Monitoring gene expression of Salmonella inside mammalian cells: comparison of luciferase and β-galactosidase fusion systems

ELSEVIER

Journal ofMicrobiological Methods

Journal of Microbiological Methods 24 (1995) 155-164

Monitoring gene expression of SaZmoneZZuinside mammalian cells: comparison of luciferase and p -galactosidase fusion systems C.G. Pfeifer”,“,

B.B. Finlay”*b

“Department of Microbiology and Immunology, University of British Columbia, 237-6174 University Boulevard, Vancouver, BC, Canada V6T 123 bDepartments of Biochemistry and Molecular Biology, and Biotechnology Laboratory, University of British Columbia, #237 - 6174 University Boulevard, Vancouver, BC, Canada V6T 123

First received 3 February 1995; revised 17 May 1995; accepted 1 June 1995

Abstract Gene expression of bacteria can be studied through the fusion of bacterial genes with reporter genes encoding assayable enzymes such as luciferase and P-galactosidase. However, quantitative measurement of gene expression from intracellular bacteria (bacteria inside eukaryotic host cells) can be difficult, due to problems such as low bacterial numbers or the presence of endogenous enzymes that mimic reporter enzyme activity. In this paper, we determined the efficacy of two reporter systems, 1uxAB (encoding luciferase from Vibrio harveyi) and 1~2 (encoding j3_galactosidase), to measure gene expression from intracellular Salmonella. One set of genes shown previously to have increased expression by intracellular Salmonella are the Salmonella plasmid virulence genes (spvRAB). The spv operon is known to be involved in Salmonella virulence in animal models of infection. Therefore, in this study the spvRAB-1acZ reporter system was compared with an spvRAB-1uxAB reporter system. Although both spv reporter systems showed comparable sensitivity, the luciferase assay provided several advantages. First, it was faster and easier to perform than the &galactosidase assay. It was not necessary to lyse bacteria containing lu.xAB fusions for measurement of luciferase activity, whereas it was necessary to lyse bacteria containing 1ucZ fusions for the measurement of P-galactosidase activity. Furthermore, mammalian host cells and Salmonella did not contain endogenous luciferase, so that any light produced was a result of expression from the 1uxAB constructs. Collectively, these results demonstrate that bacterial luciferase is a favorable alternative to P-galactosidase for determining gene expression of bacterial pathogens that reside within mammalian host cells. Keywords:

P-Galactosidase;

Gene expression;

Intracellular;

1. Introduction Many

bacterial

pathogens

have

the ability

to

* Corresponding author. Tel: +l (604) 822-2493; fax: +l 822-9830; E-mail: [email protected]; (604) [email protected]. Elsevier Science B.V. SSDZ 0167-7012(95)0006.5-8

Luciferase;

MDCK; Salmonella; spvB

invade and persist within host cells. With Sulmonella species, bacterial mutants that are unable to enter and survive within tissue culture cells (in vitro infection models) are less virulent within animal hosts (in vivo infection models) [l-3]. Therefore by monitoring bacterial gene expression throughout the invasion process in

156

CC.

Pfeifer. B. B. Finlay I Journal of Microbiological

tissue culture systems, it may be possible to identify bacterial genes that are induced intracellularly and possibly involved in virulence [4-61. Reporter enzymes make it possible to visualize the expression of bacterial genes whose products are not readily assayable. The expression of a specific gene can be monitored by first fusing that gene with a promoterless reporter gene and then measuring activity of the reporter gene product. However, the reporter enzyme must meet at least two criteria. First, it must have an activity that is distinct from endogenous cellular or bacterial enzymes in the system used. Second, the reporter enzyme assay must be very sensitive, as numbers of invasive bacteria are often low and bacterial gene expression may be moderated. Two reporter enzymes used to study the gene expression of intracellular bacteria include &galactosidase (luc2) [4,7,8] and luciferase (luxAB) [9,10]. @Galactosidase has traditionally been the most widely used bacterial reporter enzyme, although bacterial luciferases are becoming increasingly popular. The use of bacterial luciferases offers several advantages. The luciferase aldehyde substrate (n-decanal) is volatile, amphipathic, and readily crosses membranes [ll-151, unlike most P-galactosidase substrates luciferase activity can be [4,7,16]. Therefore, assayed without the need for bacterial or host cell lysis, and potentially, expression could be assayed within the same sample over time. As well, unlike P-galactosidase, most bacteria and tissue culture cells have no endogenous luciferase activity. Any light produced is the direct result of luciferase activity. Furthermore, light production is also an indicator of bacterial viability [17-231. In the absence of a sufficient supply of flavin mononucleotide (FMNH,) within the bacteria, light is not produced even if luciferase is present ~resulting in measurement of activity from viable organisms only. It is possible that some bacteria are killed within the course of the experiment, i.e. if bacteria-containing vacuoles fuse with cellular lysosomes containing degradative enzymes. With the use of p-galactosidase, enzymatic activity persisting in a killed bacterium could not be differentiated from the activity in a viable bacterium. This would result

