Reversible and Specific Extracellular Antagonism of Receptor-Histidine Kinase Signaling

Abstract

Staphylococcal pathogenesis is regulated by a twocomponent quorum-sensing system, agr, activated by a self-coded autoinducing peptide (AIP). The agr system is widely divergent and is unique in that variant AIPs cross-inhibit agr activation in heterologous combinations. Cross-inhibition, but not self-activation, is widely tolerant of structural diversity in the AIPs so that these two processes must involve different mechanisms of interaction with the respective receptors. Herein, we have utilized this naturally occurring antagonism to demonstrate that both activation and inhibition are reversible and that activators and inhibitors interact at a common site on the receptor. These results suggest that molecules designed to compete with natural agonists for binding at receptor-histidine kinase sensor domains could represent a general approach to the inhibition of receptor-histidine kinase signaling. Receptor-histidine kinases (RHKs)1 have been extensively characterized in bacteria and are present in archaea, microbial eukarya, and higher plants, where they have recently been shown (in Arabidopsis) to play a role in hormone signaling (1). In bacteria, RHKs are involved in sensing the environmental surroundings. Many of these kinases contain two transmembrane helices flanking a periplasmic domain. This domain contains the binding site for the appropriate ligand, such as metal ions (Mg 2ϩ in the case of PhoP) (2). Many RHKs have been shown to exist as preformed dimers in the inner cell membrane of Gram-negative bacteria (3). In the case of EnvZ, a pair of cytoplasmic ␣-helices from each monomer form a four-helix bundle (4). In Gram-positive bacteria, pheromone-inducible RHKs usually have a polytopic sensor domain, containing 5-8 membrane-spanning segments. RHKs predicted to possess this polytopic sensor domain include AgrC from Staphylococcus aureus (5), ComD from Streptococcus pneumoniae (6), ComP from Bacillus subtilis (7), and SapK from Lactobacillus sakei Lb706 (8). These RHKs respond to secreted signaling peptides, which bind to the sensor domain to initiate the transmembrane signal that activates the intracellular histidine kinase (HK). Notably, there is at least one RHK in Escherichia coli, UhpB, which contains 6 -8 transmembrane helices and which is also thought to function as a homodimeric complex (9).The agr system in S. aureus is the most fully characterized in terms of the receptor-ligand interaction. Density-dependent accumulation of an extracellular peptide, known as the AIP and derived from processing of the prepeptide, AgrD, triggers activation of the receptor-histidine kinase, AgrC. This leads to a downstream virulence response via the unique regulator, RNAIII (10). This signaling process is one example of densitydependent or "quorum-sensing" systems widespread in bacteria (11,12). The sequence of the AIPs is highly variable, resulting in at least four specificity groups within S. aureus and many others in other staphylococcal species (13-15). All strains within a group produce the same AIP. The agrB...

agr receptor mutants reveal distinct modes of inhibition by staphylococcal autoinducing peptides

“…In previous AIP structure-function analyses, agr cross-inhibition was determined to require the AIP ring structure, but it was relatively tolerant of amino acid replacements (6,14), as well as replacement of the thiolactone bond with a lactam (4,13,17). These results suggested that autologous and heterologous AIPs interact with AgrC via unique yet overlapping binding sites, consistent with a process of competitive inhibition.…”

“…Generally, only the cognate ligand-receptor interaction causes activation, whereas most heterologous interactions inhibit the receptor, resulting in a form of bacterial interference directly affecting accessory gene expression. Inhibition by heterologous AIPs is reversible (13), is equivalent in strength to activation (2), and is highly tolerant of AIP sequence variation (2,6,14). agr crossinhibition may drive evolutionary diversification among the staphylococci (11) and has important implications for host infections, because treatment with S. aureus AIP-II was shown to prevent the formation of an experimental murine abscess by agr-I cells (2,15).…”

Exploiting Quorum Sensing To Confuse Bacterial Pathogens

“…This may help to explain why residues critical for activity differ between AIP groups. Systematic mutation of native AIP molecules has generated analogues with various changes in activity, including reduced activation activity, enhanced activation activity, loss of all activity, conversion of self-activation to self-inhibition activity, and a unique case of conversion of cross-inhibition to cross-activation activity (218)(219)(220)(221). Combining multiple mutations known to provide antagonist activity, for example, truncating the tail of a selfinhibitory AIP-I mutant, has also been shown to have potential in the generation of stronger inhibitors that function against all agr groups (218).…”

Transmembrane Topology of AgrB, the Protein Involved in the Post-translational Modification of AgrD in Staphylococcus aureus

“…The P2 transcript, RNA II, encodes four genes, agrA, B, C, and D (6,7). AgrC, a transmembrane protein, is a sensor kinase of the classic bacterial two component signal transduction system: the N-terminal half is the input domain that interacts with a signal molecule produced by the bacteria, and the C-terminal half is a transmitter that is autophosphorylated at a conserved histidine upon stimulation by the signal molecule (2,13,14). AgrA resembles a response regulator, which is required for the activation of both agr promoters P2 and P3 (2,7,15), although it is not clear whether AgrA binds to these two promoters directly (16,17).…”