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The regulation of pathogenicity and mutualism in Photorhabdus
The regulation of pathogenicity and mutualism in Photorhabdus Susan A Joyce, Robert J Watson and David J Clarke Photorhabdus is a genus of insect-pathogenic bacteria that also maintains a mutualistic interaction with Heterorhabditid nematodes. Bacteria in this genus are members of the family Enterobacteriaceae and are, therefore, closely related to many important mammalian pathogens. This bacteria–nematode complex has been exploited as a biocontrol agent that is active against several insect pests. However, this model system is also uniquely placed to address important fundamental questions about pathogenicity and mutualism. Indeed, recent genetic studies have suggested that there is a significant overlap in the genetic requirements of Photorhabdus for these contrasting interactions. In addition, the identification of key regulators of pathogenicity and symbiosis only serves to highlight the similarities between Photorhabdus, a genus of bacteria that infects invertebrate hosts, and closely related mammalian enteric pathogens. Addresses Molecular Microbiology Laboratory, Department of Biology and Biochemistry, University of Bath, Bath, BA2 7AY, UK Corresponding author: Clarke, David J ([email protected])
Current Opinion in Microbiology 2006, 9:127–132 This review comes from a themed issue on Cell regulation Edited by Werner Goebel and Stephen Lory Available online 15th February 2006 1369-5274/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.mib.2006.01.004
Life cycle The life cycles of P. luminescens and P. temperata are complex and involve interactions with two eukaryotic hosts (Figure 1). P. luminescens and P. temperata are normally found colonising the gut of the soil-dwelling infective juvenile (IJ) stage of the Heterorhabditis nematode. The IJs represent a specialized stage of nematode development which is designed for survival and dispersal in the soil. The IJs actively seek out and penetrate potential insect larval hosts and release the bacteria into the insect haemolymph (blood) [4]. Here, the bacteria reproduce to a high cell density and secrete a wide range of extracellular hydrolytic enzymes that serve to convert the organs and tissues of the insect into an ideal niche for nematode growth and development. The insect larva generally succumbs to a general septicaemia within 48– 72 hours of the infection, during which the IJ develops into a self-fertile hermaphrodite (a process that is termed nematode recovery). The nematode uses the bacterial biomass as a source of nutrients, and nematode growth and development normally proceeds for two to three generations. At this point, it is thought that the developing juvenile nematodes receive environmental signals that stimulate their entry into diapause and the development of IJs. These IJs are colonised by Photorhabdus before the nematodes emerge from the insect cadaver into the soil. This life cycle, from infection to emergence, takes approximately 14 days under laboratory conditions, and a single infecting IJ will result in the development of > 100 000 IJs within the larva of a model insect host — for example, the greater waxmoth, Galleria mellonella. Clearly, this system represents an extremely efficient symbiosis of pathogens, and, as a result of the promiscuous and twotiming nature of the Photorhabdus life cycle, this model system provides us with a unique opportunity to study the relationship between pathogenicity and mutualism.
Introduction Photorhabdus is a genus of Gram-negative bacteria that is part of the family Enterobacteriaceae. The genus Photorhabdus is characterised by the ability of to produce light (through luciferase), and these are the only terrestrial bacteria known to bioluminesce [1]. Based on molecular analyses, the genus has been divided into three bacterial species: Photorhabdus luminescens, Photorhabdus temperata and Photorhabdus asymbiotica [2]. Both P. luminescens and P. temperata have complex life cycles that involve a pathogenic interaction with insect larvae and a symbiotic interaction with nematodes from the family Heterorhabditidiae. P. asymbiotica, by contrast, has only ever been isolated from human wounds and it is not thought to have a natural association with nematodes [3]. www.sciencedirect.com
In this review, we discuss recent developments in our understanding of the molecular mechanisms employed by Photorhabdus to control these contrasting interactions [5].
Pathogenicity In nature, pathogenicity and mutualism are intrinsically linked as part of the life cycle of Photorhabdus, and mutualism has an obligate requirement for pathogenicity (and vice versa). However, in the laboratory it is possible to uncouple mutualism and pathogenicity and this has facilitated independent genetic analyses of these interactions. When injected directly into the haemolymph of insect larvae, all strains of Photorhabdus tested are extremely virulent, with a calculated LD50 of < 5 colony-forming Current Opinion in Microbiology 2006, 9:127–132
128 Cell regulation
Figure 1
The life cycle of Photorhabdus. Photorhabdus is normally found colonising the gut of the IJ stage of the nematode Heterorhabditis. The IJ is a free-living, non-feeding stage that actively seeks out and infects soil-dwelling insect larvae. The host range of Heterorhabditis includes the larvae of insects from the Coleoptera (beetles) and the Lepidoptera (moths and butterflies). The Photorhabdus bacteria are released into the insect bloodstream, where they reproduce, resulting in larval death approximately 48–72 hours post-infection. During this time, the IJ has recovered and developed into a self-fertile hermaphrodite nematode that begins to feed on the high bacterial biomass present in the insect cadaver. Therefore, Heterorhabditis only requires that a single IJ infect a host to ensure that nematode growth and development can occur. The hermaphrodite lays eggs, which develop, through four larval moults, into male and female adults. Nematode reproduction occurs for two to three generations, until environmental conditions within the cadaver — for example, crowding and nutrient availability — stimulate the development of an alternative J3 stage nematode, the IJ. The IJ is colonised by some of the remaining Photorhabdus in the insect before emerging into the soil to seek a new insect host.
