Department of Clinical Microbiology, University Hospital (Rigshospitalet), Copenhagen Ø, DK-2100, Denmark1
Department of Microbiology, the Technical University of Denmark, Lyngby,DK-2800, Denmark2
Department of Biological Sciences, Florida International University, University Park, Miami, FL 33199, USA3
Lehrstuhl fur Mikrobiologie, Technische Universitat Munchen, Freising D-85350, Germany4
Author for correspondence: Michael Givskov. Tel: +45 45252769. Fax: +45 45932809. e-mail: immg{at}pop.dtu.dk
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ABSTRACT |
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Keywords: AHL, quorum sensing, green fluorescent protein (GFP), mouse model, lung infection
Abbreviations: AHL, N-acylhomoserine lactone (BHL, N-butanoyl-, HHL, N-hexanoyl-, OdDHL, N-(3-oxododecanoyl)-, ODHL, N-(3-oxodecanoyl)-, OHHL, N-(3-oxohexanoyl)-, OHL, N-octanoylhomoserine lactone); CF, cystic fibrosis; CSLM, confocal scanning laser microscopy; GFP, green fluorescent protein; PMN, polymorphonuclear leukocyte
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INTRODUCTION |
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Bacterial quorum-sensing systems enable the expression of target genes in response to culture density (Fuqua & Greenberg, 1998 ; Hardman et al., 1998
; Hastings & Greenberg, 1999
). Quorum-sensing systems exert their activities via small diffusible signal molecules which, in Gram-negative bacteria, are often N-acylhomoserine lactones (AHLs) (Fuqua & Greenberg, 1998
; Fuqua et al., 1996
; Salmond et al., 1995
). AHLs are synthesized from precursors by a synthetase (LuxI homologue), and they interact with a transcription activator (LuxR homologue) to induce expression of target genes (Fuqua et al., 1996
). When the bacteria reach a certain population size, the concentration of AHLs exceeds a threshold level. At this critical concentration, the AHL molecules are thought to induce a conformational change in the R-protein, which then initiates transcription of target genes (Fuqua et al., 1996
). Short- and medium-chain AHL molecules are thought to freely pass the bacterial membrane whereas the long-chain molecules appear to require active efflux (Pearson et al., 1999
).
In P. aeruginosa there are at least two different quorum-sensing systems, las (Gambello & Iglewski, 1991 ) and rhl (Ochsner & Reiser, 1995
), which consist of two signal-generating synthetases (LasI/RhlI) and two cognate transcriptional regulators (LasR/RhlR). The major products of LasI and RhlI are N-(3-oxododecanoyl)homoserine lactone (OdDHL or 3OC12-HSL or PAI-1) (Pearson et al., 1994
) and N-butanoylhomoserine lactone (BHL or C4-HSL or PAI-1) (Pearson et al., 1995
; Winson et al., 1995
), respectively. The lasIR-encoded quorum-sensor system has been shown to modulate expression of lasI itself (Seed et al., 1995
), lasB (elastase) (Passador et al., 1993
; Pearson et al., 1997
), lasA (staphylolytic protease) (Gambello et al., 1993
), apr (alkaline protease) (Gambello et al., 1993
), xcp (secretion pathway) (Chapon-Herve et al., 1997
) and rhlR (Latifi et al., 1996
; Pesci et al., 1997
) The rhlIR-encoded quorum sensor modulates expression of rhlI itself (Latifi et al., 1996
), rhlAB (rhamnolipid biosynthesis) (Ochsner & Reiser, 1995
; Pearson et al., 1997
), lasB (Brint & Ohman, 1995
; Pearson et al., 1995
, 1997
) and rpoS (Latifi et al., 1996
). Recently, the two quorum-sensing systems have been shown to regulate twitching motility (Glessner et al., 1999
) and to be involved in the differentiation of biofilm (Davies et al., 1998
). Biofilm differentiation is thought to protect P. aeruginosa from the host defence system and from the action of antibiotics (Costerton et al., 1987
). But on top of that, OdDHL interferes with the host immune system, where it specifically down-regulates the production of the cytokines IL-12 and TNF
which support the bactericidal Th-1 milieu and protect the host (Telford et al., 1998
)
Quorum-sensing-regulated gene expression probably reflects the need for the invading pathogen to reach a critical population density sufficient to overwhelm host defences and thus establish infection. It has been suggested that rising AHL levels indicate increasing preparedness for assault. Current understanding of AHL quorum-sensing systems in bacteria is mainly based on in vitro data. A few studies, however, have demonstrated the relationship between components of the quorum-sensing systems and virulence. Tang et al. (1996) demonstrated that a P. aeruginosa lasR mutant is substantially less virulent in an acute pneumonia mouse model. In plant-pathogenic bacteria of the genus Erwinia, which causes soft rot disease, production of several extracellular enzymes that degrade cell walls, such as pectate lyase, pectin lyase, polygalacturonase, cellulase and protease, is subject to quorum-sensing-regulated gene expression (Jones et al., 1993
; Pirhonen et al., 1993
). In a simple but elegant potato experiment the direct effect of exogenous AHL-induced virulence factors was demonstrated (Jones et al., 1993
).
