The FAR protein family of the nematode Caenorhabditis elegans. Differential lipid binding properties,structural characteristics,and developmental regulation

Parasitic nematodes of humans and plants secrete a structurally novel type of fatty acid- and retinol-binding protein, FAR, into the tissues they occupy. These proteins may interfere with intercellular lipid signaling to manipulate the defense reactions of the host or acquire essential lipids for the parasites. The genome of the nematode Caenorhabditis elegans encodes eight FAR-like proteins (Ce-FAR-1 to -8). These fall into three discrete groups as indicated by phylogenetic sequence comparisons and intron positions, the proteins from parasitic nematodes falling into group A. Recombinant Ce-FAR-1 to -7 were produced in Escherichia coli and tested for lipid binding in fluorescence-based assays. Ce-FAR-1 to -6 bound DAUDA (11-((5-dimethylaminonaphthalene-1-sulfonyl)amino)undecanoic acid), cis-parinaric acid, and retinol with dissociation constants in the micromolar range, whereas Ce-FAR-7 bound the latter two lipids relatively poorly. Each protein produced a characteristic shift in peak fluorescence emission of DAUDA, and one (Ce-FAR-5) produced a shift greater than has been observed previously for any lipid-binding protein. Selected Ce-FAR proteins were analyzed by circular dichroism (CD) and differential scanning calorimetry, were found to be helix-rich, and exhibited high thermal stability (transition midpoint, 82.7 degrees C). CD and secondary structure predictions, however, both indicated that Ce-FAR-7 possesses substantially less helix than the other FAR proteins. The genes encoding the Ce-FAR proteins were found to be transcribed differentially through the life cycle of C. elegans, such that Ce-far-4 was transcribed at highest levels in the fourth larval stage, and Ce-far-3 and -7 predominated in males.     

A Target Repurposing Approach Identifies N-myristoyltransferase as a New Candidate Drug Target in Filarial Nematodes

Myristoylation is a lipid modification involving the addition of a 14-carbon unsaturated fatty acid, myristic acid, to the N-terminal glycine of a subset of proteins, a modification that promotes their binding to cell membranes for varied biological functions. The process is catalyzed by myristoyl-CoA:protein N-myristoyltransferase (NMT), an enzyme which has been validated as a drug target in human cancers, and for infectious diseases caused by fungi, viruses and protozoan parasites. We purified Caenorhabditis elegans and Brugia malayi NMTs as active recombinant proteins and carried out kinetic analyses with their essential fatty acid donor, myristoyl-CoA and peptide substrates. Biochemical and structural analyses both revealed that the nematode enzymes are canonical NMTs, sharing a high degree of conservation with protozoan NMT enzymes. Inhibitory compounds that target NMT in protozoan species inhibited the nematode NMTs with IC₅₀ values of 2.5–10 nM, and were active against B. malayi microfilariae and adult worms at 12.5 µM and 50 µM respectively, and C. elegans (25 µM) in culture. RNA interference and gene deletion in C. elegans further showed that NMT is essential for nematode viability. The effects observed are likely due to disruption of the function of several downstream target proteins. Potential substrates of NMT in B. malayi are predicted using bioinformatic analysis. Our genetic and chemical studies highlight the importance of myristoylation in the synthesis of functional proteins in nematodes and have shown for the first time that NMT is required for viability in parasitic nematodes. These results suggest that targeting NMT could be a valid approach for the development of chemotherapeutic agents against nematode diseases including filariasis.     