Methods 24 (1995) 155-164

in an apparent higher activity per bacterium than really existed. Previously, P-galactosidase (luc2) was used to show that the intracellular location of S. dublin within both phagocytic and non-phagocytic cells induced the expression of the virulence plasmidencoded Salmonellu gene operon spv [7]. Similar results of spv induction were confirmed in a separate study, where the Pap fimbriae gene cluster from Escherichiu coli KS71A was placed under the transcriptional control of the Salmonella spvR, and induction of fimbrial fusion products was monitored using immunofluorescence and bacterial agglutination tests [24]. In this study, we constructed a luciferase gene fusion with spvRAB, similar to the P-galactosidase fusion, and directly compared the enzymatic activities of P-galactosidase and luciferase as reporters of gene expression in intracellular bacteria. These comparisons allowed us to determine the relative merits of each fusion system when used to measure gene expression of a bacterial pathogen inside a mammalian host cell.

2. Materials and Methods 2.1. Chemicals and media

Luria-Bertani broth and agar plates were used to culture bacteria. Tissue culture medium, consisting of minimal Eagle’s medium (MEM) plus 10% fetal bovine serum (FBS) (Gibco Life Technologies), was used to grow tissue culture cells and bacteria used in reporter gene assays. Phosphate-buffered saline (PBS) was used as a diluent where indicated. PBS consisted of 0.2 g 1-l KCl, 0.2 g 1-l KH,PO,, 8 g 1-l NaCl, and 2.16 g 1-l Na,HPO, .7H,O. PBS contained no calcium or magnesium, and the final pH was adjusted to 7.4 with HCl. n-Decanal (99%; Sigma) was used for luciferase assays and tested in a range of aldehyde concentrations. These dilutions were made by first adding 1 ~1 n-decanal into 1, 2, 4, or 10 ml of a solution of 70% (v:v) ethanol:8% (v:v) methanol. One milliliter of this solution was

C.G.

Pfeifer, B.B.

Finlay

I Journal of Microbiological Methods 24 (1995) 155-164

then added to 4.5 ml of MEM + 10% FBS, resulting in concentrations of 0.02%, O.Oll%, 0.0055%, and 0.0022% (v:v) n-decanal, respectively. Two further concentrations of aldehyde were made by adding 1 ~1 n-decanal to 100 or 200 ~1 ethanol:methanol, and then adding 100 ~1 of this solution to 1 ml of tissue culture media, resulting in aldehyde concentrations of 0.099% and 0.0495% (v:v) respectively. Use of alcohol allowed the even dispersal of the aldehyde in the solutions. The concentration of alcohol was 14.2% (v:v) for the four lowest dilutions, and 7.8% (v:v) for the two higher dilutions. Due to their long-term instability in suspension, aldehyde solutions were kept in airtight containers, at room temperature, in the dark, for no longer than 4 h. Note that aldehyde was further diluted by ten-fold when used in the luciferase assays (see below), i.e. 10 ~1 aldehyde solution was added to 90 ~1 MEM in a sample well. For P-galactosidase assays, the fluorescent compound fluorescein-di-galacto-pyranoside (FDG) and the P-galactosidase inhibitor phenylethyl-thio-galactoside (PETG) were purchased from Molecular Probes (Eugene, OR). FDG and PETG were initially dissolved in dimethyl sulfoxide (DMSO), and stored at a concentration of 50 mM; with the final concentration of DMSO at 25% (v:v). Chloramphenicol (for plasmid selection) [25] was used at a final concentration of 30 pg/ml; spectinomycin (for plasmid amplification) [26] was used at a final concentration of 100 pg/ml; gentamicin (for invasion assays) [27] was used at a final concentration of 100 pug/ml. All antibiotics were purchased from Sigma. Triton X-100 was purchased from BDH Inc. (Toronto, ON) and made up as a 1% solution in PBS. T4 DNA ligase and restriction enzymes were purchased from Boehringer Mannheim (Laval, Pa). X-ray film X-OMAT was purchased from Eastman Kodak Company (Rochester, NY). 2.2. Bacterial strains Escherichia

plasmids

for

coli DHSa

the

was used to propagate development of pSPLUX.

