately 1% in controls. Levels of TRH-[Gly(4)] were depressed in homozygotes. Because the hypothalamus contains some TRH, another carboxypeptidase must be responsible for processing. Immunocytochemical studies indicate that TRH neurons contain CPE- and carboxypeptidase D-like immunoreactivity. Recombinant CPE or carboxypeptidase D can convert synthetic TRH-[Gly(4)-Lys(5)] and TRH-[Gly(4)-Lys(5)-Arg(6)] to TRH-[Gly(4)]. When Cpe(fat/fat) mice are exposed to cold, they cannot maintain their body temperatures, and this loss is associated with hypothalamic TRH depletion and reduction in thyroid hormone. These findings demonstrate that the Cpe(fat) mutation can affect not only carboxypeptidase activity but also endoproteolysis. Because Cpe(fat/fat) mice cannot sustain a cold challenge, and because alterations in the hypothalamic-pituitary-thyroid axis can affect metabolism, deficits in pro-TRH processing may contribute to the obese and diabetic phenotype in these mice.