Enhanced nematicidal potential of the chitinase pachi from Pseudomonas aeruginosa in association with Cry21Aa

Nematodes are known to be harmful to various crops, vegetables, plants and insects. The present study reports that, chitin upregulates the activity of chitinase (20%) and nematicidal potential (15%) of Pseudomonas aeruginosa. The chitinase gene (pachi) from P. aeruginosa was cloned, and its nematicidal activity of pachi protein against Caenorhabditis elegans was studied. The mortality rate induced by pachi increased by 6.3-fold when in association with Cry21Aa from Bacillus thuringiensis. Pachi efficiently killed C. elegans in its native state (LC50 = 387.3 ± 31.7 μg/ml), as well as in association with Cry21Aa (LC50 = 30.9 ± 4.1 μg/ml), by degrading the cuticle, egg shell and intestine in a relatively short time period of 24 h. To explore the nematidal potential of chitinase, six fusion proteins were constructed using gene engineering techniques. The CHACry showed higher activity against C. elegans than others owing to its high solubility. Notably, the CHACry showed a synergistic factor of 4.1 versus 3.5 a mixture [1:1] of pachi and Cry21Aa. The present study has identified eco-friendly biological routes (e.g., mixed proteins, fusion proteins) with potent nematicidal activity, which not only can help to prevent major crop losses but also strengthen the agro-economy and increase gross crop yield.

Effects of pachi on egg shell, cuticle, and intestine. In the present study, we observed that pachi acts effectively on the egg shell and cuticle to kill nematodes. Fresh eggs were incubated with pachi at 20 °C and 37 °C for different time periods (2, 4, 6, 8, 10, 12 h). The egg surface began to show roughness and irregularities after 6 h (at 20 °C), whereas eggs incubated with the control (PBS) were smooth and remained formed after 6 h (Fig. 6A). The egg shells were destroyed in 4 h when incubated with pachi at 37 °C and disappeared after 12 h of incubation, whereas eggs incubated in the PBS control remained unaffected (Fig. 6B).
Similarly, the nematode cuticle was critically destroyed after incubation with pachi at 37 °C for 24 h, with hardly any pieces of whole cuticle observed under a microscope after 48 h (Fig. 6D). In contrast, the cuticle remained in its original state after 48 h of incubation in PBS (Fig. 6C). Furthermore, most ICPs were able to destroy the nematode intestine 8 . After incubation with pachi for 24 h, the intestine was digested and destroyed, leading to nematode death (Fig. 7C). The intestine shrunk and became difficult to identify after incubation with Cry21Aa for 36 h, whereas the cuticle remained unaffected (Fig. 7B). Moreover, the ability of Cry21Aa to destroy the intestine of nematodes was enhanced by pachi (Fig. 7D). When the catalytic domain of pachi was fused with the Cry21Aa toxin region, the intestine and the cuticles were destroyed after 60 h of incubation at 37 °C. This result was probably due to each protein killing the nematodes by different mechanisms. Here, the degradation of the cuticle and intestine demonstrated that pachi damaged the skin and intestine, whereas the ICP acted only on the intestine of the nematode.

Discussion
In various previous studies, P. aeruginosa was reported to cause disease in insects, nematodes, and mice 18,[26][27][28] . In the present study, we attempted to explore new nematicidal agents from P. aeruginosa. Chitinase from P. aeruginosa has been selected not only because of the regulation of its nematicidal activity by colloidal chitin (Fig. 1C) but also the scarcity of reports on chitinase as a bio-control agent for nematodes (Fig. 2). The chitinase pachi showed a high potential to kill nematodes, bio-control broods, and inhibit growth through digestion of the cuticle, egg shells, and intestine of C. elegans. Moreover, the chitinase pachi enhanced Cry21Aa activity against C. elegans.
