Bassler Lab Research

 

 

Overview

 

Bacterial Quorum SensingOur lab wants to understand quorum sensing: the process of cell-cell communication in bacteria. Quorum sensing involves the production, release, and subsequent detection of chemical signal molecules called autoinducers. This process enables populations of bacteria to regulate gene expression, and therefore behavior, on a community-wide scale. Quorum sensing is wide-spread in the bacterial world, so understanding this process is fundamental to all of microbiology, including industrial and clinical microbiology, and ultimately to understanding the development of higher organisms Our studies of quorum sensing are providing insight into intra- and inter-species communication, population-level cooperation, and the design principles underlying signal transduction and information processing at the cellular level. These investigations are also leading to synthetic strategies for controlling quorum sensing. Our objectives include development of anti-microbial drugs aimed at bacteria that use quorum sensing to control virulence, and improved industrial production of natural products such as antibiotics. We have pursued our goal of understanding bacterial communication by combining genetics, biochemistry, structural biology, chemistry, microarray studies, bioinformatics, modeling, and engineering approaches.

Bacterial processes such as biofilm formation, virulence factor secretion, bioluminescence, antibiotic production, sporulation, and competence for DNA uptake are often critical for survival. However, these behaviors are seemingly futile if performed by a single bacterium acting alone. Yet, we know that bacteria perform these tasks effectively. How do bacteria manage? We now understand that, through quorum sensing, bacteria synchronously control gene expression in response to changes in cell density and species complexity. Quorum sensing allows bacteria to switch between two distinct gene expression programs: one that is favored at low-cell-density for individual, asocial behaviors, and another, that is favored at high-cell-density for social, group behaviors.

The fundamental steps involved in detecting and responding to fluctuations in cell number are analogous in all known quorum-sensing systems. First, low molecular weight molecules called autoinducers are synthesized intracellularly. Second, these molecules are either passively released or actively secreted outside of the cells. As the number of cells in a population increases, the extracellular concentration of autoinducer likewise increases. Third, when autoinducers accumulate above the minimal threshold level required for detection, cognate receptors bind the autoinducers and trigger signal transduction cascades that result in population-wide changes in gene expression. Thus, quorum sensing enables cells in a population to function in unison and, in so doing; they carry out behaviors as a collective.

The Model Bacterium Vibrio harveyi and Our Studies to Understand Quorum-Sensing Signal-Transduction Mechanisms

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We focus on marine vibrios as model systems for quorum sensing. The first observation indicating that bacteria could communicate with multiple quorum-sensing autoinducers came with our definition of the quorum-sensing system of Vibrio harveyi. The V. harveyi quorum-sensing system consists of three autoinducers and three cognate receptors functioning in parallel to channel information into a shared regulatory pathway. V. harveyi produces a homoserine lactone signal termed HAI-1 (3OHC4-homoserine lactone). This molecule is made by LuxM. HAI-1 binds to a membrane bound sensor histidine kinase (LuxN). The second V. harveyi signal is a furanosyl borate diester known as AI-2, and its production requires the LuxS enzyme. In V. harveyi, AI-2 is bound in the periplasm by the protein LuxP, and the LuxP-AI-2 complex interacts with another membrane bound sensor histidine kinase, LuxQ. The third V. harveyi signal, termed CAI-1 is (S)-3-hydroxytridecan-4-one and is produced by the CqsA enzyme. Again, this signal interacts with a membrane bound sensor histidine kinase, CqsS.