157

Modified plasmids isolated from DHSa were electroporated into competent wild type S. typhimurium SL1344 [28] and S. dublin LD842 (plasmid-cured strain of S. dublin Lane) [29, 301 using a Gene PulserTM from BioRad (Richmond, CA). Transformed SL1344 and LD842 were then used in reporter assays. 2.3. Tissue culture cells Throughout this paper, the term ‘cells’ will specifically refer to eukaryotic cells, unless modified by the word bacterial. Epithelial-like MadinDarby canine kidney (MDCK) cells were grown in MEM supplemented with 10% FBS, at 37°C in an atmosphere of 95% air-5% CO,. For pgalactosidase assays, cells were grown in Microtest III tissue culture plates (96-well, flat bottom, sterilized by gamma irradiation; Falcon, Becton Dickinson, Lincoln Park, NJ). For luciferase assays, cells were grown in Immunoware 8-Well EIA strip plates (96-well microtiter plates with grids, flat bottom; Pierce, Rockford, IL). Immunoware plates were sterilized with 70% ethanol and allowed to dry in a Type II, HEPAfiltered biological safety hood prior to seeding with MDCK cells. 2.4. Plasmids The plasmid pFF14 containing an spvB-facZ translational fusion was previously described by Fang et al. [29]. The plasmid pSPLUX was made by inserting a 1uxAB gene cassette [31] into the BamHI site of pFF14, as shown in Fig. 1, to create a transcriptional fusion between spvB and &A. Both pFF14 and pSPLUX are low copy number plasmids derived from pACYC184 [26]. 2.5. Invasion assay All assays were performed in triplicate. Invasion assays were done according to the method described by Tang et al. [27], with minor modifications. Briefly, 1 ml of MEM + 10% FBS was inoculated with Salmonella previously grown on LB agar plates, and cultures were incubated overnight, not shaking, at 37”C, 5% CO,. The

C.G. Pfeifer, B.B.

Finlay I Journal of Microbiological

Methods 24 (1995) 155-164

PBS and viable bacterial above.

counts performed

as

2.6. P-Galactosidase assay

Fig. 1. Plasmid maps of pFF14 and pSPLUX. The 11.3 kbp plasmid pFF14 which contains spvR, spvA, and a translational spvB-ZacZ fusion, as shown by the circular map, was used for the P-galactosidase studies. The plasmid pSPLUX was made by inserting a 3.25 kb promoterless luxAB gene cassette into the BamHI site between the spvB and 1acZ genes, thus placing luxAB under the transcriptional control of the spvB gene. The plasmid pSPLUX was used for the luciferase studies.

P-Galactosidase assays were carried out in a similar manner to the invasion assays except that instead of treating cells with Triton X-100, the cells and bacteria were lysed with 10 ~1 0.1% SDS and 10 ~1 chloroform [4]. The resulting lysates were then incubated with the substrate FDG (1 mM final concentration) for 1 h. The reaction was stopped with the inhibitor PETG (1 mM final concentration), and the resulting fluorescence (excitation A = 480 nm; emission h = 535 nm) was measured with a Pandex fluorometer (IDEXX; Portland, OR) [4]. Extra wells for viable counts were run alongside the p-galactosidase assay wells, and the viable bacterial counts were performed as described for the invasion assay. Fluorescence was then correlated with viable counts to calculate activity as fluorescent units per bacterium per hour. 2.7. Luciferase assay

next day, bacteria were washed with PBS and added to MDCK cells at a bacteria: cell ratio of approximately 10 to 1. Bacteria were not opsonized. After 2 h, the media containing the extracellular Salmonella was removed. Viable counts of these extracellular bacteria were performed by counting bacterial colonies that formed within 24 h on LB agar plates containing no antibiotics. Serial dilutions for bacterial counts were made using PBS. The infected MDCK cells were washed three times with PBS to remove any traces of the supernatant, and 100 ~1 tissue culture media containing gentamicin was overlaid on the cells to kill any remaining extracellular bacteria. After incubation for an additional 2 h, the antibiotic-containing media was washed off. The infected epithelial cells were then lysed with 20 ~1 1.0% Triton X-100 for 10 min at room temperature; (bacteria are resistant to lysis with this non-ionic detergent). The well volumes were made up to 100 ~1 with

Luciferase assays were performed in a similar manner to the invasion assays, but with additional steps before Triton X-100 treatment. After the gentamicin was removed, 90 ~1 of MEM + 10% FBS was added to each of the microtiter plate wells. Ten microlitres of the various aldehyde dilutions was then added directly to each well and mixed by pipetting for 5 s. The resulting light production was measured using a Luminograph LB980 photon-imaging video system (EG&G Berthold; Germany) within 25-30 s of aldehyde addition, for a period of 3-5 min. Cells and bacteria remained intact during this time. Once light production was measured, the infected cells were then treated with Triton X-100 and viable bacterial counts performed as described for the invasion assay. Light production was then correlated to the resulting viable counts and luciferase activity calculated as photons per bacterium per second. Light production was also visualized using X-ray film, and the results were

C.G. Pfeifer, B.B.

quantitated (Molecular

Finlay I Journal of Microbiological

using a computing Dynamics; Sunnyvale,

densitometer CA).

