Systems and Methods for Diagnosis and Monitoring of Bacteria-Related Conditions

The presently-disclosed subject matter provides systems, methods, and kits for diagnosing and/or monitoring a bacteria-related condition of interest in a subject by providing a cell sensing system, each system containing a reporter molecule capable of detecting binding of a quorum sensing molecule and capable of generating a detectable signal

An Unexpected Stereochemical Insignificance Discovered in \u3cem\u3eAcinetobacter baumannii\u3c/em\u3e Quorum Sensing

Stereochemistry is a key aspect of molecular recognition for biological systems. As such, receptors and enzymes are often highly stereospecific, only recognizing one stereoisomer of a ligand. Recently, the quorum sensing signaling molecules used by the nosocomial opportunistic pathogen, Acinetobacter baumannii, were identified, and the primary signaling molecule isolated from this species was N-(3-hydroxydodecanoyl)-l-homoserine lactone. A plethora of bacterial species have been demonstrated to utilize 3-hydroxy-acylhomoserine lactone autoinducers, and in virtually all cases, the (R)-stereoisomer was identified as the natural ligand and exhibited greater autoinducer activity than the corresponding (S)-stereoisomer. Using chemical synthesis and biochemical assays, we have uncovered a case of stereochemical insignificance in A. baumannii and provide a unique example where stereochemistry appears nonessential for acylhomoserine lactone-mediated quorum sensing signaling. Based on previously reported phylogenetic studies, we suggest that A. baumannii has evolutionarily adopted this unique, yet promiscuous quorum sensing system to ensure its survival, particularly in the presence of other proteobacteria

Stereochemical Insignificance Discovered in Acinetobacter baumannii Quorum Sensing

Stereochemistry is a key aspect of molecular recognition for biological systems. As such, receptors and enzymes are often highly stereospecific, only recognizing one stereoisomer of a ligand. Recently, the quorum sensing signaling molecules used by the nosocomial opportunistic pathogen, Acinetobacter baumannii, were identified, and the primary signaling molecule isolated from this species was N-(3-hydroxydodecanoyl)-l-homoserine lactone. A plethora of bacterial species have been demonstrated to utilize 3-hydroxy-acylhomoserine lactone autoinducers, and in virtually all cases, the (R)-stereoisomer was identified as the natural ligand and exhibited greater autoinducer activity than the corresponding (S)-stereoisomer. Using chemical synthesis and biochemical assays, we have uncovered a case of stereochemical insignificance in A. baumannii and provide a unique example where stereochemistry appears nonessential for acylhomoserine lactone-mediated quorum sensing signaling. Based on previously reported phylogenetic studies, we suggest that A. baumannii has evolutionarily adopted this unique, yet promiscuous quorum sensing system to ensure its survival, particularly in the presence of other proteobacteria

Biosensing Systems Based on Genetically Engineered Whole Cells

Genetically engineered whole cells as biosensing systems in biosensors have been employed, in the past two decades, for the detection of a variety of analytes. In addition to being rapid, specific/selective, and sensitive, these whole-cell-based sensing systems provide information pertaining to the analyte bioavailability. This information is particularly important to study the effect of harmful/toxic chemicals on living systems. The whole cells used for designing and developing cell-based sensing systems can be either prokaryotic or eukaryotic in nature. These intact prokaryotic or eukaryotic cells can be genetically engineered to recognize the analytes of interest and respond with the production of a measurable signal in a dose-dependent manner. Generally, prokaryotic bacterial whole-cell sensing systems are developed by introducing a plasmid construct with a reporter gene fused to a promoter, which is induced by a target analyte through a regulatory protein. Similarly, a receptor, which is activated by a target analyte, is coupled with a reporter gene for the development of genetically modified eukaryotic cell-based biosensing systems. The most commonly used reporter proteins in whole-cell biosensing include luminescent proteins, such as bacterial and firefly luciferases; green fluorescent protein along with its variants; and β-galactosidase. The analytes that can be detected using genetically manipulated whole-cell sensing systems range from general toxicants and cell stress factors to specific analytes, such as metals, metalloids, organic pollutants, sugars, drugs, and bacterial signaling molecules. In order to develop self-contained sensing devices based on recombinant whole-cell sensing systems, preservation, miniaturization, and portability are important issues that need to be addressed

Autoinducer activity of a 1∶1 mixture of 3a and 3b.

<p>Values shown are relative luminescence units normalized with respect to cell viability (OD₆₀₀). Square  =  1∶1 <b>3a∶3b</b>. Closed circle  =  <b>3a</b>. Open circle  =  <b>3b</b>.</p

Autoinducer activities of AHL derivatives in an <i>abaI</i>::<i>lacZ</i> mutant <i>A. baumannii</i> strain.

<p>Values shown are relative luminescence units normalized with respect to cell viability (OD₆₀₀). A, Autoinducer activities from 0–100 µM. Closed circle  =  <b>3a</b>. Open circle  =  <b>3b</b>. Closed triangle  =  <b>1</b>. Square  =  <b>6</b>. Open triangle  =  3-oxo-C₁₂-d-HSL. B, Structure of <i>N</i>-(3-dodecenoyl)-l-homoserine lactone (<b>6</b>). C, Enhanced view of autoinducer activity of <b>3a</b> and <b>3b</b> at lower concentrations. Closed circle  =  <b>3a</b>. Open circle  =  <b>3b</b>. EC₅₀ and p values determined using GraphPad Prism v5.0b for Mac OS X.</p

Autoinducer activities of AHLs 3a and 3b in comparison to AHL 1 in <i>P. aeruginosa</i> strain PAO-JP2.

<p>Values shown are relative luminescence units normalized with respect to cell viability (OD₆₀₀). Closed square  =  <b>1</b>. Closed circle  =  <b>3a</b>. Open circle  =  <b>3b</b>.</p

<i>N</i>-Acyl-homoserine lactone autoinducers.

<p>A, <i>P. aeruginosa</i> autoinducers. B, <i>A. baumannii</i> autoinducer.</p