Abstract
Photorhabdus is a highly effective insect pathogen and symbiont of insecticidal nematodes. To exert its potent insecticidal effects, it elaborates a myriad of toxins and small molecule effectors. Among these, the Photorhabdus Virulence Cassettes (PVCs) represent an elegant self-contained delivery mechanism for diverse protein toxins. Importantly, these self-contained nanosyringes overcome host cell membrane barriers, and act independently, at a distance from the bacteria itself. In this study, we demonstrate that Pnf, a PVC needle complex associated toxin, is a Rho-GTPase, which acts via deamidation and transglutamination to disrupt the cytoskeleton. TEM and Western blots have shown a physical association between Pnf and its cognate PVC delivery mechanism. We demonstrate that for Pnf to exert its effect, translocation across the cell membrane is absolutely essential.
SIGNIFICANCE STATEMENT Here we provide an up to date analysis of the nano-scale syringe-like molecular devices that Photorhabdus use to manipulate invertebrate hosts, the PVC system. They are related to the Serratia Anti-Feeding Prophage and the Psuedoalteromonas MAC system. All these systems are in turn more distantly related to the well characterized Type VI secretion system currently receiving a great deal of attention. We demonstrate for the first time that the PVC nanosyringes are physically “loaded” with an effector protein payload before being freely released. The PVCs therefore represent bacterial molecular machines that are used as “long-range” protein delivery systems. This widespread class of toxin delivery system will likely prove of great significance in understanding many diverse bacteria/host interactions in future.
INTRODUCTION
Bacteria belonging to the Enterobacteriacae genus Photorhabdus exist in a symbiotic partnership with entomopathogenic Heterorhabditis sp. nematodes. This Entomopathogenic Nematode complex (EPN) comprises a highly efficient symbiosis of pathogens that is commonly used as a biological agent to control crop pests [1]. The Photorhabdus bacteria are delivered into the hemocoel of the insect, after regurgitation from the worm, where they resist the insect immune response and rapidly kill the host via septicaemic infection. Insect tissues are subsequently bio-converted into a dense soup of Photorhabdus bacteria, which provide a food source to support the replication of the nematode. As food resources are depleted Photorhabdus re-associates with infective juvenile nematodes, and together they emerge from the insect cadaver able to re-infect a new host [2, 3]. Three major species have been formally recognized to date within the genus -P. luminescens, P. asymbiotica, and P. temperata. It should be noted however that with increasing numbers of Photorhabdus genome sequences becoming available, the genus structure is under revision [4]. In addition to the normal insect life cycle, P. asymbiotica is also the etiological agent of a serious human infection termed Photorhabdosis, which is associated with severe ulcerated skin lesions both at the initial infection foci and later at disseminated distal sites [5-8].
The Photorhabdus genome encodes a diverse repertoire of virulence genes encoding for protein toxins, proteases and lipases for combating diverse hosts, that can be found in chromosomally encoded pathogenicity islands [9-14]. In addition the bacteria also secrete a potent cocktail of other biologically active small molecules to preserve the insect cadaver in the soil from competing saprophytes and microbial predators such as amoeba [15, 16]. Several classes of Photorhabdus protein insecticidal toxins have now been well characterised including the Toxin Complexes [17-24], the binary PirAB toxins [25-27] and the large single polypeptide Mcf (“makes caterpillars floppy”) toxins [28-30].
A fourth class of highly distinct toxin delivery systems first identified in Photorhabdus are the “Photorhabdus virulence cassettes”, or PVCs [31]. These represent operons of around 16, conserved, structural and synthetic genes (from hereon just described as the structural genes) encoding for a phage “tailocin” like structure [32] and one or more tightly linked downstream toxin-effector like genes. Genomic analysis of multiple strains of Photorhabdus revealed they often encode up to five or six copies of the operon, each with unique downstream effector genes [33].
It should be noted that PVC-like elements are not restricted to Photorhabdus as a well-characterized homologous operon can also be found on the pADAP plasmid of the insect pathogenic bacteria Serratia entomophila [34]. This system has been named the anti-feeding prophage (AFP), as it is responsible for the cessation of feeding in the New Zealand grass grub host. Recent cryo-electron microscopy studies have revealed that, morphologically, AFP resembles a simplified version of the sheathed tail of bacteriophages such as T4, including a baseplate complex. It also shares features with type-VI secretion systems, with the central tube of the structure having a similar diameter and axial width to the Hcp1 hexamer of P. aeruginosa T6SS [35]. One important difference between the PVC and T6SS machinery is that the T6SS relies upon direct contact between host and bacterial cell, and is anchored in to the membrane by a substantial membrane complex whose structure is still being elucidated [36], whereas the PVC needle complex is freely released into the surrounding milieu and so can act at a distance.
Furthermore, recent reports have indicated that other more diverse bacteria can also make similar needle complexes for manipulation of eukaryotic hosts. A well-characterized example is the production of analogous devices by the marine bacterium Pseudoalteromonas luteoviolacea (Figure 1A). These structures are involved in the developmental metamorphosis of the larvae of the tubeworm Hydroides elegans, and they are deployed in outward-facing arrays comprising about 100 contractile structures, with baseplates linked by tail fibres in a hexagonal net [37]. Interrogation of sequence databases with PVC protein sequences suggests many other more diverse tailocin-like systems are yet to be characterized [38]. These include operons closely related to the PVCs in Xenorhabdus bovienii CS03, Yersinia ruckeri ATCC29473 and Vibrio campbellii AND4. In addition, evidence of more diverse elements, like that of P. luteoviolacea, can also be seen. To address this, we have recently performed an exhaustive analysis of all available prokaryotic and archaeal genome sequences in the public databases to look at the distribution of pvc-like elements (unpublished data). This suggests that PVC-like nano-syringes and their distant cousins are of enormous ecological and perhaps biomedical significance.