3. Results 3.1. Plasmids pFF14

and pSPLUX

To directly compare the efficiency of ZuxAB and ZacZ gene fusions, we constructed isogenic reporter fusion plasmids. The spvB gene has previously been shown to be regulated by SpvR and to be induced inside mammalian cells [7,29]. Therefore, we started with the plasmid pFF14 (11.2 kb), which contains a promoterless la& gene translationally fused to spvB, to make the plasmid pSPLUX. The plasmid pSPLUX (14.5 kb) was constructed by ligating a promoterless 3.25 kb fuxAB gene cassette [31] into the BamHI site at the 5’ end of ZacZ, as shown in Fig. 1. Thus pSPLUX contains a transcriptional fusion between spvB and the 1uxAB reporter gene cassette. 3.2. Measurement

of light production

The sensitivity and linearity of different lightdetection methods was analyzed by comparing the amount of light detected to viable bacterial counts (results not shown). Using the Luminograph LB980, light production from bacteria was determined to be linear over a 10,000-fold range. However, with X-ray film, light production was only linear over a lo-fold range as determined by densitometry scans, and the sensitivity was about 5-fold lower than with the Luminograph LB980. Therefore, the Luminograph LB980 was used to quantitate luciferase activity. 3.3. The effects of aldehyde concentration on bacteria and luciferase activity Bacterial luciferase activity requires the binding of a reduced flavin mononucleotide (FMNH, - a bacterial energy source), oxygen, and a long chain aldehyde, resulting in the production of blue-green light (490 nm) [ll-131. When live bacteria containing the 1uxAB gene cassette

Methods 24 (1995) 155-164

1.59

(from K harveyi) are used, only the aldehyde substrate must be added exogenously. However, high aldehyde concentrations are inhibitory to light production by V. harveyi luciferase [32,33]. A range of concentrations was prepared to determine the optimal aldehyde concentration needed to measure light output from both intracellular and extracellular Salmonella. The final concentration of n-decanal used in the assay wells containing both intracellular and extracellular bacteria ranged from 0.00022% to 0.0099%. A low background level of light existed (from static electricity, dark noise from the camera, etc.) of about 2 X lo3 photons per well per second, which was subtracted in the calculations. The data in Fig. 2 show the duplicates of one representative experiment using S. dublin LD842 pSPLUX; the results using S. typhimurium SL1344 pSPLUX were similar. In Fig. 2A, a decrease in total light output from the wells was seen for the two highest concentrations of n-decanal (0.00495% and 0.0099%). Further investigation revealed that these concentrations of aldehyde were toxic to bacteria (Fig. 2B). Viable counts (determined as colony forming units, CFU) were reduced by 5 to 10 fold in the presence of the two highest concentrations of aldehyde, but CFUs were unaffected for aldehyde concentrations of 0.0022% or less. The toxic concentrations were similar for both intracellular and extracellular bacteria. However, the drop in the number of viable bacteria was greater than the drop in light production. Therefore, the light output per viable bacterium (Fig. 2C) appears to rise as the aldehyde concentration increases. In subsequent experiments a final aldehyde concentration of 0.0022% was used, since this concentration produced the highest amount of light without a reduction in bacterial numbers. At this aldehyde concentration, light production was linear, such that when bacteria were diluted, the resulting light output was similarly reduced (data not shown). Gentamicin-killed bacteria did not produce light (data not shown). Trypan blue exclusion studies [34] were performed to determine the extent of tissue culture cell death that occurred (data not shown). None of the aldehyde concentrations

C.G. Ffeifer,

B.B.

Finlay I Journal of Microbiological

.ool

.OOOl

.Ol

107 B

g----e--_+,~,

\

Methods 24 (1995) 1.55-164

Fig. 3. Detection of light production in Salmonella pSPLUX in an intracellular versus an extracellular environment. MDCK cells were infected with either S. typhimurium without (SL1344) or with the luxAB genes (SL1344 pSPLUX), or S. dublin with 1uxAB (LD842 pSPLUX), as described in the methods. After 2 h, extracellular bacteria were removed and placed into wells containing no MDCK cells. These wells were adjusted to contain the same number of bacteria as the wells containing intracellular bacteria. No light was produced in the absence of luciferase, although a low level of light was detected from the extracellular bacteria containing the plasmid pSPLUX. Increased light production can clearly be seen by intracellular bacteria containing the plasmid pSPLUX, indicating an increase in expression of the spvB-luxAB transcriptional fusion. This figure was obtained using the Luminograph LB980.

appeared to be toxic to the mammalian cells, even over extended periods of co-incubation (up to 1 h). 3.4. Detection of induction of IuxAB transcriptional fusions from within host cell environment