units per insect. It has also been shown that pathogenicity (as determined by LT50 calculations) is very tightly correlated with bacterial growth rate, and bacteria in this genus have been shown to grow exponentially in the insect, with a growth rate that is similar to that observed in Luria–Bertani broth [6–8]. However, to be able to grow within the insect, Photorhabdus must avoid or suppress the insect immune response. Insects have a sophisticated cellular and innate immune response that is normally capable of protecting the insect from infection — for example, injection of 1 108 cells of Escherichia coli into the insect haemolymph does not cause disease, and the bacteria are cleared from the blood within several hours [9–11]. The genome sequence of P. luminescens TT01 has been published (http://genolist. pasteur.fr/PhotoList/), and in silico analysis of this indicates the presence of several pathogenicity islands [12,13]. It is likely that these genetic elements are involved in the success of Photorhabdus as an entomopathogen and, indeed, the pathogenicity islands are predicted to encode several toxins, some of which have been studied in greater detail, for example, the Tc (toxin complex) and Mcf (makes caterpillars floppy) toxins [14,15]. Interestingly, a common mode of action of both Tc and Mcf is the induction of Current Opinion in Microbiology 2006, 9:127–132
apoptosis in eukaryotic cells, suggesting that their role in vivo might be to cripple the insect immune response, thereby facilitating bacterial growth [16,17]. Very little is known about how toxin production is regulated, although there is some evidence that Photorhabdus does express tcaB and tcdB (encoding components of the Tc toxins) during exponential growth in the insect larvae [8].
Type III secretion systems A functional type III secretion system has also been identified in Photorhabdus, and this type III secretion system has been shown to secrete LopT, a homologue of YopT from Yersinia pestis [18,19]. YopT inhibits the phagocytosis of Y. pestis by macrophages, and it has been shown that LopT also inhibits phagocytosis by insect immune cells, although a deletion of lopT does not appear to affect the virulence of Photorhabdus [19].
The regulation of pathogenicity Photorhabdus pathogenicity requires bacterial growth within the insect, and, therefore, mutations in genes required for in vivo growth will be affected in virulence [7]. However, it is likely that certain regulatory pathways www.sciencedirect.com
The regulation of pathogenicity and mutualism in Photorhabdus Joyce, Watson and Clarke 129
will be important for adaptation to specific niches within the insect, and, in support of this, it was recently shown that a mutation in phoP rendered P. luminescens avirulent to insect larvae [20]. The phoP gene encodes the response regulator of the PhoPQ two-component pathway (2CP), and this pathway, which responds to low levels of Mg2+, is an important virulence factor in many enteric bacteria [21,22]. The PhoPQ 2CP in Photorhabdus also responds to low Mg2+ levels, and, although the role phoP in Photorhabdus virulence has not been determined (other than it is required), there are clearly significant parallels in the regulation of pathogenicity in mammalian and insect pathogens [20].
