1 Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK
2 Centre for Developmental Genetics, University of Sheffield, Western Bank, Sheffield S10 2TN, UK
Correspondence
Simon J. Foster
S.Foster{at}sheffield.ac.uk
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ABSTRACT |
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INTRODUCTION |
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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 bacterial 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 protein, 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 hostpathogen interaction. D. melanogaster has previously been developed as a model for Pseudomonas 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 attenuated strains, pheP and perR, and demonstrate the use of the model to monitor in vivo expression and assess the effectiveness of antibiotics.
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METHODS |
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D. melanogaster infection.
Cells from overnight bacterial cultures (10 ml) were recovered by centrifugation at 4000 g for 5 min (room temperature); the supernatant was discarded and the pellet resuspended in PBS (10 ml). Cell suspensions were serially diluted in PBS and the concentration of cells determined by plating on appropriate media. For S. aureus, typically the inoculum was approximately 2x108 c.f.u. ml1. For injection, adult female flies were used (25 days old); for female flies pricking with a sterile needle is consistently harmless (D'Argenio et al., 2001). Flies were anaesthetized with CO2 and infected via pricking in the dorsal thorax with a needle (25 GA) dipped in the cell suspension. Flies were returned to standard fly culture vials with food and incubated at 30 °C.
Death of flies following infection.
Flies were infected in batches of ten. Initially a single batch was inoculated for each S. aureus strain. Following infection the number of surviving flies was recorded at intervals. Those strains for which fly death kinetics appeared to differ from S. aureus SH1000 were repeated with five batches (a total of 50 flies). Where appropriate, statistical significance was evaluated using Student's t-test.
Growth of S. aureus in vivo.
Flies were infected in batches of ten. At intervals following infection a single batch of infected flies was taken and flies separated into individual microfuge tubes. Dead and living flies were distinguished. Each fly was crushed in 100 µl PBS with a micropestle and the homogenate serially diluted in PBS. C.f.u. per fly was determined through growth on BHI agar plus appropriate antibiotics.
In vivo expression analysis
GFP.
Plasmid pSB2035 (Qazi et al., 2001) was phage-transduced into S. aureus SH1000 (Novick, 1967
). An inoculum of
2x1010 c.f.u. ml1 was typically used to infect flies. Approximately 16 h following infection all flies had died. A single fly was taken, and the head, wings and legs removed. The remaining tissue was fixed in 4 % (v/v) paraformaldehyde in PBS for 30 min at room temperature. The tissue was then washed for 20 min in 0·2 % (v/v) Triton X in PBS. This washing was repeated three times. The tissue was then incubated at 37 °C with 250 µg RNase A ml1 for 23 h followed by 10 µg propidium iodide ml1 for 30 min. The tissue was then washed again as described above and rinsed with PBS. Sections, 15 µm thick, were cut with a cryostat, collected on Superfrost Plus slides and allowed to air dry. Sections were mounted in 70 % (v/v) glycerol in PBS and observed by fluorescence microscopy (Zeiss: Axiophot 2).
-Galactosidase activity.
Flies were infected with a lacZ fusion strain or SH1000 in batches of five. At intervals following infection, a batch was taken and flies transferred to a single microfuge tube. The flies were crushed together in 250 µl PBS with a micropestle. Duplicate 100 µl samples of the homogenate were taken and frozen at 70 °C. Levels of -galactosidase activity were measured as described previously (Horsburgh et al., 2001a
). Fluorescence was measured using a Victor plate reader (Wallac) with a 0·1 s count time and calibrated with standard concentrations of MU (4-methylumbelliferone). Background fluorescence, measured from SH1000-infected flies, was deducted from all samples. One unit of
-galactosidase activity was defined as the amount of enzyme that catalysed the production of 1 pmol MU min1 per c.f.u. The remaining 50 µl was serially diluted in PBS. C.f.u. per fly was determined through growth on BHI agar plus appropriate antibiotics.
Antibiotic treatment of infected flies.
Flies were infected in batches of ten. Following infection, batches of flies were transferred to standard fly culture vials containing filter paper disks soaked with 10 % (w/v) sucrose plus tetracycline or methicillin at a range of concentrations. The number of surviving flies was recorded at intervals.