Visualization of the induction of the spvRAB-

.OOl

DO01 Final Akiehyde

Concentration (%;

.Ol

Fig. 2. Effect of aldehyde concentration on bacterial viability and light production. MDCK cells were grown to confluency in 96-well microtiter plates and infected with Salmonella as stated in the methods. Bacteria remaining with the cell monolayer after washing and gentamicin treatment were termed intracellular. whereas bacteria removed with the supernatant before washing were termed extracellular. This figure depicts the individual data points from one experiment. The lines represent the means of the data points. (A) Light production per 100 pl well. (B) Viable bacterial counts per 100 ~1 well. (C) Light production per bacterium per second. Closed symbols: intracellular bacteria; open symbols: extracellular bacteria. The use of higher concentrations of aldehyde (>0.00495%) resulted in less light production by the bacteria as shown in (A), but it also reduced the number of viable bacteria present in each well (B). The concentration of aldehyde needed to get efficient light output by bacteria without reducing viable counts was 0.0022%.

C.G. Pfeifer, B.B.

Finlay I Journal of Microbiological

1uxAB transcriptional

fusions is shown in Fig. 3. For purposes of this picture only, the number of bacteria in the medium alone has been diluted (using MEM + 10% FBS) such that the number of extracellular bacteria was equivalent to the number of intracellular organisms, since the number of intracellular Salmonella is only about 1% of those which remain extracellular. Otherwise, the extracellular bacteria were not diluted before assaying reporter activity. Wells containing S. typhimurium (SL1344) without the 1uxAB fusions did not produce light above the background static level mentioned previously. No light was produced from any of the other luxABminus clones: SL1344 pFF14; S. dublin (LD842); or LD842 pFF14 (data not shown). Wells containing extracellular Salmonella with the pSPLUX plasmid also did not produce significant quantities of light, although it was faintly detectable over the background level. However, intracellular Sulmonellu harboring the pSPLUX plasmid expressed the spvB-1uxAB transcriptional fusion and the amount of light production increased accordingly. 3.5. Comparison

of P-guluctosiduse and luciferuse us reporters of intracellular bacterial gene expression

Expression of the spv operon was determined by comparing the reporter enzyme activity of extracellular bacteria to that of intracellular bacteria. Activities were first normalized to ac-

161

Methods 24 (1995) 155-164

tivity per bacterium for each respective enzyme, and then the intracellular activity was divided by the extracellular activity to give the relative increase. The increase in luciferase activity was similar to the increase in /3-galactosidase activity (24 versus 22 fold), as shown in Table 1. The sensitivity of both enzymes was essentially equal. For both assays, the lower detection limit of spvB expression was approximately 5 X 104-1 X lo5 for extracellular bacteria (where spvB is repressed) and 1 X lo3 for intracellular bacteria (where spvB is induced). It was previously shown [28] that this induction of spv gene expression was not the result of an increased plasmid copy number within the intracellular bacteria.

4. Discussion Other studies have used bacterial luciferases as reporters of intracellular bacterial gene expression, however, the conditions of the studies were not physiological for mammalian cells. Langridge et al. [lo] showed that gene activation during plant-microbe interactions could be monitored, under conditions optimal for plant growth. Francis and Gallagher [9] demonstrated the induction of hydrogen peroxide-stimulated genes in S. typhimurium upon interaction with mouse macrophages. However, the luciferase used was from V. fischeri and was inactivated above 30°C [9,11,35]. This was of concern since temperature

Table 1 Induction of expression of reporter enzyme fusions by intracellular S. dublin Reporter enzyme

Bacterial locationa

Specific activityb

P-Galactosidase

Extracellular Intracellular Extracellular Intracellular

0.0084 2 0.1872? 0.0157 2 0.3820 k

Luciferase

0.0013 0.1740 0.0150 0.1300

Relative increase’

22 24

a Extracellular location refers to the media environment surrounding MDCK cells. Intracellular location refers to the environment surrounding bacteria inside MDCK cells. b Units for specific enzyme activity. P-galactosidase units are expressed as FDG fluorescence/bacterium/hour; luciferase units are expressed as photons/bacterium/second. Error was determined using sample standard deviation. ‘Relative increase for each enzyme is the specific activity of intracellular bacteria divided by specific activity of extracellular bacteria.

162

C.G. Pfeifer, B.B.