Solution structure of a repeated unit of the ABA-1 nematode polyprotein allergen of Ascaris reveals a novel fold and two discrete lipid-binding sites

Background

Nematode polyprotein allergens (NPAs) are an unusual class of lipid-binding proteins found only in nematodes. They are synthesized as large, tandemly repetitive polyproteins that are post-translationally cleaved into multiple copies of small lipid binding proteins with virtually identical fatty acid and retinol (Vitamin A)-binding characteristics. They are probably central to transport and distribution of small hydrophobic compounds between the tissues of nematodes, and may play key roles in nutrient scavenging, immunomodulation, and IgE antibody-based responses in infection. In some species the repeating units are diverse in amino acid sequence, but, in ascarid and filarial nematodes, many of the units are identical or near-identical. ABA-1A is the most common repeating unit of the NPA of Ascaris suum, and is closely similar to that of Ascaris lumbricoides, the large intestinal roundworm of humans. Immune responses to NPAs have been associated with naturally-acquired resistance to infection in humans, and the immune repertoire to them is under strict genetic control.

Methodology/Principal Findings

The solution structure of ABA-1A was determined by protein nuclear magnetic resonance spectroscopy. The protein adopts a novel seven-helical fold comprising a long central helix that participates in two hollow four-helical bundles on either side. Discrete hydrophobic ligand-binding pockets are found in the N-terminal and C-terminal bundles, and the amino acid sidechains affected by ligand (fatty acid) binding were identified. Recombinant ABA-1A contains tightly-bound ligand(s) of bacterial culture origin in one of its binding sites.

Conclusions/Significance

This is the first mature, post-translationally processed, unit of a naturally-occurring tandemly-repetitive polyprotein to be structurally characterized from any source, and it belongs to a new structural class. NPAs have no counterparts in vertebrates, so represent potential targets for drug or immunological intervention. The nature of the (as yet) unidentified bacterial ligand(s) may be pertinent to this, as will our characterization of the unusual binding sites.     

Mutation of the lbp-5 gene alters metabolic output in Caenorhabditis elegans

Intracellular lipid-binding proteins (LBPs) impact fatty acid homeostasis in various ways, including fatty acid transport into mitochondria. However, the physiological consequences caused by mutations in genes encoding LBPs remain largely uncharacterized. Here, we explore the metabolic consequences of lbp-5 gene deficiency in terms of energy homeostasis in Caenorhabditis elegans. In addition to increased fat storage, which has previously been reported, deletion of lbp-5 attenuated mitochondrial membrane potential and increased reactive oxygen species levels. Biochemical measurement coupled to proteomic analysis of the lbp-5(tm1618) mutant revealed highly increased rates of glycolysis in this mutant. These differential expression profile data support a novel metabolic adaptation of C. elegans, in which glycolysis is activated to compensate for the energy shortage due to the insufficient mitochondrial β-oxidation of fatty acids in lbp-5 mutant worms. This report marks the first demonstration of a unique metabolic adaptation that is a consequence of LBP-5 deficiency in C. elegans. [BMB Reports 2014; 47(1): 15-20]     

In silico analyses of neuropeptide-like protein (NLP) profiles in parasitic nematodes

Nematode parasite infections cause disease in humans and animals and threaten global food security by reducing productivity in livestock and crop farming. The escalation of anthelmintic resistance in economically important nematode parasites underscores the need for the identification of novel drug targets in these worms. Nematode neuropeptide signalling is an attractive system for chemotherapeutic exploitation, with neuropeptide G-protein coupled receptors (NP-GPCRs) representing the lead targets. In order to successfully validate NP-GPCRs for parasite control it is necessary to characterise their function and importance to nematode biology. This can be aided through identification of receptor activating ligand(s) via deorphanisation. Such efforts require the identification of all neuropeptide ligands within parasites. Here we mined the genomes of nine therapeutically relevant pathogenic nematodes to characterise the neuropeptide-like protein complements and demonstrate that: (i) parasitic nematodes possess a reduced complement of neuropeptide-like protein-encoding genes relative to Caenorhabditis elegans; (ii) parasite neuropeptide-like protein profiles are broadly conserved between nematode clades; (iii) five Ce-nlps are completely conserved across the nematode species examined; (iv) the extent and position of neuropeptide-like protein-motif conservation is variable; (v) novel RPamide-encoding genes are present in parasitic nematodes; (vi) novel Allatostatin-C-like peptide encoding genes are present in both C. elegans and parasitic nematodes; (vii) novel neuropeptide-like protein families are absent in C. elegans; and (viii) highly conserved nematode neuropeptide-like proteins are bioactive. These data highlight the complexity of nematode neuropeptide-like proteins and reveal the need for nomenclature revision in this diverse neuropeptide family. The identification of neuropeptide-like protein ligands, and characterisation of those with functional relevance, advance our understanding of neuropeptide signalling to support exploitation of the neuropeptidergic system as an anthelmintic target.     

