Mutational and transcriptional analyses of the Staphylococcus aureus low-affinity proline transporter OpuD during in vitro growth and infection of murine tissues

Abstract

Staphylococcus aureus continues to be a major health problem. This species' requirement for proline and proline transport from the extracellular environment is not well understood. Here, we identify a S. aureus low-affinity proline transport gene (opuD) with homology to the OpuD protein of Bacillus subtilis. Mutation of the opuD gene caused a significant decline in proline uptake under low-affinity conditions as compared with wild type, but the opuD mutant strain showed no significant attenuation in a murine abscess model of infection. The S. aureus opuD gene was transcriptionally activated during growth in moderate osmolarity media with high levels of proline or glycine betaine independent of SigB. In murine abscesses, the opuD gene was activated at a later time point, whereas the opuD expression dropped over the course of an 18-h period within murine urinary tracts. Transcriptional regulation of opuD in S. aureus appears to be coordinated within this species when grown in moderate to high NaCl environments, but the level of extracellular proline had a marked effect on expression of this proline transport gene. The differential regulation of proline transport genes in S. aureus may be an adaptation for life in a variety of environments, including survival within the human body.

FIG. 1

Effects of proline (open column), betaine (closed column), choline (striped column), and carnitine (gray column) concentration on S. aureus strains DAN2A (opuD∷luxABCDE) as determined with an luxABCDE transcriptional fusion. The RLU/bacterial cell were measured with a luminometer and then divided by viable counts; means ± standard deviations are indicated from at least three separate runs. The * denotes significance of P < 0.05 and ** denotes significance of P < 0.0002.

FIG. 2

Effects of osmolarity on S. aureus strains A) DAN2A and B) KJW4-4 as determined with a opuD∷luxABCDE transcriptional fusion. The RLU/bacterial cell were measured with a luminometer and then divided by viable counts; means ± standard deviations are indicated from at least three separate runs. Osmolarity effects were tested by using sucrose (closed column), sorbitol (striped column), or NaCl (open column) as the osmolyte. The * denotes significance of P < 0.05.

FIG. 2

Effects of osmolarity on S. aureus strains A) DAN2A and B) KJW4-4 as determined with a opuD∷luxABCDE transcriptional fusion. The RLU/bacterial cell were measured with a luminometer and then divided by viable counts; means ± standard deviations are indicated from at least three separate runs. Osmolarity effects were tested by using sucrose (closed column), sorbitol (striped column), or NaCl (open column) as the osmolyte. The * denotes significance of P < 0.05.

FIG. 3

Effects of osmolarity on E. coli DH5α determined with an opuD∷luxABCDE transcriptional fusion. The RLU/bacterial cell were measured with a luminometer and then divided by viable counts; means ± standard deviations are indicated from at least three separate runs. Osmolarity effects were tested by using NaCl as the osmolyte. The * denotes significance of P < 0.05 and ** denotes significance of P < 0.001.

FIG. 4

Growth stage and osmolarity effects on S. aureus strain DAN2A as determined with a opuD∷luxABCDE, transcriptional fusion. The conditions tested were A) DSM broth plus 0 mM NaCl, B) DSM plus 400 mM NaCl, and C) DSM broth plus 800 mM NaCl. Both RLUs (open column) and O.D.₆₀₀ (closed triangle) readings were measured. Means ± standard deviations are indicated from at least three separate runs.

FIG. 4

Growth stage and osmolarity effects on S. aureus strain DAN2A as determined with a opuD∷luxABCDE, transcriptional fusion. The conditions tested were A) DSM broth plus 0 mM NaCl, B) DSM plus 400 mM NaCl, and C) DSM broth plus 800 mM NaCl. Both RLUs (open column) and O.D.₆₀₀ (closed triangle) readings were measured. Means ± standard deviations are indicated from at least three separate runs.

FIG. 4

Growth stage and osmolarity effects on S. aureus strain DAN2A as determined with a opuD∷luxABCDE, transcriptional fusion. The conditions tested were A) DSM broth plus 0 mM NaCl, B) DSM plus 400 mM NaCl, and C) DSM broth plus 800 mM NaCl. Both RLUs (open column) and O.D.₆₀₀ (closed triangle) readings were measured. Means ± standard deviations are indicated from at least three separate runs.

FIG. 5

Expression of opuD in murine tissues after infection with S. aureus strain KJW4-4 as determined with a opuD∷luxABCDE transcriptional fusion after 0, 4 or 6, and 18 h post-inoculation. Murine tissues included A) bladder, B) kidney, and C) thigh abscess. The RLU were measured with a luminometer and then divided by viable counts; means ± standard deviations are indicated. Five animals per time were examined per tissue. The * denotes significance of P < 0.05.

FIG. 5

Expression of opuD in murine tissues after infection with S. aureus strain KJW4-4 as determined with a opuD∷luxABCDE transcriptional fusion after 0, 4 or 6, and 18 h post-inoculation. Murine tissues included A) bladder, B) kidney, and C) thigh abscess. The RLU were measured with a luminometer and then divided by viable counts; means ± standard deviations are indicated. Five animals per time were examined per tissue. The * denotes significance of P < 0.05.

FIG. 5

Expression of opuD in murine tissues after infection with S. aureus strain KJW4-4 as determined with a opuD∷luxABCDE transcriptional fusion after 0, 4 or 6, and 18 h post-inoculation. Murine tissues included A) bladder, B) kidney, and C) thigh abscess. The RLU were measured with a luminometer and then divided by viable counts; means ± standard deviations are indicated. Five animals per time were examined per tissue. The * denotes significance of P < 0.05.