Biomedical subjects

Michael J Federle

Publications and source records attributed to Michael J Federle.

7 recordsLinked to original sources

PubMed2006

Ligand-induced asymmetry in histidine sensor kinase complex regulates quorum sensing.

Bacteria sense their environment using receptors of the histidine sensor kinase family, but how kinase activity is regulated by ligand binding is not well understood. Autoinducer-2 (AI-2), a secreted signaling molecule originally identified in studies of the marine bacterium Vibrio harveyi, regulates quorum-sensing responses and allows communication between different bacterial species. AI-2 signal transduction in V. harveyi requires the integral membrane receptor LuxPQ, comprised of periplasmic binding protein (LuxP) and histidine sensor kinase (LuxQ) subunits. Combined X-ray crystallographic and functional studies show that AI-2 binding causes a major conformational change within LuxP, which in turn stabilizes a quaternary arrangement in which two LuxPQ monomers are asymmetrically associated. We propose that formation of this asymmetric quaternary structure is responsible for repressing the kinase activity of both LuxQ subunits and triggering the transition of V. harveyi into quorum-sensing mode.

Bacterial Proteins↗

PubMed2005

Regulation of LuxPQ receptor activity by the quorum-sensing signal autoinducer-2.

The extracellular signaling molecule autoinducer-2 (AI-2) mediates quorum-sensing communication in diverse bacterial species. In marine vibrios, binding of AI-2 to the periplasmic receptor LuxP modulates the activity of the inner membrane sensor kinase LuxQ, transducing the AI-2 information into the cytoplasm. Here, we show that Vibrio harveyi LuxP associates with LuxQ in both the presence and absence of AI-2. The 1.9 A X-ray crystal structure of apoLuxP, complexed with the periplasmic domain of LuxQ, reveals that the latter contains two tandem Per/ARNT/Simple-minded (PAS) folds. Thus, although many prokaryotic PAS folds themselves bind ligands, the LuxQ periplasmic PAS folds instead bind LuxP, monitoring its AI-2 occupancy. Mutations that disrupt the apoLuxP:LuxQ interface sensitize V. harveyi to AI-2, implying that AI-2 binding causes the replacement of one set of LuxP:LuxQ contacts with another. These conformational changes switch LuxQ between two opposing enzymatic activities, each of which conveys information to the cytoplasm about the cell density of the surrounding environment.

Amino Acid Sequence↗

PubMed2005

An expeditious synthesis of DPD and boron binding studies.

A practical synthesis has been developed for DPD (4,5-dihydroxypentane-2,3-dione), an unstable small molecule that is proposed to be the source of universal signaling agents for quorum sensing in bacteria. The synthesis allows preparation of isotopically labeled DPD and ent-DPD as well as detailed studies of spontaneous binding to borate to give the unusual borate complex 6, the signal for marine bacteria such as Vibrio harveyi. [reaction: see text]

Bacterial Physiological Phenomena↗

PubMed2004

Boron binding with the quorum sensing signal AI-2 and analogues.

[reaction: see text] The unstable bacterial metabolic product, DPD, and the related natural product, laurencione, are shown to have a high affinity for borate complexation, through the hydrated analogue. The boron complex of DPD is Vibrio harveyi AI-2, an interspecies quorum sensing signal in bacteria, and an affinity column with a borate resin is effective in providing the first method for concentrating and purifying V. harveyi AI-2 from the biosynthetic product.

Boron↗

PubMed2003

Interspecies communication in bacteria.

Until recently, bacteria were considered to live rather asocial, reclusive lives. New research shows that, in fact, bacteria have elaborate chemical signaling systems that enable them to communicate within and between species. One signal, termed AI-2, appears to be universal and facilitates interspecies communication. Many processes, including virulence factor production, biofilm formation, and motility, are controlled by AI-2. Strategies that interfere with communication in bacteria are being explored in the biotechnology industry with the aim of developing novel antimicrobials. AI-2 is a particularly attractive candidate for such studies because of its widespread use in the microbial kingdom.

4-Butyrolactone↗

PubMed2002

Virulence control in group A Streptococcus by a two-component gene regulatory system: global expression profiling and in vivo infection modeling.

Two-component gene regulatory systems composed of a membrane-bound sensor and cytoplasmic response regulator are important mechanisms used by bacteria to sense and respond to environmental stimuli. Group A Streptococcus, the causative agent of mild infections and life-threatening invasive diseases, produces many virulence factors that promote survival in humans. A two-component regulatory system, designated covRS (cov, control of virulence; csrRS), negatively controls expression of five proven or putative virulence factors (capsule, cysteine protease, streptokinase, streptolysin S, and streptodornase). Inactivation of covRS results in enhanced virulence in mouse models of invasive disease. Using DNA microarrays and quantitative RT-PCR, we found that CovR influences transcription of 15% (n = 271) of all chromosomal genes, including many that encode surface and secreted proteins mediating host-pathogen interactions. CovR also plays a central role in gene regulatory networks by influencing expression of genes encoding transcriptional regulators, including other two-component systems. Differential transcription of genes influenced by covR also was identified in mouse soft-tissue infection. This analysis provides a genome-scale overview of a virulence gene network in an important human pathogen and adds insight into the molecular mechanisms used by group A Streptococcus to interact with the host, promote survival, and cause disease.

Animals↗

PubMed2002

Identification of binding sites for the group A streptococcal global regulator CovR.

The CovRS two-component system (also called CsrRS) of the group A streptococcus (GAS) acts as a global regulator, influencing the transcription of at least six virulence factors. The synthesis of the hyaluronic acid capsule, a virulence factor encoded by the hasABC operon, is negatively regulated by CovRS. We confirmed that phosphorylation of CovR increases its binding to a DNA fragment containing the hasA promoter. Using DNase I footprinting, we identified five binding sites surrounding the hasA promoter from bases -79 to +73 (where +1 is the start of transcription). One pair of thymines within each binding site appears to be necessary for CovR binding in vitro, as shown by uracil interference analysis. When each of these thymine pairs was altered by site-directed mutagenesis, CovR binding was reduced in vitro, confirming the role of each thymine pair in binding. Using a transcriptional reporter system with a single chromosomal copy of PhasA-gusA, we demonstrated the importance of each of four of these binding sites for CovR repression of the hasA promoter. Based on this information, we propose a consensus sequence for CovR binding to DNA.

Bacterial Capsules↗