Chitin played an important role in the formation of the egg shell and cuticle of C. elegans by acting as an important barrier to protect nematodes from infection by pathogenic microorganisms 14 . Chitinase, due to its high chitin-degrading activity, has been used as a bio-control agent against phyto-pathogenic bacteria, fungi, insects, and nematodes (Fig. 2). Several bacteria such as Bacillus, Serratia, Pseudomonas, and Streptomyces could use chitin as a carbon source and infect hosts using chitinase [29][30][31][32] . The antifungal mechanism of chitinase was due to the hydrolysis of chitin in the cell wall, leading to cell lysis 24 . Chitinase could also destroy the intestine and cuticle of insects by digesting chitin, leading to gut shrinkage and osmotic pressure imbalance 33 . In this study, pachi effectively destroyed the C. elegans cuticle and intestine, resulting in the death of nematodes (Figs 6C,D and 7). Pachi also displayed high activity for the digestion of egg shells and the inhibition of egg hatching, which was supported by a study carried out by Gan et al. 34 . In the study of Mercer et al. 35 , the chitinase increased hatch rates of nematode eggs but caused the death of juveniles instantly. In the present study, chitin upregulated the activity of chitinase of P. aeruginosa and increased its nematicidal activity. Chitin could act as important carbon source and signal molecules to upregulate production of chitinase. However, thus far, the information about the nematicidal mechanism of chitinase is very limited in the literature 7 . The complex structure and multi-component system of chitin has made elusive the understanding of whether the mechanism of action of chitinase is different or the same in fungi, insects, and nematodes.
In this study, two different properties proteins, pachi obtained from P. aeruginosa and Cry21Aa from B. thuringiensis, functioned together as a nematicidal agent. Previous studies showed that chitinase could enhance the insecticidal activity of B. thuringiensis by penetrating the peritrophic membrane barrier The phylogenetic tree was constructed using the neighbor-joining method (MEGA6.0). All chitinase sequences were obtained from GeneBank and PDB (http://www.rcsb.org/pdb/home/home.do), and the accession numbers of chitinase were listed in the form of "AAM48520", except for pachi. These chitinases were reported to infect multiple phytopathogenic organisms such as fungi, insects, and nematodes.
in the larval midgut and aiding the ICPs to bind their receptors on epithelial cell membranes 36 . To enhance its large-scale application and use as an insecticidal agent, B. thuringiensis was used as a host for co-expressing several chitinases from Bacillus licheniformis, Bacillus circulans, Pseudomonas maltophilia, Bacillus sphaericus, Aeromonoas hydrophila, Serratia marcescens, and Nicotiana tabacum 22,[37][38][39][40][41] . Moreover, nematodes had a similar peritrophic membrane structure and composition as insects 42 . Therefore, as reported, the chitinase pachi also could enhance the toxicity of Cry21Aa against C. elegans by the similar modes of insecticidal.
Another attractive property was that the CHACry fusion protein showed higher activity than each individual protein. Six fusion proteins were constructed and tested on C. elegans, but the CHACry showed highest activity and solubility. A previous study indicated that C-terminal extension region was related to solubility of protein, and the CHA had higher solubility than pachi 23 . Recently, many fusion proteins have been constructed and co-expressed to enhance relative activity, and more data about such fusion proteins would facilitate the access to the binding sites, improve their stability and broaden the insect-resistance spectrum 43,44 . Fusion venoms were well known to resist proteolytic activity in insects, alter the shape of the original protein crystals and alter receptor binding sites present in the midgut of insects 45 . Previous studies also showed that the ICPs from Bacillus expressed in E. coli easily formed inclusion bodies and lowered the activity due to the dissolution process that was necessary for ICP activation 46 . Our previous study indicated that solubility of chitinase was increased after removing the chitin binding domain 23 . In the present study, the catalytic domain of pachi was fused with the toxin region of Cry21Aa, and the CHACry fusion protein (~50% soluble) showed better solubility than Cry21Aa, which increased Cry21Aa activity against C. elegans (Fig. 4B). Additionally, each toxin had a unique mechanism of killing hosts and damaging the host cell 11 . Many researchers believed that the ICP damaged the intestine of the host and that chitinases hydrolyzed the cuticle and egg shell 35,47 . Our results revealed that the chitinase showed high intestinal digestion activity, which would protect the ICP from proteolysis and help the ICP bind to its receptor. Moreover, the chitinase also showed high cuticle degradation activity and provided a new way for the ICP to enter into the intestine of the nematode. However, the mechanism of synergy by which a biopesticide entered the nematode and became resistant to insecticidal attack remained to be elucidated.