Mutualism Heterorhabditis has an obligate requirement for Photorhabdus for normal nematode growth and development, and nutrient provision is often a strong driving force in the evolution of mutualism. Recent work has identified a potentially important role for the essential nutrient iron in the association between Photorhabdus and Heterorhabditis [7]. A mutation in the exbD gene of P. temperata K122 resulted in a strain that was deficient in iron scavenging and could not support nematode growth and development [7]. ExbD is a component of the ExbB–ExbD– TonB complex that is required to facilitate the active uptake of small molecules, such as siderophores [23,24]. The requirement for exbD was taken to suggest that the defect in mutualism was due to lower levels of iron within the mutant bacteria and, in support of this, it was shown that the mutualism-deficient phenotype could be rescued by the addition of FeCl3 to the media [7]. However, iron also has an important regulatory role in bacteria, and it has not yet been established whether the role of iron in the Photorhabdus–Heterorhabditis interaction is nutritional, regulatory or both. Interestingly, a mutation in the ngrA gene, predicted to encode a phosphopantetheinyl transferase, resulted in a mutant that was unable to support the recovery of IJs into self-fertile hermaphrodites [25]. Analysis of the DNA sequence upstream from ngrA indicated a role for iron in the regulation of the expression of this gene. Phosphopantetheinyl transferases are enzymes required for the activity of polyketide synthases and non-ribosomal peptide synthases, which are responsible for the production of small bioactive molecules, including siderophores [26]. Therefore, iron levels, by affecting the level of phosphopantetheinyl transferase activity in the cell, might control the production of small polyketide or peptide molecules that are required for the interaction with the nematode — for example, signalling molecules. At the end of nematode growth and reproduction, Photorhabdus must be able to colonise the developing IJs. Although IJ colonisation is a highly specific process, very little is known about the mechanisms that control this important step in the symbiosis. Recently, it was reported that a mutation in pgbE1, the fifth gene of the www.sciencedirect.com
seven-gene pgbPE operon, rendered Photorhabdus unable to colonise the IJ [27]. Interestingly, this mutant was also highly attenuated for insect pathogenicity, suggesting that the pbgPE operon is required for the infection of the insect [27]. The pbgPE operon is homologous to the pmrHFIJKLM operon in Salmonella enterica encoding the proteins required for the modification of the lipid A moiety of lipopolysaccharide with L-aminoarabinose [28]. In Salmonella, the expression of pmrHFIJKLM is controlled, in a Mg2+-dependent manner, by the PhoPQ 2CP, and this has also been shown to be the case with the expression of the pbgPE operon in Photorhabdus [20,22]. Therefore, although the role of the PhoPQ pathway in mutualism has not been directly tested, it is clear that this signalling pathway controls the expression of genes that are required for infection of both the nematode and the insect host.
Phenotypic variation and mutualism Photorhabdus can exist in two stable phenotypic variants, the primary variant and the secondary variant [29]. The primary variant is characterised by the presentation of the primary-specific phenotypes during post-exponential growth (Table 1). The secondary variant does not present, or presents greatly reduced levels of, these phenotypes [30]. The production of the primary-specific phenotypes is implicated in the mutualistic interaction between Photorhabdus and the nematode host because both the primary and secondary variants are equally virulent to insect larvae and the primary variant can support nematode growth and development, whereas the secondary variant cannot. It has been suggested that the secondary variant is better adapted to life in the absence of the nematode and, therefore, phenotypic variation might be an adaptation for survival by the bacterial population that remains in the insect cadaver after the new generation of IJs has dispersed [31]. Unlike classical phase variation, phenotypic variation is unidirectional, occurring only in the primary to secondary direction. Therefore, if a significant level of phenotypic variation were to occur in the insect cadaver during nematode development, this would have serious consequences on the viability of the symbiosis. This implies that phenotypic variation must be tightly controlled during the life cycle of Photorhabdus, and an understanding of how phenotypic variation is regulated could be extrapolated into a better insight of the regulatory pathways that control mutualism.
The regulation of phenotypic variation Phenotypic variants have only been isolated in the laboratory after the extended incubation of Photorhabdus cultures, suggesting that this phenomenon is a response to environmental stress. Indeed, recent data suggest that phenotypic variation is regulated by at least two signalling pathways. The coordinated repression of the primaryspecific factors suggested that a repressor protein might be expressed exclusively in the secondary variant. Current Opinion in Microbiology 2006, 9:127–132
130 Cell regulation
Table 1
Figure 2
Primary-specific phenotypes in Photorhabdus and the regulation of these phenotypes by HexA. Phenotype
Repressed by HexA
Target gene(s)
Dye uptakey Colony morphologyz Bioluminescence Protease production Lipase production§ Pigmentation Antibiotic production Catalase Crystal proteins Motility
Yes# Yes Yes Yes Yes Yes# Yes Yes Yes No
ND * ND luxCDABE prtA lip-1 ND ND katE cipA, cipB -
Data for this table is from [32]. ND: not determined. y Phenotypic variants can be distinguished by the colour of colonies grown on Luria–Bertani agar supplemented with bromothymol blue and tetrazolium salt. z Primary variant colonies are convex and mucoid; secondary variant colonies are flat and non-mucoid. § The lipase is produced in equal amounts in both the primary and the secondary variant. However, the enzyme secreted by the secondary variant is inactive, indicating post-translational regulation [38]. # HexA repression of these phenotypes is partial [32]. *