At low cell density, in the absence of appreciable amounts of autoinducers, the three sensors, LuxN, LuxQ, and CqsA, act as kinases, autophosphorylate, and subsequently transfer the phosphate to the cytoplasmic protein LuxU. LuxU passes the phosphate to the DNA binding response regulator protein LuxO. Phospho-LuxO, in conjunction with a transcription factor calleds54, activates transcription of the genes encoding five regulatory small RNAs (sRNAs) termed Qrr1-5 (for Quorum Regulatory RNA). The Qrr sRNAs interact with an RNA chaperone called Hfq and allow translation of the mRNA encoding a transcription factor called AphA and destabilize the mRNA encoding a transcriptional activator called LuxR. Thus, at low cell density AphA is made and LuxR is not. AphA controls the genes underpinning individual behaviors. LuxR is required to control transcription of all the genes in the quorum-sensing regulon which include those for bioluminescence and type III secretion. Thus, at low cell density, because the luxR mRNA is degraded, the bacteria do not carry out group behaviors. At high cell density, when the autoinducers accumulate to the level required for detection, the three sensors switch from being kinases to being phosphatases and drain phosphate from LuxO via LuxU. Unphosphorylated LuxO cannot induce expression of the sRNAs,. Thus aphA translation is reduced while luxR mRNA tistranslated, allowing LuxR to be produced, and genes for collective behaviors to be expressed. This network architecture ensures maximal AphA production at low cell density and maximal LuxR production at high cell density. Microarray analyses reveal 180 genes are regulated by AphA at low cell density and 600 genes re controlled by LuxR at high cell density.

We also aim to understand how individual cell behaviors underpin the population-level behavior in quorum sensing. To gain insight, we use single-cell fluorescence microscopy combined with modeling and information theory in a variety of our newest studies. We quantified the signaling responses to and analyzed the integration of multiple autoinducers in V. harveyi. Our results reveal that the information encoded in the distinct autoinducers, HAI-1 and AI-2, are combined strictly additively in the shared phosphorelay pathway, with each autoinducer contributing nearly equally to the total response. We found a coherent response across the population with little cell-to-cell variation, indicating that the entire population of cells can reliably distinguish several distinct conditions of external autoinducer concentration. We speculate that the use of multiple autoinducers allows a growing population of cells to synchronize gene expression during a series of distinct developmental stages.

Discovery of Inter-Species Bacterial Communication

A1-2 Communication Interconventing Molecules
Beyond controlling gene expression on a global scale, quorum sensing allows bacteria to communicate within and between species. This notion arose with our discovery and study of the autoinducer AI-2, which is one of several signals used by V. harveyi in quorum sensing. In contrast to the HAI-1 and CAI-1 signals, which are restricted to one or a few vibrios and thus used for intra-species communication, we discovered luxS, encoding the AI-2 synthase, and showed that it is present in roughly half of all sequenced bacterial genomes. Likewise, AI-2 production has been verified in a large number of species, and AI-2 controls gene expression in a variety of bacteria. Together, these findings have led us to the hypothesis that AI-2 is used by bacteria to communicate between species.

We determined the biosynthetic pathway for AI-2 and defined the structure of the signal. LuxS functions in the S-adenosylmethionine (SAM) pathway, the major cellular methyl donor. Transfer of the methyl moiety to various substrates produces the toxic byproduct S-adenosylhomocysteine (SAH). The enzymes Pfs and LuxS act sequentially to convert SAH to adenine, homocysteine, and the signaling molecule 4,5-dihydroxy-2,3-pentanedione (DPD). The highly reactive DPD product can rearrange and undergo additional reactions suggesting that distinct but related molecules derived from DPD are the signals that different bacterial species recognize as AI-2. To gain evidence for this idea, we identified the signals conveying the information into V. harveyiand Salmonella typhimurium by trapping the active molecules in their respective receptors (LuxP for V. harveyi and LsrB for S. typhimurium), crystallizing the complexes, and solving their structures. In V. harveyi, AI-2 is (2S,4S)-2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran-borate (S-THMF borate) and in S. typhimurium AI-2 is (2R,4S)-2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran (R-THMF). Straightforward chemistry links these two molecules as DPD can cyclize with two equally feasible stereochemistries.

Our identification of boron in V. harveyi AI-2 is surprising, as few biological roles for boron are known. However, boron is present in high concentrations in the marine environment making it a reasonable element in the V. harveyi AI-2 signal. Significantly lower boron concentration is found in terrestrial environments, making it an unlikely component of the S. typhimurium AI-2 signal. Importantly, all of the chemicals we identified exist in equilibrium, rapidly interconvert, showing that, even if bacteria respond to distinct AI-2 moieties, they can communicate with one another (i.e., across species) through inter-conversion of the signals. Because only one enzyme (LuxS) is required to synthesize this family of interconverting signal molecules, this pathway could represent an especially economical method for evolving a complex bacterial lexicon.

