Structural Insights into the Cytotoxic Mechanism of Vibrio parahaemolyticus PirAvp and PirBvp Toxins.

In aquaculture, shrimp farming is a popular field. The benefits of shrimp farming include a relatively short grow-out time, high sale price, and good cost recovery. However, outbreaks of serious diseases inflict serious losses, and acute hepatopancreatic necrosis disease (AHPND) is an emerging challenge to this industry. In South American white shrimp (Penaeus vannamei) and grass shrimp (Penaeus monodon), this disease has a 70-100% mortality. The pathogenic agent of AHPND is a specific strain of Vibrio parahaemolyticus which contains PirAvp and PirBvp toxins encoded in the pVA1 plasmid. PirAvp and PirBvp have been shown to cause the typical histological symptoms of AHPND in infected shrimps, and in this review, we will focus on our structural understanding of these toxins. By analyzing their structures, a possible cytotoxic mechanism, as well as strategies for anti-AHPND drug design, is proposed.

1. Introduction

Acute hepatopancreatic necrosis disease (AHPND), which was originally known as early mortality syndrome (EMS), first broke out in China in 2009, then spreading to Vietnam, Malaysia and Thailand [1,2]. Because of this disease, shrimp production dropped to ~60% compared with 2012, and total economic losses have been estimated at more than $1 billion per year, globally [3]. The causative agent of AHPND was soon found to be a specific strain of Vibrio parahaemolyticus. V. parahaemolyticus is a halophilic Gram-negative bacterium that is commonly found in estuarine, marine and coastal environments [4], and originally it was not known how this opportunistic bacterium had become virulent and capable of causing disease in shrimps.

In addition to the readily observable symptoms in infected P. monodon and P. vannamei—lethargy, an empty stomach and midgut, and a pale to white atrophied hepatopancreas [4]—histological examination of the diseased shrimp further showed that the HP tubule epithelial cells sloughed into the HP tubule lumens [4,5]. Meanwhile, in the initial, acute stage of AHPND, even when a large number of bacteria could be found in the stomach, there were still sometimes no obvious bacterial colonies in the hepatopancreas tube lumens [1,4,6]. This led Tran et al. to propose that the symptoms of AHPND were caused by a toxin secreted by the pathogen [4]. This proposal was further supported by reverse gavage experiments in which introduction of the bacteria-free supernatant of the bacterial culture into healthy shrimp induced typical AHPND symptoms [4,7].

Subsequent investigations focused on isolating AHPND variants [8] and on comparing the draft genome sequences of AHPND-causing versus non-AHPND-causing strains [1,9,10,11,12]. By using a next-generation sequencing (NGS) platform to sequence and compare three virulent (3HP, 5HP and China) and one non-virulent (S02) V. parahaemolyticus strains [9], Yang et al. (2014) found that a 69-kb extrachromosomal plasmid was present in all AHPND-causing strains but not in the non-virulent strain. This plasmid was named pVA1, and sequence analysis showed that it contained homologs of the insecticidal Photorhabdus insect-related (Pir) binary toxin PirA/PirB [13]. The importance of these two toxins to AHPND was confirmed by subsequent studies [14,15,16], and they are now referred to as V. parahaemolyticus PirA/PirB (PirA/PirB).

2. The Structural Similarity between V. parahaemolyticus PirA/PirB and Bacillus thuringiensis Cry Toxins

Photorhabdus PirA and PirB were first reported as potential toxins by genomic sequencing of the entomopathogenic bacterium Photorhabdus luminescens W14 [17], and in 2009, Waterfield et al. reported that both Photorhabdus PirA and PirB were necessary for the insecticidal activity against caterpillars of the moth Galleria mellonella [18]. Although sequence similarity had previously led to Photorhabdus PirB being initially identified as a juvenile hormone esterase-like (JHE-like) protein [19], Waterfield et al. found that Photorhabdus PirB did not have JHE activity [20], and another study further showed that it had sequence similarity to the pore-forming domain I of the B. thuringiensis Cry toxin [21]. However, although it was established that Photorhabdus PirA/PirB was an effective insecticidal binary toxin [18,20,21,22], its cytotoxic mechanism remained unclear.