A genetic screen designed to find this repressor identified hexA, a gene predicted to encode a LysR-type transcriptional regulator [32]. An insertion in the hexA gene of the secondary variant of P. temperata K122 resulted in the derepression of the primary-specific factors and restored the ability of these bacteria to support nematode growth and development [32]. The overproduction of HexA in the primary variant is sufficient to induce conditional phenotypic variation, suggesting that a high level of HexA is sufficient for the formation of the secondary variant (Joyce et al., unpublished data). Although the regulatory mechanisms that control HexA levels in Photorhabdus have not been characterised, HexA can positively autoregulate its own expression, presumably to ensure that high levels are maintained in the secondary variant [32]. Interestingly, the hexA mutant was also attenuated in virulence, suggesting that HexA is required for pathogenicity [32]. This is in contrast to the situation in Erwinia carotovora, where a mutation in hexA was observed to increase virulence [33]. Therefore, in Photorhabdus, HexA appears to have reciprocal roles in the regulation of pathogenicity and mutualism (Figure 2). The AstRS 2CP has been shown to be involved in the temporal control of phenotypic variation [34]. Therefore, cells deleted for astR (encoding the response regulator of the pathway) undergo phenotypic variation seven days earlier than the wild-type strain. Proteomic analysis of the astR mutant revealed that the AstRS 2CP positively regulates the expression of the gene encoding the universal stress protein, UspA [34]. Studies in E. coli have shown that UspA is important for cell survival during periods of stress — for example oxidative stress and stasis [35,36]. Current Opinion in Microbiology 2006, 9:127–132
HexA and PhoPQ are involved in the regulation of pathogenicity and mutualism in Photorhabdus. HexA represses mutualism but is required for pathogenicity, and there is some evidence that a small RNA molecule is involved in this regulation (Joyce et al., unpublished data). PhoPQ has been shown to be essential for pathogenicity, and this 2CP regulates genes that are required for some aspects of mutualism. The AstRS 2CP controls the timing of phenotypic variation, and there is evidence that this pathway monitors oxygen levels in the environment [34]. When activated, this pathway represses motility (through a decrease in the expression of the flhDC operon) and increases the production of the universal stress protein UspA. UspA has been shown to protect bacteria during stasis and oxidative stress, and therefore, it would be expected that in the absence of the AstRS pathway, the cell is more vulnerable to stress. We speculate that this increased exposure to stress serves as a signal for the increased production of HexA and, ultimately, phenotypic variation. However, the presence, and molecular nature, of any connection between AstRS and HexA remains to be determined.
Therefore, the AstRS pathway might serve to prevent or delay phenotypic variation by protecting the cell from stress (Figure 2). The astR mutant was also hyper-motile as a result of an increase in the expression of flhDC, encoding the class 1 regulator of flagella production [34]. Interestingly, motility was the only primary-specific factor not restored in the secondary variant hexA mutant (Table 1) [32]. Motility is restored to the secondary variant of P. temperata K122 when the bacteria are cultured in an anaerobic environment, suggesting that there are at least two pathways controlling phenotypic variation, a HexA-dependent pathway and an O2-dependent pathway [37]. Is the AstRS pathway involved in the O2-dependent control of motility during phenotypic variation? Intriguingly, analysis of the predicted amino acid sequence of the AstS sensor protein does reveal the presence of a PAS domain, found in many proteins involved in redox sensing (Joyce et al., unpublished data). www.sciencedirect.com
The regulation of pathogenicity and mutualism in Photorhabdus Joyce, Watson and Clarke 131
Conclusions and future work How does Photorhabdus control the different interactions with its eukaryotic hosts? Remarkably, HexA and PhoPQ are involved in the regulation of both pathogenicity and mutualism, highlighting the interconnected nature of these relationships in Photorhabdus. In addition, the AstRS 2CP is required to prevent the early occurrence of phenotypic variation, thus ensuring efficient nematode growth and development. However, further genetic studies are required to build a complete understanding of the complex signalling and regulatory networks that are undoubtedly involved in the regulation of pathogenicity, mutualism and phenotypic variation. There is some evidence that the temporal control of gene expression is important in establishing a distinction between mutualism and pathogenicity — that is, pathogenicity is associated with bacterial exponential growth and mutualism is associated with the post-exponential growth of Photorhabdus. However, genetic studies have indicated that many of the genes required for mutualism are also required for pathogenicity (Figure 3). Therefore, studies of Photorhabdus have confirmed that there is a genetic overlap between pathogenicity and mutualism, and it follows that the host must have an important role in determining the outcome of a bacterial infection. But what is the full extent of this genetic overlap? The tripartite interaction between Photorhabdus, the nematode Heterorhabditis and the insect host provides us with a unique opportunity to address this important fundamental question. Moreover, invertebrates have been developed as an important and relevant model system for the study of bacterial infections, and future work aimed at understanding the molecular mechanisms that underpin pathogenicity and
mutualism in Photorhabdus will also increase our understanding of the molecular mechanisms that control bacterial infections in mammals.
