Structure/function analysis of the Staphylococcus aureus PutP protein

Staphylococcus aureus causes a variety of diseases in humans and other animals. Antibiotics used to treat the infections are becoming less effective due to drug resistance. Thus, new drugs must be found, possibly targeting proline transporters. Staphylococcus aureus must import proline and two separate proline transporters have been recognized. The putP gene encodes the high affinity proline transporter used in environments low in proline. Further characterization of the putP protein is critical for assessing which amino acids are important for proline transport. To achieve this aim, a low stringency PCR was used to randomly mutate the putP gene. Truncated versions of the putP gene at the 3’ end were also generated, giving rise to truncated PutP proteins at the carboxy terminus. The mutated and truncated putP PCR products were ligated onto high copy number plasmids and transformed into an Escherichia coli strain lacking all proline transporter genes. Three clones with a mutated putP gene showed diminished growth in minimal medium. The plasmids from each strain will be sequenced to determine where the mutation in putP has arisen. This information could be useful in determining which amino acids are essential for proper proline transport by PutP. INTRODUCTION Staphylococcus aureus infections, or “Staph infections”, are a significant cause of human and animal disease around the world. These bacteria can cause afflictions ranging from food poisoning and skin abscesses, to bacteremias and endocarditis (6). In fact, S. aureus is believed to be the cause of one third of all food borne illnesses in the U.S. Of more concern, S. aureus has become the number one cause of hospital-acquired infections (6). Unfortunately, antibiotic resistance has also become an increasing problem, with MRSA (methicillin-resistant S. aureus) and VRSA (vancomycin-resistant S. aureus) strains being isolated (3, 9). For some VRSA, no known chemotherapeutic agent is effective. This is why new antibiotics targeting S. aureus need to be developed. Gram negative enteric bacteria Escherichia coli and Salmonella typhimurium have also been shown contain a proline transporter gene similar to putP in S. aureus (8). Interestingly, these enteric bacteria have a much narrower osmotic growth range, which suggest that the proline transporter systems of S. aureus are able to function better at high and low osmotic environments. Since the ability of the proline transport systems in S. aureus may account for this ability to grow in such high osmotic conditions, an understanding of function at the molecular level would also be beneficial. Previous studies, using animal models of infection, have demonstrated that proline transport is necessary for the survival of S. aureus during an infection (1, 5). S. aureus has two different types of proline transport systems: a low-affinity transporter and a high-affinity transporter encoded by the putP gene (7, 8). It has been demonstrated that bacteria possessing a mutated putP gene survive less well in animals, suggesting that the high-affinity proline transport system is vital for causing disease in humans (1, 3). Understanding how the PutP proline transporter works is the first step in developing a new anti-staphylococcal drug targeting proline transport. Specific amino acids in the PutP protein need to be identified which are vital to its proper functioning. Once these specific amino acids are identified, a new anti-staphlococcal drug could be developed to target these important amino acids.