The first crystal structures to be reported for any PirA/PirB toxins were for V. parahaemolyticus PirA and PirB, and the accompanying structural analysis also suggested a relationship between B. thuringiensis Cry and PirA/PirB toxins [13]. Cry proteins are one of the B. thuringiensis insecticidal toxins, and they have an important potential use in agriculture [23,24]. Although Cry toxins can be divided into at least 75 primary subgroups, and can show differences in their amino acid sequences, the determined and predicted structures of almost all of the Cry toxins are similar [25]. Cry toxins have three functional domains: the pore-forming domain I, the receptor-binding domain II and the sugar-binding domain III [23,24,25,26,27,28,29]. The specificity and cytotoxic mechanisms of Cry toxins are mediated by these three domains, and they have been discussed in many review articles [23,24,25,28,29,30,31]. For example, B. thuringiensis Cry1A uses domains II and III to target receptors that are abundant in the midgut of insect larvae, such as alkaline phosphatase (ALP) or aminopeptidase N (APN). The concentrated Cry1A toxins then interact with another receptor, cadherin-like receptor (CAD), which facilitates the proteolytic cleavage of its domain I helix α1. This cleavage induces the formation of the Cry oligomer, which uses the activated domain I to form non-selective pores in the apical membrane. This causes colloidal osmotic lysis of the cells.

Figure 1 shows the crystal structure of the PirA and PirB toxins. Figure 2 shows how PirB corresponds to Cry domains I and II, while PirA has similar topology to Cry domain III. These structural similarities suggest PirA/PirB binary toxin is a Cry-like, three-domain toxin, but with a dissociated domain III [13,27]. The following sections discuss this idea in more detail.

Figure 1

Crystal structures of PirA (left) and PirB (right) toxins. The α-helices and β-strands are shown in red and yellow, respectively. PirA has a jelly-roll topology which is folded into an eight-stranded antiparallel β-barrel. PirB has two domains with distinct structural features: the N-terminal of PirB (PirBN; residues 12–256) forms a seven-α-helix bundle; while the C-terminal (PirBC; residues 279–436) contains two pairs of four-stranded antiparallel β-sheets. PirBN and PirBC are connected by a long loop. The PDB codes 3X0T and 3X0U were used to produce the figures for PirA and PirB, respectively.

Figure 2

A detailed comparison between structures of B. thuringiensis Cry and PirA/PirB toxins. The α-helices and â-sheets of Cry domain I and PirA/PirB are colored cyan and magenta, and red and yellow, respectively. (A) A comparison between Cry domain I and PirBN; (B) Inside the α-helical bundle of PirBN. The hydrophobic residues Leu, Ile, Val, Met, Phe, Trp and Cys are shown in yellow; (C) A comparison between Cry domain II and PirBC showing the receptor binding loops of Cry domain II. A possible receptor-binding region of PirBC is proposed based on a structural comparison to Cry domain II; (D) A comparison between Cry domain III and PirA; (E) A potential ligand-binding site of PirA. GalNAc is shown docked into the structure of PirA using the docking tool iGEMDOCK [35]. Briefly, each atom of the residues and the compound was first assigned an atom type (e.g., donor or acceptor) and formal charge based on their physiochemical properties. The scoring function of iGEMDOCK was then used to measure intermolecular interactions between PirA and GalNAc. In this docking model, the oxygen heteroatom of GalNAc forms hydrogen bonds with residue Lys29. Residue Glu36 yields a hydrogen bond with one of GalNAc’s hydroxyl groups. Gly38 is a non-polar residue that is sandwiched in close proximity to two hydroxyl groups. Residues Val37 and Arg84 interact with the compound via van der Waals forces. The PDB code 1CIY was used to produce the figures for the Cry toxin.