Characterization of Three Novel Fatty Acid- and Retinoid-Binding Protein Genes (Ha-far-1, Ha-far-2 and Hf-far-1) from the Cereal Cyst Nematodes Heterodera avenae and H. filipjevi

Heterodera avenae and H. filipjevi are major parasites of wheat, reducing production worldwide. Both are sedentary endoparasitic nematodes, and their development and parasitism depend strongly on nutrients obtained from hosts. Secreted fatty acid- and retinol-binding (FAR) proteins are nematode-specific lipid carrier proteins used for nutrient acquisition as well as suppression of plant defenses. In this study, we obtained three novel FAR genes Ha-far-1 ({"type":"entrez-nucleotide","attrs":{"text":"KU877266","term_id":"1008457416","term_text":"KU877266"}}KU877266), Ha-far-2 ({"type":"entrez-nucleotide","attrs":{"text":"KU877267","term_id":"1008457418","term_text":"KU877267"}}KU877267), Hf-far-1 ({"type":"entrez-nucleotide","attrs":{"text":"KU877268","term_id":"1008457420","term_text":"KU877268"}}KU877268). Ha-far-1 and Ha-far-2 were cloned from H. avenae, encoding proteins of 191 and 280 amino acids with molecular masses about 17 and 30 kDa, respectively and sequence identity of 28%. Protein Blast in NCBI revealed that Ha-FAR-1 sequence is 78% similar to the Gp-FAR-1 protein from Globodera pallida, while Ha-FAR-2 is 30% similar to Rs-FAR-1 from Radopholus similis. Only one FAR protein Hf-FAR-1was identified in H. filipjevi; it had 96% sequence identity to Ha-FAR-1. The three proteins are alpha-helix-rich and contain the conserved domain of Gp-FAR-1, but Ha-FAR-2 had a remarkable peptide at the C-terminus which was random-coil-rich. Both Ha-FAR-1 and Hf-FAR-1 had casein kinase II phosphorylation sites, while Ha-FAR-2 had predicted N-glycosylation sites. Phylogenetic analysis showed that the three proteins clustered together, though Ha-FAR-1 and Hf-FAR-1 adjoined each other in a plant-parasitic nematode branch, but Ha-FAR-2 was distinct from the other proteins in the group. Fluorescence-based ligand binding analysis showed the three FAR proteins bound to a fluorescent fatty acid derivative and retinol and with dissociation constants similar to FARs from other species, though Ha-FAR-2 binding ability was weaker than that of the two others. In situ hybridization detected mRNAs of Ha-far-1 and Ha-far-2 in the hypodermis. The qRT-PCR results showed that the Ha-far-1and Ha-far-2 were expressed in all developmental stages; Ha-far-1 expressed 70 times more than Ha-far-2 in all stages. The highest expression level of Ha-far-1 was observed in fourth-stage juvenile (J4), whereas the highest expression level of Ha-far-2 occurred in second-stage juvenile (J2). In conclusion, we have identified two novel far genes from H. avenae and one from H. filipjevi and have provided further indication that nematode far genes are present in a variety of nematode species, where the FAR proteins share similar basic structure, expression pattern and biochemical activities.     

