New Model Of Staph Drug Resistance Implicates Unlikely Protein; Findings May Lead To New Drugs That Overcome Resistance

The new study, which is co-authored by Mariana Pinho, Hermínia de Lencastre, Ph.D, and Alexander Tomasz, Ph.D., and appears in the August 21 online edition of the Proceedings of the National Academy of Sciences, emphasizes the importance of looking for new antibiotics that target this protein.

"Mariana's work shows that the old model of drug resistance in staphylococci is no longer tenable," says Tomasz, principal investigator of the research and professor at Rockefeller.

Since the introduction of antibiotics in the 1940s, Staphylococcus aureus, the number one cause of potentially life-threatening hospital-borne infections, has developed several mechanisms to beat the potentially lethal effects of chemotherapeutic agents. Of these bacterial tricks, the most important was the acquisition of a foreign gene - the mecA gene - that confers resistance to methicillin in addition to penicillin and a host of other antibiotics, all belonging to what scientists refer to as the beta-lactam class of antibiotics.

Today, almost half of all staphylococcal infections in U.S. hospitals are caused by these methicillin-resistant S. aureus (MRSA) strains. By studying the bacteria's resistance mechanisms at the molecular level, researchers in the Laboratory of Microbiology at Rockefeller, working with researchers in the Laboratory of Molecular Genetics at the Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Portugal, hope to come up with new strategies for fighting such resilient microbes.

The beta-lactam class of antibiotics kills bacteria by binding to and inactivating proteins, called penicillin-binding proteins (PBP), that help build the bacterial cell wall. Without a proper cell wall, the bacteria essentially cannot hold themselves together and burst. However, MRSA strains are impervious to antibiotics because the acquired mecA gene codes for a slightly different PBP - PBP2A - which is not destroyed by beta-lactam antibiotics. Formerly, scientists believed that this foreign PBP conferred drug resistance by completely taking over the role of its native counterparts in cell wall synthesis, thereby allowing the bacteria to survive despite the presence of antibiotics.

But a major study led by de Lencastre in 1994 punched a hole in this model of beta-lactam resistance: she and Tomasz showed that a large number of other proteins native to staphylococci, termed auxiliary proteins, were also necessary for PBP2A to work. Using genetic techniques to sort out these proteins, Pinho discovered that one of them was PBP2. The observation was perplexing: how could a protein exquisitely sensitive to beta-lactam antibiotics be involved in resistance against these drugs?

Now, the researchers have identified the solution to this riddle. They show that the acquired PBP2A in MRSA strains cooperates with a specific region of the native PBP2 to build cell walls in the presence of beta-lactam antibiotics.

"This is the first time we have seen an imported protein learn to cooperate with a native protein," says Tomasz.

Furthermore, the researchers demonstrate that the two proteins functionally complement each other in cell wall synthesis. PBPs tend to exhibit two separate functions when building the cross-hatched network of cell walls - linking sugars and linking short protein pieces called peptides. The recent work finds that the acquired PBP2A serves to link peptides while the native PBP2 links sugars.

This explains why it is possible for PBP2 to aid in resistance while still being inactivated by the antibiotic. Beta-lactam antibiotics are known to inactivate PBPs by binding to the peptide-linking region of the protein, hence the sugar-linking region remains free to cooperate in the resistance mechanism.

The findings indicate that the sugar-linking region or domain of PBP2 is a good target for new antibiotics. Drugs designed to block this activity could be used in combination with beta-lactam antibiotics to disable resistance mechanisms and successfully kill the staphylococci bacteria.

"Our results immediately suggest a drug target that until now has gone largely unexplored," says Pinho.

The emergence of antibiotic resistance in infectious microbes throughout the world is a serious public health threat. According to a recent report by the World Health Organization, "Drug-resistant infections in rich and developing nations alike are threatening to make once treatable diseases incurable." The development of new antibiotics along with the modification of old ones is of utmost importance.

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This paper is available online at the Proceedings of the National Academy of Sciences Web site: http://www.pnas.org.

The research was funded by a grant from the National Institutes of Health to Tomasz and by a grant from the Fundação para a Ciência e Tecnologia (FCT), Ministry of Science and Technology, Lisbon, Portugal to de Lencastre.

Tomasz is the Plutarch Papamarkou Professor of Microbiology and Infectious Diseases at The Rockefeller University. Pinho is a visiting scientist at Rockefeller from the Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, and Hermínia de Lencastre is a senior research associate at Rockefeller and head of the Laboratory of Molecular Genetics at the Instituto de Tecnologia Química e Biológica.

John D. Rockefeller founded Rockefeller University in 1901 as The Rockefeller Institute for Medical Research. Rockefeller scientists have made significant achievements, including the discovery that DNA is the carrier of genetic information. The University has ties to 21 Nobel laureates, six of whom are on campus. Rockefeller University scientists have received the award in two consecutive years: neurobiologist Paul Greengard, Ph.D., in 2000 and cell biologist Günter Blobel, M.D., Ph.D., in 1999, both in physiology or medicine. At present, 33 faculty are elected members of the U.S. National Academy of Sciences, including the president, Arnold J. Levine, Ph.D. Celebrating its centennial anniversary in 2001, Rockefeller the nation's first biomedical research center continues to lead the field in both scientific inquiry and the development of tomorrow's scientists.