Here we focus on a single Photorhabdus pvc operon (which elaborates the PVCpnf needle complex [31] to understand the relationship between the structural genes and the tightly linked effector gene, pnf. We confirm in vivo expression during insect infection and reveal a high level of population heterogeneity of expression in vitro. We demonstrate for the first time the physical association of the Pnf effector toxin protein with the secreted structural needle complex using Western blot and electron microscopy. Furthermore, we prove that the cognate Pnf effector needs to be delivered into the eukaryote cell cytoplasm to exert any measurable effect and confirm its predicted activity targeting small Rho-GTPase target proteins. Taken together this work describes an important new class of protein toxin secretion and injection delivery systems which, unlike the well-described Types III, IV and VI systems, can act “at a distance”, requiring no intimate contact between bacteria and host cells.
RESULTS
A bioinformatic analysis of pvc structural operon sequences
A comparison of pvc structural operons identified in the genome sequences of Photorhabdus and certain members of other genera, available at the time of publication ([12, 14, 39] and our unpublished data), allowed us to define three distinct genetic sub-types. The PVCpnf operon belongs to class I, which has 16 structural genes and three translationally coupled gene blocks, and is of the type typically seen in non-Photorhabdus genera. Class II and III operons differ in the number of structural genes and translationally coupled gene blocks (Figure 1B). Given the diversity of pvc-operons, and their typically poor annotation in genome sequences, it is necessary here to define a nomenclature protocol to allow reference to any given operon. An example of the method we have adopted is as follows; [Pa ATCC43949 PVCpnf], where Pa ATCC43949 is species and strain, in this case Photorhabdus asymbiotica strain ATCC43949 and PVCpnf is the specific operon within that genome with the suffix referring to one of the tightly linked effectors, in this case the pnf effector gene. We will also include gene identifiers for either end of the operon where appropriate, which in this case would be PAU_03353-PAU_03332, which are the genes for pvc1 and pnf respectively.
With reference to published literature and a detailed bioinformatic analysis of promoter regions upstream of the pvc1 genes, we can identify two distinct, potential cis-operator sequences. Firstly operons belonging to classes I (e.g. PVCpnf) and III (e.g. PVCPaTox) typically encode the highly conserved RfaH operator sequence, GGCGGTAGNNT [40]. It is possible that more degenerate RfaH operator sequences exist in other operons although this remains unclear. Secondly, all class II operons (e.g. PVClopT) and certain class I operons (e.g. PVCunits1-4) encode a minimal cryptic conserved sequence motif, CAGGTTGXTGCGGTAGCTAT. In both cases these conserved cis-encoded sequences are located between the pvc1 gene and the transcription start sites, as defined by previous RNA-seq analysis ([41] and unpublished data).
Several observations suggest that horizontal gene transfer has been responsible for the dissemination of many observed pvc-operons. These include; the location of the S. entomophila afp on a horizontally transmissible plasmid, the presence of four pvc operons in tandem in P. luminescens TT01 (directly adjacent to a type IV DNA conjugation pilus operon), the presence of multiple pvc operons in any given genome and the suggestion that several operons are regulated by RfaH. While there is no experimental evidence to confirm an exact mechanism by which this may occur, a closer inspection of the sequences flanking [PlTT01PVCu4] suggests that at least this operon was acquired as a composite transposon. Remnants of insertion sequence (IS) elements can be seen flanking this operon, with only the outer inverted repeats remaining intact [TTATATTGAA(t/g)GAATATTAAGCAAGAAAC], and YhgA-like IS transposase genes belonging to the (transposase_31 superfamily) still associated with both the 5’ and 3’ flanks of the PVCu4 operon. It should be noted that IS element remnants could also be seen flanking many other pvc operons suggesting that IS dependant transposition has been a common mechanism involved in pvc horizontal dissemination. However, our own phylogenetic studies suggest that pvc-operons have been co-evolving with their host genomes for some time, indicating that horizontal transfer is likely the method of original acquisition, but may not be as active presently. This is supported by the fact that an automatic prediction of horizontal gene transfer regions (HGTs) using Alien Hunter 1.7 [42] either did not detect any HGT elements spanning the structural regions of PVCs or in the cases where such an element was detected it was assigned a low confidence score (Figure S2).
An analysis of the conservation of individual genes across different pvc operons at both DNA and protein sequence levels suggests that either recombination or diversifying selection is more likely to have occurred in the more 3’ regions of the operons (Figure S1A). This is perhaps no surprise as each pvc operon can be seen to encode different effector genes in the 3’ payload region of the operons. An analysis of conservation of protein sequences of the pvc operons showed that within pvc-operons a good deal of variability is possible while presumably retaining the ability to produce a similar macromolecular structure (Figure S1B). This is supported by HHPRED structural homology comparisons for equivalent PVC proteins across different operons, despite often-variable primary amino acid sequences (data not shown). We note that the most diverse protein seen in pvc-operons is that of the predicted tail fibre proteins, Pvc13, which we may expect if different pvc-operons are adapted for different host cell targets. Paralogous genes within pvc-operons include pvc1 and pvc5 which encode homologs of Hcp, the inner tube protein of contractile tube mechanisms such as T6SS and phage protein Gp27 and pvc2, -3 and -4 which encode homologues of the outer sheath proteins of phage [43] and T6SS [44]. Figure S1C illustrates the organisation of the [PaATCC43949 PVCpnf] operon used as a model system in our experimental studies described here, showing the top HHPRED structural homology hits and predicted roles for each encoded protein at the time of writing.
