OppA of Listeria monocytogenes, an oligopeptide-binding protein required for bacterial growth at low temperature and involved in intracellular survival

Abstract

We identified a new oligopeptide permease operon in the pathogen Listeria monocytogenes. This opp operon consists of five genes (oppA, oppB, oppC, oppD, and oppF) and displays the same genetic organization as those of several bacterial species. The first gene of this operon, oppA, encodes a 62-kDa protein sharing 33% identity with OppA of Bacillus subtilis and is expressed predominantly during exponential growth. The function of oppA was studied by constructing an oppA deletion mutant. The phenotype analysis of this mutant revealed that OppA mediates the transport of oligopeptides and is required for bacterial growth at low temperature. The wild-type phenotype was restored by complementing the mutant with oppA. We also found that OppA is involved in intracellular survival in macrophages and in bacterial growth in organs of mice infected with L. monocytogenes, although the level of virulence was not altered in the mutant. These results show the major role of OppA in the uptake of oligopeptides and the pleiotropic effects of this oligopeptide-binding protein on the behavior of this pathogen in the environment and in its host.

FIG. 1

Genetic organization of the opp operons of L. monocytogenes, B. subtilis, S. pyogenes, and S. enterica serovar Typhimurium. Similarities between the Opp proteins are given as percent amino acid (aa) identities.

FIG. 2

Northern blot analysis of the opp operon of L. monocytogenes LO28. Bacterial strains were cultured at 37°C to mid-log exponential phase, and total RNA was extracted and hybridized with an oppA probe (A) or an oppB probe (B). Lanes 1 and 3, LO28 (wild type); lane 2, the oppA mutant.

FIG. 3

The oppA mutant is resistant to bialaphos. Bacteria were grown overnight in BHI broth and washed three times in minimal defined medium before being spread onto solid minimal defined medium plates at 37°C, with bialaphos (10 μg) spotted on a filter disc in the center of the plate. After 24 h, the zone of inhibition was measured. (A) LO28; (B) oppA mutant; (C) oppA-complemented mutant.

FIG. 4

Growth of L. monocytogenes in minimal defined medium lacking valine and/or supplemented with valine or valine-containing peptides. Wild-type or oppA bacteria were grown at 37°C, and the optical density (O.D.) at 600 nm was measured after 36 h of incubation. Controls include bacteria grown with valine (V) or without valine (−).

FIG. 5

The OppA protein of L. monocytogenes is essential for growth at 5°C. Bacteria were cultured in BHI broth, and growth was monitored by measuring optical density (O.D.) at 600 nm. (A) Growth at 5°C. ●, LO28; ▴, oppA mutant. (B) Bacteria were grown to mid-log exponential phase at 37°C and transferred to 5°C until the end of growth. Growth at 5°C is restored in an oppA-complemented mutant. ●, LO28/pAT18; ▴, oppA mutant/pAT18; ▪, oppA-complemented mutant. (C) Northern blot analysis of the oppA gene of L. monocytogenes expressed at 37°C (lane 1) or 5°C (lane 2). LO28 bacteria were cultured at 37 or 5°C to midlog exponential phase, and total RNA was extracted and hybridized with an oppA probe.

FIG. 6

Growth of L. monocytogenes in macrophages. Bone marrow-derived macrophages from C57/BL6 mice were exposed for 15 min (time zero) to bacteria (1 bacterium per cell), and bacterial survival was monitored for 10 h after the infection. ●, LO28 (wild type); ▴, oppA mutant. Bacterial growth of the oppA mutant was delayed compared to that of wild-type bacteria. Error bars indicate standard deviations.

FIG. 7

Confocal microscopy of bone marrow-derived macrophages infected (15 bacteria per cell) with LO28 (A to C) or the oppA mutant (D to F). Macrophages were observed at time zero (A to D), at 4 h (B and E), and at 8 h (C and F) postinfection. F-actin was stained with phalloidin (green). Bacteria were labeled with anti-Listeria antibodies (red). In the absence of OppA, there is a reduction of intracellular growth.

FIG. 8

Growth of L. monocytogenes LO28 (wild type) (●) or the oppA mutant (▴) in the spleens (A) or livers (B) of mice inoculated i.v. with 8 × 10⁵ bacteria. Error bars indicate standard deviations.