Unité de Biochimie Microbienne, CNRS (URA2172), Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris cedex, France1
Unité de Lutte Biologique, INRA, La Minière, 78285 Guyancourt cedex, France2
Centre dEtudes du Bouchet, Laboratoire de Microbiologie, 91710 Vert-Le-Petit, France3
Laboratoire de Pathologie Comparée, INRA-CNRS (URA 2209), Université Montpellier II, 34095 Montpellier, France4
Laboratoire de Biologie, HIA Bégin, 94160 Saint Mandé, France5
Author for correspondence: Didier Lereclus. Tel: +33 1 45 68 88 13. Fax: +33 1 45 68 89 38. e-mail: lereclus{at}pasteur.fr
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ABSTRACT |
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Keywords: Bacillus, cytolysin, haemolysin, insect pathogen, plcR regulon
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INTRODUCTION |
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B. thuringiensis and B. cereus cells produce several extracellular degradative enzymes such as phospholipases C, enterotoxins and haemolysins, which are putative virulence factors (Beecher et al., 1995a ; Drobniewski, 1993
). A transcriptional activator that positively regulates the expression of phospholipase C genes, during the late vegetative growth of B. thuringiensis cells, has been identified and named PlcR (Lereclus et al., 1996
). This regulator governs the expression of a large regulon encoding extracellular proteins including degradative enzymes, cell-surface proteins and enterotoxins (Agaisse et al., 1999
). Hence, PlcR appears to be a pleiotropic regulator of genes encoding extracellular factors potentially involved in pathogenicity. In this paper, we report experiments with two animal models (a lepidopteran insect, Galleria mellonella, and a mammal, BALB/c mice) to evaluate the opportunistic properties of B. thuringiensis and B. cereus. In both models, the pathogenicity of B. thuringiensis and B. cereus was similar and was highly reduced or abolished by the disruption of the plcR gene.
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METHODS |
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For nasal instillation in mice, the vegetative cells were prepared from cultures in Tryptic Soy Broth (Difco) at 37 °C. The cells were recovered after culture for 18 h (late stationary phase) by centrifugation at 7000 g for 15 min. The pellets were washed and suspended in PBS (phosphate-buffered saline, pH 7·2; Sigma). Spore suspensions used for nasal instillation were prepared from a 10-d-old culture on agar medium containing 10 g yeast extract l-1, 5 g NaCl l-1 and 20 g agar l-1. Spores were washed and suspended in sterile water, and incubated for 1 h at 65 °C to kill the vegetative forms.
Spores and vegetative cells were counted before bioassays by plating serial dilutions on LB agar plates. Spore preparations could be stored at 4 °C for 10 d; vegetative cells were prepared for each experiment.
The B. thuringiensis 407 Cry- spo0A mutant strain has been described elsewhere (Lereclus et al., 1995
). This strain is an asporogenic acrystalliferous mutant. pHTF3-1C, carrying the cry1C gene fused to the cry3A promoter region, was introduced into this strain, resulting in the production of Cry1C crystals in a Spo- background (Sanchis et al., 1996
). The resulting 407 Cry- [
spo0A, pHTF3-1C] strain was designated 407 Cry- 0A-1C. Cry1C toxins were prepared from the 407 Cry- 0A-1C strain grown in LB medium at 30 °C for 4 d. The culture was centrifuged at 10410 g for 20 min and the pellet was washed twice in sterile distilled water (2x100 ml). It was resuspended in 10 ml sterile distilled water. The Cry1C crystal preparation was briefly sonicated before use. Crystal protein was determined with the Bio-Rad protein assay procedure (Bradford), using bovine serum albumin as the standard.
For evaluation of the cytolytic activity of the culture supernatants, B. thuringiensis strains were grown at 30 °C in LB medium for the tests with insect haemocytes or in Tryptic Soy Broth (Difco) for the tests with human erythrocytes. The culture supernatants were recovered 2 h after the end of the exponential-growth phase. Cells were pelleted by centrifugation (10410 g for 10 min) and the supernatants were sterilized by filtration using filters with 0·22 µm pores (Sartorius).
In vivo experiments.
