Opinion
Bacillus thuringiensis: an impotent pathogen? Ben Raymond1,*, Paul R. Johnston2, Christina Nielsen-LeRoux3, Didier Lereclus3 and Neil Crickmore2 1
Department of Zoology, University of Oxford, South Parks Rd, Oxford, OX1 3PS, UK School of Life Sciences, University of Sussex, Falmer, Brighton, BN1 9QG, UK 3 INRA, Institut Micalis, La Minie`re, 78285 Guyancourt cedex, France 2
Bacillus thuringiensis (Bt) is an insecticidal bacterium that has successfully been used as a biopesticide for many years. It is usually referred to as a soil-dwelling organism, as a result of the prevalence of its spores in this environment, but one that can act as an opportunistic pathogen under appropriate conditions. Our understanding of the biology of this organism has been challenged further by the recent publication of two reports that claim that Bt requires the co-operation of commensal bacteria within the gut of a susceptible insect for its virulence. It is our opinion that Bt is not primarily a saprophyte and does not require the assistance of commensal bacteria but is a true pathogen in its own right and furthermore that its primary means of reproduction is in an insect cadaver. The ecology of Bacillus thuringiensis Spores of Bacillus thuringiensis (Bt) can be isolated from diverse environments such as soil, the rhizosphere, the phylloplane, fresh water, grain dusts and from insects, crustaceans, annelids and insectivorous mammals (Figure 1). Although there is evidence that the bacterium is present in a vegetative form in many of these environments, it is unclear whether these vegetative cells are contributing to significant rates of reproduction in all these cases [1,2]. Strains of Bt such as the kurstaki HD1 strain present in many Bt-based biopesticides (Box 1), are well equipped to kill a range of leaf-eating insects [3], yet are found predominantly in the soil (http://pubmlst.org/bcereus/), with only low densities being present on the phylloplane [1]. One of the reasons that Bt is regularly referred to as being an opportunistic pathogen [4] could be that these leaf-eatinginsect specific strains are found in such large numbers in environments not inhabited by their targets. This has also resulted in Bt having a rather confounded ecology. Although Bt strains are active against a diverse range of insects, in this article we will concentrate on those active against leaf-eating insects, as these are the best studied. The role of insect midgut bacteria in the pathogenicity of Bt Recent reports [4,5] have challenged the widely held view that Bt can establish lethal infections in susceptible Corresponding author: Crickmore, N. (
[email protected]) Present address: School of Biological Sciences, Royal Holloway University of London, Egham, Surrey, TW20 0EX, UK. *
invertebrates by breaching the intestine and replicating within the living host’s haemolymph. Prior exposure of larvae from four species of Lepidoptera to a cocktail of four antibiotics eliminated the larvae’s native gut bacteria and attenuated Bt toxicity after subsequent Bt infection. The toxicity of Bt toward antibiotic-treated hosts could be rescued by inoculating larvae with a strain of Enterobacter isolated from Lymantria dispar (gypsy moth) larvae. Furthermore, whereas Enterobacter could replicate in cell-free L. dispar haemolymph, Bt could not. Thus it was proposed that Bt is unable to infect the haemolymph of the live insect but instead relies upon host gut bacteria to establish a fatal septicaemia [4,5]. Cooperation between Bt and gut bacteria is inconsistent with previous reports of competitive exclusion of Bt from lepidopteran hosts by enterococcal gut bacteria [6], of competition between gut bacteria and Bt for exploitation of Lepidopteran cadavers [7], and of the pathogeneic synergism between Bt and B. cereus via suppression of the bacterial gut community [8]. Indeed, much of the work described in this section arose from the observation that the virulence of B. thuringiensis could be synergised by zwittermycin A, most likely by altering the composition of the insect gut microbiota [9]. Reliance upon apparent competitors for virulence by Bt would seem unnecessary, given both the formidable assortment of known Bt virulence factors and the ability of Bt to kill a wide range of invertebrates after intrahaemocoelic inoculation (see below). Additionally, entomopathogenesis seems to be coupled to antibiosis in Bt. For example, the PlcR virulence regulon includes antimicrobial peptides [10]. Such coupling is found in other entomopathogens such as Xenorhabdus and Photorhabdus, in which antibiosis is thought to suppress gut bacteria that co-invade the haemocoel and compete for consumption of the host cadaver [11]. Because dietary antibiotics are known to reduce the toxicity of Bt towards many species of Lepidoptera [12], it is possible that attenuation of Bt in antibiotictreated hosts is not attributable to the absence of gut bacteria but rather to an effect of prior antibiotic exposure upon Bt. Recent work used aseptic insect larvae with or without antibiotic exposure to demonstrate that prior antibiotic exposure, not the absence of gut bacteria, attenuates Bt toxicity towards two species of Lepidoptera, Manduca sexta (tobacco hornworm) [13] and Plutella xylostella
0966-842X/$ – see front matter ß 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.tim.2010.02.006 Available online 24 March 2010
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Enterococcus within both P. xylostella and M. sexta reduced the toxicity of the Bt toxin Cry1Ac [13,14]. These findings contrast starkly with the hypothesis that gut bacteria are obligatory for Bt toxicity in M. sexta [5], but are in agreement with a previous report of enterococcal gut bacteria that confer protection against Bt [6]. Indeed, such a protective effect might be expected when one considers that intestinal enterococci are transmitted vertically between hosts on the surface of M. sexta eggs [16].
