M. F. Semmelhack

The major Vibrio cholerae autoinducer and its role in virulence factor production

Vibrio cholerae, the causative agent of the human disease cholera, uses cell-to-cell communication to control pathogenicity and biofilm formation. This process, known as quorum sensing, relies on the secretion and detection of signalling molecules called autoinducers. At low cell density V. cholerae activates the expression of virulence factors and forms biofilms. At high cell density the accumulation of two quorum-sensing autoinducers represses these traits. These two autoinducers, cholerae autoinducer-1 (CAI-1) and autoinducer-2 (AI-2), function synergistically to control gene regulation, although CAI-1 is the stronger of the two signals. V. cholerae AI-2 is the furanosyl borate diester (2S,4S)-2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran borate. Here we describe the purification of CAI-1 and identify the molecule as (S)-3-hydroxytridecan-4-one, a new type of bacterial autoinducer. We provide a synthetic route to both the R and S isomers of CAI-1 as well as simple homologues, and we evaluate their relative activities. Synthetic (S)-3-hydroxytridecan-4-one functions as effectively as natural CAI-1 in repressing production of the canonical virulence factor TCP (toxin co-regulated pilus). 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.

Autoinducer 2: a concentration‐dependent signal for mutualistic bacterial biofilm growth

4,5‐dihydroxy‐2,3‐pentanedione (DPD), a product of the LuxS enzyme in the catabolism of S‐ribosylhomocysteine, spontaneously cyclizes to form autoinducer 2 (AI‐2). AI‐2 is proposed to be a universal signal molecule mediating interspecies communication among bacteria. We show that mutualistic and abundant biofilm growth in flowing saliva of two human oral commensal bacteria, Actinomyces naeslundii T14V and Streptococcus oralis 34, is dependent upon production of AI‐2 by S. oralis 34. A luxS mutant of S. oralis 34 was constructed which did not produce AI‐2. Unlike wild‐type dual‐species biofilms, A. naeslundii T14V and an S. oralis 34 luxS mutant did not exhibit mutualism and generated only sparse biofilms which contained a 10‐fold lower biomass of each species. Restoration of AI‐2 levels by genetic or chemical (synthetic AI‐2 in the form of DPD) complementation re‐established the mutualistic growth and high biomass characteristic for the wild‐type dual‐species biofilm. Furthermore, an optimal concentration of DPD was determined, above and below which biofilm formation was suppressed. The optimal concentration was 100‐fold lower than the detection limit of the currently accepted AI‐2 assay. Thus, AI‐2 acts as an interspecies signal and its concentration is critical for mutualism between two species of oral bacteria grown under conditions that are representative of the human oral cavity.

A quorum-sensing inhibitor blocks Pseudomonas aeruginosa virulence and biofilm formation.

Significance In this study, we prepare synthetic molecules and analyze them for inhibition of the Pseudomonas quorum-sensing receptors LasR and RhlR. Our most effective compound, meta-bromo-thiolactone, not only prevents virulence factor expression and biofilm formation but also protects Caenorhabditis elegans and human A549 lung epithelial cells from quorum-sensing–mediated killing by Pseudomonas aeruginosa. This anti–quorum-sensing molecule is capable of influencing P. aeruginosa virulence in tissue culture and animal models. Our findings demonstrate the potential for small-molecule modulators of quorum sensing as therapeutics. Quorum sensing is a chemical communication process that bacteria use to regulate collective behaviors. Disabling quorum-sensing circuits with small molecules has been proposed as a potential strategy to prevent bacterial pathogenicity. The human pathogen Pseudomonas aeruginosa uses quorum sensing to control virulence and biofilm formation. Here, we analyze synthetic molecules for inhibition of the two P. aeruginosa quorum-sensing receptors, LasR and RhlR. Our most effective compound, meta-bromo-thiolactone (mBTL), inhibits both the production of the virulence factor pyocyanin and biofilm formation. mBTL also protects Caenorhabditis elegans and human lung epithelial cells from killing by P. aeruginosa. Both LasR and RhlR are partially inhibited by mBTL in vivo and in vitro; however, RhlR, not LasR, is the relevant in vivo target. More potent antagonists do not exhibit superior function in impeding virulence. Because LasR and RhlR reciprocally control crucial virulence factors, appropriately tuning rather than completely inhibiting their activities appears to hold the key to blocking pathogenesis in vivo.