The ability to monitor the production of AHL molecules from pathogenic Gram-negative bacteria during infection will make an important contribution to the understanding of host and micro-organism interactions. In principle, the presence of exogenous AHL molecules can be detected by a reporter gene fused to any quorum-sensing target gene. The prototypic quorum sensor is encoded by the Photobacterium fischeri luxRluxI (Fuqua & Greenberg, 1998 ; Salmond et al., 1995
). The cognate AHL signal is N-(3-oxohexanoyl)homoserine lactone (OHHL). In order to perform on-line studies of AHL communication among bacteria, components of the Ph. fischeri quorum sensor encoded by luxR-PluxI have been fused to gfp genes encoding green fluorescent proteins (GFPs) with different half-lives (J. B. Andersen and others, unpublished). The sensitivity of the GFP reporter enables visualization of cellcell communication at the single-cell level. The present study reports on this biological approach to detect AHLs produced by P. aeruginosa in mouse lung tissues.
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METHODS |
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AHL monitor strains.
The GFP derivatives GFPmut3* and its unstable variant were used as reporters (Andersen et al., 1998 ). The gfpmut3* gene encodes a 238 amino acid protein that emits fluorescent light at 511 nm when excited by light with wavelength around 488 nm (Cormack et al., 1996
). GFPmut3* requires trace amounts of oxygen to mature and does not need any substrate addition in order to fluoresce. A number of gfpmut3* derivatives whose products have different half-lives are also available (Andersen et al., 1998
). The unstable versions are useful for monitoring temporal expression of AHL molecules in vivo (J. B. Andersen and others, unpublished). One such variant, GFP(ASV), containing alanine (A), serine (S) and valine (V) at the C-terminus, was used in our study. All GFP-based monitor plasmids carried the P. fischeri region encoding luxR and a fusion between PluxI and gfp. The plasmids pJBA88 and pJBA89 are pUC18NotI derivatives that contain this promoter fused to the stable gfpmut3* and the unstable gfp(ASV), respectively. The E. coli JM105 monitor strains containing these plasmids were denoted JB353 and JB357, respectively. The luxR, PluxIgfpmut3* fusion and the luxR, PluxIgfp(ASV) (unstable GFP) fusion were cloned into a stable broad-host-range vector pME6031 (pVS1 replicon, accession no. AF118811), giving rise to pJBA130 and pJBA132, respectively (J. B. Andersen and others, unpublished). The E. coli MT102 monitor strains containing these plasmids were denoted JB524 and JB525, respectively. Henceforth, the monitor strains are referred to as JB353-gfpmut3*, JB357-gfp(ASV), JB524-gfpmut3* and JB525-gfp(ASV). Two additional AHL monitor strains were used, JM105/pSB536 and JM105/pMH297, which encode bioluminescent AHL monitors. pSB536 carries the Aeromonas hydrophila ahyR, PahyIluxAB fusion to detect exogenous BHL (Swift et al., 1997
), whereas pMH297 carries the P. aeruginosa lasR, PlasBluxAB fusion to detect OdDHL and N-(3-oxodecanoyl)homoserine lactone (ODHL).
Extraction of AHL signals from culture supernatants.
Extraction was performed essentially as described by Shaw et al. (1997) . A 100 ml culture of P. aeruginosa PAO579 was grown in Luria broth to an OD600 of 2·0. The culture was extracted with an equal volume of HPLC-grade ethyl acetate, and the aqueous phase was separated using a separating funnel. The organic phase was dried with anhydrous magnesium sulphate, filtered, and evaporated almost to dryness. The residue was then resuspended in 200 µl ethyl acetate.
Separation and identification of AHL signals by TLC.