Previous studies indicated that P. aeruginosa PA14 work in two different modes to kill C. elegans. At low salt concentration medium, PA14 showed a mild infection and killed the nematodes in 72-96 h ('slow killing') via production of hydrolytic enzyme, like protease, which was due to the mass accumulation of bacteria in the intestine and enzymatic degradation. But at high salt medium, PA14 produced diffusible toxins like cyanongen, phenazines, and pycoyanin, and killed nematodes within a short time period ('fast killing') by inhibiting metabolic pathways 48 . In this study, P. aeruginosa killed C. elegans also by producing hydrolytic enzyme (chitinase) and toxin. It was interesting to note that the strain (P. aeruginosa) did not produce protease, which was different from some previous studies 17 , while chitinase played a vital role in nematicidal activity. Recent studies of Pseudomonas infection in nematodes were based upon the quorum sensing system (QS) 49 . It was a complex cell-to-cell signaling system allowing the bacteria to sense and regulated their own cell densities. There were three types of secretion systems (type I, type II and type III) in QS of P. aeruginosa to secrete extracellular hydrolytic enzyme into the cytoplasm of hosts 50 . Type II secretion system regulated the production of extracellular hydrolases, like elastase, alkaline phosphatase, phospholipase C, and chitinase etc. 51 . In the present study, the genome analysis of P. aeruginosa predicted that some extracellular hydrolases (like serine protease, collagenase, phospholipase C, chitinase) might play an important role in killing nematodes. The mechanisms of different hydrolases in killing nematodes would be an interesting topic in future studies.

Materials and Methods
Strains and culture. The strains and plasmids used in this study are listed in Supplementary data Table 1. The bacterial strain was isolated from a mud soil sample from South Lake near Huazhong Agricultural University, Wuhan, China. The culture was grown in nutrient agar medium containing peptone 1%, yeast extract 0.5%, NaCl 1%, and agar 1.5% (m/v). The selected bacterial strain was identified as P. aeruginosa by 16S rRNA sequencing. Escherichia (E.) coli strains were maintained in Luria-Bertani medium containing ampicillin (100 μ g/ml). All bacterial strains were kept in 20% (v/v) glycerol suspension at − 80 °C. The C. elegans N2 wild-type strain was provided by the Caenorhabditis Genetics Center (CGC) and maintained on nematode growth medium (NGM) agar plates with E. coli OP 50 as its food at 20 °C and stored at − 80 °C. Chitinase activity. P. aeruginosa was collected and resuspended in PBS buffer (final OD 600 ~ 3.0), followed by ultrasonic disruption. The supernatant was selected to test its chitinase and nematicidal activity. Chitinase activity was measured according to method described by Chen et al. 23 , while nematicidal activity was measured according to the method of Bischof et al. 52 .
Cloning of gene pachi and Cry21Aa. Genomic DNA extracted from P. aeruginosa was used as a template to amplify pachi using the primers pachi-f and pachi-r (Supplementary data Table 2). The Cry21Aa gene was amplified from the plasmid pHT304-Cry21Aa using the primer pair Cry21Aa-f and Cry21Aa-r. The pachi gene was digested by EcoR I and Xho I and cloned into the pGEX-6p-1 vector to construct the pGEX-6p-pachi expression vector, whereas Cry21Aa was digested by BamH I and Xho I to construct the pGEX-6p-Cry21Aa expression vector. The recombinant plasmids were then transformed into E. coli BL21 (DE3) cells.
