Downloaded from www.microbiologyresearch.org by IP: 54.224.121.223 On: Sat, 07 May 2016 01:54:08 Drosophila melanogaster as a model host for Staphylococcus aureus infection Andrew J. Needham, 1 Monica Kibart, 2 Howard Crossley, 1 Philip W. Ingham 2 and Simon J. Foster 1 Correspondence Simon J. Foster S.Foster@sheffield.ac.uk Department of Molecular Biology and Biotechnology 1 and Centre for Developmental Genetics 2 , University of Sheffield, Western Bank, Sheffield S10 2TN, UK Received 24 February 2004 Revised 13 April 2004 Accepted 19 April 2004 Staphylococcus aureus is an important pathogen of humans, causing a range of superficial and potentially life-threatening diseases. Infection of the fruit fly Drosophila melanogaster with S. aureus results in systemic infection followed by death. Screening of defined S. aureus mutants for components important in pathogenesis identified perR and pheP, with fly death up to threefold slower after infection with the respective mutants compared to the wild-type. Infection of D. melanogaster with reporter gene fusion strains demonstrated the in vivo expression levels of the accessory gene regulator, agr, a-toxin, hla, and a manganese transporter, mntA. The use of the green fluorescent protein as a reporter under the control of the agr promoter (P3) showed S. aureus microcolony formation in vivo. The disease model also allowed the effect of antibiotic treatment on the flies to be determined. D. melanogaster is a genetically tractable model host for high-throughput analysis of S. aureus virulence determinants. INTRODUCTION Staphylococcus aureus is an opportunist pathogen, causing a variety of diseases in humans ranging from superficial skin lesions and wound infections to more serious con- ditions, such as osteomyelitis, endocarditis and septicaemia (Waldvogel, 1995). In order to study host–pathogen inter- action a number of animal models have been developed (Abdelnour et al., 1993; Cheung et al., 1998; Tarkowski et al., 2001). The use of mouse models of infection has allowed the identification of numerous virulence determinants. However, for functional genomic studies the use of large numbers of mammals is difficult for logistical, ethical and financial reasons. Recently a number of non-mammalian models of infection for human pathogens have been deve- loped. Model hosts include: zebrafish, Danio rerio (Neely et al., 2002; van der Sar et al., 2003); the cress Arabidopsis thaliana (Rahme et al., 1997); the soil-living amoeba Dictyostelium discoideum (Solomon et al., 2000) and the nematode Caenorhabditis elegans (Kurz & Ewbank, 2003; Darby et al., 1999; Tan & Ausubel, 2000; Garsin et al., 2001). C. elegans was used as a model host for S. aureus by Sifri et al. (2003), who found that previously characterized virulence determinants important for mammalian infec- tion are required for full pathogenicity in nematodes. Thus key virulence determinants are necessary for disease in a diverse range of hosts. S. aureus infection has also been modelled in silkworm larvae, Bombyx mori (Kaito et al., 2002), the use of which identified this as a possible screen for antibiotics. The fruit fly Drosophila melanogaster is genetically well defined, has a short generation time and possesses an innate immune system which is remarkably similar to that of humans (Takeda & Akira, 2003; Leulier et al., 2003). Infection by Gram-positive bacteria induces the Toll signalling cascade, comparable to the Toll-like receptor (TLR) cascade in vertebrates (Takeda & Akira., 2003). In insects this cascade leads to the expression of a number of antimicrobial and antifungal peptides. Previously, the expression of antimicrobial peptides in response to bac- terial infection, including S. aureus, has been assessed in D. melanogaster (Lemaitre et al., 1997). A recent study showed the importance of two pattern recognition receptors (PRRs) for the detection of Gram-positive bacteria in D. melanogaster : PGRP-SA, a peptidoglycan recognition pro- tein, and GNBP1, a Gram-negative binding protein now found to be required for Toll activation following infection by the Gram-positive bacteria S. aureus and Enterococcus faecalis (Pili-Floury et al., 2004). The induction of these pathways has been examined in more detail through the injection of purified bacterial cell components of Escherichia coli, Pseudomonas aeruginosa and Bacillus thuringiensis (Leulier et al., 2003). These factors make D. melanogaster a suitable model for studies of human host–pathogen interaction. D. melanoga- ster has previously been developed as a model for Pseudo- monas aeruginosa (D’Argenio et al., 2001; Fauvarque et al., 2002), Mycobacterium marinum (Dionne et al., 2003) and Listeria monocytogenes (Mansfield et al., 2003). This study investigates the use of D. melanogaster as a model for S. aureus infection. We describe the identification of two 0002-7116 G 2004 SGM Printed in Great Britain 2347 Microbiology (2004), 150, 2347–2355 DOI 10.1099/mic.0.27116-0