Analytical TLC was done essentially as described by Shaw et al. (1997) . C18-reversed-phase TLC plates (aluminium sheets RP-18 F254s (20 x 20 cm), Merck Chrom line) were used to chromatograph 10 µl of the AL preparation with methanol/water (60:40, v/v). After separation, the solvent was evaporated, and the dried plates were overlaid with AHL monitor strains, prepared as follows. A 0·7 ml overnight culture was added to 450 ml Luria broth containing 1% agar at 42 °C. The media/culture mix was overlaid on top of the TLC plate, until the entire plate was covered. Following solidification, the plate was incubated overnight at 30 °C. The positions of AHL spots were visualized as described previously (Shaw et al., 1997
; J. B. Andersen and others, unpublished). Chemically synthesized OHHL and OdDHL were obtained from P. Williams, University of Nottingham, UK. All other AHLs were from Fluka Chemie.
Animals and experimental groups.
Two mouse strains, NMRI and CBA/J, were used. The strains were obtained from The Panum Institute, Copenhagen University, Denmark. The number of animals used, and the experimental protocols, are shown in Table 1. All animal experiments were performed after authorization from the National Animal Ethics Committee. Where appropriate, the animals were given subcutaneous injections of 200 µg ampicillin per g body weight once a day.
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Challenge procedures.
Before challenge, all mice were anaesthetized by subcutaneous injection of a 1:1 mixture of etomidat (Janssen) and midazolam (Roche) at a dose of 10 ml per kg body weight and tracheotomized (Johansen et al., 1993 ; Moser et al., 1997
). Intratracheal challenge with 0·04 ml (1·0 x 109 c.f.u. ml1) of alginate beads was performed as described previously (Moser et al., 1997
). The incision was sutured with silk and healed without any complications. The animals were killed on different days by administering 20% pentobarbital (DAK) at 2 ml per kg body weight.
Freeze microtomy.
The lung tissues with pathological changes from each group (Table 1) were embedded with Tissue-Tek and frozen at -20 °C to -40 °C immediately after death. Frozen sections 4050 µm thick were made at different levels of the lung tissues by freeze microtomy. The frozen sections were mounted with saline to prevent drying.
Epifluorescence microscopy.
An axioplan epifluorescence microscope (Leitz ARISTOPLAN E Camera System, type 307-148.002) was used to visualize green fluorescence of the AHL monitor strains. The microscope was equipped with a 100 W mercury lamp, and filter set no. 10 (Carl Zeiss) to visualize GFP. A slow-scan charge-coupled device (CCD) camera CH250 (Photometrics) equipped with a KAF 1400 chip (pixel size 608 x 608 µm) was used for capturing digital images. The camera was operated at -40 °C, and the chip was read out in 12 bits (4096 intensity levels) at a rate of 200 kHz.
Confocal scanning laser microscopy (CSLM).
Microscopic inspection and image acquisition of the frozen lung sections were performed by CSLM (model TCS4D, Leica Lasertechnik) equipped with the I3 filter set (Leica Lasertechnik) for detection of green fluorescence emitted by the monitor bacteria. Simulated fluorescence projections and vertical cross-sections through the lung tissues were generated using the IMARIS (Bitplane AG) software package running on a Silicon Graphics Indigo 2 workstation. Images were further processed for display using Adobe Photoshop software.
Macroscopic pathology of the lungs.
The macroscopic lung pathology was expressed as the lung index of macroscopic pathology (LIMP) as described previously (Song et al., 1998 ) according to the following modified formula: LIMP=the lung area with pathological change divided by the area of the whole lung.
Histopathology of the lungs.
Frozen sections (thickness 10 µm) of the lung samples from different days after challenge were chosen for haematoxylin/eosin staining to evaluate the severity of pathological changes by light microscopy. The pathology includes lung consolidation, where lung tissues and bronchi are filled by inflammatory cells and oedema or haemorrhage, and atelectasis, where the alveoli and airways have collapsed.
Lung bacteriology.
Lung samples from 10 of the animals in each group were prepared for quantitative bacteriological examination as previously described (Johansen et al., 1993 ). Lungs were homogenized in 5 ml PBS, and appropriately diluted samples were plated on Blue agar plates (a solid medium for Gram-negative rods containing lactose, pH 7·0; State Serum Institute, Copenhagen, Denmark) to determine the number of c.f.u. after 2024 h incubation at 37 °C. Bacteria from lung tissues containing mixed inocula of P. aeruginosa and E. coli (Pa+Ec group) were discriminated based on the colony colour on the blue agar plates combined with the ability of E. coli colonies to express green fluorescence in the presence of exogenous AHLs.