A bioinformatic analysis of pvc-operon effector gene sequences
A comparison of the 3’ effector “payload regions” of different pvc operons reveals a large diversity of effector genes, with a range of predicted activities, covering a large range of sizes and isoelectric point values (data not shown). Some operons encode only a single putative effector, e.g. [PaATCC43949 PVCPaTox PAU_02249-02230] while others have several, either tandem homologues of one another, e.g. [PaATCC43949 PVCu4 PAU_02790-02808] or entirely unrelated putative effector genes, e.g. [PaATCC43949 PVClopT PAU_02112-02095]. Many effector genes are also tightly linked to transposase gene remnants suggesting they are typically exchanged by horizontal acquisition. This is further supported by the observation that orthologous pvc-operons in the same chromosomal context may have different effector genes in different strains. A good example of this being the unrelated effector genes seen in the orthologous structural “PVCpnf” operon loci of PaKingscliff and PaATCC43949 which carry a tyrosine glycosylase and Pnf (this paper) respectively. Analysis with Alien Hunter 1.7, suggests that certain pvc-operon / effector associations are ancestral to any given species. For example the association of the pvc17 effector with PVCu4, and the multiple linked effectors with the PVClopT operon in both PaATCC43949 and PlTT01. Conversely other pvc-operons show evidence of recent horizontal acquisition of their 3’linked effectors, e.g. PVCcif and PVCpnf (not shown)
Expression of PVC pnf in vitro and in vivo
A previous RNA-seq analysis of global transcription in three strains; P. asymbiotica ATCC43949 [41], P. asymbiotica Kingscliff and P. luminescens TT01 (unpublished) showed condition dependent expression of certain pvc-operons but not all. Therefore, due to the diversity of pvc operons and effectors in Photorhabdus, we focused on a single model class I pvc operon, [PaATCC43949 PVCpnf], to elucidate the relationship between the conserved structural and effector proteins. This operon was selected as it elaborates a well-defined needle complex structure (as observed by electron microscopy) which has potent insect killing activity when heterologously expressed in E. coli [31]. This operon has two putative effector genes in the downstream “payload region”, PAU_03337, which shows similarity to adenylate cyclase toxins (e.g. the anthrax Edema Factor and Pseudomonas ExoY toxin) and pnf (PAU_03332). While the predicted activity of PAU_03337 has not been tested directly, when expressed in the NIH-3T3 cell cytoplasm (in transient transfection experiments) it did produce a highly unusual cytoskeleton phenotype [31]. Pnf (Photorhabdus necrosis factor) is a homologue of the active site domain of the Yersinia CNF2 (Cyto Necrosis Factor 2) toxin, which has small-GTPase deamidase and transglutaminase activities [45].
In order to confirm the expression of this model pvc-operon in Photorhabdus during an insect infection we constructed transcription-translation reporter plasmids in which the promoter regions and the first 150 bp of coding sequence of pvc1, pnf [both from PaATCC43949 PVCpnf] and the P. asymbiotica chromosomal rpsM ribosomal “housekeeping” gene (as a positive control) were genetically fused in frame to a gfpmut2 gene with no start codon (referred to hereon as pvc1::gfp, pnf::gfp and rpsM::gfp reporters). Note, the genomic context and our previous unpublished RT-PCR studies suggested that pnf had its own promoter and could be transcribed independently of the pvc structural genes. As we are unable to transform PaATCC43949 itself, these plasmids were transformed into the well-characterised and genetically tractable strain P. luminescensTT01 to provide suitable reporter strains for in vitro and in vivo expression studies. For in vitro studies we cultured the bacteria in LB medium supplemented with Manduca sexta clarified hemolymph and grown to late stationary phase, before microscopic examination. For in vivo studies, we injected the reporter strains into M. sexta, and allowed the infection to establish before macroscopic examination of insect tissues in situ using a (fluorescence) dissecting microscope. We also took hemolymph samples from these insects and visualised the hemocytes and bacteria microscopically using confocal microscopy.
Figure 2A shows expression of GFP reporter from the rpsM positive control and both the pvc1::gfp and pnf::gfp reporters in LB supplemented with M. sexta hemolymph, although not in all cells of the bacterial population (see below). Furthermore, we also saw expression in bacteria in the ex vivo hemolymph samples taken during infection of live insects (Figure 2A). It was also possible to confirm expression of pnf::gfp in bacteria attached to the insect trachea in localised putative biofilm masses. In this case, while the expected insect melanisation immune response could be seen to have occurred elsewhere on the trachea, it was notably absent from the pnf expressing bacterial biomass (Figure 2B). In order to corroborate the observations made using the plasmid based reporter constructs in P. luminescens TT01 we also performed RT-PCR analysis of transcription of the PVCpnf chromosomal operon in the original PaATCC43939 strain. This confirmed transcription across the operon in vitro when the bacteria were grown at either 28°C or 37°C, although transcription of certain genes was difficult to detect in vivo during Manduca sexta infections (Figure S3).
We subsequently expanded this analysis to include a transcription-translation reporter plasmid for the promoter and pvc1 gene of an orthologue of PVCpnf from a different P. asymbiotica strain, [PaPB68 PVCpnf]. In this case, we used fluorescence microscopy to assess the expression pattern across the growth phases of the originator PaPB68 strain harbouring the reporter plasmid, when grown in LB with aeration and maintaining plasmid marker selection. Interestingly we observed a high level of population heterogeneity in expression with only very few cells expressing GFP at any one time (Figure S4). A similar level of heterogeneity in expression was also seen for reporter constructs from seven other pvc-operons from both PaPB68 and PlTT01 (data not shown). We also assessed expression in biofilms grown statically on glass slides and observed the same pattern, though with even fewer cells seen to express GFP (data not shown).
The Pnf effector protein is physically associated with the PVC needle complex
We investigated if the Pnf effector protein actually becomes physically associated with the pvc-encoded needle complex we had previously visualised by electron microscopy [31]. To do this we raised anti-peptide antibodies against synthetic peptides representing amino acids 206-219 of Pnf (TGQKPGNNEWKTGR) and amino acids 130-143 (DGPETELTINGAEE) of predicted outer sheath protein Pvc2. Previously we used 2D-SDS PAGE analysis of PVCpnf needle complex produced by an E. coli cosmid clone to confirm the presence of Pvc2, along with Pvc1, 3 5, 11, 14 and 16 proteins ([31] and unpublished data). We confirmed specificity of the Pnf antibody using western blot analysis of extracts of E. coli heterologously expressing Pnf alone.