Acknowledgements Research on Photorhabdus has been supported with grants from the Leverhulme Trust and the Biotechnology and Biological Sciences Research Council (BBSRC) of the UK. The authors would also like to acknowledge past members of the laboratory for their contributions to the work described in this review.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
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The genetic overlap between pathogenicity and mutualism in Photorhabdus. Genetic studies have clearly shown that there is some genetic overlap between pathogenicity and mutualism (e.g. pbgE1 and exbD are required for both interactions). However, this overlap is not complete, and mutations that are affected in one or other interaction have also been characterised. The ngrA gene is not required for pathogenicity but is required for mutualism (perhaps as a result of a requirement in the production of a signal molecule). In addition, the toxins produced by Photorhabdus are involved in pathogenicity but no role for these molecules has yet to be determined in mutualism. www.sciencedirect.com
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Current Opinion in Microbiology 2006, 9:127–132 This review comes from a themed issue on Cell regulation Edited by Werner Goebel and Stephen Lory Available online 15th February 2006 1369-5274/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.mib.2006.01.004
Life cycle The life cycles of P. luminescens and P. temperata are complex and involve interactions with two eukaryotic hosts (Figure 1). P. luminescens and P. temperata are normally found colonising the gut of the soil-dwelling infective juvenile (IJ) stage of the Heterorhabditis nematode. The IJs represent a specialized stage of nematode development which is designed for survival and dispersal in the soil. The IJs actively seek out and penetrate potential insect larval hosts and release the bacteria into the insect haemolymph (blood) [4]. Here, the bacteria reproduce to a high cell density and secrete a wide range of extracellular hydrolytic enzymes that serve to convert the organs and tissues of the insect into an ideal niche for nematode growth and development. The insect larva generally succumbs to a general septicaemia within 48– 72 hours of the infection, during which the IJ develops into a self-fertile hermaphrodite (a process that is termed nematode recovery). The nematode uses the bacterial biomass as a source of nutrients, and nematode growth and development normally proceeds for two to three generations. At this point, it is thought that the developing juvenile nematodes receive environmental signals that stimulate their entry into diapause and the development of IJs. These IJs are colonised by Photorhabdus before the nematodes emerge from the insect cadaver into the soil. This life cycle, from infection to emergence, takes approximately 14 days under laboratory conditions, and a single infecting IJ will result in the development of > 100 000 IJs within the larva of a model insect host — for example, the greater waxmoth, Galleria mellonella. Clearly, this system represents an extremely efficient symbiosis of pathogens, and, as a result of the promiscuous and twotiming nature of the Photorhabdus life cycle, this model system provides us with a unique opportunity to study the relationship between pathogenicity and mutualism.
Introduction Photorhabdus is a genus of Gram-negative bacteria that is part of the family Enterobacteriaceae. The genus Photorhabdus is characterised by the ability of to produce light (through luciferase), and these are the only terrestrial bacteria known to bioluminesce [1]. Based on molecular analyses, the genus has been divided into three bacterial species: Photorhabdus luminescens, Photorhabdus temperata and Photorhabdus asymbiotica [2]. Both P. luminescens and P. temperata have complex life cycles that involve a pathogenic interaction with insect larvae and a symbiotic interaction with nematodes from the family Heterorhabditidiae. P. asymbiotica, by contrast, has only ever been isolated from human wounds and it is not thought to have a natural association with nematodes [3]. www.sciencedirect.com
In this review, we discuss recent developments in our understanding of the molecular mechanisms employed by Photorhabdus to control these contrasting interactions [5].
Pathogenicity In nature, pathogenicity and mutualism are intrinsically linked as part of the life cycle of Photorhabdus, and mutualism has an obligate requirement for pathogenicity (and vice versa). However, in the laboratory it is possible to uncouple mutualism and pathogenicity and this has facilitated independent genetic analyses of these interactions. When injected directly into the haemolymph of insect larvae, all strains of Photorhabdus tested are extremely virulent, with a calculated LD50 of < 5 colony-forming Current Opinion in Microbiology 2006, 9:127–132
128 Cell regulation
Figure 1
The life cycle of Photorhabdus. Photorhabdus is normally found colonising the gut of the IJ stage of the nematode Heterorhabditis. The IJ is a free-living, non-feeding stage that actively seeks out and infects soil-dwelling insect larvae. The host range of Heterorhabditis includes the larvae of insects from the Coleoptera (beetles) and the Lepidoptera (moths and butterflies). The Photorhabdus bacteria are released into the insect bloodstream, where they reproduce, resulting in larval death approximately 48–72 hours post-infection. During this time, the IJ has recovered and developed into a self-fertile hermaphrodite nematode that begins to feed on the high bacterial biomass present in the insect cadaver. Therefore, Heterorhabditis only requires that a single IJ infect a host to ensure that nematode growth and development can occur. The hermaphrodite lays eggs, which develop, through four larval moults, into male and female adults. Nematode reproduction occurs for two to three generations, until environmental conditions within the cadaver — for example, crowding and nutrient availability — stimulate the development of an alternative J3 stage nematode, the IJ. The IJ is colonised by some of the remaining Photorhabdus in the insect before emerging into the soil to seek a new insect host.