Addition of carbon nucleophiles to arene-chromium complexes

Abstract The electrophilic reactivity of arenes coordinated to the chromium tricarbonyl unit has been developed into several distinct methods for coupling carbon nucleophiles with aromatic rings. Addition of the nucleophile produces stable η 5 -cyclohexadienyl chromium complexes which can be oxidized to induce loss of the endo hydrogen and the metal, overall nucleophilic substitution for hydrogen. Alternatively, the intermediate can be protonated and the resulting cyclohexa-1,3-diene can be detached from the chromium, effecting nucleophilic addition with reduction of one double bond. If a halogen (F, Cl) is present as a ring substituent, and if the nucleophile can migrate about the arene ligand, then loss of halide can occur parallel with classical nucleophilic aromatic substitution for halogen in electron-deficient haloarenes. With substituted arenes, the regioselectivity of addition becomes important and is often very high. Particularly useful are strong resonance donor substituents (RO-, R 2 N-, F-) where selectivity for meta attack is high. Indole provides an excellent example of selective activation, as the six-membered ring complexes selectively and is then susceptible to nucleophilic substitution, predominantly at the 4 and 7 positions. Substitution for halogen is a somewhat limited process and depends upon the nature of the nucleophile. Very reactive nucleophiles add to unsubstituted positions and are often slow to isomerize to the ipso position from which loss of halide can occur.

Mechanism of the oxidation of alcohols by 2,2,6,6-tetramethylpiperidine nitrosonium cation

Abstract Simple Hammett studies, hydrogen isotope effects, and attempted preparation of proposed transient intermediates are used to probe the mechanism of oxidation of alcohols by 2,2,6,6-tetramethylpiperidine nitrosonium ion.

Signal production and detection specificity in Vibrio CqsA/CqsS quorum-sensing systems

Quorum sensing is a process of bacterial cell–cell communication that enables populations of cells to carry out behaviours in unison. Quorum sensing involves detection of the density‐dependent accumulation of extracellular signal molecules called autoinducers that elicit population‐wide changes in gene expression. In Vibrio species, CqsS is a membrane‐bound histidine kinase that acts as the receptor for the CAI‐1 autoinducer which is produced by the CqsA synthase. In Vibrio cholerae, CAI‐1 is (S)‐3‐hydroxytridecan‐4‐one. The C170 residue of V. cholerae CqsS specifies a preference for a ligand with a 10‐carbon tail length. However, a phenylalanine is present at this position in Vibrio harveyi CqsS and other homologues, suggesting that a shorter CAI‐1‐like molecule functions as the signal. To investigate this, we purified the V. harveyi CqsS ligand, and determined that it is (Z)‐3‐aminoundec‐2‐en‐4‐one (Ea‐C8‐CAI‐1) carrying an 8‐carbon tail. The V. harveyi CqsA/CqsS system is exquisitely selective for production and detection of this ligand, while the V. cholerae CqsA/CqsS counterparts show relaxed specificity in both production and detection. We isolated CqsS mutants in each species that display reversed specificity for ligands. Our analysis provides insight into how fidelity is maintained in signal transduction systems.

ARENE-METAL COMPLEXES IN ORGANIC SYNTHESIS

In the 20 or so years that have passed since the study of x-arene-metal complexes began in a general way, hundreds of stable organometallic compounds with arene ligands have been prepared (for comprehensive reviews of the subject, see Reference 1). The structures and chemical properties are now well established, but application of x-arene complexes to organic synthesis has lagged many years behind the inorganic interest. Recent developments, however, suggest that this situation is changing, that new areas of arene chemistry are becoming available that involve selective reactions on or adjacent to metal-complexed aromatic rings. This paper focuses on the aromatic ring; it attempts to summarize the changes in reactivity induced by metal coordination, the examples of conversions with significance for organic synthesis, and the patterns of reactivity that portend valuable synthetic methods. In general, with new methods of synthesis that involve transition metals, three aspects must be examined: introduction of the activating metal unit, induction of the appropriate organic transformation at the ligand of interest, and removal of the metal unit. This paper begins with a summary of the techniques available for formation of arene-metal complexes and for eventual release of the modified arene unit. The emphasis is on transformations of the coordinated arene.