We first used the anti-Pnf peptide antibody to test for the presence of Pnf protein in supernatants from the native bacterial strain PaATCC43949. We tested for the presence of Pnf in needle complex enriched particulate preparations and clarified supernatants. We could detect Pnf in preparations enriched for the complexes but not in clarified supernatants. More specifically, the Pnf protein could only be detected in the needle complex fraction, if it was first either chemically or physically disrupted before electrophoresis (Figure 3B). Taken together these findings are consistent with the hypothesis that the Pnf protein is sequestered inside the needle complex or in some other configuration such that the TGQKPGNNEWKTGR epitope is physically hidden from access by the antibody.
Secondly we enriched needle complexes from insect toxic supernatants of an E. coli cosmid clone that encodes the PaATCC43949 PVCpnf operon, as previously described [31]. The anti-Pnf antibody was used for in situ labelling of Pnf on Transmission Electron Microscopy grids, visualised with negative staining and an anti-rabbit gold-conjugate secondary antibody. It was only possible to detect Pnf protein near the ends of either contracted or damaged needle complexes (Figure 3A). Note we saw no non-specific labelling when the gold-conjugate secondary antibody was used alone. In the case of the Pvc2 antibody, we only saw a signal associated with what appeared to be disrupted fragments of needle complexes suggesting the Pvc2 epitope is not normally solvent exposed in intact needle complexes.
The Pnf protein requires delivery into the eukaryotic cell cytoplasm to exert its effect
In a previous publication we reported that injection of an enriched PaATCC43949 PVCpnf needle complex preparation; heterologously produced by an E. coli cosmid clone, caused melanisation and death of Galleria mellonella larvae within 30 minutes. In addition, microscopic analysis of phalloidin stained hemocytes taken from these dying animals revealed the cells were shrunken with highly condensed cytoskeletons, and likely already dead. This effect was abolished by heat denaturing the preparation. In this same publication [31] we demonstrated that transient cytoplasmic expression of the Pnf protein caused extensive cytoskeleton re-arrangement and likely cell death in cultured human HeLa cells, similar to that observed in the ex vivo G. mellonella hemocytes. In an attempt to directly visualise the interaction of the heterologously produced PVCpnf needle complex with insect hemocytes and to determine the initial effects on the cellular morphology, we injected intact or heat denatured PVCpnf needle complex preparations into 5th instar Manduca sexta larvae before bleeding the animals and preparing their circulating hemocytes for surface examination by cryo-SEM. The surface of hemocytes injected with intact complex showed membrane ruffling consistent with the predicted mode of action of the Pnf protein (see below). Furthermore, we could also see linear structures approximately 150nm in length on the surface of the cells near the sites of membrane ruffles consistent with attached needle complexes. The surface of the control hemocytes injected with heat-denatured complex remained relatively smooth and homogeneous and we saw no equivalent linear structures. Figure 4 shows representative images from these experiments.
We wished to know if the Pnf effector could exert this toxic effect independently of the needle complex, when applied externally to eukaryotic cells. Therefore we heterologously expressed (in E. coli) and purified the Pnf protein in addition to a predicted toxoid derivative. The toxoid was designed based on homology between Pnf and the CNF2 toxin active site, wherein we mutated the cysteine at amino acid position 190 into an alanine (Pnf C190A). Firstly, neither purified wild type nor toxoid proteins had any obvious toxic effect when injected into cohorts of G. mellonella, even at high doses (data not shown). We subsequently used bioPORTER, a liposome based transfection system, to introduce the purified proteins directly into cultured human cells. We visualised effects on the cytoskeleton and nucleus using TRITC-phalloidin and DAPI staining respectively. The wild type Pnf protein had a very clear effect on the cells, producing phenotypes consistent with those predicted by similarity to the CNF2 toxin. CNF2 is known to modify the cellular Rho GTPases, RhoA, Rac1 and Cdc42. Pnf delivery as a bioPORTER formulation lead to the formation of F-actin filaments within 24h followed by multi-nucleation by 48h, phenotypes consistent with the modification of the Rho GTPases. The toxoid derivative, delivered at the same dose using the same approach, produced no changes, giving cellular phenotypes consistent with that of the negative control or of the wild-type Pnf protein topically applied without the bioPORTER transfection agent (Figure 5).
The Pnf protein effector modifies small Rho GTPases
Based on homology to CNF2 the effect of Pnf on target cell proteins is predicted to include the modification of several Rho-family GTPases. Therefore we used western blot assays to examine in vitro transglutamination and deamidation effects of purified heterologously produced Pnf on purified small GTPases RhoA, Rac1 and Cdc42. Transglutamination is the formation of a covalent bond between a free amine group, as may be found on a lysine residue, and the gamma-carboxamide group of glutamine. As a result protein electrophoretic mobility of the protein is altered. Deamidation is a chemical reaction in which an amide functional group is removed from the protein, which may be detected using deamidated protein specific antibodies. These experiments demonstrated that Pnf induced transglutamination and deamidation of both RhoA and Rac1 (Figure 6), although unlike the reported activity of CNF2, had no effect on Cdc42. As predicted the active site toxoid mutant had no enzymic activity on any of the three Rho GTPases confirming it was a true toxoid derivative.
DISCUSSION
An analysis of the different subunit proteins of PVCs show they share several elements in common with other contractile phage-tail derived systems, including the Type VI secretion system (T6SS) [46] and to a lesser extent R-type pyocins [47]. However PVC-like elements are distinct in two important ways. Firstly, unlike the T6SS, they are freely released from the producing bacterial cell and so, in common with R-type pyocins, they can act at a distance. Secondly, like T6SS but unlike R-type Pyocins, they are evolved to inject bioactive protein effectors into other cells. We hypothesise that the PVCs are evolved to specifically target eukaryotic cells, unlike T6SS, which have been shown to be able to deliver to both eukaryotes and prokaryotic competitors. However, while our previous attempts to show PVCpnf attachment to a range of bacterial species from different genera showed no binding we could detect (data not shown), we cannot rule out the possibility that homologues exist which are able to target prokaryotes.