Our Investigation of Small-RNA Control of Quorum Sensing

Small RNA Control of Quorum Sensing
As mentioned, small regulatory RNAs (sRNAs) lie at the core of the Vibrio quorum-sensing cascade. Until recently, bacteria were not known to use sRNAs as a major form of gene regulation. However, hundreds of sRNAs have recently been identified and shown to control major developmental processes in a wide variety of bacteria. These studies show that sRNA-directed regulation is a highly important but under-studied phenomenon. In the case of quorum sensing, we are especially curious what features sRNAs, acting post-transcriptionally, provide the quorum sensing circuits that traditional transcription factors do not provide. We found that that, the Qrr sRNAs are functionally redundant. That is, expression of any one of them is sufficient for wild-type quorum-sensing behavior. This is because the combined action of two feedback loops, one involving the sRNA-activator LuxO and one involving the sRNA-target LuxR, promotes gene dosage compensation between the four qrr genes. Gene dosage compensation adjusts the total Qrr sRNA pool so that it is exquisitely sensitive to small perturbations in Qrr levels. We further found that precisely maintained Qrr levels are required to direct the proper timing and correct patterns of expression of quorum-sensing-regulated target genes. Other regulators appear to be involved in control of V. harveyi qrr expression, allowing the integration of additional sensory information into the regulation of quorum-sensing gene expression.

We used microarrays to identify sixteen new targets of the Qrr sRNAs. Mutagenesis demonstrated that particular sequence differences among the Qrr sRNAs determine their target specificities. Modeling coupled with computational, biochemical, and genetic analyses allowed us to find that all of the Qrr sRNAs possess four stem-loops: the first stem-loop is crucial for base-pairing with a subset of targets. This stem-loop also protects the Qrr sRNAs from RNase E-mediated degradation. The second stem-loop contains conserved sequences required for base-pairing with the majority of the target mRNAs. The third stem-loop plays an accessory role in base-pairing. The fourth stem-loop functions as a rho-independent terminator. In the quorum-sensing regulon, the genes that are controlled by the Qrr sRNAs are the most rapid to respond to quorum-sensing autoinducers and presumably encode components required to instigate subsequent phases of the quorum-sensing program. The Qrr sRNAs are conserved throughout vibrios, thus insights from this work could apply generally to vibrio quorum sensing.

Studies of Quorum Sensing Control of Pathogenesis: Vibrio cholerae

Vibrio Cholerae Quorum Sensing
We also work on the human pathogen Vibrio cholerae, the causative agent of the endemic diarrheal disease cholera. We showed that V. cholerae possesses a quorum-sensing network similar to that of V. harveyi. V. cholerae has no equivalent of the HAI-1/LuxN branch of the system but does posses the AI-2/LuxPQ and CAI-1/CqsS branches as well as LuxU, LuxO, four Qrr sRNAs, and a V. harveyi LuxR-like protein called HapR. The V. cholerae systems function analogously to V. harveyi but the system controls virulence and biofilm formation. Surprisingly, quorum sensing promotes V. cholerae virulence factor expression and biofilm formation at low cell density and represses these traits at high cell density. Quorum sensing commonly controls bacterial virulence factor expression, but typically, induction occurs at high cell density. This opposite pattern of regulation in the case of V. cholerae can be understood in terms of the specific disease the bacterium causes. Following a successful V. cholerae infection, the ensuing diarrhea washes huge numbers of bacteria from the human intestine into the environment. Repression of virulence factor production and biofilm formation genes at high cell density promotes dissemination of V. cholerae.

