Anita Nielsen

Inhibition of Virulence Gene Expression in Staphylococcus aureus by Novel Depsipeptides from a Marine Photobacterium

During a global research expedition, more than five hundred marine bacterial strains capable of inhibiting the growth of pathogenic bacteria were collected. The purpose of the present study was to determine if these marine bacteria are also a source of compounds that interfere with the agr quorum sensing system that controls virulence gene expression in Staphylococcus aureus. Using a gene reporter fusion bioassay, we recorded agr interference as enhanced expression of spa, encoding Protein A, concomitantly with reduced expression of hla, encoding α-hemolysin, and rnaIII encoding RNAIII, the effector molecule of agr. A marine Photobacterium produced compounds interfering with agr in S. aureus strain 8325-4, and bioassay-guided fractionation of crude extracts led to the isolation of two novel cyclodepsipeptides, designated solonamide A and B. Northern blot analysis confirmed the agr interfering activity of pure solonamides in both S. aureus strain 8325-4 and the highly virulent, community-acquired strain USA300 (CA-MRSA). To our knowledge, this is the first report of inhibitors of the agr system by a marine bacterium.

Solonamide B Inhibits Quorum Sensing and Reduces Staphylococcus aureus Mediated Killing of Human Neutrophils

Methicillin-resistant Staphylococcus aureus (MRSA) continues to be a serious human pathogen, and particularly the spread of community associated (CA)-MRSA strains such as USA300 is a concern, as these strains can cause severe infections in otherwise healthy adults. Recently, we reported that a cyclodepsipeptide termed Solonamide B isolated from the marine bacterium, Photobacterium halotolerans strongly reduces expression of RNAIII, the effector molecule of the agr quorum sensing system. Here we show that Solonamide B interferes with the binding of S. aureus autoinducing peptides (AIPs) to sensor histidine kinase, AgrC, of the agr two-component system. The hypervirulence of USA300 has been linked to increased expression of central virulence factors like α-hemolysin and the phenol soluble modulins (PSMs). Importantly, in strain USA300 Solonamide B dramatically reduced the activity of α-hemolysin and the transcription of psma encoding PSMs with an 80% reduction in toxicity of supernatants towards human neutrophils and rabbit erythrocytes. To our knowledge this is the first report of a compound produced naturally by a Gram-negative marine bacterium that interferes with agr and affects both RNAIII and AgrA controlled virulence gene expression in S. aureus.

Method for Screening Compounds That Influence Virulence Gene Expression in Staphylococcus aureus

ABSTRACT We present a simple assay to examine effects of compounds on virulence gene expression in the human pathogen Staphylococcus aureus. The assay employs transcriptional reporter strains carrying lacZ fused to central virulence genes. Compounds affecting virulence gene expression and activity of the agr locus are scored based on color change in the presence of a chromogenic β-galactosidase substrate. The assay can be used to screen for novel antivirulence compounds from many different sources, such as fungi, as demonstrated here.

Identification of four new agr quorum sensing-interfering cyclodepsipeptides from a marine Photobacterium.

During our search for new natural products from the marine environment, we discovered a wide range of cyclic peptides from a marine Photobacterium, closely related to P. halotolerans. The chemical fingerprint of the bacterium showed primarily non-ribosomal peptide synthetase (NRPS)-like compounds, including the known pyrrothine antibiotic holomycin and a wide range of peptides, from diketopiperazines to cyclodepsipeptides of 500–900 Da. Purification of components from the pellet fraction led to the isolation and structure elucidation of four new cyclodepsipeptides, ngercheumicin F, G, H, and I. The ngercheumicins interfered with expression of virulence genes known to be controlled by the agr quorum sensing system of Staphylococcus aureus, although to a lesser extent than the previously described solonamides from the same strain of Photobacterium.

Nigribactin, a Novel Siderophore from Vibrio nigripulchritudo, Modulates Staphylococcus aureus Virulence Gene Expression

Staphylococcus aureus is a serious human pathogen that employs a number of virulence factors as part of its pathogenesis. The purpose of the present study was to explore marine bacteria as a source of compounds that modulate virulence gene expression in S. aureus. During the global marine Galathea 3 expedition, a strain collection was established comprising bacteria that express antimicrobial activity against Vibrio anguillarum and/or Staphylococcus aureus. Within this collection we searched colony material, culture supernatants, and cell extracts for virulence modulating activity showing that 68 out of 83 marine bacteria (affiliated with the Vibrionaceae and Pseudoalteromonas sp.) influenced expression of S. aureus hla encoding α-hemolysin toxin and/or spa encoding Protein A. The isolate that upon initial screening showed the highest degree of interference (crude ethyl acetate extract) was a Vibrio nigripulchritudo. Extraction, purification and structural elucidation revealed a novel siderophore, designated nigribactin, which induces spa transcription. The effect of nigribactin on spa expression is likely to be independent from its siderophore activity, as another potent siderophore, enterobactin, failed to influence S. aureus virulence gene expression. This study shows that marine microorganisms produce compounds with potential use in therapeutic strategies targeting virulence rather than viability of human pathogens.