We speculate that these large protein complexes are costly for the cell to produce, consistent with the observation of population heterogeneity of pvc-operon expression. Indeed, uncontrolled heterologous over expression in E. coli, as cosmid clones, results in deletion of regions of the pvc-operon and loss of viability (unpublished data). It should be noted that in a natural insect infection the vast majority of the Photorhabdus bacterial population are sacrificial. The majority of the population act as a food source for the replicating nematodes, with very few cells passing into the next generation of infective juvenile nematodes [2]. As such the population may restrict PVC production to a limited number of sacrificial cells. The method of PVC release remains unclear, although to date we have not observed cell lysis associated with pvc expression. The finding that PaATCC43949 PVCpnf, and seven other pvc-operons from PaPB68 and PlTT01, all show population heterogeneity in expression, at least in vitro, suggests that they are likely deployed in a highly regulated and conservative manner. While it is difficult to fully characterise this heterogeneity in vivo, the PVCpnf GFP reporter strain did show restricted expression to one specific tissue, the spiracles, and not throughout the body of the insect. In regards to these experiments, it should be noted that we did not see any melanisation response around the bacterial biomass showing GFP. The insect melanisation immune response is typically activated at sites of encapsulation. This is mediated by the recruitment of hemocytes, surrounding and enclosing foreign bodies, and entombing them in melanin. The absence of melanisation around this GFP expressing bacterial mass is consistent with the expression of anti-hemocyte virulence genes, which are likely to include the native PlTT01 pvc-operons.
Examination of the promoter regions has provided no clue as to the mechanism of population heterogeneity of expression. Nevertheless the identification of RfaH and a second cryptic conserved potential operator sequence upstream of the pvc1 genes provides a starting point for addressing this in future. RfaH is a conserved anti-termination protein that is known to regulate large operons encoding for extracellular factors in E. coli. It is also believed to be important in ensuring appropriate transcriptional control of horizontally acquired operons [40]. Many of the pvc-operons in Photorhabdus and members of other genera (including Xenorhabdus and Yersinia) encode this operator sequence. An unusual example is the pADAP plasmid encoded Serratia entomophila anti feeding prophage (afp) [34]. While the afp promoter also encodes an RfaH operator sequence, it has been demonstrated that it is positively regulated by a tightly linked specific regulator protein, AnfA1 [48, 49]. This protein is a distant homologue of RfaH suggesting that other class I or III pvc operons are not necessarily under the regulation of the chromosomal RfaH orthologue, but might also be controlled by other diverse regulators that utilise this same operator sequence [50]. Operons containing the second cryptic putative regulatory sequence include [Pl TT01PVClopT] and [Pl TT01 PVCu4]. Analysis of the supplementary data from a recently published RNA-seq study [51], suggests that these operons may be dependent upon Hfq/HexA activity [52].
Unlike many of the other genera in which we see pvc-like operons, Photorhabdus genomes encode multiple copies, typically around 5 to 6, suggesting they play important and diverse roles in the life cycle. With this in mind, we examined the conservation of the different subunit genes between operons. We observe a “break point” in conservation, toward the 3’ end of the operons. We postulate this may be due to imprecise recombination events in the 3’ payload regions of pvc-operons, where incoming sequences, which have a GC-content that is distinct from the host genome, gradually ‘erode’ the upstream sequence. Alternatively, it is plausible that the lower GC at the distal end of these long operons (each of ∼25kb) may assist in strand separation during transcription, maintaining stoichiometry for these large, multi-subunit structures. Indeed low GC stretches of DNA are common origins of replication because of their reduced strand separation energy [53]. However, as yet we do not know whether the pvc1 promoter serves the whole operon, or if there are additional promoters internal to the operon.
Each of the pvc-operons in a Photorhabdus genome encodes multiple paralogous copies of pvc1/5 and pvc2/3/4 genes. We were therefore surprised not to see any operons showing signs of genetic degradation. This suggests there is sufficient positive selection for maintaining these multiple operons, with each operon potentially adapted for a specific role. This hypothesis is supported by the high variation in the Pvc13 protein sequences, which we speculate represent the host cell binding fibres. The need to maintain multiple copies of pvc-operons may also have arisen if the structural genes for the needle complexes are specifically adapted for delivery of their cognate cis-linked effector proteins in some way.
Circumstantial evidence from genomic sequences and previous work on the related AFP system of Serratia has suggested the needle complexes serve to deliver the cis-encoded effector proteins. We present here for the first time direct evidence that a linked effector protein does in fact become physically associated with the needle complex. Western blot detection of Pnf from preparations enriched for needle complexes taken from the native PaATCC43949 supernatants confirmed it was being expressed in vitro and suggested it was physically associated with the complexes. In addition, physical or chemical disruption was required to release the Pnf protein for detection. When taken alongside the immuno-gold EM observations, showing Pnf could only be seen near contracted or damaged needle complexes, it confirms the protein is either sequestered inside the complex or physically associated in such as way that the TGQKPGNNEWKTGR epitope is not solvent accessible. The anti-Pvc2 antibody is able to specifically detect the protein in Western blots, however it only showed binding to what appeared to be disrupted fragments of needle complexes, again suggesting the relevant epitope is not accessible in the intact native needle complex structure. Indeed, iTasser structural model simulations of a PVC outer sheath Pvc2 protein, using the homologous Pseudomonas 3J9Q PDB structure of an R-type pyocin outer sheath as a model [54], supports this idea, suggesting the epitope is partially occluded between adjacent subunits.
While we have not yet directly demonstrated injection of Pnf into host cells by the needle complex, the results of the topical application and bioPORTER transfection experiments confirmed that the Pnf effector absolutely requires a mechanism to facilitate entry into the host cell cytoplasm to exert its effect. We argue the evidence for injection by the needle complex is very strong, and is corroborated by the SEM visualisation of needle-like structures of the correct dimensions on the surface of intoxicated hemocytes. Finally, we have confirmed that Pnf acts in a manner similar to the Yersinia CNF2 toxin, modifying two of the same Rho-GTPases, which correlates with the observed phenotypic effects on the cell.