We showed that of the two V. cholerae autoinducers, CAI-1 and AI-2, CAI-1 is the stronger of the two signals. We purified and identified CAI-1 as (S)-3-hydroxytridecan-4-one, a new type of bacterial autoinducer. We developed a synthetic route to both the R and S isomers of CAI-1 as well as simple homologues, and we evaluated their relative activities. Synthetic (S)-3-hydroxytridecan-4-one functions as well as natural CAI-1 in repressing production of the canonical virulence factor toxin co-regulated pilus (TCP). These findings suggest that CAI-1 could be used as a therapy to prevent cholera infection and, furthermore, that strategies to manipulate bacterial quorum sensing hold promise in the clinical arena.

We analyzed the CAI-1 autoinducer-CqsS receptor interaction in V. cholerae For these studies, we exploited the fact that, unlike in most bacterial-two-componet systems, the quorum-sensing signals are known molecules and so we could identify mutants in the V. cholerae quorum-sensing receptor CqsS that display altered responses to natural and synthetic ligands. Using this chemical-genetics approach, we assigned particular amino acids of the CqsS sensor to particular roles in recognition of the native ligand, CAI-1 (S-3 hydroxytridecan-4-one) as well as ligand analogs. Amino acids W104 and S107 dictate receptor preference for the carbon-3 moiety. Residues F162 and C170 specify ligand head size and tail length, respectively. By combining mutations, we can build CqsS receptors responsive to ligand analogs altered at both the head and tail. This work suggests that rationally designed ligands can be employed to study and ultimately to control histidine kinase activity. We reconstituted the CqsS->LuxU->LuxO phosphorylation cascade in vitro. We found that CAI-1 inhibits the initial auto-phosphorylation of CqsS whereas subsequent phosphotransfer steps and CqsS phosphatase activity are not CAI-1-controlled. CAI-1 binding to CqsS causes a conformational change that renders His194 in CqsS inaccessible to the CqsS catalytic domain. CqsS mutants with altered ligand detection specificities from our above analysis of the CAI-1-CqsS interaction, are faithfully controlled by their corresponding modified ligands in vitro. Likewise, pairing of agonists and antagonists allows in vitro assessment of their opposing activities.

Manipulating Quorum Sensing in Pseudomonas aeruginosa to Control Virulence and Biofilm Formation

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We recently expanded our studies to include P. aeruginosa a pathogen that is devastating in cystic fibrosis patients, hospital burn units, and on sub-epithelial inserted medical devices such as intubation tubes, stents, and other long-term submerged prosthetics. In all of these infection settings, quorum-sensing-directed virulence factor production and biofilm formation appear to be critical. We are studying and manipulating P. aeruginosa quorum-sensing with small molecules with the aim of reduce or preventing virulence and/or biofilm formation and thereby improve the outcome in an animal host or in devices that model industrial and medical apparatus. P. aeruginosa uses two homoserine lactone quorum sensing circuits called the LasI/R and RhlI/R quorum-sensing systems. We have identified and characterized a series of small molecule signal mimics that target either LasR, RhlR or both in simplified E. coli systems. Our most potent qntagonsits inhibit quorum-sensing controlled phenotypes in P. aeruginosa and protects C. elegans and A549 human lung epithelial cells from quorum-sensing-mediated death by P. aeruginosa, demonstrating the potential for small molecule modulators of quorum snesing to be developed into therapeutics.

Our Development of a Novel P. aeruginosa Biofilm Assay that Mimics Realistic Conditions

Biofilm Streamers in Stents

In its natural habitat, both in the environment and as a pathogen, P. aeruginosa predominantly lives in biofilms. Consequently, P. aeruginosa biofilms have been studied in the lab for decades. Two features that have not been captured in laboratory biofilm studies – but that are common to all natural environments – are the presence of rough surfaces, which at the microscopic level reduce to surfaces with many corners, and a pressure-driven flow. To understand how quorum sensing controls P. aeruginosa biofilms, how quorum-sensing-directed biofilm formation plays out in pathogenicity, and whether molecules we discover can interfere with the process, we developed a new microfluidic system that combines the two shared features of realistic P. aeruginosa habitats, a sequence of corners and a flow driven by a constant pressure. In our microfluidic system, biofilm streamers form and cause rapid clogging transitions. The biofilm streamers initiate on corners and rapidly expand to cause a catastrophic disruption of the flow on time scales as short as 30 minutes. Streamers initially consist of extracellular polymeric substances (EPS). Over time, the EPS filaments bridge the distances between corners and capture cells as they flow past. It is these trapped cells that cause the catastrophic clog.

