Synthesis of d-desthiobiotin-AI-2 as a novel chemical probe for autoinducer-2 quorum sensing receptors.

In processes regulated by quorum sensing (QS) bacteria respond to the concentration of autoinducers in the environment to engage in group behaviours. Autoinducer-2 (AI-2) is unique as it can foster interspecies communication. Currently, two AI-2 receptors are known, LuxP and LsrB, but bacteria lacking these receptors can also respond to AI-2. In this work, we present an efficient and reproducible synthesis of a novel chemical probe, d-desthiobiotin-AI-2. This probe binds both LuxP and LsrB receptors from different species of bacteria. Thus, this probe is able to bind receptors that recognise the two known biologically active forms of AI-2, presenting the plasticity essential for the identification of novel unknown AI-2 receptors. Moreover, a protocol to pull down receptors bound to d-desthiobiotin-AI-2 with anti-biotin antibodies has also been established. Altogether, this work highlights the potential of conjugating chemical signals to biotinylated derivatives to identify and tag signal receptors involved in quorum sensing or other chemical signalling processes.

Identification of novel autoinducer-2 receptors in Clostridia reveals plasticity in the binding site of the LsrB receptor family.

Autoinducer-2 (AI-2) is unique among quorum-sensing signaling molecules, as it is produced and recognized by a wide variety of bacteria and thus facilitates interspecies communication. To date, two classes of AI-2 receptors have been identified: the LuxP-type, present in the Vibrionales, and the LsrB-type, found in a number of phylogenetically distinct bacterial families. Recently, AI-2 was shown to affect the colonization levels of a variety of bacteria in the microbiome of the mouse gut, including members of the genus Clostridium, but no AI-2 receptor had been identified in this genus. Here, we identify a noncanonical, functional LsrB-type receptor in Clostridium saccharobutylicum. This novel LsrB-like receptor is the first one reported with variations in the binding-site amino acid residues that interact with AI-2. The crystal structure of the C. saccharobutylicum receptor determined at 1.35 Å resolution revealed that it binds the same form of AI-2 as the other known LsrB-type receptors, and isothermal titration calorimetry (ITC) assays showed that binding of AI-2 occurs at a submicromolar concentration. Using phylogenetic analysis, we inferred that the newly identified noncanonical LsrB receptor shares a common ancestor with known LsrB receptors and that noncanonical receptors are present in bacteria from different phyla. This led us to identify putative AI-2 receptors in bacterial species in which no receptors were known, as in bacteria belonging to the Spirochaetes and Actinobacteria phyla. Thus, this work represents a significant step toward understanding how AI-2-mediated quorum sensing influences bacterial interactions in complex biological niches.

Synthesis and biological activity of a potent optically pure autoinducer-2 quorum sensing agonist.

Quorum sensing (QS) regulates population-dependent bacterial behaviours, such as toxin production, biofilm formation and virulence. Autoinducer-2 (AI-2) is to date the only signalling molecule known to foster inter-species bacterial communication across distantly related bacterial species. In this work, the synthesis of pure enantiomers of C4-propoxy-HPD and C4-ethoxy-HPD, known AI-2 analogues, has been developed. The optimised synthesis is efficient, reproducible and short. The (4S) enantiomer of C4-propoxy-HPD was the most active compound being approximately twice as efficient as (4S)-DPD and ten-times more potent than the (4R) enantiomer. Additionally, the specificity of this analogue to bacteria with LuxP receptors makes it a good candidate for clinical applications, because it is not susceptible to scavenging by LsrB-containing bacteria that degrade the natural AI-2. All in all, this study provides a new brief and effective synthesis of isomerically pure analogues for QS modulation that include the most active AI-2 agonist described so far.

Combined transcriptomics-metabolomics profiling of the heat shock response in the hyperthermophilic archaeon Pyrococcus furiosus.

Pyrococcus furiosus is a remarkable archaeon able to grow at temperatures around 100 °C. To gain insight into how this model hyperthermophile copes with heat stress, we compared transcriptomic and metabolomic data of cells subjected to a temperature shift from 90 °C to 97 °C. In this study, we used RNA-sequencing to characterize the global variation in gene expression levels, while nuclear magnetic resonance (NMR) and targeted ion exchange liquid chromatography-mass spectrometry (LC-MS) were used to determine changes in metabolite levels. Of the 552 differentially expressed genes in response to heat shock conditions, 257 were upregulated and 295 were downregulated. In particular, there was a significant downregulation of genes for synthesis and transport of amino acids. At the metabolite level, 37 compounds were quantified. The level of di-myo-inositol phosphate, a canonical heat stress solute among marine hyperthermophiles, increased considerably (5.4-fold) at elevated temperature. Also, the levels of mannosylglycerate, UDP-N-acetylglucosamine (UDPGlcNac) and UDP-N-acetylgalactosamine were enhanced. The increase in the pool of UDPGlcNac was concurrent with an increase in the transcript levels of the respective biosynthetic genes. This work provides the first metabolomic analysis of the heat shock response of a hyperthermophile.