MATERIALS AND METHODS
Insects, bacterial strains and growth conditions
Manduca sexta (Lepidoptera: Sphingidae) were individually reared as described [55]. Briefly, larvae were maintained individually at 25°C under a photoperiod of 17 h light: 7 h dark and fed on an artificial diet based on wheat germ. Larvae 1 day after ecdysis to the 5th instar were used for all experiments. Batches of wax moth larvae (75 g; Livefood UK Ltd, Rooks Bridge, UK) in their final instar stage were stored in the dark at 4°C and used within a week of receipt. DH5α™ E. coli (containing various plasmid constructs) were grown on LB agar at 37°C or in LB liquid, shaking at 200 rpm. Spontaneous rifampicin-resistant mutants of Photorhabdus asymbiotica subsp. asymbiotica Thai (strain PB68.1) [56] and Photorhabdus luminescens subsp. laumondii TTO1 [12] were used in these studies as hosts for reporter plasmids. Photorhabdus were routinely cultured in LB broth or on LB agar supplemented with 0.1 % (w/v) pyruvate at 30°C or 37°C (for P. asymbiotica). When required antibiotics were added at the following concentrations: ampicillin (Amp): 100 μg ml-1, kanamycin (Km): 25 μg ml-1, chloramphenicol (Cm): 25 μg ml-1, rifampicin (Rif): 25 μg ml-1. HeLa ATCC CCL2 cells were cultured for 10 passages in Dulbecco’s modified Eagle medium (Sigma-Aldrich) containing 4.5 g/L glucose (Sigma-Aldrich), 10% heat-inactivated fetal bovine serum (Sigma-Aldrich), 2 mM glutamine (Sigma-Aldrich), 100 μg /mL penicillin, and 100 μg/mL streptomycin (Sigma-Aldrich) and incubated at 37°C and 5% CO2.
PVC gene reporter plasmid construction
Translational fusions with the gfpmut2 gene were constructed by PCR in a pACYC184 vector containing the gfpmut2 (pACYC-GFP) [57] as follows. The pvc1, pnf and rpsM genes (consisting of promoter regions and the first 150 bp of coding sequence) were amplified from P. asymbiotica ATCC43949 genomic DNA and cloned into pACYC-gfp to generate pACYC-afp1-gfp, pACYC-pnf-gfp and pACYC-rpsM-gfp. The constructs were further digested to release the pvc1, pnf or rpsM genes in frame with gfp and the fusion fragments were cloned into pBBR1-MCS [58] to generate pBBR1-pvc1-gfp, pBBR1-pnf-gfp and pBBR1-rpsM-gfp. Mating experiments were performed as previously described [59] to transfer plasmid constructs into P. luminescens TTO1 resulting in PlTTO1-pvc1-gfp, PlTTO1-pnf-gfp and PlTTO1-rpsM-gfp. Plasmid stability was confirmed in bacteria harbouring the various constructs isolated after in vivo passages. For the expanded panel of gfp-reporter fusions, the promoter regions for the operons selected, inclusive of the putative RfaH operator sites, and the native RBS and first codon of the pvc1 gene (approximately 500 bp upstream), were cloned in to the pAGAG vector. pAGAG was derivatised from the promoterless pGAG1 gfp bearing plasmid., In brief, pGAG1 was used as a template to amplify gfpmut3* without a start codon using primers pG_GFPfor (5’-AATGTCGACCGTAAAGGAGAAGAACTTTTC-3’) and pG_GFPrev (5’-AATACTAGTGGATCTATTTGTATAGTTCATCCATG-3’). The resulting product and the pGAG1 vector were cut by digestion with SalI-HF and SpeI and ligated together, thus replacing the original intact gfpmut3* gene with one that lacks a ribosome binding site and the first ATG codon. Thus, 5’ regions introduced subsequently restored the construct. All upstream regions were incorporated between the KpnI and BamHI sites of the resulting pAGAG vector. The Pnf reporter discussed in this paper specifically (Figure S4), was amplified using the primers (PB68.1Pnf-BamHI F 5’-ATAGGATCCATCCCAACGTATCTTGTCC-3’ and PB68.1Pnf-KpnI R 5’-ATTGGTACCTGTACTTGTAGACATAAAAGCCC-3’
Fluorescent reporter strain assays
In vitro experiments
Reporter strains were cultured with shaking aeration in LB liquid medium supplemented with 20% (v/v) freshly harvested 5th instar M. sexta clarified hemolymph. To obtain the hemolymph, insects were chilled on ice for 20 minutes before being bled (by cutting the tip of the tail horn) into a tube on ice, containing 10 μl of saturated Phenol Thio Urea (PTU) solution, which prevents melanisation. Hemolymph was clarified by centrifugation to remove hemocytes and other debris. Bacteria were grown to late stationary phase, before microscopic visualisation using a Leica inverted epi-fluorescent microscope. In vivo experiments, we injected ca. 100 cells of the reporter strains into cohorts of 5th instar M. sexta, and allowed the infection to establish before macroscopic examination of insect tissues using a (fluorescence) dissecting microscope. We also took hemolymph samples from these insects and performed microscopic examination of fixed ex vivo hemocytes stained with phalloidin conjugate and confocal microscopy to visualise host cell cytoskeleton and any GFP expression from the recombinant bacteria. Images were acquired with a LSM510 confocal microscope (Leica).