Finlay I Journal of Microbiological

is the basis for induction of numerous virulenceassociated genes over a broad range of pathological bacteria [5,6,36,37], with many virulence factors optimally expressed at 37°C. The lcucAB gene cassette that we used encodes for a heterodimeric luciferase from V. harveyi, which remains active at 37°C [11,35,38]. Both bacteria and tissue culture cells were grown and assayed at physiological temperatures (37°C)) with minimal disruption to interactions occurring between the intracellular bacteria and the mammalian host cells. Although an alternative luciferase from Xenorhabdis Zuminescens remains thermostable up to 45°C [12,38], it has a lower specific activity than V. harveyi luciferase [38]. As well, V. harveyi 1uxAB genes were contained within a convenient 3.25 kb BumHI gene cassette which did not contain its own endogenous promoter. Not included within this cassette were the aldehyde biosynthetic genes luxCDE, encoded by an extra 4 kb DNA, While the presence of substrate synthesizing genes may appear to be an advantage, a fusion with high expression promoters would result in overproduction of aldehyde, and increased bacterial mortality. Even though the aldehyde substrate must be exogenously supplied when the aldehyde synthesis genes are not present, studies have indicated that recombinant Zux products exhibit high activity in the presence of externally-added decyl aldehyde [12,38]. We further addressed the effects of aldehyde concentration on both bacteria and MDCK cells. While Langridge et al. [lo] found that vapors from high decanal concentrations (10% or more) ‘resulted in increased levels of mortality among young plantlets’, they did not determine the effect of n-decanal on bacteria. Our results indicated that none of the aldehyde concentrations we used killed the mammalian cells, but we found that higher aidehyde concentrations (>0.0022%) were toxic to the bacteria (Fig. 2B). It is possible that the aldehyde has a direct toxic effect on Salmonella. We also found that the drop in numbers of colony-forming bacteria was greater than the drop in total light output per well (Fig. 2A,B), indicating that higher aldehyde concentrations elicited more light production from the remaining viable bacteria (Fig. 2C). It

Methods 24 (1995) 155-164

may be that the concentrations of aldehyde we tested did not reach the substrate saturation range of the luciferase enzyme. The use of higher amounts of aldehyde would probably result in more efficient activity of the enzyme, but at the same time would reduce bacterial viability. As the mechanism of aldehyde toxicity remains unknown at this time, we decided to use the concentration of aldehyde (0.0022%) which permitted high light production by luciferase without concomitant bacterial death. We would further like to address some recent findings concerning the use of bacterial luciferase as a reporter of gene expression. An article by Forsberg et al. [39] reported that an intrinsically curved segment of DNA in the 5’ coding end of the luxA gene may influence promoter activity of the target gene. We found that our luciferase data showed the same amount of activity as the previously established p-galactosidase data, which indicated that the 5’ end of the LuxA gene did not interfere with spv regulation. Likewise, an article by Gonzalez-Flecha and Demple [40] linked luciferase activity (in the absence of ndecanal) with an increase in oxidative stress to bacteria. However, it has been previously shown that the spv operon is not influenced by the redox state of the bacteria [29]. We therefore concluded that both the IuxAB genes and the luciferase enzyme activity did not interfere with spvB gene expression. An assay based on the use of living organisms often results in greater variability than found in a controlled in vitro situation. It was suggested by Meighen [12] that changes in intensity of luminescence in vivo may depend not only on the amount of functional luciferase available, but also on the availability of substrates (FMNH,, aldehyde, and 0,). This implies that luciferase would be an inaccurate reporter in situations where either oxygen or energy was lacking. We found that the environment that Salmonella encounters upon invasion of MDCK cells contains enough oxygen to support luciferase activity (Figs. 2 and 3; and Table 1). As well, intracellular Salmonella remain viable (not energy depleted), providing the aldehyde concentration is not too high. These results indicate

C.G. Pfeifer, B.B.

Finlay I Journal of Microbiological

that use of luciferase for future Safmoneffa studies will not be inhibited by either the lack of oxygen or bacterial energy available to the intracellular bacteria. Furthermore, Francis and Gallagher [9] showed that the luciferase enzyme was variably expressed within infected cells suggesting that the number of bacteria per cell or the intracellular environment may fluctuate significantly between individual cells or within a cellular subpopulation. Along a similar line, Abshire and Neidhardt [41] demonstrated that there may actually be two populations of Salmonella that exist within cells, one static and the other rapidly dividing. These variables together may account for the variation in Table 1. No reporter system is ideal for all situations and each system has its advantages and disadvantages. In this study, the use of luciferase as a reporter of intracellular bacterial gene expression was assessed using conditions optimal for Salmonella invasion of non-phagocytic mammalian cells. We demonstrated that bacterial luciferase, encoded by promoterless 1uxAB genes from I/. harveyi, provided an alternative reporter system to P-galactosidase, with several advantages. We showed that luciferase is an accurate and sensitive reporter of intracellular Salmonella spv gene expression in that it confirmed data from a previous study using P-galactosidase [7]. Moreover, the luciferase assay was faster and easier to perform. Sample activity was measured immediately after substrate addition. Since bacterial lysis was not needed to detect luminescence, it was possible to determine bacterial gene expression while the bacteria were still within the intracellular environment. Moreover, the number of bacteria could be determined from the same well from which luciferase was assayed. Another advantage of using luciferase was the absence of background activity from the host cells, whereas within MDCK cells, low levels of &galactosidase activity could be detected. Mammalian cells and bacteria have no endogenous luciferase activity, and any light detected was a direct result of the IuxAB constructs. The ability to monitor luciferase activity without disruption to either the bacteria or the cells also suggests the possibility of monitoring

