Penicillin resistance of the bacterium that causes pneumonia, the pneumococcus, is a growing global health problem. Although S. pneumoniae was once considered to be routinely susceptible to penicillin, since the mid-1980s the incidence of resistance of this organism to penicillin and other antimicrobial agents has been increasing in the United States and throughout the world. Now, researchers at The Rockefeller University, reporting in the April 25 issue of the Proceedings of the National Academy of Sciences, show that resistance can be stopped by inactivating a pair of genes responsible for producing molecules called branched muropeptides, the availability of which appears to be essential for the bacterium to survive in the presence of penicillin. The finding suggests that the branched peptides may be a new drug target for fighting penicillin-resistant bacteria.
"We have known for some time about the connection between the branched muropeptides -- structural elements of the pneumococcal cell wall -- and penicillin resistance in pneumococcus," says senior author Alexander Tomasz, Ph.D., professor and head of the Laboratory of Microbiology at Rockefeller. "We have now identified two genes that are responsible for making these branched muropeptides, and we have shown for the first time that by inactivating these genes we can restore penicillin's potency. This opens the door to the development of new drugs that would act synergistically with penicillin by blocking the production of the branched peptides."
S. pneumoniae represents the most important microbial pathogen, causing a number of frequent community-acquired infections, some life threatening. In the United States alone, S. pneumoniae is estimated to cause at least 6,000 cases of meningitis, 50,000 cases of blood infections, a half million cases of pneumonia and several million cases of childhood ear infections annually. The global annual rate of mortality from pneumococcal disease is estimated at one million. A powerful threat to the elderly, young children and people with underlying medical conditions, including HIV infection, drug-resistant strains of pneumococcus are spreading from day-care centers to hospital rooms, raising the concern of public health officials and physicians about the possible failure of antibiotic therapy against the resistant bacteria.
For the last 20 years, researchers from Tomasz's lab have been studying the biochemical and genetic basis of penicillin resistance in the pneumococcus. The mechanism of penicillin resistance in clinical isolates of pneumococci was first identified in the Tomasz lab in 1980. He and former graduate student Sonia Zighelboim, Ph.D., studied South African strains of pneumococci that were a thousand times more resistant to penicillin than any other previous strains and discovered a new bacterial ploy. Instead of producing an enzyme that destroyed penicillin -- a tactic seen in resistant strains of another bacterial pathogen, Staphylococcus aureus -- the South African pneumococci rebuilt the target proteins of the antibiotic, enzymes called penicillin-binding proteins (PBPs).
Working in assembly-line fashion, PBPs play an important role in building the bacterial cell wall, an uninterrupted protective network of molecules that maintains the integrity of the cell. Penicillin produces its devastating effect on bacteria by inactivating PBPs and thus inhibiting synthesis of the cell wall. A thorough study by the researchers revealed the PBPs in the penicillin-resistant pneumococci had undergone subtle alterations in their DNA blueprints, reducing the ability of PBPs to bind penicillin, thus providing penicillin resistance to the bacteria.
By 1990, Tomasz and former Rockefeller postdoctoral fellow Jose Garcia-Bustos, Ph.D., discovered that the resistant pneumococci not only had altered low-affinity PBPs, but the altered PBPs appeared to build a chemically unusual cell wall enriched with the branched muropeptides. In penicillin-susceptible pneumococcal strains, most of the muropeptides are linear in shape. But in penicillin-resistant strains, "branched" muropeptides‹so-called because of the presence of two additional amino acids, either a serine and an alanine or two alanines, that branch off from the main peptide‹are abundant in the cell wall.
In the new work, Filipe and Tomasz identified two genes, called murM and murN, that work in concert and are involved in the synthesis of branched cell wall muropeptides. The two genes work in tandem in a genetic system called an operon. They showed that inactivation of the murMN operon in penicillin-resistant strains causes the disappearance of the branched muropeptides from the cell wall and also a complete loss of penicillin resistance.
"These studies show that penicillin resistance in the pnemococcus requires not only the alterations in the PBPs but intact murMN genes as well," says Filipe.
How exactly the murMN genes contribute to the expression of penicillin is not clear yet. Branched muropeptides may compete with penicillin for a site on the resistant PBPs, or they may perform some yet-to-be-identified signaling function in cell wall synthesis or occupy strategic sites within the cell wall that are important for the continued growth of bacteria in the presence of penicillin.
Tomasz and Filipe think that the synthesis of branched muropeptides is a good target for the design of new antibacterial drugs that would work synergistically with penicillin to treat resistant pneumococcal disease. Inhibitors of the synthesis of branched muropeptides may also reduce virulence of pneumococcal infections. The principle of using a combination of two drugs is already in use in such successful and widely marketed antimicrobial drugs as Augmentin, a combination of the antibiotic amoxicillin and clavulanate, which inhibits beta-lactamase, an enzyme that inactivates penicillin.
This research was supported in part by the National Institute of Allergy and Infections Diseases, part of the federal government's National Institutes of Health, and by the Irene Diamond Foundation.
Journal
Proceedings of the National Academy of Sciences