Statistical analysis.
The KruskalWallis test was used to compare the data of macroscopic lung pathology between three groups, and the MannWhitney U test was employed to compare the data of lung bacteriology and macroscopic lung pathology between two groups in the study.
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RESULTS |
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Lung bacteriology
To determine the number of bacteria and verify if the lung tissues harboured functional monitor strains (especially in the control experiments where mice were challenged with the E. coli strains alone), we examined the lungs on different days after intratracheal challenge (Table 2). In experiments II and III, the E. coli JB524-gfpmut3* or JB525-gfp(ASV) counts were significantly reduced after day 3. P. aeruginosa PAO579 remained more stable in the lungs than did the E. coli strains. In experiment II (lungs with a single inoculum), bacterial clearance with the AHL monitor strains (Ec group) was significantly faster compared to the P. aeruginosa strain (Pa group) (P<0·001). In experiment III, the bacterial counts in the Ec group were significantly reduced compared to the Pa group except for day 3 (P<0·01). The experiments showed that it was hard to detect E. coli cells in the lung samples 5 d post-challenge (Table 2
).
The presence of functional AHL monitor bacteria in the lungs of mice challenged with E. coli alone (the Ec group) was confirmed by plating and incubation on agar plates supplemented with 10 nM OHHL (data not shown). As judged from the expression of green fluorescence this test demonstrated that the mice on day 3 carried functional monitor bacteria that were simply not turned on in the lung tissue (data not shown).
Macroscopic lung pathology
In experiment II, the lung index of macroscopic pathology on day 14 was significantly milder compared to day 3 (P<0·05) in all three groups. The differences among the three groups were significant (P<0·001) on day 3, 5, 7 and 14 after challenge. The pathological changes in the Ec group were all remarkably milder than in the other two groups (P<0·02) on the four different days (Table 3), whereas in experiment III the three groups did not significantly differ from each other in macroscopic lung pathology. In general, the macroscopic lung pathology in the Pa+Ec group was not significantly different from the Pa group in either experiment II or experiment III (Table 3
). Introduction of alginate beads without bacteria did not lead to any significant pathological changes (data not shown).
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DISCUSSION |
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The use of alginate-entrapped bacteria enables the establishment of a chronic lung infection (Pedersen et al., 1990 ; Moser et al., 1997
) and in combination with the GFP-based monitor systems it makes the study of bacterial cross-talk in the lungs of experimental animals possible. In the course of a P. aeruginosa co-infection, all versions of the AHL monitor strains [E. coli JB357-gfp(ASV), JB524-gfpmut3* and JB525-gfp(ASV)] were induced to express green fluorescence. From 1 to 3 d after intratracheal bacterial challenge, production of AHLs from P. aeruginosa was detected by the monitor cells as expression of green fluorescence when frozen sections of the lung tissue were observed under the epifluorescence microscope. In experiment II, fully induced, green fluorescent bacteria could easily be found in the lung tissues exhibiting severe pathological changes (large areas of consolidation) but were rarely found in the lung tissues exhibiting minor pathological changes. The lung bacteriology showed that both P. aeruginosa and E. coli JB524-gfpmut3* or JB525-gfp(ASV) were found at high densities up to day 3. Five days after bacterial challenge, GFP-expressing bacteria could not be detected in the lung tissues. Taken together with colony counts, and since E. coli JB524-gfpmut3* cells express stable GFP (signal stable for days), this shows that the monitor strain is cleared from the lung tissues. Two of the monitor strains, E. coli JB357-gfp(ASV) and JB525-gfp(ASV), express an unstable variant of GFP which is rapidly degraded in exponentially growing as well as in stationary-phase cells. In the absence of exogenous AHL molecules, the fluorescent signals of the monitor cells will be below detection level after 4 h (Fig. 3b
). These results indicate that AHL molecules are being produced during infection until both types of bacteria are cleared. Although we have no data on the actual half-life of GFP in bacteria infecting the lungs, our results suggest that the inoculated P. aeruginosa produced AHL molecules from at least 24 h to 3 d of infection. In addition to functioning as AHL indicators in vivo, the E. coli monitor strains directly demonstrate that intercellular communication and quorum sensing take place in the infected animal lung.