2.1. PirBvp Contains Both Cry-Like Pore-Forming and Receptor Domains

Both the N-terminal domain of PirB (PirBN) and Cry domain I contain a bundle of α-helices (Figure 2A) [13]. Figure 2B further shows that there are abundant hydrophobic residues located in the center of the PirBN α-bundle, and that the hydrophobic α-helix 8 of PirBN is sheltered within a bundle of amphipathic α-helices. This “inside-out membrane fold” is consistent with other pore-forming toxins that can switch between soluble and transmembrane conformations [32]. After triggering conformational change, these hydrophobic residues become exposed on the surface of the protein, where they are able to interact with membrane lipids. It should also be noted that these helices are generally longer than 40 Å, which is sufficient to cross the cell membrane (the length of the lipid bilayer is ~40 Å). Similar features are seen in the pore-forming domain I of Cry toxin, as well as in other pore-forming toxins, like colicin [28,29,32,33]. All of this strongly suggests that PirBN has the ability to form a pore on the cell membrane that causes cell death. Meanwhile, the C-terminal domain of PirB (PirBC) has three antiparallel β-sheets arranged in a manner similar to that seen in Cry domain II (Figure 2C) [13]. Since the Cry domain II contains an immunoglobulin-like folding that is involved in protein–protein or protein–ligand interactions [34], it seems likely that the PirBC domain plays a similar functional role. Further, since Cry domain II could interact with insect receptors [23,24,25,26,29,30,31], the structural similarity suggests PirBC is also a receptor binding domain.

2.2. PirAvp Contains a Possible Sugar-Binding Pocket

Like PirB, the biological functions of PirA may also be revealed by its structural features. PirA contains two antiparallel β-sheets that are packed together in a jelly-roll topology [13]. This folding is similar to domain III of the Cry toxin (Figure 2D). Cry domain III contains a galactose-binding domain-like fold [36,37]; this is thought to be related to the toxin’s specificity via its recognition of receptor-bound N-acetylgalactosamine (GalNAc) [23,24,25,26,29,30,31,36,37,38]. In the interaction between Cry1Ac and APN, Cry1Ac domain III first interacts with the GalNAc sugar on the APN receptor to facilitate the subsequent toxin-receptor binding [23]. PirA does indeed play a similar role to Cry domain III, then it should facilitate target-specific recognition by binding to certain ligands on the cell membrane/receptor. Interestingly, a potential sugar-binding cavity formed by three loops was found in PirA (Figure 2E). The docking model shows that when the GalNAc molecule was fitted into this cavity, it could potentially interact with the PirA residues Lys29, Glu36, Val37, Gly38 and Arg84 (Figure 2E). We further note that, since the potential binding cavity of PirA is deep and narrow (Figure 2E), it may be possible that PirA not only targets the monosaccharides like GalNAc, but also oligosaccharides.

2.3. Unanswered Questions Relating to the Cytotoxic Mechanism of PirAvp/PirBvp

We have shown that the PirA/PirB toxin has structures that are similar to the functional domains of Cry. This further suggests that PirA/PirB might also induce cell death via the respective Cry-like steps of receptor binding, oligomerization and pore forming. To explore this model, identification of the cell receptors that might interact with PirA/PirB is a logical place to start. We note that although the main folding of Cry domain II and PirBC is similar, the loop regions between these two domains are quite different (Figure 2C). Since the loop á-8, loop 2 and loop 3 of Cry Domain II are very important to aminopeptidase N (APN)-, alkaline phosphatase (ALP)- and cadherin (CAD)-receptor binding [26,39,40,41], these divergent loop regions suggest either that the toxin-interacting regions on shrimp’s APN, ALP and CAD receptors are different to those found in insects, or else that PirB targets different receptors on the shrimp cell’s membrane. In either case, given that PirA/PirB toxin induces cell death in the shrimp’s hepatopancreas, but not in the stomach or other organs, it seems very likely that these putative PirA/PirB receptors will be found exclusively in the hepatopancreas membrane. However, we caution that there is as yet no experimental evidence in support of this; at present, the structure of these shrimp receptors remains unknown. We also note that several other critical processes still need to be investigated experimentally. For example, we do not yet know whether the cleavage of N-terminal á-helices on PirB is important for toxin activation, or whether PirA/PirB forms an oligomer in order to make a pore in the membrane.

Determination of the binding ligand of PirA is also worth investigating. Although the binding model between PirA and GalNAc seems reasonable, this interaction still needs to be confirmed by experiments such as surface plasmon resonance. To explore more possibilities, a high-throughput screening of PirA bound ligands would be useful, and we note that a feasible chip platform designed for carbohydrate-protein interactions has recently been developed [42,43,44].