Figure 1. The complex ecology of Bt. The various locations from which Bt has been isolated and might replicate are shown diagrammatically. Possible locations for reproduction include the soil, the rhizosphere, the phylloplane or other plant tissues, or within living or dead insects, or within other invertebrates. Bt is shown either in its vegetative form (red rectangle) or in the form of spores (cream ovals) and crystals (pink diamonds).
(diamondback moth) [14]. It is notable that both studies demonstrated antibiotic attenuation of Bt after hosts were reared on much lower concentrations of antibiotics than those reported previously [4,5]. In addition, in P. xylostella, prior antibiotic exposure did not attenuate antibioticresistant Bt mutants, implying that attenuation is the result of a direct effect of the antibiotics upon Bt [14]. This conclusion is supported by recent work on L. dispar itself, in which a range of different antibiotics were used to remove the gut bacteria, but only the cocktail used previously [4,5] was found to inhibit the activity of Bt [15]. The presence of Enterococcus in host guts actually reduced the toxicity of Bt towards M. sexta, whereas the presence of
Box 1. Bacillus thuringiensis: a commercial biopesticide Owing to the insecticidal nature of Bt, the bacterium has been commercialised as an insecticide. As the bacterium sporulates, it produces, under limited nutrient conditions, one or more crystals containing proteins known as delta endotoxins or Cry toxins that, when ingested by a susceptible insect, cause destruction of the epithelial cells lining the insect gut. It is generally believed that these toxins act by creating pores in the cell membrane [24]. Products containing mixtures of spores and crystals of Bt have been commercialised to control a range of different insects during their larval stages, including caterpillars, beetles and mosquitoes [48]. Although the bacterium contributes to the death of the insect, the delta endotoxins alone are enough to kill some species if produced at sufficiently high doses. This fact has been exploited by expressing the delta endotoxin genes in systems such as alternative bacteria that are better adapted to survive in particular environmental niches, or in bacteria such as the pseudomonads that are then rendered non-viable, resulting in an encapsulated Bt crystal that is not living and therefore considered a lower environmental risk [49]. The most important non-bacterial expression system for delta endotoxins in recent years has been in major crop plants targeted by agricultural insect pests. Toxin-expressing cotton and maize varieties, among others, are now widely and successfully, used around the world to prevent insect damage [50].
