Anjali K. Struss

Toward Implementation of Quorum Sensing Autoinducers as Biomarkers for Infectious Disease States

The opportunistic bacterial pathogen Pseudomonas aeruginosa causes chronic lung infections in cystic fibrosis (CF) patients. Importantly, virulence factor expression and biofilm formation in P. aeruginosa is coordinated by quorum sensing (QS) and one of the key QS signaling molecules is 3-oxo-C12-HSL. Remarkably, a tetramic acid, (C12-TA), with antibacterial properties is formed spontaneously from 3-oxo-C12-HSL under physiological conditions. Seeking to better understand this relationship, we sought to investigate whether 3-oxo-C12-HSL and C12-TA may be contributing factors to the overall pathogenicity of P. aeruginosa in CF individuals and if their detection and quantitation in sputum samples might be used as an indicator to assess disease states and monitor therapy success in CF patients. To this end, 3-oxo-C12-HSL and C12-TA concentrations were initially analyzed in P. aeruginosa flow cell biofilms using liquid chromatography coupled with mass spectrometry (LC-MS). A liquid chromatography tandem mass spectrometry (LC-MS/MS)-based method was then developed and validated for their detection and quantification in the sputa of CF patients. To the best of our knowledge, this is the first report to show the presence of both the quorum sensing molecule (3-oxo-C12-HSL) and its rearranged product (C12-TA) in human clinical samples such as sputum. A total of 47 sputum samples from 20 CF and 2 non-CF individuals were analyzed. 3-Oxo-C12-HSL was detected and quantified in 45 samples with concentrations ranging from 20 to >1000 nM; C12-TA was found in 14 samples (13-900 nM). On the basis of our findings, quorum sensing autoinducers merit further investigation as biomarkers for infectious disease states.

Antagonism of a zinc metalloprotease using a unique metal-chelating scaffold: tropolones as inhibitors of P. aeruginosa elastase.

Tropolone emerged from the screening of a chelator fragment library (CFL) as an inhibitor of the Zn(2+)-dependent virulence factor, Pseudomonas aeruginosa elastase (LasB). Based on this initial hit, a series of substituted tropolone-based LasB inhibitors was prepared, and a compound displaying potent activity in vitro and in a bacterial swarming assay was identified. Importantly, this inhibitor was found to be specific for LasB over other metalloenzymes, validating the usage of tropolone as a viable scaffold for identifying first-in-class LasB inhibitors.

A Multivalent Probe for AI-2 Quorum Sensing Receptors

Multivalency is a common principle in the recognition of cellular receptors, and multivalent agonists and antagonists have played a major role in understanding mammalian cell receptor biology. The study of bacterial cell receptors using similar approaches, however, has lagged behind. Herein we describe our efforts toward the development of a dendrimer-based multivalent probe for studying AI-2 quorum-sensing receptors. From these studies, we have discovered a chemical probe specific for Lsr-type AI-2 quorum-sensing receptors with the potential for enabling the identification of new bacterial species that utilize AI-2 as a quorum-sensing signaling molecule.

Synthesis of “clickable” acylhomoserine lactone quorum sensing probes: unanticipated effects on mammalian cell activation

Alkynyl- and azido-tagged 3-oxo-C(12)-acylhomoserine lactone probes have been synthesized to examine their potential utility as probes for discovering the mammalian protein target of the Pseudomonas aeruginosa autoinducer, 3-oxo-C(12)-acylhomoserine lactone. Although such substitutions are commonly believed to be quite conservative, from these studies, we have uncovered a drastic difference in activity between the alkynyl- and azido-modified compounds, and provide an example where such structural modification has proved to be much less than conservative.

The Fe(III) and Ga(III) coordination chemistry of 3-(1-hydroxymethylidene) and 3-(1-hydroxydecylidene)-5-(2-hydroxyethyl)pyrrolidine-2,4-dione: novel tetramic acid degradation products of homoserine lactone bacterial quorum sensing molecules.

Bacteria use small diffusible molecules to exchange information in a process called quorum sensing (QS). An important class of quorum sensing molecules used by Gram-negative bacteria is the family of N-acylhomoserine lactones (HSL). It was recently discovered that a degradation product of the QS molecule 3-oxo-C(12)-homoserine lactone, the tetramic acid 3-(1-hydroxydecylidene)-5-(2-hydroxyethyl)pyrrolidine-2,4-dione, is a potent antibacterial agent, thus implying roles for QS outside of simply communication. Because these tetramic acids also appear to bind iron with appreciable affinity it was suggested that metal binding might contribute to their biological activity. Here, using a variety of spectroscopic tools, we describe the coordination chemistry of both the methylidene and decylidene tetramic acid derivatives with Fe(III) and Ga(III) and discuss the potential biological significance of such metal binding.