To become a true three-domain toxin, PirA and PirB must first form a complex. Although the complex formation of PirA/PirB was confirmed using gel filtration [13], the resulting structure is still unknown, so how these two toxins bind to each other is still unclear. Based on the locations of the corresponding domains in the Cry toxin, a possible binding model of PirA and PirB was proposed (Figure 3; [13]). In this model, á-helices 1, 2, 12 and 13, and loops 12 and 13 of PirB create a potential binding cavity for PirA, while the â-sheets 1, 3 and 9 of PirA interact with PirB (Figure 3A). Figure 3B shows how the surface charges on the PirA/PirB interface are complementary to each other, further suggesting that this model is reasonable. However, as noted above, this PirA/PirB binding model still needs to be verified experimentally.

Figure 3

Possible binding mode and interface between PirA and PirB toxins. (A) Cry and proposed PirA/PirB complex. The PirA/PirB complex was predicted by reference to the positions of the three Cry domains. The possible binding regions of PirA and PirB are colored orange and blue; (B) The surface charges on the complex interfaces of PirA and PirB. Red and blue respectively indicate negatively and positively charged regions.

Furthermore, although there are many structural similarities, some physiological characteristics between Cry and PirA/PirB toxins may be different. For example, the Cry protoxins generally form crystals in the mother cell compartment [45,46]. Since the crystals have to be solubilized in the gut of insect larvae to become biologically active, this ability of the protoxins to crystallize may decrease their susceptibility to premature proteolytic degradation [45]. Previous reports have shown that the solubility of these Cry crystals is dependent on pH [45,47,48]; the crystals that form in the neutral pH of the mother cells subsequently dissolve in the acidic environment (PirB, and although in vivo crystallization of Cry toxins is an important control step of their toxicities, it seems unlikely that PirA/PirB would use a similar control mechanism. Nevertheless this has not yet been demonstrated experimentally.

A more complete understanding of the cytotoxic mechanisms of PirA/PirB toxins is likely to be important for AHPND research, but could also be important for agricultural applications. Although there is genetic distance between PirA/PirB and the PirA/PirB homologs that are found in other bacteria such as Photorhabdus asymbiotica (WP_015835800/WP_015835799) [18], Photorhabdus luminescens (ABE68878/ABE68879) [19], Xenorhabdus doucetiae (CDG18638/CDG18639), Xenorhabdus cabanillasii (CDL79383/CDL79384), Xenorhabdus nematophila (WP013183676/WP010845483) and Alcaligenes faecalis (WP003801867/WP003801865), these insecticidal PirA and PirB toxins have allowed Photorhabdus and Xenorhabdus to be used in biological pest control [18,21,22]. The study of PirA/PirB should also therefore provide useful information for insecticidal applications.

3. Strategies for Designing Drugs to Block the Cytotoxic Effects of V. parahaemolyticus PirA and PirB Toxins

Although AHPND-detection methods that can monitor the shrimps and the environment during cultivation have already been developed [7,49,50], there are still no available drugs that can be used in the treatment of AHPND. It has already been clearly established that PirA and PirB toxins are the main cytotoxic source of AHPND; for example, the deletion/mutation of their pirA and pirB genes from pVA1 can decrease AHPND severity and reduce the mortality of the shrimps [12,13,15,16]. Additionally, PirA and PirB are both secreted proteins [13,15], which means that they could be easily targeted by drugs/inhibitors. Neutralization of PirA and/or PirB toxicity is therefore a rational direction for AHPND drug design. Further, since the structures of PirA and PirB are both available, a structure-based drug design can be used to achieve this goal more efficiently.