PVC purification from E. coli cosmid clone supernatants and electron microscopy
Cosmid libraries of P. asymbiotica ATCC43949 were prepared in E. coli EC100 and arrayed into 96-well microtiter plates by MWG Biotech, Munich, Germany, as described previously [13, 28]. A 250ml overnight culture of E. coli with the PaATCC43949 PVCpnf cosmid (c4DF10) was grown in LB medium supplemented with 100 μg ml-1 ampicillin at 28°C with aeration in the dark. The culture was centrifuged at 6800 x g at 4°C for 30 min at 4°C. The supernatant was decanted to remove each cell pellet, and the centrifugation procedure was repeated to remove any remaining cells. Cell-free supernatants were then centrifuged, in small batches, at 150,000 × g for 90 min at 4°C to harvest particulate material. The particulate pellets were washed by gentle re-suspension in 1× Phosphate Buffered Saline (PBS) before a second centrifugation at 150,000 × g for 90 min at 4°C to pellet the particulate material. Each pellet was further separated by DEAE-Sepharose chromatography. 10 ml of particulate material in ice-cold PBS were mixed with an equivalent volume of DEAE-Sepharose CL-6B anion exchanger (in PBS) and the preparation was incubated at room temperature for 15 min. The Sepharose resin was harvested by centrifugation (3,000 × g), and the supernatant was discarded. The resin was resuspended in 40 ml of ice-cold PBS and again harvested by centrifugation. This washing step was repeated another three times, and the resin was finally resuspended in 10 ml of elution buffer (0.5 M NaCl, 50 mM phosphate buffer [pH 7.4]). The resin was removed by centrifugation, and the supernatant containing the PVCs was again centrifuged at 150,000 × g for 90 min at 4°C to pellet the particulate material and concentrate the needle structures in 500 μl of ice-cold PBS.
For transmission electron microscopy (TEM) pioloform-covered 300-mesh copper grids that were coated with a fine layer of carbon were used as substrates for the protein fractions. The following four aqueous negative stains were tested with the protein samples: 1% uranyl acetate, 3% ammonium molybdate, 3% methylamine tungstate, and 2% sodium silicotungstate. The preferred stain, 3% methylamine tungstate, produced acceptable contrast and minimum artefacts and was subsequently used for all samples viewed by TEM. The coated grids were exposed to UV light for 16 h immediately prior to use to ensure adequate wetting of the substrate. A 10 μl drop was applied to the TEM grid, and the protein was allowed to settle for 5 min. Liquid was absorbed with filter paper from the edge of the grid and replaced immediately with 10 μl of filtered negative stain. The drop was partially removed with filter paper, and the grids were allowed to air dry thoroughly before they were viewed with a JEOL 1200EX transmission electron microscope (JEOL, Tokyo, Japan) operating at 80 kV.
Pnf cloning and heterologous expression for Galleria injection and antibody specificity test
Pnf gene was amplified from P. asymbiotica ATCC43949 genomic DNA (using primers Pnf_NdeI 5’-ATATATCATATGATGTTAAAATATGCTAATCCT-3’, Pnf_BamHI 5’-ATATATGGATCCTTATAACAACCGTTTTTTAAG-3’) and the PCR product was purified and cloned in-frame with a His-tag into the IPTG-inducible expression plasmid pET-15b (Novagen) to create construct pET15b-Pnf. The clone was verified by sequencing and transformed into Arctic Express competent cells (Agilent) for protein expression. A site-directed mutant of Pnf (toxoid) was generated with either the QuikChange site-directed mutagenesis kit (Agilent). To construct the Pnf mutant plasmid pET15b-PnfC190A, pET15b-Pnf was amplified with FPLC-purified primers designed to generate a Cys to Ala substitution at position 190 (PnfC190A_for 5’-TCACCGAATATACCATAGTAGCACCGCTCAATGCTCCAGAC-3’, PnfC190A_rev 5’-GTCTGGAGCATTGAGCGGTGCTACTATGGTATATTCGGTGA-3’) using the following thermal profile (step 1: 95°C for 30 s, step 2: 95°C for 30 s, 55°C for 60 s, 68°C for 6 min 45 s for 16 cycles). The identity of six different positive clones was confirmed by sequencing. Subsequently, −80°C glycerol stocks were used to inoculate 5 ml of fresh LB medium supplemented with 0.2% (w/v) glucose and 100 μg ml-1 ampicillin. Bacteria were grown overnight at 30°C with shaking, and 1 ml of the culture was then harvested, re-suspended in 100 ml of the same medium, and incubated in an orbital incubator at 37°C until the optical density at 600 nm was 0.7 to 0.9. Cells were then harvested at room temperature by centrifugation at 4,000 rpm for 10 min. The pellet was re-suspended in 100 ml of fresh LB medium supplemented with the 100 μg ml-1 ampicillin and 0.1 mM of the inducer isopropyl-β-d-thiogalactopyranoside (IPTG). Optimized times for inductions were determined experimentally, and cells were then harvested. The bacterial cell pellet was re-suspended in 10 ml of 1x PBS and sonicated (four 20-s sonications at 45 mA using a Branson 450 digital Sonifier) fitted with a tapered probe. The freshly sonicated samples were then diluted in 1x PBS for injection into Galleria larvae and for SDS-polyacrylamide gel electrophoresis analysis to confirm expression of the target protein. For toxicity testing cohorts of Galleria larvae (n=20) were chilled on ice before injection with 10 μl of a dilution series (in sterile PBS) of sonicated cells expressing Pnf or vector control. Insects were then returned to room temperature and observed for 5 days or mortality or morbidity.
Recombinant Pnf and small Rho-GTPase purification
ArcticExpress containing pET-15b-Pnf were initially grown in LB broth supplemented with 100 μg ml-1 ampicillin at 37°C until OD 0.6 when Pnf expression was induced with a final concentration of 0.1 mM of IPTG at 12°C for 16 h to produce soluble Pnf. Pnf was purified over HisTrap™ Ni2+-affinity column with the fast phase liquid chromatography (FPLC) AKTA system as per the manufacturer’s protocol (GE Healthcare). Plasmids pGEX-2T-wtRhoA, pGEX-2T-wtRac1 and pGEX-2T-G25K (Cdc42) were gifts from Prof Alan Hall (University College London, London, UK) and were maintained in E. coli DH5α grown on LB agar or in LB broth supplemented with 50 μg ml-1 ampicillin. RhoA, Rac1 and Cdc42 were purified over GSTrap HP™ affinity columns with the FPLC AKTA system as per the manufacturer’s protocol (GE Healthcare).
