Bile Salts Regulate Zinc Uptake and Capsule Synthesis in a Mastitis-Associated Extraintestinal Pathogenic Escherichia coli Strain

Abstract

Extraintestinal pathogenic Escherichia coli (ExPEC) strains are major causes of urinary and bloodstream infections. ExPEC reservoirs are not completely understood. Some mastitis-associated E. coli (MAEC) strains carry genes associated with ExPEC virulence, including metal scavenging, immune avoidance, and host attachment functions. In this study, we investigated the role of the high-affinity zinc uptake (znuABC) system in the MAEC strain M12. Elimination of znuABC moderately decreased fitness during mouse mammary gland infections. The ΔznuABC mutant strain exhibited an unexpected growth delay in the presence of bile salts, which was alleviated by the addition of excess zinc. We isolated suppressor mutants with improved growth in bile salts, several of which no longer produced the K96 capsule made by strain M12. The addition of bile salts also reduced capsule production by strain M12 and ExPEC strain CP9, suggesting that capsule synthesis may be detrimental when bile salts are present. To better understand the role of the capsule, we compared the virulence of mastitis strain M12 with that of its unencapsulated ΔkpsCS mutant in two models of ExPEC disease. The wild-type strain successfully colonized mouse bladders and kidneys and was highly virulent in intraperitoneal infections. Conversely, the ΔkpsCS mutant was unable to colonize kidneys and was unable to cause sepsis. These results demonstrate that some MAEC strains may be capable of causing human ExPEC illness. The virulence of strain M12 in these infections is dependent on its capsule. However, capsule may interfere with zinc homeostasis in the presence of bile salts while in the digestive tract.

Keywords: ExPEC; ZnuABC; bile salts; group III capsule; mastitis-associated E. coli; zinc transport.

FIG 1

Role of the znuABC zinc transporter in the mastitis-associated E. coli M12 strain. (A) The znu locus of strain M12 and the 2,560-bp region that was deleted to create the ΔznuABC mutant strain. The normalized TnSeq fitness scores predicted for each gene as calculated by Rendon et al. (34) are indicated. (B) Growth of the M12 wild-type, ΔznuABC mutant, and ΔznuABC(pMO3) complemented strain in LB broth containing 1.0 mM EDTA (means ± standard errors of the means [SEM]) (n = 3). The mutant displayed a significant delay in growth that was fully restored with the addition of 100 μM zinc or by complementation with the plasmid pMO3. The addition of supplemental zinc enhanced the growth of the wild-type strain. (C) Competitive fitness of the ΔznuABC mutant compared with the M12 wild-type strain during growth in milk (n = 6) or in mouse mammary glands (n = 6). Equal ratios of both strains were inoculated in unpasteurized bovine milk or injected through the teat canal of lactating mice. Bacterial numbers were determined after 8 h and 24 h, respectively. The mutant strain was significantly less fit (*, P = 0.03125 by a Mann-Whitney test) than the wild-type strain in mammary glands but not milk.

FIG 2

Loss of znuABC delays the growth of strain M12 specifically in the presence of bile salts. (A) Growth of M12 or the ΔznuABC mutant in LB medium containing 2% bile salts (means ± SEM) (n = 3). The mutant strain entered the exponential growth phase several hours later than the wild type (wt) but had similar absorbances at 600 nm and during the saturation phase. The addition of 100 μM zinc to the medium decreased the time that it took for the ΔznuABC mutant to enter exponential phase. (B and C) Identical growth curves of wild-type M12 and the ΔznuABC mutant in 5% SDS (B) or 2 μg/ml of polymyxin E (C) showing that the mutation does not confer a generalized membrane defect.

FIG 3

Suppressor mutations in the ΔznuABC background that restore growth in bile salts also eliminate capsule synthesis. (A) Saturated cultures of the wild-type strain and the ΔznuABC mutant grown in LB medium with bile salts and subcultured in the same medium exhibited similar growth (means ± SEM) (n = 3), suggesting the possibility of suppressor mutations arising in the population. (B) Ten independently derived suppressor mutants had increased growth in LB medium containing 2% bile salts compared to the wild-type strain. Whole-genome sequencing indicated that suppressor mutants 2, 8, and 10 contained SNPs that mapped to predicted capsule synthesis genes. (C) Gel electrophoresis and alcian blue staining (image representative of an experiment performed three times) showing that 8 of 10 suppressor mutants produce less capsule than the wild-type strain. (D and E) A ΔkpsCS mutant unable to produce capsule reaches exponential phase faster than the wild-type strain when grown in LB medium with bile salts, but growth in LB medium is indistinguishable from that of the wild-type strain.

FIG 4

Growth in bile salts reduces capsule expression by strain M12 and ExPEC strain CP9. (A) Gel electrophoresis and alcian blue staining of capsule produced by M12 and CP9 grown in LB medium or LB medium containing 2% bile salts. (B) Capsule measurement by flow cytometry. The M12 ΔkpsCS mutant strain was used to gate for cells that reacted with K54 anticapsule serum. In both M12 and CP9, the proportion of bacteria with detectable capsule was lower when grown in bile salts than when grown in LB medium alone. Shown is a representative image of an experiment that was repeated three times.

FIG 5

Virulence of mastitis-associated strain M12 in ExPEC infections depends on capsule production. (A) M12 wild-type (n = 6), M12 ΔkpsCS mutant (n = 6), and ExPEC CP9 (n = 4) strains (∼5 × 10⁵ CFU) were injected into the intraperitoneal cavity of C57BL/6 mice, and bacterial loads in the spleens were determined at 24 h. Both M12 wild-type and CP9 strains were recovered at high levels in the spleen, while the ΔkpsCS mutant was severely attenuated (P = 0.0303 by a Mann-Whitney test). The dotted line represents the limit of detection (∼100 CFU). (B) Adult female Swiss-Webster mice were inoculated via transurethral catheterization and sacrificed 72 h later. Both M12 wild-type (n = 8) and ΔkpsCS (n = 7) strains colonized the bladders of the mice to similar levels, but in the kidney, the mutant strain was not detected (P = 0.0003 by a Mann-Whitney test). Black and gray dotted lines represent the limits of detection for the bladder (∼293 CFU) and kidney (∼30 CFU) infections, respectively.