Two zinc uptake systems contribute to the full virulence of Listeria monocytogenes during growth in vitro and in vivo

Abstract

We report here the identification and characterization of two zinc uptake systems, ZurAM and ZinABC, in the intracellular pathogen Listeria monocytogenes. Transcription of both operons was zinc responsive and regulated by the zinc-sensing repressor Zur. Deletion of either zurAM or zinA had no detectable effect on growth in defined media, but a double zurAM zinA mutant was unable to grow in the absence of zinc supplementation. Deletion of zinA had no detectable effect on intracellular growth in HeLa epithelial cells. In contrast, growth of the zurAM mutant was significantly impaired in these cells, indicating the importance of the ZurAM system during intracellular growth. Notably, the deletion of both zinA and zurAM severely attenuated intracellular growth, with the double mutant being defective in actin-based motility and unable to spread from cell to cell. Deletion of either zurAM or zinA had a significant effect on virulence in an oral mouse model, indicating that both zinc uptake systems are important in vivo and establishing the importance of zinc acquisition during infection by L. monocytogenes. The presence of two zinc uptake systems may offer a mechanism by which L. monocytogenes can respond to zinc deficiency within a variety of environments and during different stages of infection, with each system making distinct contributions under different stress conditions.

Fig 1

Schematic of the zinc uptake operons of L. monocytogenes EGDe (A) and schematic of strains used in the present study (B).

Fig 2

Transcription of the zurA-zurM-zur operon is regulated by Zur and zinc. The β-galactosidase activity generated by zur-lacZ, Δzur::lacZ, and Δzur::lacZ pZur strains was determined after 22 h of growth in either defined medium alone (black) or in defined medium supplemented with 50 μM ZnSO₄ (dark gray bars), 600 nM TPEN (light gray bars), or both (white bars), as indicated. The data are the means of three independent experiments ± the SE.

Fig 3

Metal content analysis of L. monocytogenes. ICP-MS analysis of nitric acid-digested, chelate-washed cell pellets of L. monocytogenes wild type (WT), ΔzurAM, and ΔzinA strains following growth in defined medium with (+) or without (−) 1 μM TPEN supplementation for 22 h. Values are the numbers of atoms of manganese (black bars), iron (gray bars), or zinc (white bars) per CFU. The data are the means of three independent experiments ± the SE.

Fig 4

Transcription of the zinABC operon is regulated by Zur and zinc. The β-galactosidase activity generated by zin-lacZ, Δzur zin-lacZ, and Δzur zin-lacZ pZur strains was determined after 22 h of growth in either defined medium (black bars) or in defined medium supplemented with 50 μM ZnSO₄ (dark gray bars), 600 nM TPEN (light gray bars), or both (white bars), as indicated. The data are the means of three independent experiments ± the SE.

Fig 5

Growth of zinc uptake mutants in vitro. Growth (A₆₀₀) of wild-type (squares), ΔzurAM (triangles), ΔzinA (diamonds), or ΔzurAM ΔzinA (circles) strains in defined medium (A) or in defined medium supplemented with 50 μM ZnSO₄ (B) or TSB (D). Final A₆₀₀ values of cultures of wild-type (WT), ΔzurAM ΔzinA mutant, or the complemented ΔzurAM ΔzinA mutant (ΔΔcomp) strains were determined in defined medium after 22 h of growth (C) or TSB after 5 h of growth (E) either with (white bars) or without (black bars) 50 μM ZnSO₄ supplementation. In all cases, the data are the means of at least three independent experiments ± the SE.

Fig 6

Comparison of intracellular growth of zinc uptake mutant strains in HeLa cells. HeLa cells were infected with an MOI of 10 with either the wild-type (squares), ΔzurAM (triangles), ΔzinA (diamonds), ΔzurAM ΔzinA (circles) or Δhly ΔplcB (crosses) strain, and growth was assessed by lysing HeLa cells at intervals, followed by serial dilution and viable counting of bacteria. The data are the means of at least three independent experiments ± the SE.

Fig 7

Immunofluorescence analysis of HeLa cells at 4.5 h (A to H) or 24 h (I to K) postinfection. Images are of uninfected HeLa cells (A, B, and I) or HeLa cells infected with a wild-type (C, D, and J), Δhly ΔplcB (E and F), or ΔzurAM ΔzinA (G, H, and K) strain. Bacteria appear red due to labeling of the bacterial cell surface by the anti-listeria antiserum, cellular F-actin appears green due to phalloidin-FITC, and HeLa cell nuclear material appears blue due to staining with Hoechst 33342 DNA dye.

Fig 8

Survival of MF-1 mice orally infected with either wild-type L. monocytogenes (solid line) or ΔzurAM (dashes), ΔzinA (dots and dashes), or ΔzurAM ΔzinA (dots) mutant strains. Mice were infected as described in Materials and Methods and monitored over a 240-h period.