units per insect. It has also been shown that pathogenicity (as determined by LT50 calculations) is very tightly correlated with bacterial growth rate, and bacteria in this genus have been shown to grow exponentially in the insect, with a growth rate that is similar to that observed in Luria–Bertani broth [6–8]. However, to be able to grow within the insect, Photorhabdus must avoid or suppress the insect immune response. Insects have a sophisticated cellular and innate immune response that is normally capable of protecting the insect from infection — for example, injection of 1 108 cells of Escherichia coli into the insect haemolymph does not cause disease, and the bacteria are cleared from the blood within several hours [9–11]. The genome sequence of P. luminescens TT01 has been published (http://genolist. pasteur.fr/PhotoList/), and in silico analysis of this indicates the presence of several pathogenicity islands [12,13]. It is likely that these genetic elements are involved in the success of Photorhabdus as an entomopathogen and, indeed, the pathogenicity islands are predicted to encode several toxins, some of which have been studied in greater detail, for example, the Tc (toxin complex) and Mcf (makes caterpillars floppy) toxins [14,15]. Interestingly, a common mode of action of both Tc and Mcf is the induction of Current Opinion in Microbiology 2006, 9:127–132
apoptosis in eukaryotic cells, suggesting that their role in vivo might be to cripple the insect immune response, thereby facilitating bacterial growth [16,17]. Very little is known about how toxin production is regulated, although there is some evidence that Photorhabdus does express tcaB and tcdB (encoding components of the Tc toxins) during exponential growth in the insect larvae [8].
Type III secretion systems A functional type III secretion system has also been identified in Photorhabdus, and this type III secretion system has been shown to secrete LopT, a homologue of YopT from Yersinia pestis [18,19]. YopT inhibits the phagocytosis of Y. pestis by macrophages, and it has been shown that LopT also inhibits phagocytosis by insect immune cells, although a deletion of lopT does not appear to affect the virulence of Photorhabdus [19].
The regulation of pathogenicity Photorhabdus pathogenicity requires bacterial growth within the insect, and, therefore, mutations in genes required for in vivo growth will be affected in virulence [7]. However, it is likely that certain regulatory pathways www.sciencedirect.com
The regulation of pathogenicity and mutualism in Photorhabdus Joyce, Watson and Clarke 129
will be important for adaptation to specific niches within the insect, and, in support of this, it was recently shown that a mutation in phoP rendered P. luminescens avirulent to insect larvae [20]. The phoP gene encodes the response regulator of the PhoPQ two-component pathway (2CP), and this pathway, which responds to low levels of Mg2+, is an important virulence factor in many enteric bacteria [21,22]. The PhoPQ 2CP in Photorhabdus also responds to low Mg2+ levels, and, although the role phoP in Photorhabdus virulence has not been determined (other than it is required), there are clearly significant parallels in the regulation of pathogenicity in mammalian and insect pathogens [20].
Mutualism Heterorhabditis has an obligate requirement for Photorhabdus for normal nematode growth and development, and nutrient provision is often a strong driving force in the evolution of mutualism. Recent work has identified a potentially important role for the essential nutrient iron in the association between Photorhabdus and Heterorhabditis [7]. A mutation in the exbD gene of P. temperata K122 resulted in a strain that was deficient in iron scavenging and could not support nematode growth and development [7]. ExbD is a component of the ExbB–ExbD– TonB complex that is required to facilitate the active uptake of small molecules, such as siderophores [23,24]. The requirement for exbD was taken to suggest that the defect in mutualism was due to lower levels of iron within the mutant bacteria and, in support of this, it was shown that the mutualism-deficient phenotype could be rescued by the addition of FeCl3 to the media [7]. However, iron also has an important regulatory role in bacteria, and it has not yet been established whether the role of iron in the Photorhabdus–Heterorhabditis interaction is nutritional, regulatory or both. Interestingly, a mutation in the ngrA gene, predicted to encode a phosphopantetheinyl transferase, resulted in a mutant that was unable to support the recovery of IJs into self-fertile hermaphrodites [25]. Analysis of the DNA sequence upstream from ngrA indicated a role for iron in the regulation of the expression of this gene. Phosphopantetheinyl transferases are enzymes required for the activity of polyketide synthases and non-ribosomal peptide synthases, which are responsible for the production of small bioactive molecules, including siderophores [26]. Therefore, iron levels, by affecting the level of phosphopantetheinyl transferase activity in the cell, might control the production of small polyketide or peptide molecules that are required for the interaction with the nematode — for example, signalling molecules. At the end of nematode growth and reproduction, Photorhabdus must be able to colonise the developing IJs. Although IJ colonisation is a highly specific process, very little is known about the mechanisms that control this important step in the symbiosis. Recently, it was reported that a mutation in pgbE1, the fifth gene of the www.sciencedirect.com
seven-gene pgbPE operon, rendered Photorhabdus unable to colonise the IJ [27]. Interestingly, this mutant was also highly attenuated for insect pathogenicity, suggesting that the pbgPE operon is required for the infection of the insect [27]. The pbgPE operon is homologous to the pmrHFIJKLM operon in Salmonella enterica encoding the proteins required for the modification of the lipid A moiety of lipopolysaccharide with L-aminoarabinose [28]. In Salmonella, the expression of pmrHFIJKLM is controlled, in a Mg2+-dependent manner, by the PhoPQ 2CP, and this has also been shown to be the case with the expression of the pbgPE operon in Photorhabdus [20,22]. Therefore, although the role of the PhoPQ pathway in mutualism has not been directly tested, it is clear that this signalling pathway controls the expression of genes that are required for infection of both the nematode and the insect host.