We find that, in contrast to what is observed in standard assays, a non-motile flagellar mutant (ΔflgK) forms streamers/clogs similarly to wild type suggesting that swimming is not required and that, instead, flow provides the necessary transport of cells to the clog-forming biofilm. By contrast, other previously identified genes that are important for biofilm formation do have large effects in our model system. For example, EPS production is required, as a ΔpelA mutant is unable to initiate biofilms or clog the channel. Most importantly, in our view, quorum sensing – which up-regulates EPS production – is required for streamers and efficient clogging. Disruption of quorum-sensing with our small molecule inhibitors prevents clogging in this device. Thus our findings show that that, in a system that mimics the naturally occurring environment of P. aeruginosa, quorum sensing is critical for clogging and that our lead inhibitor is an effective preventative treatment. Going forward, we will use this apparatus to assay the promising antagonists we discover as part of this work.

To probe the extent to which our system simulates natural P. aeruginosa environments, we investigated three flow systems in which biofilm-induced clogging is well-documented, but a mechanistic understanding is lacking: soil, water filtration devices containing spiral-wound reverse osmosis filters, and biliary stents. We documented P. aeruginosa streamers leading to clogs in all three cases. These examples illustrate that biofilm streamers are likely a major feature of biofilms in natural, industrial, and medical environments, causing rapid clogging without warning. As we move forward, we will test potent anti-quorum-sensing molecules identified in this work in these and other devices.

Development of Pro- and Anti-Quorum Sensing Therapies

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A more applied side of our research is focused on developing pro- and anti-quorum sensing molecules to be used as new therapeutics. We are carrying out large-scale high-throughput screens for chemicals that agonize or antagonize different quorum-sensing circuits. In one such effort, we identified a small molecule that competitively antagonizes the V. harveyi HAI-1 receptor LuxN. We found that this molecule is also a potent antagonist of many other bacterial receptors for homoserine lactone type autoinducers. We synthesized derivatives of the original molecule and optimized them for potency. In each case, we characterized the mode of action of antagonism using genetics, biochemistry, and crystallography. Our most potent antagonist (called CL for chlorolactone) protects the model nematode C. elegans from quorum-sensing-mediated killing by a bacterial pathogen, validating the notion that targeting quorum sensing has potential for antimicrobial drug development.

As described in the P. aeruginosa work, we have used analogous strategies to identify and develop molecules that inhibit LasR and RhlR and these molecules are effective at prevented quorum-sensing mediated killing of C. elegans and also human tissue culture cells. The anti-Pseudomonas quorum-sensing molecules also prevent biofilms and clogging in microfluidics models of natural conditions for Pseudomonas such as soil, water filtration devices and stents (described above).

Regarding V. cholerae and manipulation of quorum sensing, as mentioned above, this pathogen causes an acute disease, quorum sensing repress virulence factor production and biofilm formation. Thus, molecules that activate quorum sensing in V. cholerae have the potential to control pathogenicity in this globally important bacterium. We have identified molecules that activate V. cholerae quorum sensing: some are CqsS receptor agonists and some are antagonists of LuxO, the central NtrC-type response regulator that controls the global V. cholerae quorum-sensing cascade. We analyzed the LuxO inhibitors and found that they act by an uncompetitive mechanism by binding to the pre-formed LuxO-ATP complex to inhibit ATP hydrolysis. Our genetic analyses suggest that the inhibitors bind in close proximity to the Walker B motif. The inhibitors display broad-spectrum capability in activation of quorum sensing in Vibrio species that employ LuxO. To the best of our knowledge, these are the first molecules identified that inhibit the ATPase activity of a NtrC-type response regulator. Our discovery supports the idea that exploiting pro-quorum-sensing molecules is a promising strategy for the development of novel anti-infectives.

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