Norlichexanthone Reduces Virulence Gene Expression and Biofilm Formation in Staphylococcus aureus

Staphylococcus aureus is a serious human pathogen and antibiotic resistant, community-associated strains, such as the methicillin resistant S. aureus (MRSA) strain USA300, continue to spread. To avoid resistance, anti-virulence therapy has been proposed where toxicity is targeted rather than viability. Previously we have shown that norlichexanthone, a small non-reduced tricyclic polyketide produced by fungi and lichens, reduces expression of hla encoding α-hemolysin as well as the regulatory RNAIII of the agr quorum sensing system in S. aureus 8325–4. The aim of the present study was to further characterise the mode of action of norlichexanthone and its effect on biofilm formation. We find that norlichexanthone reduces expression of both hla and RNAIII also in strain USA300. Structurally, norlichexanthone resembles ω-hydroxyemodin that recently was shown to bind the agr two component response regulator, AgrA, which controls expression of RNAIII and the phenol soluble modulins responsible for human neutrophil killing. We show that norlichexanthone reduces S. aureus toxicity towards human neutrophils and interferes directly with AgrA binding to its DNA target. In contrast to ω-hydroxyemodin however, norlichexanthone reduces staphylococcal biofilm formation. Transcriptomic analysis revealed that genes regulated by the SaeRS two-component system are repressed by norlichexanthone when compared to untreated cells, an effect that was mitigated in strain Newman carrying a partially constitutive SaeRS system. Our data show that norlichexanthone treatment reduces expression of key virulence factors in CA-MRSA strain USA300 via AgrA binding and represses biofilm formation.

Dereplication-guided isolation of depsides thielavins S-T and lecanorins D-F from the endophytic fungus Setophoma sp.

Dereplication methodology using UHPLC-DAD-QTOFMS was applied during the metabolic profiling investigation of the endophyte Setophoma sp., a fungus isolated from symptomless guava fruits. The approach performed allowed a fast analysis of the microbial secondary metabolites. From this fungus, seven highly C-alkylated depsides were isolated and identified as polyketides thielavins S, T, U and V and lecanorins D, E and F. Their structures were elucidated through spectroscopic methods including NMR, HRMS and especially with assistance of HRMS/MS experiments. The compounds were tested for quorum sensing regulation activity in the virulence gene expression of Staphylococcus aureus, but no inhibitory effect was detected. Nevertheless, moderate antibacterial activity was encountered in three of tested depsides, particularly with thielavin T, whose MIC was 6.25 μg/mL against S. aureus.

Akademi og industri. Kjemiutdanning og -forskning ved NTNU gjennom 100 år

The American entrepreneur Gordon Moore is famous for “Moore’s Law” for the doubling of components on chips every year (or every 2 years) and for his role in the founding of Fairchild Semiconductors in 1957 and Intel in 1968. While he has clearly played an important role in the development of microchips and hence computing, this does not seem to have much to do with chemistry. But Moore was originally a chemist, studying chemistry at San Jose State University, University of California at Berkeley, and Caltech, where he took a Ph.D. in physical chemistry. He then undertook postgraduate work at Applied Physics Laboratory, a government-funded institute of Johns Hopkins University. His move into the new field of transistors began in 1956 when he was hired out of the blue byWilliam Shockley for Shockley’s new semiconductor development laboratory funded by Beckman Instruments. The Caltech connection was important here: both Arnold Beckman – who admired (and funded) Shockley – and Shockley himself were Caltech alumni. Shockley and most of his scientists soon fell out over whether to pursue the silicon microchip or the four-layer diode (which Shockley regarded as being the future). Moore was one of the eight “traitors”who left Shockley and Beckman to found Fairchild Semiconductors with the financial backing of Sherman Fairchild. Eleven years later Moore left Fairchild with Robert Noyce to set up Intel, which became the major player in the industry. This book is the work of three authors –Arnold Thackray, David C. Brock, and Rachel Jones – but one does not get the sense of three different hands at work. With Thackray, the wellknown historian of chemistry, on board, there was no danger of overlooking Moore’s background as a chemist, the importance of chemistry, and the role of Arnold Beckman. Unusually for a scientific biography, there is considerable material about Moore’s personal life and thoughts. He was strongly conservative, and when the students were rioting in the Californian campuses in the late 1960s he believed that it was he, not the student radicals, whowas the quiet revolutionary that was going to transform the world. There is also much on his wife Betty (née Whitaker), and this is as much a biography of her life as his. In particular, the book opens with Moore’s ancestors taking the overland trail in 1847 to California, which was just on the point of becoming part of the United States. His Californian background (and that of his wife) was an important aspect of Moore’s career. It meant that he went to such important centres of chemical education as Berkeley and Caltech, and that he was happy to move back fromNew Jersey with Shockley toMountain View, which was near Shockley’s home town of Palo Alto. Subsequently, the area around the southern side of San Francisco Bay became famous as “Silicon Valley” close to the childhood homes of Gordon in Redwood City (he was initially bought up in isolated Pescadero on the coast), and of Betty in Los Gatos. This is a good read; the text is very clear and moves smoothly. The book is recommended to anyone interested in the history of chemistry in California, the history of the semiconductor and transistor industry, and the links between chemistry and electronics.