G. mellonella eggs were hatched at 30 °C and the larvae reared on beeswax and pollen (Naturalim). Last instar larvae (150350 mg) were force-fed using 0·5x25 mm needles (Burkard Manufacturing) and a microinjector (Automatic Microapplicator; Burkard Manufacturing) with spore/crystal suspensions in sterile water (10 µl per larva). Thirty larvae were used for each dose. Infection of G. mellonella larvae (last instar) by intrahaemocoelic injection was performed as follows. Groups of 30 larvae were injected at the base of last proleg with 10 µl spore preparation using the Burkard microinjector with a 1 ml hypodermic syringe and 0·45x12 mm needles (Terumo). After force-feeding and injection experiments, the larvae were kept individually in boxes containing beeswax and pollen at 25 °C. They were checked daily and casualties were recorded over 7 d. For injection experiments, mortality data were analysed using the log-probit program of Raymond et al. (1993 ). This program tests the linearity of dosemortality curves, provides lethal doses (LD50) and the slope of each dosemortality curve. Mortality curves were considered identical when their parallelism was not rejected at the 0·05 level and the 95% confidence limits of the susceptibility ratio included the value 1. To count B. thuringiensis cells in the living and dead insects, 10 larvae were crushed and homogenized in 10 ml sterile water; dilutions were plated onto LB agar plates containing appropriate antibiotics. The parental strain, B. thuringiensis 407 Cry-, is resistant to oxacillin (10 µg ml-1) and the mutant strain, B. thuringiensis 407 Cry-
plcR, is resistant to oxacillin and kanamycin (250 µg ml-1).
We used 5-week-old female BALB/c mice (Charles River) kept in a biosafety containment facility in groups of five, with sterile water and food. For each strain, groups of 510 mice were infected intranasally under slight ether anaesthesia with 50 µl of the appropriate suspension. The spore or bacterial suspension was carefully deposited at the corner of the nostril. The mouse inhaled the inoculum naturally by breathing, so there was therefore no mechanical traumatism.
Construction of mutant strains.
To obtain the plcR strains, the B1 insert cloned in pHT304 (Lereclus et al., 1996
) was inserted (SmaI/HindIII) into the pKS vector. The resulting plasmid isolated from E. coli transformants was designated pKS
B1. The KmR cassette of pDG783, conferring resistance to kanamycin (Km, 250 µg ml-1), was isolated as a 1·5 kb EcoRI fragment carrying the aphA3 gene from Enterococcus faecalis (Trieu-Cuot & Courvalin, 1983
). This KmR cassette was then inserted into pKS
B1, between the internal EcoRI sites of plcR, yielding pKS
B1KmR. The disrupted plcR gene was then inserted between the BamHI and HindIII sites of the thermosensitive plasmid pRN5101, conferring resistance to erythromycin (Em, 5 µg ml-1). The resulting plasmid, pRNB1KmR, was introduced into B. thuringiensis and B. cereus by electroporation (Lereclus et al., 1989
) and the chromosomal wild-type copy of plcR was replaced by the disrupted copy as previously described (Lereclus et al., 1995
). Mutant colonies had an EmS, KmR phenotype. The genotype was checked by PCR.
Degradative enzyme activity measurements.
Columbia medium agar plates (BioMérieux) containing either 5% horse blood or 5% sheep blood, and 5% human blood agar plates (Sanofi Pasteur) were used to evaluate the haemolytic activity of the B. thuringiensis and B. cereus strains. Tests for cytotoxic activity were performed as previously described (Ribeiro et al., 1999 ). Briefly, chilled 2-d-old last-instar larvae of G. mellonella were surface sterilized before collection of haemolymph in anticoagulant buffer (62 mM NaCl, 100 mM glucose, 10 mM EDTA, 30 mM trisodium citrate, 26 mM citric acid, pH 4·5). The haemolymph was centrifuged and the haemocyte pellet was rinsed and resuspended in PBS. Haemocyte suspension (20 µl) was layered on heat-sterilized (220 °C, 2 h) coverslips and haemocytes were allowed to spread and to adhere to the glass for 15 min in a wet chamber at room temperature. The monolayers were rinsed, 20 µl of the solution under study was added and the monolayers were incubated for 1 h at 23 °C for microscopy and counting of dead cells. Haemocyte mortality was checked by adding 2 µl Trypan Blue dye (0·4% in PBS) and incubating for a further 5 min.
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RESULTS AND DISCUSSION |
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A clear pattern of synergism was observed in analysis of the force-feeding assays (Fig. 1). Very low mortality levels (<10%) were obtained with crystals or spores alone. In contrast, mixing B. thuringiensis spores and crystals strongly increased mortality, demonstrating synergism (70% mortality). Killing the spores by autoclaving before force-feeding them to G. mellonella abolished synergism (not shown), suggesting that either heat-labile toxic components are associated with the spores or the spores germinate. As co-ingested crystals and vegetative cells of B. thuringiensis were also synergistic (47% mortality, not shown), the spores are almost certainly living and able to develop into vegetative cells. This is consistent with the increase in bacterial count observed on collection of the contents of the haemocoel from living larvae when, due to transit elimination, a decrease in spore count was expected (Fig. 2a
).