Identification of Amino Acids Conferring Chain Length Substrate Specificities on Fatty Alcohol-forming Reductases FAR5 and FAR8 from Arabidopsis thaliana

Fatty alcohols play a variety of biological roles in all kingdoms of life. Fatty acyl reductase (FAR) enzymes catalyze the reduction of fatty acyl-coenzyme A (CoA) or fatty acyl-acyl carrier protein substrates to primary fatty alcohols. FAR enzymes have distinct substrate specificities with regard to chain length and degree of saturation. FAR5 (At3g44550) and FAR8 (At3g44560) from Arabidopsis thaliana are 85% identical at the amino acid level and are of equal length, but they possess distinct specificities for 18:0 or 16:0 acyl chain length, respectively. We used Saccharomyces cerevisiae as a heterologous expression system to assess FAR substrate specificity determinants. We identified individual amino acids that affect protein levels or 16:0-CoA versus 18:0-CoA specificity by expressing in yeast FAR5 and FAR8 domain-swap chimeras and site-specific mutants. We found that a threonine at position 347 and a serine at position 363 were important for high FAR5 and FAR8 protein accumulation in yeast and thus are likely important for protein folding and stability. Amino acids at positions 355 and 377 were important for dictating 16:0-CoA versus 18:0-CoA chain length specificity. Simultaneously converting alanine 355 and valine 377 of FAR5 to the corresponding FAR8 residues, leucine and methionine, respectively, almost fully converted FAR5 specificity from 18:0-CoA to 16:0-CoA. The reciprocal amino acid conversions, L355A and M377V, made in the active FAR8-S363P mutant background converted its specificity from 16:0-CoA to 18:0-CoA. This study is an important advancement in the engineering of highly active FAR proteins with desired specificities for the production of fatty alcohols with industrial value.     

Biochemical Analysis of Caenorhabditis elegans Surface Mutants

A collection of Caenorhabditis elegans mutants that show ectopic surface lectin binding (Srf mutants) was analyzed to determine the biochemical basis for this phenotype. This analysis involved selective removal or labeling of surface components, specific labeling of surface glycans, and fractionation of total protein with subsequent detection of wheat germ agglutinin (WGA) binding proteins. Wild-type and mutant nematodes showed no differences in their profiles of extractable surface glycoproteins or total WGA-binding proteins, suggesting that the ectopic lectin binding does not result from the novel expression of surface glycans. Instead, these results support a model in which ectopic lectin binding results from an unmasking of glycosylated components present in the insoluble cuticle matrix of wild-type animals. To explain the multiple internal defects found in some surface mutants, we propose that these mutants have a basic defect in protein processing. This defect would interfere with the expression of the postulated masking protein(s), as well as other proteins required for normal development.     

Molecular Characterization and Functions of Fatty Acid and Retinoid Binding Protein Gene (Ab-far-1) in Aphelenchoides besseyi

Rice white tip nematode, Aphelenchoides besseyi, is a kind of plant parasitic nematodes that cause serious losses in rice and many other crops. Fatty acid and retinoid binding protein (FAR) is a specific protein in nematodes and is related to development, reproduction, infection to the host, and disruption of plant defense reactions, so the inhibition of FAR function is the potential approach to control A. besseyi. The full-length of Ab-far-1 cDNA is 805 bp, including 546 bp of ORF that encodes 181 amino acids. Software analysis revealed that the Ab-FAR-1 was rich in α-helix structure, contained a predicted consensus casein kinase II phosphorylation site and a hydrophobic secretory signal peptide, but did not have glycosylation sites. The Ab-FAR-1 had 52% homology to Gp-FAR-1 protein. The Ab-FAR-1 and Gp-FAR-1 were grouped in the same branch according to the phylogenetic tree. Fluorescence-based ligand binding analysis confirmed that the recombinant Ab-FAR-1 (rAb-FAR-1) bound with the fluorescent analogues 11-((5-dimethylaminonaphthalene-1-sulfonyl) amino) undecannoic acid (DAUDA), cis-parinaric acid and retinol, but the oleic acid would compete with the binding site. Quantitative PCR (qPCR) was used to assess the expression level of Ab-far-1 at different development stages of A. besseyi, the highest expression was found in the females, followed by eggs, juveniles and males. Using in situ hybridization technique, Ab-far-1 mRNA was present in the hypodermis of juveniles and adults, the ovaries of females and the testes of males. When A. besseyi was treated with Ab-far-1 dsRNA for 48 h, the silencing efficiency of Ab-far-1 was the best and the number of nematodes on the carrot was the least. Thus FAR plays important roles in the development and reproduction of nematodes. This study is useful and helpful to figure out a new way to control the plant parasitic nematodes.     