Structure-based drug design has been successfully used before. For example, in the well-studied pore-forming toxins, such as colicin and hemolysin, structural biology provided a wealth of useful knowledge regarding conformation rearrangement, receptor/ligand binding regions and oligomerization [32]. Structural insights into toxins also enables the development of novel therapeutic strategies [32]. For example, small molecules or engineered antibodies can be designed to interact with specific sites on the toxins. In the case of Aeromonas hydrophila aerolysin, which targets glycosylphosphatidylinisotol (GPI)-anchored proteins, synthetic GPI molecules and GPI analogues have been proposed as inhibitors [51]. It has also recently been shown that Staphylococcus aureus hemolysin can be neutralized by an antibody that targets the receptor binding site of this toxin [52]. Similarly, with other pore-forming toxins, receptors that bind these toxins, such as CCR5 and ADAM10, can also be considered in a reverse strategy for drug design [53,54]. For example, Leukotoxin ED pore-forming toxin targets human CCR5 receptor, and CCR5 receptor antagonists such as maraviroc were shown to block Leukotoxin ED-induced cell death [53]. The structural characteristics of PirA and PirB suggests three regions that are potentially suitable for structure-based drug design: (1) the potential receptor-binding region of PirB; (2) ligand-binding region of PirA and (3) the interacting region between PirA and PirB (Figure 4, Table 1). Interface information such as amino acid sequences and structural motifs can be used for antibody engineering, as well as for in silico compound screening.

Figure 4

Strategies for designing drugs to block the cytotoxic effects of PirA and PirB toxins.

Table 1

Potential interacting regions on PirA and PirB that may be suitable targets for structure-based drug design.

Potential Function	Regions Involved in Possible Interactions	Amino Acid Sequences
Receptor binding	PirBvp Loop 12	274-VGFPS-278
	PirBvp Loop 14	316-SIEIHYYNRV-325
	PirBvp Loop 18	369-GPE-371
	PirBvp Loop 22	413-QEGSDKV-419
PirAvp/PirBvp complex formation	PirAvp β-sheet 1	11-YSHDWTV-17
	PirAvp β-sheet 3	26-VDSKH-30
	PirAvp β-sheet 9	104-GFCTIYY-110
	PirBvp α-helix 1	35-YAFKAMVSFG-43
	PirBvp α-helix 2	45-LSN-47
	PirBvp α-helix 12	247-MILWQKIKEL-256
	PirBvp α-helix 13	260-DVFVHSNLISY-270
	PirBvp Loop 12	298-PNMFGERR-305
	PirBvp Loop 13	431-PDEF-434
Ligand binding	PirAvp β-sheet 3	26-VDSKH-30 *
	PirAvp Loop 4	31-TPIIPEVGRS-40 *
	PirAvp Loop 8	83-QRPDNAFY-90 *

* GalNAc interacting residues are shown in bold.

Although engineered antibodies can be used to investigate the importance of these various PirA and PirB regions in the laboratory, they are probably too expensive and difficult to use in the field. By contrast, small compounds may be more suitable for AHPND treatment in aquaculture. Recently, in silico screening approaches have been used to identify small molecules that disrupt protein–protein interactions [55,56], and currently, data is available for over 35 million compounds on databases such as ZINC (http://zinc.docking.org/; [57]). By using in silico screening approaches, these compounds can be virtually docked into specific sites on PirA or PirB. Furthermore, the binding affinities between PirA/PirB and compounds can be predicted using molecular docking tools, such as iGEMDOCK [35] and AutoDock Vina [58]. Ligand-based screening is another approach that can be used to identify inhibitors [59]. On the assumption that similar compounds can mimic physicochemical properties of the interacting regions and occupy the interface of the target protein, this approach uses online chemistry tools (e.g., Open Babel; [60]) to search for compounds that are similar to interacting peptides (e.g., a loop) of partner proteins (e.g., the PirA binding interface on PirB). Ultimately, compounds with high docking scores that predict greater affinity can be considered as potential inhibitors, and these can then be validated through bioassays, as well as shrimp challenge assays. Using these approaches, we are hopeful that a potential PirA/PirB drug/inhibitor can be discovered in the near future.

4. Conclusions

In this review, we have presented structural views of the major pathogenic factors of AHPND: V. parahaemolyticus PirA and PirB. Based on the structural similarity to B. thuringiensis Cry pore-forming toxin, we hypothesized that PirA and PirB may use similar mechanisms to cause cell death in shrimps. Furthermore, strategies for drug/inhibitor design against these two toxins were proposed. As more details are discovered, we anticipate that the future safety and usefulness of the insecticidal applications of this toxin family will also be improved.