Quorum-sensing, intra- and inter-species competition in the staphylococci.

In Gram-positive bacteria such as Staphylococcus aureus and the coagulase-negative staphylococci (CoNS), the accessory gene regulator (agr) is a highly conserved but polymorphic quorum-sensing system involved in colonization, virulence and biofilm development. Signalling via agr depends on the interaction of an autoinducing peptide (AIP) with AgrC, a transmembrane sensor kinase that, once phosphorylated activates the response regulator AgrA. This in turn autoinduces AIP biosynthesis and drives target gene expression directly via AgrA or via the post-transcriptional regulator, RNAIII. In this review we describe the molecular mechanisms underlying the agr-mediated generation of, and response to, AIPs and the molecular basis of AIP-dependent activation and inhibition of AgrC. How the environment impacts on agr functionality is considered and the consequences of agr dysfunction for infection explored. We also discuss the concept of AIP-driven competitive interference between S. aureus and the CoNS and its anti-infective potential.

Conformational analysis and interaction of the Staphylococcus aureus transmembrane peptidase AgrB with its AgrD propeptide substrate.

Virulence gene expression in the human pathogen, S. aureus is regulated by the agr (accessory gene regulator) quorum sensing (QS) system which is conserved in diverse Gram-positive bacteria. The agr QS signal molecule is an autoinducing peptide (AIP) generated via the initial processing of the AgrD pro-peptide by the transmembrane peptidase AgrB. Since structural information for AgrB and AgrBD interactions are lacking, we used homology modelling and molecular dynamics (MD) annealing to characterise the conformations of AgrB and AgrD in model membranes and in solution. These revealed a six helical transmembrane domain (6TMD) topology for AgrB. In solution, AgrD behaves as a disordered peptide, which binds N-terminally to membranes in the absence and in the presence of AgrB. In silico, membrane complexes of AgrD and dimeric AgrB show non-equivalent AgrB monomers responsible for initial binding and for processing, respectively. By exploiting split luciferase assays in Staphylococcus aureus, we provide experimental evidence that AgrB interacts directly with itself and with AgrD. We confirmed the in vitro formation of an AgrBD complex and AIP production after Western blotting using either membranes from Escherichia coli expressing AgrB or with purified AgrB and T7-tagged AgrD. AgrB and AgrD formed stable complexes in detergent micelles revealed using synchrotron radiation CD (SRCD) and Landau analysis consistent with the enhanced thermal stability of AgrB in the presence of AgrD. Conformational alteration of AgrB following provision of AgrD was observed by small angle X-ray scattering from proteodetergent micelles. An atomistic description of AgrB and AgrD has been obtained together with confirmation of the AgrB 6TMD membrane topology and existence of AgrBD molecular complexes in vitro and in vivo.

A Pseudomonas aeruginosa PQS quorum-sensing system inhibitor with anti-staphylococcal activity sensitizes polymicrobial biofilms to tobramycin.

As single- and mixed-species biofilms, Staphylococcus aureus and Pseudomonas aeruginosa cause difficult-to-eradicate chronic infections. In P. aeruginosa, pseudomonas quinolone (PQS)-dependent quorum sensing regulates virulence and biofilm development that can be attenuated via antagonists targeting the transcriptional regulator PqsR (MvfR). Here, we exploited a quinazolinone (QZN) library including PqsR agonists and antagonists for their activity against S. aureus alone, when co-cultured with P. aeruginosa, and in combination with the aminoglycoside tobramycin. The PqsR inhibitor, QZN 34 killed planktonic Gram-positives but not Gram-negatives. QZN 34 prevented S. aureus biofilm formation, severely damaged established S. aureus biofilms, and perturbed P. aeruginosa biofilm development. Although P. aeruginosa protected S. aureus from tobramycin in mixed biofilms, the combination of aminoglycoside antibiotic with QZN 34 eradicated the mixed-species biofilm. The mechanism of action of QZN 34 toward Gram-positive bacteria is shown to involve membrane perturbation and dissipation of transmembrane potential.

Actinomadura graeca sp. nov.: A novel producer of the macrocyclic antibiotic zelkovamycin.