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RESULTS AND DISCUSSION |
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Infection of D. melanogaster was obtained through pricking of the dorsal thorax with a needle dipped in a bacterial suspension; flies were not injected with a defined amount of the inoculum. Approximately 25 nl was inoculated into the fly. This value is calculated from the determination of c.f.u. per fly immediately after infection, typically 104 c.f.u. with a 2x108 c.f.u. ml1 inoculum. However, upon inoculation some cells may adhere to the surface of the fly; the infective dose may therefore be less than 104 cells. S. aureus SH1000 cells at concentrations between 1·9x107 and 1·9x109 c.f.u. ml1 were shown to kill flies, whilst those flies inoculated with sterile PBS survived (Fig. 1). Those flies inoculated with autoclaved S. aureus SH1000 (4·6x109 c.f.u. ml1) also survived (data not shown). As for C. elegans, this indicates that death of infected flies is due to the presence of live bacteria and not due to shock. The rate of death was proportional to the number of bacteria inoculated, with 100 % death achieved after 23 h with an inoculum of 1·9x108 c.f.u. ml1 (Fig. 2
). Infection of D. melanogaster with Mycobacterium marinum (Dionne et al., 2003
) or Listeria monocytogenes (Mansfield et al., 2003
), through a similar method of infection, also causes fly death; flies take up to a week to die with an inoculum approximately 10-fold lower than that used in this study (M. marinum, 500 c.f.u.; L. monocytogenes, 100400 c.f.u.). Death kinetics of D. melanogaster infected with Pseudomonas aeruginosa (4002000 c.f.u.) are comparable to those seen in this study, with 100 % fly death 28 h following infection (D'Argenio et al., 2000
). The lethal effect of S. aureus is not strain specific, as 8325-4, COL and S6 also kill flies, although with varying kinetics (Fig. 2
). Similarly, injection of silkworm larvae Bombyx mori with S. aureus also causes death, due to growth in larval haemolymph and tissues (Kaito et al., 2002
). S. aureus grows exponentially in D. melanogaster, with a doubling time of
80 min and a yield of approximately 2x107 c.f.u. per fly (Fig. 3
). For S. aureus SH1000, stationary phase is reached approximately 15 h after infection. Infection of D. melanogaster infection with P. aeruginosa results in a comparable bacterial titre of 1x10640x106 c.f.u. per fly before fly death (D'Argenio et al., 2000
).
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Interestingly, none of the well-characterized regulators of virulence appears to have a major role in the D. melanogaster model. Both agr and sarA have been shown to be required for full virulence in several mammalian model systems. This shows that although components like perR and pheP are important in both the fly and the mouse there is disparity between the systems. Thus the fly model is not directly comparable to mammalian models but does allow the identification of potential novel components involved in pathogenesis.
Bacterial infection is systemic in the fly
The green fluorescent protein (GFP) is encoded on the plasmid pSB2035 with expression under the control of P3 (agr) (Qazi et al., 2001). P3, in vitro, is turned on in post-exponential phase. pSB2035 was phage-transduced from S. aureus 8325-4 to SH1000 (Novick, 1967
). The resulting strain (SJF1219) was used to inoculate flies. An inoculum of
2x1010 c.f.u. ml1 was used to ensure that infection progressed in all flies. Fig. 6
shows a section through the mid-thorax of a fly infected with SJF1219. The fly was fixed and sectioned 16 h after infection. Fig. 6
is typical of sections throughout the fly, where S. aureus forms microcolonies on a range of tissues. Individual cells can be seen embedded in a matrix of material. SH1000-infected flies were also dissected into head, thorax and abdomen and the c.f.u. per section determined (data not shown). S. aureus cells were present throughout the fly in numbers proportional to the volume of tissue present, supporting the histological results indicating systemic infection. Such pathology may be analogous to infection observed in other models: in S. aureus-infected silkworm larvae, bacterial cells proliferate in blood and tissues, particularly at the epithelial surface of the midgut (Kaito et al., 2002
); and in S. aureus-fed C. elegans, death is correlated with the accumulation of bacteria within the digestive tract (Sifri et al., 2003
). A systemic infection in the fly is not surprising considering the nature and range of disease caused by S. aureus in humans. The development of infection in D. melanogaster has previously been observed using GFP-expressing P. aeruginosa (Fauvarque et al., 2002
) and M. marinum (Dionne et al., 2003
); infection is characterized by bacterial growth at the site of infection followed by systemic infection and fly death.
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Conclusions
D. melanogaster forms a convenient high-throughput model of S. aureus infection with a defined end point. It is versatile in that microbial status in terms of growth rate and gene expression can be easily measured. Whilst the model is engineered, and it is unlikely that D. melanogaster can be viewed as a natural host for S. aureus, it has the capability of identifying novel virulence determinants. This is important, as the role of a large number of genes in S. aureus is unknown. The D. melanogaster model provides an initial screen for virulence determinants whose role in mammalian infection can subsequently be analysed. The model can also be used to assay gene expression throughout the course of infection and allows high-throughput in vivo screening for the action of novel antimicrobials. Finally, analysis of the host innate response to S. aureus in this defined model will allow hostpathogen interaction to be determined at the molecular level.
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ACKNOWLEDGEMENTS |
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Received 24 February 2004;
revised 13 April 2004;
accepted 19 April 2004.
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