Methodc 24 (1995) 155-164

163

bacterial-cell interactions over time. Furthermore, the product (light) does not build up and low dose applications of the aldehyde substrate were found to be non-toxic to both bacteria and tissue culture cells. Collectively, the results demonstrated that luciferase gene fusions are an easy and sensitive way to monitor gene expression of bacterial pathogens that reside within mammalian host cells.

Acknowledgements This work was made possible through an operating grant from the Medical Research Council of Canada. We thank Murry Stein and Annette Siebers for their critical reading of the manuscript.

References [II Fields, P.I., Swanson, R.V., Haidaris, C.G. and Heffron, F. (1986) Mutants of Salmonella typhimurium that cannot survive within the macrophage are avirulent. Proc. Natl. Acad. Sci. USA 83, 5189-5193. PI Finlay, B.B. and Falkow, S. (1989) Salmonella as an intracellular parasite. Mol. Microbial. 3, 1833-1841. [31 Leung, K.Y. and Finlay, B.B. (1991) Intracellular replication is essential for the virulence of Salmonella typhimurium. Proc. Nat]. Acad. Sci. USA 88, 1147011474. [41 Garcia del-Portillo, F., Foster, J.W., Maguire, M.E. and Finlay, B.B. (1992) Characterization of the micro-environment of Salmonella typhimurium-containing vacuoles within MDCK epithelial cells. Mol. Microbial. 6, 3289-3297. PI Abshire, K.Z. and Neidhardt, F.C. (1993) Analysis of proteins synthesized by Salmonella typhimurium during growth within a host macrophage. J. Bacterial. 175, 3734-3743. PI Buchmeier, N.A. and Heffron, F. (1990) Induction of Salmonella stress proteins upon infection of macrophages. Science 248, 730-732. [71 Fierer, J., Eckmann, L., Fang, F., Pfeifer, C., Finlay, B.B. and Guiney, D. (1993) Expression of the Salmonella virulence plasmid gene spvB in cultured macrophages and nonphagocytic cells. Infect. Immun. 61, 5231-5236. PI Mahan, M.J., Slauch, J.M. and Mekalanos, J.J. (1993) Selection of bacterial virulence genes that are specifically induced in host tissues. Science 259, 686-688.

164

C.G. Pfeifer, B.B.

Finlay I Journal of Microbiological

[9] Francis, K.P. and Gallagher, M.P. (1993) Light emission from a mulux transcriptional fusion in Salmonella typhimurium is stimulated by hydrogen peroxide and by

interaction with the mouse macrophage cell line 5774.2. Infect. Immun. 61, 640-649. [lo] Langridge, W., Escher, A., Wang, G., Ayre, B., Fodor, I. and Szalay, A. (1994) Low-light image analysis of transgenic organisms using bacterial luciferase as a marker. J. Biolumin. Chemilumin. 9, 185-200. [ll] Hill, P.J., Rees, C.E.D., Winson, M.K. and Stewart, G.S.A.B. (1993) Review: the application of lux genes. Biotechnol. Appl. Biochem. 17, 3-14. [12] Meighen, E.A. (1991) Molecular biology of bacterial bioluminescence. Microbial. Rev. 55, 123-142. [13] Schauer, A.T. (1988) Visualizing gene expression with luciferase fusions. TIBTECH 6, 23-27. [14] Park, S.F., Stewart, G.S.A.B. and Kroll, R.G. (1992) The use of bacterial luciferase for monitoring the environmental regulation of expression of genes encoding virulence factors in Listeria monocytogenes. J. Gen. Microbial. 138, 2619-2627. [15] Peabody, D.S., Andrews, C.L., Escudero, K.W., Devine, J.H., Baldwin. T.O. and Bear, D.G. (1989) A plasmid vector and quantitative techniques for the study of transcription termination in Escherichia coli using bacterial luciferase. Gene 75, 289-296. (161 Silhavy, T.J. and Beckwith, J.R. (1985) Uses of lac fusions for the study of biological problems. Microbial. Rev. 49, 398-418. [17] Bulich, A.A., Tung, K.K. and Scheibner, G. (1990) The luminescent bacteria toxicity test: its potential as an in vitro alternative. J. Biolumin. Chemilumin. 5, 71-77. [18] Jassim, S.A.A., Ellison, A., Denyer, S.P. and Stewart, G.S.A.B. (1990) In vivo bioluminescence: a cellular reporter for research and industry. J. Biolumin. Chemlumin. 5, 115-122. [19] Kodikara, C.P., Crew, H.H. and Stewart, G.S.A.B. (1991) Near on-line detection of enteric bacteria using lux recombinant bacteriophage. FEMS Microbial. Lett. 83, 261-266. [ZO] Korpela, M., Mantsala, P., Lilius, E.M. and Karp, M. (1989) Stable-light-emitting Escherichia coli as a biosensor. J. Biolumin. Chemilumin. 4, 551-554. [21] Stewart, G.S.A.B. and Williams, P. (1992) lux genes and the applications of bacterial bioluminescence. J. Gen. Microbial. 138, 1289-1300. [22] Van Dyk, T.K., Majarian, W.R., Konstantinov, K.B., Young, R.M., Dhurjati, P.S. and LaRossa, R.A. (1994) Rapid and sensitive pollutant detection by induction of heat shock gene-bioluminescence gene fusions. Appl. Environ. Microbial. 60, 1414-1420. 1231 Walk, C.P., Cai, Y. and Panoff, J.M. (1991) Use of a transposon with luciferase as a reporter to identify environmentally responsive genes in a cyanobacterium. Proc. Natl. Acad. Sci. USA 88, 5355-5359. [24] Rhen, M., Riikonen, P. and Taira, S. (1993) Transcriptional regulation of Salmonella enterica virulence plasmid genes in cultured macrophages. Mol. Microbial. 10, 45-56.