Construction of fusion proteins. The pGEx-6p-1 plasmid was used as the template to construct the pGEX-6p-H plasmid by inserting an additional Hind III restriction enzyme site between BamH I and EcoR I using the primer pair 6p-H-f and 6p-H-r. Rapid polymerase chain reaction (PCR)-based site-directed mutagenesis was used to add the two restriction enzyme sites. Fusion proteins were constructed by using over-lapping PCR. The CHACry (3087 bp; 114.5 kDa; 3 bp for termination codon) was constructed by fusing the catalytic domain of pachi (1026 bp; 37.7 kDa) with the toxin region of Cry21Aa (Cry) (2058 bp; 76.8 kDa). The Cry contained the N-terminus extension region, endotoxin N and delta-endotoxin C. Cloning of the Cry21Aa N-terminus was directed by using the primer pair Cry21Aa-f 2 and Cry21Aa-r 2 . The catalytic domain (CHA) of pachi was cloned with the primer pair CHA-f and CHA-r. Finally, the recombinant CHACry gene was cloned with the primer pair CHA-f and Cry21Aa-r 2 by overlapping PCR. Recombinant pGEX-6p-CHACry was transformed into E. coli BL21 (DE3) cells to express the CHACry fusion protein.  Protein expression and purification. E. coli BL21 (DE3) cells harboring pGEX-6p-pachi, named DE3/pGEX-6p-pachi, were inoculated into LB broth (ampicillin; 100 μ g/ml) with shaking at 37 °C for 12 h. The seed culture was used to inoculate production broth (v/v, 2/100), and growth was induced by adding IPTG (1 mM) after 2-3 h. The IPTG-induced production broth was incubated at 18 °C for 14 h with shaking (250 rpm/min). Finally, the induced cells were collected by centrifugation, resuspended in phosphate-buffered saline (PBS) buffer (NaCl 0.8%, KCl 0.02%, Na 2 HPO 4 0.14%, KH 2 PO 4 0.03%; pH 7.0) and homogenized using a high-pressure homogenizer (NS100IL 2K, Niro Soavi, Germany). The target proteins (Cry21Aa, pachi and CHACry) were purified using a glutathione S-transferase (GST) Gene Fusion System (GE Healthcare, USA) and eluted from the GST tag by 3C proteases (PreScission, Pharmacia). The molecular weight was analyzed by SDS-PAGE with 10% polyacrylamide gels. The protein concentration was measured by the Bradford method using bovine serum albumin (BSA) as a standard.
Quantitative analysis of nematicidal activity. The purified proteins were used for bioassays including quantitative mortality tests, brood size assays, and growth analyses. The bioassay procedures and 50% lethal/inhibition/growth concentration (LC 50 /IC 50 /GC 50 ) evaluations were undertaken according to the method of Bischof et al. 52 .
Cuticle and egg shell digestion. The cuticle was separated from the nematode body as described by Cox et al. 14 . Cuticles (eggs) was incubated with pachi at 20 °C and 37 °C for various time periods. The L4 worms were incubated with pachi, Cry21Aa, and the CHACry fusion protein at 20 °C and 37 °C for various time periods. Pachi was used to study complete digestion at 1 mg/ml for the cuticle and at 200 μ g/ml for the egg shell and intestine. The effects of Cry21Aa on the intestine were assessed at a final concentration of 150 μ g/ml, while CHACry was assessed at 100 μ g/ml. Pachi (1 mg/ml) was used as an effective nematicidal dose to ensure the complete killing of C. elegans. Synergistic activity assays for pachi and Cry21Aa in L4 worms of the N2 strain. The synergistic factor was calculated by the formula of Tabashnik et al. 53 : 1/LC 50(m) = Ra/LC 50(a) + Rb/LC 50(b) , where Ra and R b indicate the percentage of toxin A and toxin B proteins used in the final mixture; LC 50(a) and LC 50(b) represent the LC 50 values for each toxin, and LC 50(m) is the expected theoretical value of LC 50 calculated from the formula above. The real LC 50 value was calculated from the bioassay for the observed toxicity of the mixture. Synergism was indicated with a SF value of greater than 1.
Statistical analysis. Independent experiments were repeated at least three times. All of the data obtained were analyzed using GraphPad Prism 5.0 and Excel 2003 software for figures and LC 50 values. All of the values were expressed as the mean values ± standard deviation, with the statistical significance set at p < 0.05.

Image analysis by confocal microscopy.
To study the effect of each protein on nematodes and their body parts, images were captured using a 20X objective lens with confocal laser scanning microscopy on a Zeiss LSM 510 microscope (CLSM; Zeiss LSM 510) imaging. Mixture of diethyl ether/ethanol absolute (1:1) was used as anaesthetic treatment to keep worms static during image capture. All pictures were processed using Photoshop 7.0 software, and the worm sizes were calculated using ImageJ 2.4.1.7.
Nucleotide sequence accession number. The nucleotide sequences of Pseudomonas aeruginosa 16S and pachi have been submitted to GeneBank under accession numbers KR007310 (16S) and KR007311 (pachi).