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We assessed the extent to which the PlcR-regulated genes are involved in pathogenicity by evaluating the entomopathogenic properties of B. thuringiensis 407 Cry- plcR and B. cereus
plcR mutant strains in in vivo experiments. Spores from these two mutant strains did not have a synergistic effect against the G. mellonella larvae (Fig. 1
): the lethality was similar to that of Cry1C crystals alone. On agar plates, the germination rate of the 407 Cry-
plcR strain did not appear to differ from that of the parental strain, thus excluding a major defect in germination. This is consistent with the co-infection experiments (see below and Fig. 2a
, b
), showing that the
plcR mutants are able to germinate in insect larvae.
The pathogenicity of the B. thuringiensis parental strain, 407 Cry-, and of the 407 Cry- plcR mutant was also assessed by introducing the spores into the larval haemocoel by injection. The mortality curves for the parental and mutant strains were not significantly different (parallelism not rejected:
2=0·68, d.f.=5, P>0·98; the ratio of the LD50 included the value 1). The LD50 (and the confidence intervals), determined 2 d after injection of the spores, were 1·7x104 (8·4x1033·7x104) for the parental strain, 407 Cry-, and 1·8x104 (8·5x1034·1x104) for the 407 Cry-
plcR mutant strain. In addition, mortality kinetics were similar for the two B. thuringiensis strains: in both cases, the larvae began to die 16 h after infection and mortality reached a plateau 2 d later. These results indicate that the plcR regulon is not required for pathogenicity if the spores are mechanically introduced into the haemocoel. Hence, PlcR appears to play a key role in the regulation of the synergistic properties of B. thuringiensis (and B. cereus) if spores enter the larvae via the digestive tract, suggesting a role in the early stages of infection (for example, by allowing the cells to gain access to the haemocoel from the gut).
The role of the plcR regulon in the entomopathogenicity of B. thuringiensis
PlcR-regulated genes encode proteins containing sequences similar to secretory signal peptides (Agaisse et al., 1999 ; Økstad et al., 1999
). Thus, the 407 Cry-
plcR strain may be less able to cause opportunistic infections due to its inability to produce one or several of these secreted factors. Thus, the parental strain may provide the mutant with extracellular factors, enabling it to multiply in co-infected larvae. To test this hypothesis, we first compared the fate of the B. thuringiensis parental strain, 407 Cry-, and of the 407 Cry-
plcR mutant strain in G. mellonella larvae, following mono-infection experiments with spore/crystal preparations (Fig. 2a
). At 24 h after infection, the two strains performed similarly with bacterial counts of about 9x106 c.f.u. larva-1 and 107 c.f.u. larva-1, respectively. Two days later, substantial multiplication (109 c.f.u. larva-1) had occurred in dead larvae infected with the parental strain. No dead larvae were obtained from the insects infected with the
plcR mutant. After 72 h, 2·4x106 c.f.u. larva-1 were isolated from living larvae infected with the parental strain and 3·7x105 c.f.u. larva-1 were isolated from larvae infected with 407 Cry-
plcR spores. This suggests that bacterial multiplication essentially occurs in the dead larvae. Thus, both the wild-type and mutant cells are slowly eliminated from larvae surviving the infection (compare bacterial counts from living larvae at 24 h and 72 h, in Fig. 2a
).
We next assessed the multiplication of the parental and mutant strains following co-infection with spore/crystal preparations. Larvae were fed with a 50% initial ratio of the two types of spores (Fig. 2b). At 24 h after ingestion, 5·1x106 of the recovered c.f.u. were of the 407 Cry-
plcR type and 1·5x106 were of the 407 Cry- type. The ratio (77% versus 23%) is significantly different from the expected 50% ratio (Students t-test; P<0·01). This may indicate that the
plcR mutant has a selective advantage during the first few hours of infection. The ratio significantly decreased over time (one-way ANOVA; P=0·0073) and similar amounts of both strains were recovered from dead larvae at 48 h and from living larvae at 72 h. These results show that the parental B. thuringiensis 407 Cry- strain may provide extracellular factors enabling the
plcR mutant to multiply in the infected larvae. Thus, the results for mono- and co-infections suggest two possible roles for PlcR: (1) the plcR regulon may be required to cause the death of the larvae so that the bacterial can multiply, or, conversely, (2) the plcR regulon may be required to create favourable conditions for bacterial multiplication, resulting in the death of the larvae by septicaemia.
Cytotoxic and haemolytic activities are controlled by PlcR
Previous studies have indicated a possible correlation between phospholipase activity and the entomopathogenic properties of B. thuringiensis (Heimpel, 1955 ; Krieg, 1971
; Stephens, 1952
). Two phosphatidylcholine esterases are known to be encoded by PlcR-regulated genes (Agaisse et al., 1999
). They are phospholipase C and sphingomyelinase, which together form cereolysin AB (Gilmore et al., 1989
). We checked that the 407 Cry-
plcR strain did not form a halo on lecithin-agar plates, consistent with the genetic data (results not shown). In addition, the gene encoding the haemolytic HblC component of the Hbl enterotoxin of B. thuringiensis and B. cereus is also regulated by PlcR (Agaisse et al., 1999
; Økstad et al., 1999
). As HblC is enterotoxigenic and haemolytic (Lindbäck et al., 1999
), and phospholipases C are known to degrade the phospholipid constituents of cell membranes, we investigated the activity of culture supernatants from the 407 Cry- parental strain and from the 407 Cry-
plcR mutant strain against G. mellonella haemocytes.