As part of a screening programme for antibiotic-producing bacteria, a novel Actinomadura species was discovered from a soil sample collected in Santorini, Greece. Preliminary 16S rRNA gene sequence comparisons highlighted Actinomadura macra as the most similar characterised species. However, whole-genome sequencing revealed an average nucleotide identity (ANI) value of 89% with A. macra, the highest among related species. Further phenotypic and chemotaxonomic analyses confirmed that the isolate represents a previously uncharacterised species in the genus Actinomadura, for which the name Actinomadura graeca sp. nov. is proposed (type strain 32-07T). The G+C content of A. graeca 32-07 is 72.36%. The cell wall contains DL-diaminopimelic acid, intracellular sugars are glucose, ribose and galactose, the predominant menaquinone is MK-9(H6), the major cellular lipid is phosphatidylinositol and fatty acids consist mainly of hexadecanoic acid. No mycolic acid was detected. Furthermore, A. graeca 32-07 has been confirmed as a novel producer of the non-ribosomal peptide antibiotic zelkovamycin and we report herein a provisional description of the unique biosynthetic gene cluster.

Clostridioides difficile: innovations in target discovery and potential for therapeutic success.

INTRODUCTION: Clostridioides difficile infection (CDI) remains a worldwide clinical problem. Increased incidence of primary infection, occurrence of hypertoxigenic ribotypes, and more frequent occurrence of drug resistant, recurrent, and non-hospital CDI, emphasizes the urgent unmet need of discovering new therapeutic targets. AREAS COVERED: We searched PubMed and Web of Science databases for articles identifying novel therapeutic targets or treatments for C. difficile from 2001 to 2021. We present an updated review on current preclinical efforts on designing inhibitory compounds against these drug targets and indicate how these could become the focus of future therapeutic approaches. We also evaluate the increasing exploitability of gut microbial-derived metabolites and host-derived therapeutics targeting VEGF-A, immune targets and pathways, ion transporters, and microRNAs as anti-C. difficile therapeutics, which have yet to reach clinical trials. Our review also highlights the therapeutic potential of re-purposing currently available agents . We conclude by considering translational hurdles and possible strategies to mitigate these problems. EXPERT OPINION: Considerable progress has been made in the development of new anti-CDI drug candidates. Nevertheless, a greater comprehension of CDI pathogenesis and host-microbe interactions is beginning to uncover potential novel therapeutic targets, which can be exploited to plug gaps in the CDI drug discovery pipeline.

Timing Is Everything: Impact of Naturally Occurring Staphylococcus aureus AgrC Cytoplasmic Domain Adaptive Mutations on Autoinduction.

Mutations in the polymorphic Staphylococcus aureusagr locus responsible for quorum sensing (QS)-dependent virulence gene regulation occur frequently during host adaptation. In two genomically closely related S. aureus clinical isolates exhibiting marked differences in Panton-Valentine leukocidin production, a mutation conferring an N267I substitution was identified in the cytoplasmic domain of the QS sensor kinase, AgrC. This natural mutation delayed the onset and accumulation of autoinducing peptide (AIP) and showed reduced responsiveness to exogenous AIPs. Other S. aureus strains harboring naturally occurring AgrC cytoplasmic domain mutations were identified, including T247I, I311T, A343T, L245S, and F264C. These mutations were associated with reduced cytotoxicity, delayed/reduced AIP production, and impaired sensitivity to exogenous AIP. Molecular dynamics simulations were used to model the AgrC cytoplasmic domain conformational changes arising. Although mutations were localized in different parts of the C-terminal domain, their impact on molecular structure was manifested by twisting of the leading helical hairpin α1-α2, accompanied by repositioning of the H-box and G-box, along with closure of the flexible loop connecting the two and occlusion of the ATP-binding site. Such conformational rearrangements of key functional subdomains in these mutants highlight the cooperative response of molecular structure involving dimerization and ATP binding and phosphorylation, as well as the binding site for the downstream response element AgrA. These appear to increase the threshold for agr activation via AIP-dependent autoinduction, thus reducing virulence and maintaining S. aureus in an agr-downregulated "colonization" mode.IMPORTANCE Virulence factor expression in Staphylococcus aureus is regulated via autoinducing peptide (AIP)-dependent activation of the sensor kinase AgrC, which forms an integral part of the agr quorum sensing system. In response to bound AIP, the cytoplasmic domain of AgrC (AgrC-cyt) undergoes conformational changes resulting in dimerization, autophosphorylation, and phosphotransfer to the response regulator AgrA. Naturally occurring mutations in AgrC-cyt are consistent with repositioning of key functional domains, impairing dimerization and restricting access to the ATP-binding pocket. Strains harboring specific AgrC-cyt mutations exhibit reduced AIP autoinduction efficiency and a timing-dependent attenuation of cytotoxicity which may confer a survival advantage during established infection by promoting colonization while restricting unnecessary overproduction of exotoxins.

