Identification and characterization of the PutP proline permease that contributes to in vivo survival of Staphylococcus aureus in animal models

Abstract

Staphylococcus aureus is an important pathogen of humans and other animals, causing bacteremia, abscesses, endocarditis, and other infectious syndromes. A signature-tagged mutagenesis (STM) system was adapted for use in studying the genes required for in vivo survival of S. aureus. An STM library was ultimately created in S. aureus RN6390, with Tn917 being used to create the transposon mutations. Pools of S. aureus RN6390 mutants were screened in mouse abscess, bacteremia, and wound infection models for growth attenuation after in vivo passage. One of the mutants that was identified displayed marked attenuation following large-pool screening in all three animal models, which was confirmed in bacteremia and endocarditis models of infection with a smaller pool of mutants. Sequence analysis of the entire open reading frame showed a 99% identity to the high-affinity proline permease (putP) gene characterized in another strain of S. aureus. In wound and murine abscess infection models, the putP mutant was approximately 10-fold more attenuated than was wild-type strain RN6390. Another S. aureus strain transduced with the putP mutation also displayed an attenuated phenotype after passage in the wound model. A [3H]proline uptake assay showed that less proline was specifically transported into the putP mutant than into strain RN6390. The reduced viability of the bacteria possessing the mutation in the S. aureus high-affinity proline permease suggests that proline scavenging by the bacteria is important for in vivo growth and proliferation and that analogs of proline may serve as potential antistaphylococcal therapeutic agents.

FIG. 1

Identification of attenuated genes by large-pool DNA dot blot hybridizations. PCR-amplified DNAs from S. aureus cells passaged through culture (in vitro), an abscess model, an i.v. (IV) systemic model (spleen and liver), and a wound model were radiolabelled and used to probe a 96-well array. The hybridization patterns from the array for the putP::Tn917 mutant strain 16F-157 and an unrelated (UR) Tn917 mutant of strain RN6390 are depicted.

FIG. 2

Limiting-dilution PCR analyses of DNA from S. aureus cells isolated from infected cardiac vegetations compared to in vitro-grown cells. New Zealand White rabbits were infected with a pool of 11 S. aureus ST mutants, and the infected cardiac tissue was collected 1 day postinoculation. DNAs were serially diluted 1/4, starting at a concentration of 10 ng/μl, and each dilution was PCR amplified with either a 157-plus-SIG-BLOT-R primer pair (16F-157) or a 123-plus-SIG-BLOT-R primer pair (unrelated [UR] Tn917 mutant). A 100-bp molecular size marker (MW; Gibco/BRL) was used to measure the sizes of the UR Tn917 PCR product (295 bp) and the 16F-157 PCR product (299 bp).

FIG. 3

Proline uptake by the high-affinity system of S. aureus. Transport measurements were performed after 2, 6, or 10 min by the filtration method outlined in the text. Proline was present at 5 μM. Cells were suspended in transport buffer at 40 μg of total cellular protein per ml. Symbols: □, RN6390; ⧫, RN6390 plus 10 mM AZ; ○, RN6390 plus 10 mM DHP; ▴, 16F-157 (putP::Tn917). This is a representative graph from three independent experiments.

FIG. 4

Growth curves of 16F-157 (putP::Tn917) and the parental strain RN6390 with or without proline analogs in BHI broth. Symbols: □, RN6390; ⧫, RN6390 plus 10 mM DHP; ○, RN6390 plus 10 mM AZ; ▴, 16F-157 (putP::Tn917). This is a representative graph from two independent experiments.

FIG. 5

Comparison of the putP mutant and two other mutants to the wild-type strain RN6390 in the murine abscess model. Viable counts were determined for the initial inocula and at 3 or 7 days postinoculation. Symbols: □, RN6390; ⧫, 16F-157 (putP::Tn917); ○, an unrelated (UR) Tn917 SigTag mutant; ▴, an agr mutant (agr::TetM) of strain RN6390.

FIG. 6

Comparison of the putP mutant and another Tn917 mutant to the wild-type strain RN6390 in the murine burn wound model. Viable counts were determined at 1, 4, and 7 days postinoculation. Symbols: □, RN6390; ░⃞, 16F-157 (putP::Tn917); ▪, an unrelated (UR) Tn917 SigTag mutant.