Synthesis and Molecular Modeling Provide Insight Into a Pseudomonas aeruginosa Quorum Sensing Conundrum

The triphenyl amide/ester 12 was originally reported to be a potent mimic of the natural 3-oxo-dodecanoyl homoserine lactone quorum sensing molecule in Pseudomonas aeruginosa. However, explicit synthesis/chemical characterization was lacking, and a later report providing protein crystallographic data inferred 12 to be incorrect, with 9 now being the surmised structure. Because of these inconsistencies and our interest in quorum sensing molecules utilized by gram-negative bacteria, we found it necessary to synthesize 9 and 12 to test for agonistic activity in a P. aeruginosa reporter assay. Despite distinct regiochemical differences, both 9 and 12 were found to have comparable EC(50) values. To reconcile these unanticipated findings, modeling studies were conducted, and both compounds were revealed to have comparable properties for binding to the LasR receptor.

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.

Immunomodulation and the quorum sensing molecule 3-oxo-C12-homoserine lactone: the importance of chemical scaffolding for probe development

As a guide for chemical probe design, focused analogue synthetic studies were undertaken upon the lactone ring of 3-oxo-C(12)-homoserine lactone. We have concluded that hydrolytic instability of the heterocyclic ring is pivotal for its ability to modulate immune signaling and probe preparation was aligned with these findings.

Retraction: Stereochemical insignificance discovered in Acinetobacter baumannii quorum sensing.

The authors request the retraction of the manuscript, “Stereochemical Insignificance Discovered in Acinetobacter baumannii Quorum Sensing,” as the main conclusion reporting a case of stereochemical insignificance in A. baumannii is not supported by more recent data generated by our and another group. Subsequent to our publication, another laboratory reported the synthesis and biological evaluation of the acylhomoserine lactones (AHLs), N-((R)-3-hydroxydodecanoyl)-L-homoserine lactone (3a) and N-((S)-3-hydroxydodecanoyl)-L-homoserine lactone (3b), described in our manuscript (see Stacy, D. M.; Welsh, M. A.; Rather, P. N.; Blackwell, H. E. Attenuation of Quorum Sensing in the Pathogen Acinetobacter baumannii Using Non-native N-Acyl Homoserine Lactones. ACS Chem. Biol. 2012, 7, 1719-1728.). In our case, these AHLs were synthesized using CBS reduction methodology followed by HPLC purification to yield what we believed to be the pure diastereomers. During our biological characterization of these compounds, we found the AHLs to exhibit very similar autoinducer activities in Acinetobacter baumannii. In the report in ACS Chem. Biol., the authors prepared these same AHLs via Noyori reduction and in their hands, the 3b diastereomer exhibited a 40-fold decrease in autoinducer activity; thus, we sought to repeat our biological analyses to confirm our results. The diastereomeric AHLs prepared for our publication no longer existed in the laboratory and when we attempted to repeat the reported synthetic and purification methods, pure compounds could not be obtained, instead a mixture of 3a and 3b co-eluted as confirmed using chiral HPLC. Pure 3-hydroxy AHLs were obtained using the Noyori reduction conditions as reported by Blackwell and colleagues. To examine the effect of mixtures of 3a and 3b on autoinducer activity in Acinetobacter baumannii, we measured the EC50 values of pure AHLs prepared from the Noyori reduction methodology and varying mixtures of these diastereomers (1:1-1:9). Upon examination, little difference was found to exist between the EC50 values of pure 3a and a 1:1 mixture of 3a and 3b (0.173 mM and 0.498 mM, respectively; 2.9-fold difference). Moreover, our reported values of 0.67 mM and 0.82 mM for 3a and 3b, respectively, align with a ~1:1-1:2 mixture of the compounds. In the light of the results obtained, it is likely that the preparations employed for the work described in the PLOS ONE article included a mixture of the 3a and 3b diastereomers; the novel data do not support the conclusion that stereochemistry is not significant for the signaling activity of the two compounds. The attached figure http://www.plosone.org/attachments/pone.0037102.comment1.pdf reports the results of our further analyses showing how the stereochemistry and the mixture of the two stereoisomers affect the biological activity.