Rational Design and Synthesis of Modified Teixobactin Analogues: In Vitro Antibacterial Activity against Staphylococcus aureus, Propionibacterium acnes and Pseudomonas aeruginosa.

Teixobactin, a recently discovered depsipeptide that binds to bacterial lipid II and lipid III, provides a promising molecular scaffold for the design of new antimicrobials. Herein, we describe the synthesis and antimicrobial evaluation of systematically modified teixobactin analogues. The replacement of the Ile11 residue with aliphatic isosteres, the modification of the guanidino group at residue 10 and the introduction of a rigidifying residue, that is, dehydroamino acid, into the macrocyclic ring generated useful structure-activity information. Extensive antimicrobial susceptibility assessment against a panel of clinically relevant Staphylococcus aureus and Propionibacterium acnes strains led to the identification of the new lead compound, [Arg(Me)10 ,Nle11 ]teixobactin, with an excellent bactericidal activity (minimum inhibitory concentration (MIC)=2-4 µg mL-1 ). Significantly, the antimicrobial activity of several of the teixobactin analogues against the pathogenic Gram-negative Pseudomonas aeruginosa was "restored" when combined with the sub-MIC concentration of the outer membrane-disruptive antibiotic colistin. The antimicrobial effectiveness of a [Tfn10 ,Nle11 ]teixobactin (32 µg mL-1 )-colistin (2 µg mL-1 ; 0.5×MIC) combination against P. aeruginosa PAO1 reveals, for the first time, an alternative therapeutic option in the treatment of Gram-negative infections.

5-Hydroxyethyl-3-tetradecanoyltetramic acid represents a novel treatment for intravascular catheter infections due to Staphylococcus aureus.

Objectives: Biofilm infections of intravascular catheters caused by Staphylococcus aureus may be treated with catheter lock solutions (CLSs). Here we investigated the antibacterial activity, cytotoxicity and CLS potential of 5-hydroxyethyl-3-tetradecanoyltetramic acid (5HE-C14-TMA) compared with the related compounds 3-tetradecanoyltetronic (C14-TOA) and 3-tetradecanoylthiotetronic (C14-TTA), which are variants of quorum sensing signalling molecules produced by Pseudomonas aeruginosa . Methods: Antibacterial activity and mechanism of action of 5HE-C14-TMA, C14-TOA and C14-TTA were determined via MIC, bacterial killing, membrane potential and permeability assays. Susceptibility of S. aureus biofilms formed in the presence of plasma in vitro was investigated, MTT cytotoxicity testing was undertaken and cytokine release in human blood upon exposure to 5HE-C14-TMA and/or S. aureus biofilms was quantified. The effectiveness of 5HE-C14-TMA as CLS therapy in vivo was assessed using a rat intravascular catheter biofilm infection model. Results: MICs of 5HE-C14-TMA, C14-TOA and C14-TTA ranged from 2 to 4 mg/L. 5HE-C14-TMA and C14-TTA were bactericidal; all three compounds perturbed the staphylococcal membrane by increasing membrane permeability, depolarized the transmembrane potential and caused ATP leakage. Cytotoxicity and haemolytic activity were compound and target cell type-dependent. 5HE-C14-TMA reduced S. aureus biofilm viability in a dose-dependent manner in vitro and in vivo and did not trigger release of cytokines in human blood, but inhibited the high levels of IL-8 and TNF-α induced by S. aureus biofilms. Conclusions: 5HE-C14-TMA, C14-TOA and C14-TTA are membrane-active agents. 5HE-C14-TMA was the most potent, eradicating S. aureus biofilms at 512-1024 mg/L both in vitro and in vivo as a CLS.

New Found Hope for Antibiotic Discovery: Lipid II Inhibitors.

Research into antibacterial agents has recently gathered pace in light of the disturbing crisis of antimicrobial resistance. The development of modern tools offers the opportunity of reviving the fallen era of antibacterial discovery through uncovering novel lead compounds that target vital bacterial cell components, such as lipid II. This paper provides a summary of the role of lipid II as well as an overview and insight into the structural features of macrocyclic peptides that inhibit this bacterial cell wall component. The recent discovery of teixobactin, a new class of lipid II inhibitor has generated substantial research interests. As such, the significant progress that has been achieved towards its development as a promising antibacterial agent is discussed.

Controlled intracellular generation of reactive oxygen species in human mesenchymal stem cells using porphyrin conjugated nanoparticles.