The presence of fully induced E. coli JB357-gfp(ASV) cells indicates that the AHL concentration locally exceeds a minimum of 5 nM, but due to sensitivity limitations of the TLC overlay technique we were unable to determine which AHL molecule is the major species in the lungs of the infected animals. An analysis of this is in progress. It is likely that the production as well as the variety of AHLs from P. aeruginosa is affected by many factors such as specific antibodies, phagocytes and other host-defence mechanisms. The conditions in the lung may also affect the sensitivity of the E. coli monitor strains. The monitor bacteria present inside the lung tissues were covered by alginate, lung tissue and blood cells, etc. Thus, the induction of GFP in the monitor bacteria might require a much higher AHL level in vivo than in vitro. Although all three monitors functioned well and allowed detection of GFP-expressing bacteria at the single-cell level, E. coli JB357-gfp(ASV), producing the unstable version of GFP from a high-copy-number vector, gave the strongest signal compared to the other two monitor strains, which contained stabilized, low-copy-number vectors. The usefulness of the JB357-gfp(ASV) system in chronic infections is somewhat limited since persistence of plasmid-carrying cells in the lungs required administration of ampicillin to the mice.
The histopathological changes seen in the mouse lung tissues were similar to what we found in the lungs of CF patients, i.e. inflammation was dominated by the presence of PMNs as demonstrated in Fig. 4 (Hoiby et al., 1993
). The lung bacteriology and pathology results indicate that the AHL monitor strains inside the alginate beads were cleared more easily and induced less severe pathological changes compared with P. aeruginosa PAO579. Macroscopic lung pathology studies showed that changes were still observed on days 7 and 14 in the E. coli monitor group even though the lung bacteriology indicated that the monitor strains had been cleared by day 5. Histopathology showed that lung atelectasis and beads blocking the bronchi were the major changes observed from day 5. Therefore mechanical blocking by the beads might be partly responsible for the lung atelectasis in the Ec group after day 5.
In favour of using the E. coli strains as AHL monitor is the fact that these strains induce minor lung pathological changes compared to P. aeruginosa. Furthermore, the E. coli monitor system is not controlled by a number of complex regulatory factors and media effects, as are the quorum sensors of P. aeruginosa. E. coli can therefore be employed as a useful AHL monitor strain in short-term experiments of mouse lung infections with P. aeruginosa. We also found that the E. coli monitors are 50- to 100-fold more sensitive to AHL molecules compared with the JP2 strain of P. aeruginosa PAO1 (lasI rhlI) (Pearson et al., 1997 ) harbouring pJB130 and pJB132 (data not shown). However, for extended studies more appropriate monitor strains will have to be employed and work is in progress to construct modified P. aeruginosa and Burkholderia cepacia strains with inactivating mutations in the AHL signal-generating systems carrying lasR, PlasB-based monitors in combination with unstable versions of GFP. The ideal strain would be expected to persist for a long time in the lungs co-infected with virulent P. aeruginosa strains.
Geisenberger et al. (2000) recently reported that neither the amounts nor the AHL profiles of P. aeruginosa isolates (when grown under standard in vitro conditions) from five CF patients who were monitored over periods of up to 11 years changed significantly during long-term colonization. These results suggest that phenotypic adaptations to the CF lung environment during chronic colonization do not affect the ability to synthesize AHLs. The present study provides direct evidence of AHL expression in vivo in mouse lung tissues using live AHL monitor strains. This is in accordance with the finding of Hardman et al. (1999)
that the sputum of CF patients infected with P. aeruginosa activated AHL monitor strains, strongly suggesting that AHL signals are being produced during infection. Expression of the P. aeruginosa virulence factors is modulated by AHL molecules. Hence it was not very surprising to detect the presence of AHL mainly in the lung tissues exhibiting severe pathological changes. This correlation between AHL exposure and severity of infection supports the view of Tang et al. (1996)
that quorum sensing plays a critical role in the virulence of P. aeruginosa. In addition, Telford et al. (1998)
suggested that OdDHL not only functions to regulate bacterial virulence gene expression through cellcell communication but also, by virtue of its immuno-modulatory properties, it may be a virulence determinant per se. There is clearly a role for live AHL monitors for in vivo studies with quorum-sensing inhibitors. Furanone compounds produced by the macro-alga Delisea pulchra are powerful inhibitors of quorum-sensing systems (Givskov et al., 1996
; Manefield et al., 1999
) and their antagonist activity in vivo can be judged from their ability to switch off AHL-controlled GFP expression in the lung. This may provide a basis for the development of anti-AHL therapy, as postulated by Finch et al. (1998)
and Hartman & Wise (1998)
.
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ACKNOWLEDGEMENTS |
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Received 30 March 2000;
revised 10 July 2000;
accepted 21 July 2000.