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Bacillus thuringiensis is built like a pathogen Despite claims that Bt might only be an opportunistic pathogen and might even be incapable of full virulence without the aid of other bacteria, it appears to express a sufficiently wide range of factors to equip it well for a pathogenic lifestyle. Figure 2 illustrates some of the host barriers and defences with which an insect pathogen must cope [17], and some of the processes that Bt must undertake to be infectious and achieve large-scale reproduction. In Bt, many of these processes are elicited by virulence factors whose expression is controlled by PlcR, a pleiotropic regulator that is well conserved in Bt and B. cereus strains [18]. Deletion of the plcR gene drastically reduces the virulence of Bt in orally infected insects [19]. Ingested spores are able to germinate in the gut [19,20], although this environment is hostile to ingested cells or germinated spores. To combat this environment, Bt synthesizes a modified cell surface that helps protect it from antimicrobial peptides (AMPs) synthesized by the host or competing bacteria [21]. To be able to compete with other gut bacteria, Bt produces its own AMPs and bacteriocins [10,22] and it is likely that it also expresses a number of drug efflux transporters to combat antibiotics produced by competitors [10]. Ingestion of Bt often results in midgut paralysis and cessation of feeding [23]. The mechanism for this remains elusive although it undoubtedly prevents the loss of the bacterium through peristaltic action. Having survived the gut environment, Bt must attack the host. The best known of the Bt virulence factors are the specific protein toxins formed as crystalline inclusions during sporulation. These toxins target, and cause the destruction of, the epithelial cells lining the midgut [24]. For some insects, the action of these toxins alone is sufficient for mortality, whereas for others the spore must also be present, and death can be caused by septicaemia [23]. Germination of the spore allows the expression and production of other virulence factors involved in the destruction of the midgut tissues, which include an array of phospholipases, enterotoxins and proteases including the InhA2 metalloprotease [10,25]. To invade the insect further, Bt must cross the peritrophic matrix, a largely chitinous web that separates the midgut cells from the gut contents [26]. Although the mechanism by which Bt breaches the peritrophic membrane is not clear, this process might be facilitated by the production of chitinases and enhancins [27,28]. Once the midgut has been breached, the Bt cells or spores can enter the haemocoel. Potentially, spores and vegetative cells might also enter the haemocoel through spiracles or breaches in the cuticle. Spores are clearly able to germinate and replicate in the haemolymph because their
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Figure 2. Interactions between Bt and its target insect, following ingestion or introduction into the haemocoel. The various compartments, barriers and some innate immune defences encountered by Bt, are shown diagrammatically from a section across a caterpillar. The section is seen from the lumen of the central midgut to the peritrophic matrix (blue line), the midgut epithelial cells, the haemocoel and then finally the cuticle (green line). Bt is shown either in its vegetative form (red rectangle) or in the form of spores (cream ovals) and crystals (pink diamonds).
injection directly into the haemocoel results in septicaemia [13,25,29]. Within the haemolymph, the bacteria must avoid destruction by the innate humoral immune system and circulating haemocytes. Production of the metalloproteases InhA1 and InhA3 might help to protect Bt from the innate immune system through cleavage of AMPs [30,31] or by facilitating its escape from haemocytes [32]. The expression of enterotoxins by Bt during septicaemia has been reported, and these might play an active role in pathogenesis [33]. Additionally, the production of iron acquisition systems by Bt counters iron sequestration by the host and thus facilitates colonization [29,34,35]. Increased reproduction of Bt within the haemolymph results in acute septicaemia. Furthermore, Bt has been shown to reproduce within an insect cadaver at the expense of intestinal enterococci that were otherwise found to rapidly exploit hosts killed in the absence of Bt [13]. This ability to compete might be facilitated by the production of quorum quenchers and antibiotics [22,36,37].
Transmission of infection An essential part of the ecology of every pathogen is the process of transmission, that is, the transfer of infection from one host to another. Experimental work suggests that vertical transmission (transfer of a sublethal infection from a parent to their offspring) is not a major mechanism of Bt transmission [38]. Horizontal transmission (transfer of infection within a single generation) fits more readily with the biology of a highly virulent bacterium that needs to kill its host in order to proliferate. There are two possible routes of horizontal transmission for a Bt strain active against a leaf-eating insect. The first is indirect transmission via a reservoir in which the spores released from a cadaver are quickly deposited in soil. Thereafter, bacteria must recolonize plant material to infect hosts at a later date. This could be achieved by co-migration of Bt with the growing plant [1], or transfer by biotic or abiotic means or even through endophytic transport [39]. Crystals are readily degraded or lost in soil, and Bt spores will have to germinate and undergo 191