Mechanism of Vibrio cholerae Autoinducer-1 Biosynthesis

Vibrio cholerae, the causative agent of the disease cholera, uses a cell to cell communication process called quorum sensing to control biofilm formation and virulence factor production. The major V. cholerae quorum-sensing signal CAI-1 has been identified as (S)-3-hydroxytridecan-4-one, and the CqsA protein is required for CAI-1 production. However, the biosynthetic route to CAI-1 remains unclear. Here we report that (S)-adenosylmethionine (SAM) is one of the two biosynthetic substrates for CqsA. CqsA couples SAM and decanoyl-coenzyme A to produce a previously unknown but potent quorum-sensing molecule, 3-aminotridec-2-en-4-one (Ea-CAI-1). The CqsA mechanism is unique; it combines two enzymatic transformations, a β,γ-elimination of SAM and an acyltransferase reaction into a single PLP-dependent catalytic process. Ea-CAI-1 is subsequently converted to CAI-1, presumably through the intermediate tridecane-3,4-dione (DK-CAI-1). We propose that the Ea-CAI-1 to DK-CAI-1 conversion occurs spontaneously, and we identify the enzyme responsible for the subsequent step: conversion of DK-CAI-1 into CAI-1. SAM is the substrate for the synthesis of at least three different classes of quorum-sensing signal molecules, indicating that bacteria have evolved a strategy to leverage an abundant substrate for multiple signaling purposes.

The free and bound forms of Lpp occupy distinct subcellular locations in Escherichia coli

The lipoprotein Lpp is the most numerically abundant protein in Escherichia coli, has been investigated for over 40 years, and has served as the paradigmatic bacterial lipoprotein since its initial discovery. It exists in two distinct forms: a ‘bound‐form’, which is covalently bound to the cells peptidoglycan layer, and a ‘free‐form’, which is not. Although it is known that the carboxyl‐terminus of bound‐form Lpp is located in the periplasm, the precise location of free‐form Lpp has never been determined. For decades, it has been widely assumed that free‐form Lpp is associated with bound‐form. In this work, we show that the free and bound forms of Lpp are not largely associated with each other, but are found in distinct subcellular locations. Our results indicate that free‐form Lpp spans the outer membrane and is surface‐exposed, whereas bound‐form Lpp resides in the periplasm. Thus, Lpp represents a novel example of a single lipoprotein that is able to occupy distinct subcellular locations, and challenges models in which the free and bound forms of Lpp are assumed to be associated with each other.

The Vibrio cholerae quorum-sensing autoinducer CAI-1: analysis of the biosynthetic enzyme CqsA

Vibrio cholerae, the bacterium that causes the disease cholera, controls virulence factor production and biofilm development in response to two extracellular quorum-sensing molecules, called autoinducers. The strongest autoinducer, called CAI-1 (for cholera autoinducer-1), was previously identified as (S)-3-hydroxytridecan-4-one. Biosynthesis of CAI-1 requires the enzyme CqsA. Here, we determine the CqsA reaction mechanism, identify the CqsA substrates as (S)-2-aminobutyrate and decanoyl coenzyme A, and demonstrate that the product of the reaction is 3-aminotridecan-4-one, dubbed amino-CAI-1. CqsA produces amino-CAI-1 by a pyridoxal phosphate (PLP)-dependent acyl-CoA transferase reaction. Amino-CAI-1 is converted to CAI-1 in a subsequent step via a CqsA-independent mechanism. Consistent with this, we find cells release ≥100 times more CAI-1 than amino-CAI-1. Nonetheless, V. cholerae responds to amino-CAI-1 as well as CAI-1, whereas other CAI-1 variants do not elicit a quorum-sensing response. Thus, both CAI-1 and amino-CAI-1 have potential as lead molecules in the development of an anti-cholera treatment.