BioPORTER assay and actin stress fibre analysis
For BioPORTER assays, 80 μl of purified wild type and mutant Pnf proteins (500 μg ml-1), or PBS as a negative control, were added to one BioPORTER tube (Genlantis) and resuspended in 920 μl of DMEM. The samples were added to HeLa cells grown in 6-well plates and incubated for 4 h. BioPORTER/protein or PBS mixes were replaced by fresh complete medium and the cells were incubated for 20–48 h. To visualize cell morphology and actin cytoskeleton, cells were fixed for 15 min in 4% PBS-formaldehyde, permeabilized with 0.1% Triton X-100 and stained with Tetramethylrhodamine B isothiocyanate (TRITC)-phalloidin (Sigma) and DAPI dihydrochloride (Sigma). Images were acquired with a LSM510 confocal microscope (Leica).
Deamidation and Transglutamination of Rho GTPases
Deamidation assay
Deamidation assays were done according to previously described procedures [60] with the following modifications. Briefly, a 20:1 molar ratio of GTPase (RhoA, Rac1 or Cdc42) to toxin was incubated in deamidation buffer (50 mM NaCl, 50 mM Tris-HCl pH 7.4, 5 mM MgCl2, 1 mM DTT, 1 mM phenylmethanesulphonyl fluoride) for either 30 min or 2.5 h at 37°C. Untreated RhoA served as a negative control. After toxin treatment, samples were concentrated by the addition of 10% trichloroacetic acid and stored overnight at 4°C. Precipitated proteins were pelleted, washed with acetone, air-dried and resuspended in 20 mM Tris-HCl pH 7.4. Samples were subjected to SDS-PAGE and analysed by Western blotting using either an anti-RhoA (1:1500, Santa Cruz Biotechnology), anti-Rac1 (1:5000, Upstate Biotechnology), or anti-Cdc42 (1:1000, Santa Cruz Biotechnology) monoclonal antibody or rabbit polyclonal antisera (1:2000) that had been raised against a peptide antigen specifically designed to detect modified/deamidated RhoA/Rac1/Cdc42 [61], provided by Prof A. D. O’Brien, Department of Microbiology and Immunology at Uniformed Services University, Maryland, USA). Reactive proteins were detected with either the HRP-conjugated goat anti-mouse IgG (Sigma) or donkey anti-rabbit IgG (1:3000, Sigma) followed by visualization with DAB (Sigma). Transglutamination assay: Transglutamination assays were done as previously described [62] with several modifications. Briefly, a 2:1 molar ratio of RhoA to toxin was incubated in transglutamination buffer (50 mM Tris-HCl pH 7.4, 8 mM CaCl2, 5 mM MgCl2, 1 mM DTT, 1 mM EDTA) in the presence of ethylenediamine (50 mM, pH 9) for 10 min or 1 h at 37°C. As a negative control, RhoA was incubated with ethylenediamine but without toxin. Samples (0.25 μg RhoA/well) were subjected to SDS-PAGE and then processed for Western blot analyses as described above. Immunoblots were probed with a mouse anti-RhoA monoclonal antibody (1:1500, Santa Cruz Biotechnology) and reactive proteins visualized with DAB after incubation with the HRP-conjugated goat anti-mouse IgG secondary antibody.
SUPPLIMENTARY INFORMATION
SUPPLIMENTARY METHODS
A bioinformatic analysis of pvc structural operon sequences
DNA sequences for each of the 16 conserved structural loci were clustered syntenically (all pvc1s, all pvc2’s etc.). % GC content for each CDS in each syntenic position was calculated (up to 16 observations per locus), and plotted as a boxplot via ggplot2 (Figure S1A). The average GC content across the full operon, as well as for the whole genome, were plotted as intervals in the plot background to show the PVC loci %GC in contrast. The breakpoint was defined by use of the “cumSEG” package in R [63]. Amino acid similarity scores (Figure S1B) were generated by CLUSTAL Omega [64] multiple sequence alignment, using default parameters. Resulting pairwise alignment scores were plotted as boxplots using ggplot.
RNA purification and RT-PCR
For in vitro transcription analysis, overnight cultures of P. asymbiotica were sub-cultured into liquid LB medium and grown with aeration at 28°C or 37°C 200 rpm in the dark. Planktonic cultures were collected at 4, 8 and 24 h and mixed with a double volume of RNAlater (Ambion) and after 5 minute incubation, bacteria were harvested by centrifugation and the pellets stored at −80°C. For in vivo transcription analysis, overnight cultures of P. asymbiotica were extensively washed in PBS and diluted in Grace’s insect media (GIM) to achieve 1000 bacteria per 50 μl of culture. Each M. sexta larvae was injected with 50 μl of P. asymbiotica culture and they were placed in a humid temperature controlled room at 28°C. After 3h or 6 h of incubation, insects were bled in equal volume of GIM containing 20mM phenylthiocarbamide (PTC). The sample was initially fractionated into plasma and total hemocytes by centrifugation at 200 x g at 4°C for 5 min, and plasma was further centrifuged at 6800 x g at 4°C for 5 min to form a bacterial pellet. For each condition, total RNA was extracted using the RNeasy Mini Kit (Qiagen) and 2 μg total RNA was treated with TURBO DNA-free Kit (Ambion) and subjected to RT-PCR using the Qiagen OneStep RT-PCR kit. Each RT-PCR reaction performed in a volume of 50 μl (containing 100 ng template RNA, 1x QIAGEN OneStep RT-PCR buffer, 400 μM dNTPs, 0.6 μM gene specific primers, 5U RNase inhibitor and 2 μl of QIAGEN OneStep RT-PCR enzyme mix) for 28 cycles.
SUPPLIMENTARY FIGURES
ACKNOWLEDGEMENTS
This work would not have been possible without the much appreciated funding by BBSRC grants BB/C008367/1 and BB/E021328/1, The Leverhulme Trust grant RPG-2015-194, EPSRC (MOAC) DTP EP/F500378/1, MRC DTP in Interdisciplinary Biomedical Research MR/N014294/1 and the Warwick University Medical School. We would also like to acknowledge Chris Apark for maintaining and supplying the Manduca sexta insects from the University or Bath colony.