Four haemocyte types are present in the haemolymph of G. mellonella larvae. Two of these cell types (Fig. 3a and 3b
), plasmatocytes (Pl) and granular haemocytes 1 (GH1) (Brehélin & Zachary, 1986
), are directly involved in defence reactions such as phagocytosis. They account for more than 80% of the total haemocyte population. The supernatant of the parental 407 Cry- strain was strongly cytolytic to Pl and GH1 (Fig. 3c
). Both cell types lost their shape and were stained with Trypan Blue, indicating cell death on a large scale. The supernatant of the 407 Cry-
plcR mutant strain had a different activity: cells were clearly not killed but extensive vacuole formation was observed (Fig. 3d
). There was a highly significant difference in the number of dead cells between these two haemocyte preparations (Students t-test; P<10-17). Indeed, 99·0% of the cells were dead after incubation with supernatant from the parental strain, 407 Cry-, whereas only 5·3% of cells were dead after treatment with a supernatant from the 407 Cry-
plcR mutant. The vacuole formation observed with the supernatant of the 407 Cry-
plcR mutant was not dependent on PlcR-regulated functions and may have been due to other compounds, such as the B. cereus emetic toxins, which are known to cause vacuole formation in eukaryotic cells (Agata et al., 1995
). In contrast, sterile LB and the supernatant of a B. subtilis strain, used as controls, had no cytolytic effect on G. mellonella haemocytes (Fig. 3a
and 3b
).
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The opportunistic properties of B. thuringiensis and B. cereus in mice
Hernandez et al. (1998 , 1999
) recently showed that some B. thuringiensis strains are pathogenic to mice by nasal instillation. We first determined whether the B. thuringiensis 407 Cry- strain (subspecies thuringiensis) and B. cereus ATCC 14579 belonged to this group. Nasal instillation of 108 or 5x107 spores of the 407 Cry- strain and of the B. cereus strain per mouse rapidly resulted in high mortality rates (Table 1
). Mice died within 24 h, with syndromes similar to those described for the B. thuringiensis strain konkukian (Hernandez et al., 1999
). However, instillation of 107 spores per mouse in the same volume (50 µl) resulted in no deaths with 407 Cry- spores and a high mortality rate with the B. cereus strain, which suggests that the two strains differ in pathogenicity. Similar assays were performed with the Bacillus anthracis vaccine strain, Sterne, which lacks the virulence plasmid, pXO2. B. anthracis is a member of the B. cereus group, in which plcR is present in a truncated inactive form (Agaisse et al., 1999
). B. anthracis Sterne, instilled at the same concentration, did not kill mice (Table 1
). This indicates the existence in B. thuringiensis and B. cereus of specific opportunistic traits, not functional in a pX02- strain of B. anthracis (i.e. the plcR regulon). B. thuringiensis vegetative forms instilled in mice at a dose of 108 or 2x107 cells per mouse caused 100% mortality within 46 h, with haemorrhagic symptoms (nose bleeding and lungs clearly haemorrhagic on autopsy).
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Conclusions |
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Here we show that in specific infection conditions, mammals may act as alternative hosts for insect pathogens. This is reminiscent of the multi-host system of the opportunistic bacterium Pseudomonas aeruginosa (Finlay, 1999 ; Jander et al., 2000
; Rahme et al., 1997
; Tan et al., 1999
). Similarly, the B. thuringiensis/insect pathogenesis model can be used to determine the factors involved in the opportunistic properties of bacilli.
In practical terms, these results clearly show that B. thuringiensis and B. cereus are opportunistic pathogens. As with B. cereus, B. thuringiensis strains produce various food poisoning toxins (Granum & Lund, 1997 ) and a B. thuringiensis strain has been found associated with an outbreak of food poisoning (Jackson et al., 1995
). However, by comparison with chemical pesticides, B. thuringiensis is generally recognized as a safe product and there is no evidence to suggest that its use for insect control should be reduced. Nevertheless, more detailed knowledge of the pathogenicity of each commercial strain may be required to improve the safety of B. thuringiensis-based biopesticides. It is therefore important to identify the PlcR-regulated functions specifically responsible for the opportunistic properties of B. thuringiensis and B. cereus.
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ACKNOWLEDGEMENTS |
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Received 26 May 2000;
revised 28 July 2000;
accepted 1 August 2000.