Phenotypic variation and mutualism Photorhabdus can exist in two stable phenotypic variants, the primary variant and the secondary variant [29]. The primary variant is characterised by the presentation of the primary-specific phenotypes during post-exponential growth (Table 1). The secondary variant does not present, or presents greatly reduced levels of, these phenotypes [30]. The production of the primary-specific phenotypes is implicated in the mutualistic interaction between Photorhabdus and the nematode host because both the primary and secondary variants are equally virulent to insect larvae and the primary variant can support nematode growth and development, whereas the secondary variant cannot. It has been suggested that the secondary variant is better adapted to life in the absence of the nematode and, therefore, phenotypic variation might be an adaptation for survival by the bacterial population that remains in the insect cadaver after the new generation of IJs has dispersed [31]. Unlike classical phase variation, phenotypic variation is unidirectional, occurring only in the primary to secondary direction. Therefore, if a significant level of phenotypic variation were to occur in the insect cadaver during nematode development, this would have serious consequences on the viability of the symbiosis. This implies that phenotypic variation must be tightly controlled during the life cycle of Photorhabdus, and an understanding of how phenotypic variation is regulated could be extrapolated into a better insight of the regulatory pathways that control mutualism.
The regulation of phenotypic variation Phenotypic variants have only been isolated in the laboratory after the extended incubation of Photorhabdus cultures, suggesting that this phenomenon is a response to environmental stress. Indeed, recent data suggest that phenotypic variation is regulated by at least two signalling pathways. The coordinated repression of the primaryspecific factors suggested that a repressor protein might be expressed exclusively in the secondary variant. Current Opinion in Microbiology 2006, 9:127–132
130 Cell regulation
Table 1
Figure 2
Primary-specific phenotypes in Photorhabdus and the regulation of these phenotypes by HexA. Phenotype
Repressed by HexA
Target gene(s)
Dye uptakey Colony morphologyz Bioluminescence Protease production Lipase production§ Pigmentation Antibiotic production Catalase Crystal proteins Motility
Yes# Yes Yes Yes Yes Yes# Yes Yes Yes No
ND * ND luxCDABE prtA lip-1 ND ND katE cipA, cipB -
Data for this table is from [32]. ND: not determined. y Phenotypic variants can be distinguished by the colour of colonies grown on Luria–Bertani agar supplemented with bromothymol blue and tetrazolium salt. z Primary variant colonies are convex and mucoid; secondary variant colonies are flat and non-mucoid. § The lipase is produced in equal amounts in both the primary and the secondary variant. However, the enzyme secreted by the secondary variant is inactive, indicating post-translational regulation [38]. # HexA repression of these phenotypes is partial [32]. *
A genetic screen designed to find this repressor identified hexA, a gene predicted to encode a LysR-type transcriptional regulator [32]. An insertion in the hexA gene of the secondary variant of P. temperata K122 resulted in the derepression of the primary-specific factors and restored the ability of these bacteria to support nematode growth and development [32]. The overproduction of HexA in the primary variant is sufficient to induce conditional phenotypic variation, suggesting that a high level of HexA is sufficient for the formation of the secondary variant (Joyce et al., unpublished data). Although the regulatory mechanisms that control HexA levels in Photorhabdus have not been characterised, HexA can positively autoregulate its own expression, presumably to ensure that high levels are maintained in the secondary variant [32]. Interestingly, the hexA mutant was also attenuated in virulence, suggesting that HexA is required for pathogenicity [32]. This is in contrast to the situation in Erwinia carotovora, where a mutation in hexA was observed to increase virulence [33]. Therefore, in Photorhabdus, HexA appears to have reciprocal roles in the regulation of pathogenicity and mutualism (Figure 2). The AstRS 2CP has been shown to be involved in the temporal control of phenotypic variation [34]. Therefore, cells deleted for astR (encoding the response regulator of the pathway) undergo phenotypic variation seven days earlier than the wild-type strain. Proteomic analysis of the astR mutant revealed that the AstRS 2CP positively regulates the expression of the gene encoding the universal stress protein, UspA [34]. Studies in E. coli have shown that UspA is important for cell survival during periods of stress — for example oxidative stress and stasis [35,36]. Current Opinion in Microbiology 2006, 9:127–132
