MSTP Fellow Edward Geisinger Investigates the Molecular Dynamics of How Pathogenic Staphylococci Communicate with Each Other


Edward Geisinger^a, Tom W. Muir^b and Richard P. Novick^a
agr receptor mutants reveal distinct modes of inhibition by staphylococcal autoinducing peptides
Proc Natl Acad Sci U S A. 2009 Jan 27;106(4):1216-21

^aDepartments of Medicine and Microbiology, Kimmel Center for Biology and Medicine of the Skirball Institute, New York University Medical Center, New York, NY 10016; and ^bLaboratory of Synthetic Protein Chemistry, The Rockefeller University, New York, NY 10065
Correspondence: novick@saturn.med.nyu.edu



Although unicellular by nature, bacteria have evolved clever ways to communicate, enabling social functioning within coordinated, democratic communities. This occurs through a biological process known as quorum sensing (QS), in which bacteria secrete a pheromone and detect its concentration. When enough of these molecular "votes" are present—reflecting a critical density of bacteria, or quorum—the bugs are signaled to assume a phenotype that is specially suited for group behavior.

In the case of the pathogen Staphylococcus aureus, in which the QS pheromone is a small peptide (the AIP), QS mediates a switch from a sticky, attachment lifestyle early in infection, to an invasive, toxin-secreting group phenotype that facilitates bacterial dissemination. Interestingly, different staphylococcal species and strains each make their own unique AIPs, which cause interference of QS in divergent cells, a kind of "molecular filibustering." The staphylococcal QS receptor, AgrC, is highly variable in its AIP-binding domain across the staphylococci, and residue motifs that determine activation by the specific cognate ligand have been described. How cross-inhibition by non-cognate ligands occurs is unclear.

In their recent article, Edward Geisinger and his co-authors examined the phenomenon of QS interference by manipulating AgrC genetically. Using random mutagenesis, they first isolated constitutively active mutants of S. aureus AgrC. The constitutive mutations clustered to two general regions of the receptor (Figure) and shed light on possible conformational changes involved in receptor function. Then they asked what would happen when these mutant receptors were challenged with cross-inhibitory AIPs from other staphylococci: would they lose their constitutive activity or would they be indifferent to the inhibitory effect of these divergent peptides?

The answer was both. They found that certain divergent AIPs reversed the mutants' constitutive activity (that is, they behave pharmacologically as inverse agonists), whereas others had no effect, and only act as purely competitive inhibitors (that is, they are neutral antagonists). This demonstrated that QS interference can occur through two distinct mechanisms, one of which represents an unexpectedly active process.

Finally Geisinger's team sought to identify residues in AgrC that mediated the inhibitory effect of divergent ligands. Again using random mutagenesis, they selected for mutants that could be activated by an antagonist AIP and identified one mutation in the receptor's third extracellular loop (Figure) that indeed enabled this dramatically altered ligand specificity. On further characterization, this mutation essentially eliminated the ability of the receptor to be inhibited by any AIP, thus identifying a single residue as a determinant of inhibition by heterologous ligands, and indicating that inhibition, like activation, involves critical, evolutionarily selected residue contacts.

Together these findings uncover several molecular details of QS interference among the staphylococci, information that may be useful for the future design of novel therapeutics against staphylococcal infections.





Figure:
AgrC-I topology and mutagenized residues. S. aureus AgrC-I has a transmembrane sensor domain and a cytoplasmic histidine kinase domain. Residues in which mutation (listed at right) resulted in constitutive activity are shown in black. Residue I171, in which mutation to lysine resulted in broadened specificity, is also highlighted. (Inset) Helical wheel analysis of the first HK domain α-helix.

Geisinger et al. PNAS January 27, 2009 vol. 106(4). Reprinted by Permission © 2009 by The National Academy of Sciences of the USA