Methods 24 (1995) 155-164

[25] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular cloning: A laboratory manual, second edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pp.1.21-1.32. [26] Chang, A.C.Y. and Cohen, S.N. (1978) Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J. Bacterial. 134, 1141-1156. [27] Tang, P.. Foubister, V., Pucciarelli, M.G. and Finlay, B.B. (1993) Methods to study bacterial invasion. J. Microbial. Methods 18, 227-240. [28] Hoiseth, S.K. and Stocker, B.A.D. (1981) Aromaticdependent Salmonella typhimurium are non-virulent and effective as live vaccines. Nature 291, 238-239. (291 Fang, F.C., Krause, M., Roudier, C., Fierer, J. and Guiney, D.G. (1991) Growth regulation of a Salmonella plasmid gene essential for virulence. J. Bacterial. 173, 6783-6789. [30] Chikami. G.K., Fierer, J. and Guiney, D.G. (1985) Plasmid-mediated virulence in Salmonella dublin demonstrated by use of a TnS-oriT construct. Infect. Immun. 50, 420-424. [31] Guzzo, A. and DuBow. M.S. (1991) Construction of stable, singie-copy luciferase gene fusions in Escherichia coli. Arch. Microbial. 156, 444-448. [32] Fransisco. W.A.. Abu-Soud, H.M., Baldwin, T.O. and Raushel, F.M. (1993) Interaction of bacterial luciferase with aldehyde substrates and inhibitors. J. Biol. Chem. 268, 24734-24741. [33] Lei, B., Cho, K.W. and Tu, S.C. (1994) Mechanism of aldehyde inhibition of Vibrio harveyi luciferase: identification of two aldehyde sites and relationship between aldehyde and flavin binding. J. Biol. Chem. 269, 56125618. [34] Jakoby, W.B. and Pastan, I.H. (1979) Cell Culture Methods in Enzymology Volume LVIII. Academic Press, Toronto, ON, pp.150-152. [35] Engebrecht, J.. Simon, M. and Silverman, M. (1985) Measuring gene expression with light. Science 227, 1345-1347. [36] Finlay, B.B. and Falkow, S. (1989) Common themes in microbial pathogenicity. Microbial. Rev. 53, 210-230. [37] Mekalanos, J.J. (1992) Minireview - Environmental signals controlling expression of virulence determinants in bacteria. J. Bacterial. 174, 1-7. [38] Szittner, R. and Meighen, E. (1990) Nucleotide sequence, expression, and properties of luciferase coded by lux genes from a terrestrial bacterium. J. Biol. Chem. 265, 16581-16587. [39] Forsberg, A.J., Pavitt, G.D. and Higgins, C.F. (1994) Use of transcriptional fusions to monitor gene expression: a cautionary tale. J. Bacterial. 176, 21282132. [40] Gonzalez-Flecha, B. and Demple, B. (1994) Intracellular generation of superoxide as a by-product of Vibrio harveyi luciferase expressed in Escherichia coli. J. Bacterial. 176, 2293-2299. [41] Abshire, K.Z. and Neidhardt, F.C. (1993) Growth rate paradox of Salmonella typhimurium with host macrophages. J. Bacterial. 175, 3744-3748.