Polyamine-mediated resistance of uropathogenic Escherichia coli to nitrosative stress

Abstract

During the course of a urinary tract infection, substantial levels of nitric oxide and reactive nitrogen intermediates are generated. We have found that many uropathogenic strains of Escherichia coli display far greater resistance to nitrosative stress than the K-12 reference strain MG1655. By selecting and screening for uropathogenic E. coli transposon mutants that are unable to grow in the presence of acidified nitrite, the cadC gene product was identified as a key facilitator of nitrosative stress resistance. Mutation of cadC, or its transcriptional targets cadA and cadB, results in loss of significant production of the polyamine cadaverine and increased sensitivity to acidified nitrite. Exogenous addition of cadaverine or other polyamines rescues growth of cad mutants under nitrosative stress. In wild-type cells, the concentration of cadaverine produced per cell is substantially increased by exposure to acidified nitrite. The mechanism behind polyamine-mediated rescue from nitrosative stress is unclear, but it is not attributable solely to chemical quenching of reactive nitrogen species or reduction in mutation frequency.

FIG. 1.

The effect of 3 mM ASN on growth of E. coli strains UTI89 and MG1655. Fresh stationary phase starter cultures were subcultured 1:100 into 100 mM MES-buffered LB (pH 5) with or without added ASN. The cultures were grown with constant shaking at 37°C, and the OD₆₀₀ values were determined at the indicated times.

FIG. 2.

CadC expression from pPH2200 complements the mutant phenotype of the cadC transposon mutant, and ΔcadA, -B, and -C UTI89 mutants are attenuated for growth in the presence of 3 mM ASN. (A) ODs were determined 24 h after subculture into media containing 3 mM ASN and 1 mM IPTG, plus 50 μg of ampicillin/ml for the plasmid-bearing strain. (B) ODs were determined 24 h after subculture into medium containing 3 mM ASN. Bars show means ± the standard deviation of three replicates.

FIG. 3.

Rescue of the ΔcadA mutant by polyamines. (A) ODs of cultures with (+) or without (−) 3 mM ASN and with or without the indicated polyamines at 17 h after subculture with UTI89 or the isogenic ΔcadA mutant. This time point captures wild-type UTI89 (+ASN) in exponential growth phase and shows that spermine and spermidine are more effective than either cadaverine or putrescine in rescuing bacterial growth in ASN. Wild-type UTI89 and the polyamine-supplemented ΔcadA mutant all eventually recover fully in the presence of ASN, whereas the unsupplemented ΔcadA mutant invariably fails to multiply appreciably with ASN present. Bars show means ± the standard deviation of three replicates. (B) In the absence of ASN, wild-type and ΔcadA mutant cultures have very similar growth kinetics, and the addition of 3 mM cadaverine (cad) has little effect on bacterial growth. The datum points show the means of three replicates. For clarity, error bars are not shown, but the standard deviation for all points is less than 0.03 OD₆₀₀ units.

FIG. 4.

Quantitation of cadaverine production. (A) Comparison of total cadaverine generated by MG1655, UTI89, and the ΔcadA UTI89 mutant grown for 3 h in unsupplemented MES-buffered LB (pH 5). (B) Time course of cadaverine produced per CFU of wild-type UTI89 with or without added 1 mM ASN in the media. Bars show means ± the standard deviation of three replicates. ✽, Cadaverine levels below the minimal level of detection for the assay.

FIG. 5.

Cadaverine does not rescue growth of ΔcadA UTI89 in ASN by quenching RNI activity. (A) Diagram of the experimental protocol used. The final concentrations of both ASN and cadaverine in the indicated cultures were 3 mM. (B) ΔcadA UTI89 grows normally in MES-LB (pH 5) (⧫) but fails to multiply in the presence of 3 mM ASN (▪). Coincubating ASN with cadaverine for 12 h prior to addition of bacteria did not reduce its inhibitory activity (○) and actually rendered the medium slightly more attenuating to growth of the ΔcadA mutant than media in which there was no prior coincubation of ASN with cadaverine (▴). These results were consistent in three separate experiments and indicate that the protective effect of cadaverine is not via an RNI quenching mechanism.

FIG. 6.

Cadaverine does not abrogate the mutagenic effects of ASN. Wild-type UTI89 and the ΔcadA mutant were grown in MES-LB (pH 5) with or without 1 mM ASN for 24 h prior to plating on selective (plus 100 μg of rifamycin/ml) and nonselective LB agar. Mutation frequencies were determined by dividing the number of rifamycin-resistant colonies by the total number of bacteria present in each sample. Bars show the means ± the standard deviation of three replicates.