Nanoparticles capable of generating controlled amounts of intracellular reactive oxygen species (ROS), that advance the study of oxidative stress and cellular communication, were synthesized by functionalizing polyacrylamide nanoparticles with zinc(II) porphyrin photosensitisers. Controlled ROS production was demonstrated in human mesenchymal stem cells (hMSCs) through (1) production of nanoparticles functionalized with varying percentages of Zn(II) porphyrin and (2) modulating the number of doses of excitation light to internalized nanoparticles. hMSCs challenged with nanoparticles functionalized with increasing percentages of Zn(II) porphyrin and high numbers of irradiations of excitation light were found to generate greater amounts of ROS. A novel dye, which is transformed into fluorescent 7-hydroxy-4-trifluoromethyl-coumarin in the presence of hydrogen peroxide, provided an indirect indicator for cumulative ROS production. The mitochondrial membrane potential was monitored to investigate the destructive effect of increased intracellular ROS production. Flow cytometric analysis of nanoparticle treated hMSCs suggested irradiation with excitation light signalled controlled apoptotic cell death, rather than uncontrolled necrotic cell death. Increased intracellular ROS production did not induce phenotypic changes in hMSC subcultures.

A facile approach to tryptophan derivatives for the total synthesis of argyrin analogues.

A facile route has been established for the synthesis of indole-substituted (S)-tryptophans from corresponding indoles, which utilizes a chiral auxiliary-facilitated Strecker amino acid synthesis strategy. The chiral auxiliary reagents evaluated were (S)-methylbenzylamine and related derivatives. To illustrate the robustness of the method, eight optically pure (S)-tryptophan analogues were synthesized, which were subsequently used for the convergent synthesis of a potent antibacterial agent, argyrin A and its analogues.

Targeting Staphylococcus aureus quorum sensing with nonpeptidic small molecule inhibitors.

A series of 3-oxo-C12-HSL, tetramic acid, and tetronic acid analogues were synthesized to gain insights into the structural requirements for quorum sensing inhibition in Staphylococcus aureus. Compounds active against agr were noncompetitive inhibitors of the autoinducing peptide (AIP) activated AgrC receptor, by altering the activation efficacy of the cognate AIP-1. They appeared to act as negative allosteric modulators and are exemplified by 3-tetradecanoyltetronic acid 17, which reduced nasal cell colonization and arthritis in a murine infection model.

Attenuating Staphylococcus aureus virulence gene regulation: a medicinal chemistry perspective.

Virulence gene expression in Staphylococcus aureus is tightly regulated by intricate networks of transcriptional regulators and two-component signal transduction systems. There is now an emerging body of evidence to suggest that the blockade of S. aureus virulence gene expression significantly attenuates infection in experimental models. In this Perspective, we will provide insights into medicinal chemistry strategies for the development of chemical reagents that have the capacity to inhibit staphylococcal virulence expression. These reagents can be broadly grouped into four categories: (1) competitive inhibitors of the accessory gene regulator (agr) quorum sensing system, (2) inhibitors of AgrA-DNA interactions, (3) RNAIII transcription inhibitors, and (4) inhibitors of the SarA family of transcriptional regulators. We discuss the potential of specific examples of antivirulence agents for the management and treatment of staphylococcal infections.

Enhanced uptake of nanoparticle drug carriers via a thermoresponsive shell enhances cytotoxicity in a cancer cell line.

Polymer particles consisting of a biodegradable poly[lactide-co-glycolide] (PLGA) core and a thermoresponsive shell have been formulated to encapsulate the dye rhodamine 6G and the potent cytotoxic drug paclitaxel. Cellular uptake of these particles is significantly enhanced above the thermal transition temperature (TTT) of the polymer shells in the human breast carcinoma cell line MCF-7 as determined by flow cytometry and fluorescence microscopy. Paclitaxel-loaded particles display reduced and enhanced cytotoxicity below and above the TTT respectively compared to unencapsulated drug. The data suggests a potential route to enhanced anti-cancer efficacy through temperature-mediated cell targeting.

Synthetic peptides derived from an N-terminal domain of the E2 protein of GB virus C in the study of GBV-C/HIV-1 co-infection.