HexA and PhoPQ are involved in the regulation of pathogenicity and mutualism in Photorhabdus. HexA represses mutualism but is required for pathogenicity, and there is some evidence that a small RNA molecule is involved in this regulation (Joyce et al., unpublished data). PhoPQ has been shown to be essential for pathogenicity, and this 2CP regulates genes that are required for some aspects of mutualism. The AstRS 2CP controls the timing of phenotypic variation, and there is evidence that this pathway monitors oxygen levels in the environment [34]. When activated, this pathway represses motility (through a decrease in the expression of the flhDC operon) and increases the production of the universal stress protein UspA. UspA has been shown to protect bacteria during stasis and oxidative stress, and therefore, it would be expected that in the absence of the AstRS pathway, the cell is more vulnerable to stress. We speculate that this increased exposure to stress serves as a signal for the increased production of HexA and, ultimately, phenotypic variation. However, the presence, and molecular nature, of any connection between AstRS and HexA remains to be determined.
Therefore, the AstRS pathway might serve to prevent or delay phenotypic variation by protecting the cell from stress (Figure 2). The astR mutant was also hyper-motile as a result of an increase in the expression of flhDC, encoding the class 1 regulator of flagella production [34]. Interestingly, motility was the only primary-specific factor not restored in the secondary variant hexA mutant (Table 1) [32]. Motility is restored to the secondary variant of P. temperata K122 when the bacteria are cultured in an anaerobic environment, suggesting that there are at least two pathways controlling phenotypic variation, a HexA-dependent pathway and an O2-dependent pathway [37]. Is the AstRS pathway involved in the O2-dependent control of motility during phenotypic variation? Intriguingly, analysis of the predicted amino acid sequence of the AstS sensor protein does reveal the presence of a PAS domain, found in many proteins involved in redox sensing (Joyce et al., unpublished data). www.sciencedirect.com
The regulation of pathogenicity and mutualism in Photorhabdus Joyce, Watson and Clarke 131
Conclusions and future work How does Photorhabdus control the different interactions with its eukaryotic hosts? Remarkably, HexA and PhoPQ are involved in the regulation of both pathogenicity and mutualism, highlighting the interconnected nature of these relationships in Photorhabdus. In addition, the AstRS 2CP is required to prevent the early occurrence of phenotypic variation, thus ensuring efficient nematode growth and development. However, further genetic studies are required to build a complete understanding of the complex signalling and regulatory networks that are undoubtedly involved in the regulation of pathogenicity, mutualism and phenotypic variation. There is some evidence that the temporal control of gene expression is important in establishing a distinction between mutualism and pathogenicity — that is, pathogenicity is associated with bacterial exponential growth and mutualism is associated with the post-exponential growth of Photorhabdus. However, genetic studies have indicated that many of the genes required for mutualism are also required for pathogenicity (Figure 3). Therefore, studies of Photorhabdus have confirmed that there is a genetic overlap between pathogenicity and mutualism, and it follows that the host must have an important role in determining the outcome of a bacterial infection. But what is the full extent of this genetic overlap? The tripartite interaction between Photorhabdus, the nematode Heterorhabditis and the insect host provides us with a unique opportunity to address this important fundamental question. Moreover, invertebrates have been developed as an important and relevant model system for the study of bacterial infections, and future work aimed at understanding the molecular mechanisms that underpin pathogenicity and
mutualism in Photorhabdus will also increase our understanding of the molecular mechanisms that control bacterial infections in mammals.
Acknowledgements Research on Photorhabdus has been supported with grants from the Leverhulme Trust and the Biotechnology and Biological Sciences Research Council (BBSRC) of the UK. The authors would also like to acknowledge past members of the laboratory for their contributions to the work described in this review.
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The genetic overlap between pathogenicity and mutualism in Photorhabdus. Genetic studies have clearly shown that there is some genetic overlap between pathogenicity and mutualism (e.g. pbgE1 and exbD are required for both interactions). However, this overlap is not complete, and mutations that are affected in one or other interaction have also been characterised. The ngrA gene is not required for pathogenicity but is required for mutualism (perhaps as a result of a requirement in the production of a signal molecule). In addition, the toxins produced by Photorhabdus are involved in pathogenicity but no role for these molecules has yet to be determined in mutualism. www.sciencedirect.com
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