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.

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.

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 ␤-galactosidase substrate. The assay can be used to screen for novel antivirulence compounds from many different sources, such as fungi, as demonstrated here.Staphylococcus aureus is a significant human pathogen that causes a variety of diseases ranging from minor skin infections to life-threatening endocarditis and septicemia (2). A contributing factor to the severity of staphylococcal infections is the ability of the organism to acquire resistance to numerous antibiotics (22). The growing problems of antibiotic-resistant microorganisms have necessitated alternative therapeutic strategies, including antivirulence therapy (3, 6). The aim of antivirulence therapy is to silence virulence gene expression, allowing the host immune system time to act and eradicate the pathogen. In S. aureus, the ability to cause disease relies on the timely production of an impressive collection of virulence factors. During exponential growth, the cell surface-located virulence factors are expressed, including spa, encoding protein A, while upon entry into stationary phase, transcription of hla, encoding ␣-hemolysin, and other genes encoding extracellular factors are induced (15). This regulation is mediated partly by the agr quorum sensing system, composed of a two-component system and a regulatory RNA molecule, RNAIII, that is synthesized in response to increasing concentrations of autoinducer peptide (AIP), also encoded by the agr locus (17, 18). In order to search for new agents that interfere with S. aureus virulence gene expression, we have developed a simple plate assay that allows an easy screen of compounds from a diverse range of biological sources.Results and Discussion. In order to examine the influence of novel compounds on hla and spa gene expression in S. aureus, we incorporated the 8325-4 (16)-derived hla::lacZ (PC322; Ery r ) (5) and spa::lacZ (PC203; Ery r ) (5) fusion strains in tryptic soy agar (TSA) (Oxoid) plates containing erythromycin and 5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside (X-Gal). Briefly, 1 ml of 10 Ϫ3 -diluted overnight culture (tryptone soya broth [TSB]; Oxoid) was placed in a petri dish to which 25 ml of TSA (ϳ40°C) containing 150 g ml Ϫ1 X-Gal and 5 g ⅐ ml Ϫ1 erythromycin was added and mixed by careful whirling. The plates were dried, and wells were formed with a sterile drill. When incubated at 37°C for optimized periods of time, the plates containing PC203 (spa::lacZ) became light blue while those containing PC322 (hla::lacZ) became intensely blue, consistent with the Agr-dependent reciprocal regulation of cell-associated and secreted virulence determinants (15) (Fig. 1).To verify that the assay can visualize established e...

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.

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.