Synthetic peptides derived from GB virus C (GBV-C) have previously been studied in our group for the development of new systems capable of diagnosing diseases caused by this humanotropic virus. We also recently described specific peptide domains of the E2 envelop protein of GBV-C that have the capacity to interfere with the HIV-1 fusion peptide, produce a notable decrease in cellular membrane fusion, and perturb HIV-1 infectivity in a dose-dependent manner. The present work discloses the design and synthesis of both linear and cyclic branched peptides based on a previously reported N-terminal sequence of the GBV-C E2 protein. Immunoassays and cell-cell fusion assays were performed to evaluate their diagnostic value to detect anti-GBV-C antibodies in HIV-1 patients, as well as their putative anti-HIV-1 activity as entry inhibitors. Our results showed that chemical modifications of the selected E2(7-26) linear peptide to afford cyclic architecture do not result in an enhanced inhibition of gp41 HIV-1-mediated cell-cell fusion nor improved sensitivity in the detection of GBV-C antibodies in HIV-1 co-infected patients. Thus, the ELISA data reinforce the potential utility of linear versions of the E2(7-26) region for the development of new peptide-based immunosensor devices for the detection of anti-GBV-C antibodies in HIV-1 co-infected patients.

Methicillin resistance reduces the virulence of healthcare-associated methicillin-resistant Staphylococcus aureus by interfering with the agr quorum sensing system.

The difficulty in successfully treating infections caused by methicillin-resistant Staphylococcus aureus (MRSA) has led to them being referred to as highly virulent or pathogenic. In our study of one of the major healthcare-associated MRSA (HA-MRSA) clones, we show that expression of the gene responsible for conferring methicillin resistance (mecA) is also directly responsible for reducing the ability of HA-MRSA to secrete cytolytic toxins. We show that resistance to methicillin induces changes in the cell wall, which affects the bacteria's agr quorum sensing system. This leads to reduced toxin expression and, as a consequence, reduced virulence in a murine model of sepsis. This diminished capacity to cause infection may explain the inability of HA-MRSA to move into the community and help us understand the recent emergence of community-associated MRSA (CA-MRSA). CA-MRSA typically express less penicillin-binding protein 2a (encoded by mecA), allowing them to maintain full virulence and succeed in the community environment.

Thermoresponsive polymer colloids for drug delivery and cancer therapy.

Many difficulties in treating cancer arise from the problems in directing highly cytotoxic agents to the deseased tissues, cells and intracellular compartments. Many drug delivery systems have been devised to address this problem, including those that show a change in properties in response to a temperature stimulus. In particular, colloidal materials based on thermoresponsive polymers offer a means to transport drugs selectively into tumour tissues that are hyperthermic, either intrinsically or through the application of clinical procedures such as localised heating. In this paper, the key attributes of thermoresponsive polymer colloids are considered, a number of important recent examples are discussed and the possible future developments of these materials are evaluated.

Protease sensing with nanoparticle based platforms.

Nanoparticulate systems in various unique configurations are highly effective at detecting protease activity both in vivo and in vitro. In this article, we have summarised the conventional modern methods for monitoring protease activity, and critically appraised recent advances in protease-responsive nanosensors.

End-stapled homo and hetero collagen triple helices: a click chemistry approach.

A CuAAC reaction was established for modular synthesis of end-stapled homo- and hetero-triple helical peptides, generating "clicked" macro-assemblies with enhanced thermal stability.

Regulation of neurotoxin production and sporulation by a Putative agrBD signaling system in proteolytic Clostridium botulinum.

A significant number of genome sequences of Clostridium botulinum and related species have now been determined. In silico analysis of these data revealed the presence of two distinct agr loci (agr-1 and agr-2) in all group I strains, each encoding putative proteins with similarity to AgrB and AgrD of the well-studied Staphylococcus aureus agr quorum sensing system. In S. aureus, a small diffusible autoinducing peptide is generated from AgrD in a membrane-located processing event that requires AgrB. Here the characterization of both agr loci in the group I strain C. botulinum ATCC 3502 and of their homologues in a close relative, Clostridium sporogenes NCIMB 10696, is reported. In C. sporogenes NCIMB 10696, agr-1 and agr-2 appear to form transcriptional units that consist of agrB, agrD, and flanking genes of unknown function. Several of these flanking genes are conserved in Clostridium perfringens. In agreement with their proposed role in quorum sensing, both loci were maximally expressed during late-exponential-phase growth. Modulation of agrB expression in C. sporogenes was achieved using antisense RNA, whereas in C. botulinum, insertional agrD mutants were generated using ClosTron technology. In comparison to the wild-type strains, these strains exhibited drastically reduced sporulation and, for C. botulinum, also reduced production of neurotoxin, suggesting that both phenotypes are controlled by quorum sensing. Interestingly, while agr-1 appeared to control sporulation, agr-2 appeared to regulate neurotoxin formation.