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 ␤-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...

Antibiotic resistance—the need for global solutions

“…Inhibition of quorum sensing disrupts this process. Several such inhibitory compounds have been identifi ed that block virulence and quorum sensing in P aeruginosa 362 and S aureus, 363,364 for example. Bacterial colonisation of tissue is essential to the establishment of infection and interference with this process is a high-quality target for antivirulence compounds.…”

Amphibian antimicrobial peptide fallaxin analogue FL9 affects virulence gene expression and DNA replication in Staphylococcus aureus

“…The expression of recA was tested in an agar diffusion assay as described previously (Nielsen et al, 2010). The strain HI2682 was used to monitor expression from the recA promoter.…”

“…Virulence gene expression was tested in an agar diffusion assay as described previously (Nielsen et al, 2010). To monitor the promoter activity of hla and rnaIII, the agar plates contained PC322 or SH101F7 (Chan & Foster, 1998;Horsburgh et al, 2002), which harbour an hla : : lacZ and an rnaIII : : lacZ fusion, respectively.…”

“…To analyse the effect of FL9 on the SOS response, we examined the expression of a LexA-controlled gene, recA, using a S. aureus recA : : lacZ fusion and an agar diffusion assay (Nielsen et al, 2010). The result clearly demonstrated an increased expression of recA in response to FL9 monitored as a blue ring in the agar reporter assay (Fig.…”

Exploiting Quorum Sensing To Confuse Bacterial Pathogens

“…A clear demonstration of this system can be found in studies that tested 100 extracts from 50 species of Penicillium, finding that one-third of species produce QSI compounds, including penicillic acid and patulin (232). Staphylococcal AIP reporters that make use of genetic reporters, such as ␤-galactosidase fused to central virulence genes of the agr QS system, have also been developed, which have allowed, for example, the identification of trihydroxy methyl xanthones from fungal sources as inhibitors of AIP signaling (233).…”