Identification and Characterization of the PutP Proline Permease That Contributes to In Vivo Survival ofStaphylococcus aureusin 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. aureusRN6390 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 theputP 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 theputP mutant than into strain RN6390. The reduced viability of the bacteria possessing the mutation in the S. aureushigh-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.

Identification and characterization of genes encoding sex pheromone cAM373 activity in Enterococcus faecalis and Staphylococcus aureus

“…No inverted or direct repeats were found flanking this segment, indicating that it is probably not a mobile DNA element. Genes for proline permease are found in both strains; however, the inverted repeat apparently serving as a bidirectional transcription terminator for the opposing genes in COL, and associated with the proline permease genes of other S. aureus strains (Wengender and Miller, 1995; Schwan et al , 1998), is only found adjacent to the camS gene in strain 879R4S, indicating that 879R4S may be somewhat unique in this regard. The organization of genes of the S. aureus chromosome including camS and upstream regions bears striking simi‐larity to that of Staphylococcus epidermidis , Bacillus subtilis and Listeria monocytogenes.…”

Development and Characterization of a Staphylococcus aureus Nasal Colonization Model in Mice

“…Our model might be used to observe the possible inhibitory impact of mutations or antibodies directed against such putative adhesins on the establishment of nasal colonization. It might also serve as a model to identify genes expressed during or essential to the colonization process by methods such as in vivo expression technology or signature-tagged mutagenesis, both of which have proven to be successful in identifying virulence factors expressed during experimental S. aureus infection of animals (10,19,25).…”

Staphylococcus aureus genetic loci impacting growth and survival in multiple infection environments

“…Therefore, such transport mechanisms might be viable as targets for novel inhibitors or exploited for drug uptake of novel antibiotic conjugates. In addition to the Opp transporters described here, other transport functions impacting in vivo survival were also identified, including a proton motive force-dependent di-tri-peptide transporter (dtp) (Steiner et al, 1995) and amino acid permease systems for proline (putP ) (Schwan et al, 1998) and alanine (alsT ) uptake. The assortment of independent mutations recovered in peptide and amino acid transport mechanisms emphasizes the biological significance of these loci on S. aureus survival in multiple in vivo environments.…”