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Laboratory of Microbiology

Alexander Tomasz
Plutarch Papamarkou Professor

We are interested in the chemical structure, mode of assembly, and biological functions of bacterial cell walls and associated cell surface components. Cell walls may be envisioned as vast networks of covalent bonds organized into macromolecular sheets with the shape and size of the entire bacterial cell. This is a dynamic structure undergoing profound and highly regulated structural changes related to cell division, growth rate, and mutations affecting penicillin resistance. Specific interactions between bacterial surface components and the immune system make up many of the molecular events of disease in a host invaded by bacteria. In our studies interest in issues of basic microbiology interlock with important problems of medicine, chemotherapy, and infectious disease. A special focus of our current studies is the mechanism of antibiotic resistance and virulence in multidrug-resistant pneumococci, staphylococci, and enterococci, as well as molecular epidemiology of drug-resistant clones.

Penicillin-binding Proteins (PBPs) and Penicillin Resistance in Pneumococci. Bacteria assemble the macromolecular sheets of cell walls on the outer surface of the cell membrane, i.e., essentially in outer space, and some of the enzymes catalyzing this process are sensitive to penicillin and may be detected as proteins covalently binding radioactive penicillin.

A novel mechanism of antibiotic resistance among clinical strains of pneumococci involves reengineering of PBP structure to lower antibiotic affinity. Most interestingly, such resistant PBPs also build structurally abnormal cell walls. The mechanism of PBP reengineering involves import of blocks of foreign DNA from as-yet-unidentified DNA donors and the generation of "mosaic" PBP genes containing nonpneumococcal sequences. Such mosaics may be used as specific fingerprints of a particular, resistant PBP gene. The combination of such fingerprints with methods that identify chromosomal backgrounds was used to demonstrate the extensive geographic speed of multidrug resistant pneumococcal clones.

The lab’s discovery of PBP reengineering built on much earlier work. In 1965, while studying genetic competence, a state in which bacteria can take up DNA and undergo genetic transformation, Dr. Tomasz and his colleagues found that pneumococci manufacture and excrete a modified peptide that acts like hormone, synchronizing the competence of a population. This discovery was the first evidence bacterial cells ‘talk’ to one another to coordinate their activities, a concept later known as quorum sensing (Bassler and Losick, 2006).

Methicillin-resistant Staphylococci (MRSA). MRSA strains have become the most important causative agents of hospital-borne infections worldwide. The molecular mechanism involves acquisition of a foreign genetic element (mec) carrying the structural gene for a low-affinity PBP. Full expression of high level resistance also requires a surprisingly large number of domestic auxiliary genes which may be inactivated by Tn551 mutagenesis. A new transposon library in the background of a highly resistant strain has identified a large number of new chromosomal sites (representing at least 12 new genes) the inactivation of which leads to the drastic reduction of methicillin resistance. These sites are located on the SmaI fragments A, B, C, D, E, F, and I. Several of the new mutants showed novel cell wall defects: block in the amidation of the alpha carboxyl group in the D-glutamic acid residues of the cell wall stem peptides; partial block in the addition of diamino acid residue; incomplete oligoglycine cross-links.

Murein Hydrolases, Autolysins, and Antibiotic Tolerance. Enlargement of the cell wall must include at least a transient rupture of some covalent bonds. Candidates for the catalysis of such a process are bond-transferring enzymes (transpeptidases). Another kind of bond-rupturing enzymes, autolysins, are involved with the lysis of bacteria during treatment with penicillin. As a follow-up to the previously described Staphylococcus aureus mutant with Tn551-inactivated autolytic system, the autolysin gene was cloned and sequenced in Escherichia coli. The data indicate that the autolysin is a bifunctional protein which contains both an amidase and an endo--N-acetylglucosaminidase domain, and is processed to generate these two enzymes in extracellular form. A pneumococcal transposon mutant with greatly increased endoglycosidase activity was isolated. In contrast to the major autolysin, the endoglycosidase does not require choline residues for its cell wall degrading activity.

Biological Activities of Cell Walls. Chemical components built into the wall polymers are known to be responsible for invoking a whole spectrum of host responses related to inflammation and disease. The ability of cell walls prepared from gram-positive bacteria to induce TNF-alpha and IL-6 production by human monocytes awas demonstrated. Optimal activity required a protein cofactor. A search is on to identify chemical structures responsible for the induction of inflammation in an experimental model of meningitis. The early vasodilation effect of cell walls in this model could be distinguished from later pathophysiological effects (edema, inflammation) by the use of certain antiinflammatory agents.

Molecular Epidemiology. The Tomasz lab is also involved with tracking the spread of antibiotic resistant clones of MRSA in hospitals and community health centers.

 

Alexander Tomasz Plutarch Papamarkou ProfessorWe are interested in the chemical structure, mode of assembly, and biological functions of bacterial cell walls and associated cell surface components.