Opinion at least one round of vegetative growth epiphytically or endophytically in order to produce Cry toxins and have full pathogenicity. The second route is direct transmission, in which new infections are produced by larvae feeding on spores and crystals recently released onto leaf material from a Btkilled cadaver. The direct route of transmission must be reconciled with what we know of the ecology of Bt. Previous measurements of the transmission rate of the biopesticidal DiPel strain of Bt in the laboratory suggested that direct horizontal transmission from dead cadavers was incapable of propagating Bt in diet-reared populations of Plodia interpunctella [40], partly because of the anti-feedant effects associated with the presence of Cry toxins on larval food. By contrast, an experiment demonstrating horizontal transmission of Bt on a plant surface showed that the presence of a Bt-infected cadaver on a leaf surface caused the death of around a third of uninfected larvae added to the same leaf (Figure 3). Interestingly, cadavers produced by DiPel contained fewer spores and were significantly less infectious than cadavers produced by a recently isolated strain (M.S. Naryanan, MSc Thesis, University of Oxford, 2006), supporting the idea that this biopesticidal strain is attenuated for virulence compared with some environmental isolates. An additional factor that has not yet been considered is the extent of local adaptation or local coevolution between Bt and its hosts. Some Cry toxins can be broadly pathogenic across insect families and genera [3], and it is not really known whether Bt requires additional adaptations to ensure efficient replication within a particular species. Local differentiation in bacterial populations and their
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bacteriophage parasites can occur at the scale of centimetres in soil bacteria [41,42]. Therefore, it is plausible that reproduction (and transmission) of Bt strains that are adapted to a locally abundant host will be even more efficient than transmission rates measured in laboratory experiments. Another puzzling aspect of the biology of Bt is the ease with which metabolically expensive toxins can be separated from the spore, that is, the clone that invested all the resources in constructing these proteins. Why produce a crystal at all, instead of secreting an active virulence factor in the insect gut? In evolutionary terms, Cry toxins represent a ‘durable public good’ [43]. This is a substance that persists in the environment (is durable) but that can provide a benefit to a local population rather than an individual. In this case of Cry toxins, their presence in the insect mid-gut provides the population of bacteria within the gut access to the fat and protein within the insect host. This good is public because both toxin- producing and non-toxin-producing bacteria in the gut can benefit from the toxin [44]. Durability of this highly costly public good is particularly important because there is only periodic access to high- quality resources for growth. The discussion of the possible routes of transmission above suggests there are only two important places where toxin production takes place: in the host or on/within the plants. Access to hosts certainly occurs periodically. However, vegetative growth of Bt on leaves is not only poor but also periodic [45] and might be linked to fluctuation in leaf chemistry or ambient humidity. If transmission occurs indirectly, then Cry toxin production must take place in situ on the leaf surface in a relatively dry environment,
Figure 3. Horizontal transmission of Bt on a leaf. Cadavers infected by two strains of Bt were each placed on a leaf and their position marked (red line). Healthy second instar larvae were then added to the leaf and left for 7 days, after which live larvae were removed to fresh leaves. The proportion of larvae that died before pupating were calculated, as were the number of spores per cadaver. Cadavers produced with the environmental Bt contained more spores than DiPel cadavers (F1,17 = 9.4, P = 0.007) and produced more new infections (d.f. = 2, x2 = 12.4, P < 0.001) (M.S. Naryanan, MSc Thesis, University of Oxford, 2006).
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Opinion possibly in protected micro-sites. The separation of spore and crystal might therefore not be an issue unless spores are washed off plants into the soil to re-enter dormancy. Sporulation also occurs after replication within host cadavers when limiting resources are exhausted. Again, this is a relatively dry environment, and spores and crystals might be protected from rain and UV radiation when associated with the insect cuticle and other tissue fragments. Therefore, in either environment – within the host or on the plant – spores and crystals are unlikely to be readily separated. Concluding remarks: Bt is a bona fide insect pathogen The pathogenic lifestyle of Bt has been questioned because of its presence and ability to replicate in environments in which it is unlikely to encounter a susceptible insect host. Furthermore, it has been claimed that within the insect, cooperation from commensal gut bacteria is required for Bt to be fully pathogenic. We suggest that Bt is an insect pathogen, that its primary means of reproduction is in an insect cadaver, and that it does not require the assistance of other microbes for its pathogenicity. Although many facets of the organism’s lifecycle are not fully understood, genomic and proteomic studies are certainly consistent with it being a pathogen rather than an epiphyte or soildwelling saprophyte [46,47]. As more putative adaptation and virulence factors are identified through genome sequencing or other techniques, future studies should fully elucidate the role of these in the pathogenic process within the insect and also shed more light on the ecology of this bacterium. Acknowledgements We would like to thank COST862 for providing the opportunity to meet and to discuss this topic with colleagues. NC would like to thank the Royal Society for funding. BR would like to thank the NERC for funding. CNL and DL thank the MICA department from INRA for funding.
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