The antimicrobial activity of Sub3 is dependent on membrane binding and cell-penetrating ability.

Because of their high activity against microorganisms and low cytotoxicity, cationic antimicrobial peptides (AMPs) have been explored as the next generation of antibiotics. Although they have common structural features, the modes of action of AMPs are extensively debated, and a single mechanism does not explain the activity of all AMPs reported so far. Here we investigated the mechanism of action of Sub3, an AMP previously designed and optimised from high-throughput screening with bactenecin as the template. Sub3 has potent activity against Gram-negative and Gram-positive bacteria as well as against fungi, but its mechanism of action has remained elusive. By using AFM imaging, ζ potential, flow cytometry and fluorescence methodologies with model membranes and bacterial cells, we found that, although the mechanism of action involves membrane targeting, Sub3 internalises inside bacteria at lethal concentrations without permeabilising the membrane, thus suggesting that its antimicrobial activity might involve both the membrane and intracellular targets. In addition, we found that Sub3 can be internalised into human cells without being toxic. As some bacteria are able to survive intracellularly and consequently evade host defences and antibiotic treatment, our findings suggest that Sub3 could be useful as an intracellular antimicrobial agent for infections that are notoriously difficult to treat.

Design and characterization of novel antimicrobial peptides, R-BP100 and RW-BP100, with activity against Gram-negative and Gram-positive bacteria.

BP100 is a short cationic antimicrobial peptide with a mechanism of action dependent on peptide-lipid interactions and microbial surface charge neutralization. Although active against Gram-negative bacteria, BP100 is inactive against Gram-positive bacteria. In this study we report two newly designed BP100 analogues, RW-BP100 and R-BP100 that have the Tyr residue replaced with a Trp and/or the Lys residues replaced with an Arg. The new analogues in addition to being active against Gram-negative bacteria, possess activity against all tested Gram-positive bacteria. Mechanistic studies using atomic force microscopy, surface plasmon resonance and fluorescence methodologies reveal that the antibacterial efficiency follows the affinity for bacterial membrane. The studies suggest that the activity of BP100 and its analogues against Gram-negative bacteria is mainly driven by electrostatic interactions with the lipopolysaccharide layer and is followed by binding to and disruption of the inner membrane, whereas activity against Gram-positive bacteria, in addition to electrostatic attraction to the exposed lipoteichoic acids, requires an ability to more deeply insert in the membrane environment, which is favoured with Arg residues and is facilitated in the presence of a Trp residue. Knowledge on the mechanism of action of these antimicrobial peptides provides information that assists in the design of antimicrobials with higher efficacy and broader spectra of action, but also on the design of peptides with higher specificity if required.

Antimicrobial properties of analgesic kyotorphin peptides unraveled through atomic force microscopy.

Antimicrobial peptides (AMPs) are promising candidates as alternatives to conventional antibiotics for the treatment of resistant pathogens. In the last decades, new AMPs have been found from the cleavage of intact proteins with no antibacterial activity themselves. Bovine hemoglobin hydrolysis, for instance, results in AMPs and the minimal antimicrobial peptide sequence was defined as Tyr-Arg plus a positively charged amino acid residue. The Tyr-Arg dipeptide alone, known as kyotorphin (KTP), is an endogenous analgesic neuropeptide but has no antimicrobial activity itself. In previous studies new KTP derivatives combining C-terminal amidation and Ibuprofen (Ib) - KTP-NH(2), IbKTP, IbKTP-NH(2) - were designed in order to improve KTP brain targeting. Those modifications succeeded in enhancing peptide-cell membrane affinity towards fluid anionic lipids and higher analgesic activity after systemic injection resulted therefrom. Here, we investigated if this affinity for anionic lipid membranes also translates into antimicrobial activity because bacteria have anionic membranes. Atomic force microscopy revealed that KTP derivatives perturbed Staphylococcus aureus membrane structure by inducing membrane blebbing, disruption and lysis. In addition, these peptides bind to red blood cells but are non-hemolytic. From the KTP derivatives tested, amidated KTP proves to be the most active antibacterial agent. The combination of analgesia and antibacterial activities with absence of toxicity is highly appealing from the clinical point of view and broadens the therapeutic potential and application of kyotorphin peptides.