Needles in the EST haystack: large-scale identification and analysis of excretory-secretory (ES) proteins in parasitic nematodes using expressed sequence tags (ESTs)

Background

Parasitic nematodes of humans, other animals and plants continue to impose a significant public health and economic burden worldwide, due to the diseases they cause. Promising antiparasitic drug and vaccine candidates have been discovered from excreted or secreted (ES) proteins released from the parasite and exposed to the immune system of the host. Mining the entire expressed sequence tag (EST) data available from parasitic nematodes represents an approach to discover such ES targets.

Methods and Findings

In this study, we predicted, using EST2Secretome, a novel, high-throughput, computational workflow system, 4,710 ES proteins from 452,134 ESTs derived from 39 different species of nematodes, parasitic in animals (including humans) or plants. In total, 2,632, 786, and 1,292 ES proteins were predicted for animal-, human-, and plant-parasitic nematodes. Subsequently, we systematically analysed ES proteins using computational methods. Of these 4,710 proteins, 2,490 (52.8%) had orthologues in Caenorhabditis elegans, whereas 621 (13.8%) appeared to be novel, currently having no significant match to any molecule available in public databases. Of the C. elegans homologues, 267 had strong “loss-of-function” phenotypes by RNA interference (RNAi) in this nematode. We could functionally classify 1,948 (41.3%) sequences using the Gene Ontology (GO) terms, establish pathway associations for 573 (12.2%) sequences using Kyoto Encyclopaedia of Genes and Genomes (KEGG), and identify protein interaction partners for 1,774 (37.6%) molecules. We also mapped 758 (16.1%) proteins to protein domains including the nematode-specific protein family “transthyretin-like” and “chromadorea ALT,” considered as vaccine candidates against filariasis in humans.

Conclusions

We report the large-scale analysis of ES proteins inferred from EST data for a range of parasitic nematodes. This set of ES proteins provides an inventory of known and novel members of ES proteins as a foundation for studies focused on understanding the biology of parasitic nematodes and their interactions with their hosts, as well as for the development of novel drugs or vaccines for parasite intervention and control.     

Bacillus thuringiensis DB27 Produces Two Novel Protoxins,Cry21Fa1 and Cry21Ha1, Which Act Synergistically against Nematodes

Bacillus thuringiensis has been widely used as a biopesticide, primarily for the control of insect pests, but some B. thuringiensis strains specifically target nematodes. However, nematicidal virulence factors of B. thuringiensis are poorly investigated. Here, we describe virulence factors of nematicidal B. thuringiensis DB27 using Caenorhabditis elegans as a model. We show that B. thuringiensis DB27 kills a number of free-living and animal-parasitic nematodes via intestinal damage. Its virulence factors are plasmid-encoded Cry protoxins, since plasmid-cured derivatives do not produce Cry proteins and are not toxic to nematodes. Whole-genome sequencing of B. thuringiensis DB27 revealed multiple potential nematicidal factors, including several Cry-like proteins encoded by different plasmids. Two of these proteins appear to be novel and show high similarity to Cry21Ba1. Named Cry21Fa1 and Cry21Ha1, they were expressed in Escherichia coli and fed to C. elegans, resulting in intoxication, intestinal damage, and death of nematodes. Interestingly, the effects of the two protoxins on C. elegans are synergistic (synergism factor, 1.8 to 2.5). Using purified proteins, we determined the 50% lethal concentrations (LC₅₀s) for Cry21Fa1 and Cry21Ha1 to be 13.6 μg/ml and 23.9 μg/ml, respectively, which are comparable to the LC₅₀ of nematicidal Cry5B. Finally, we found that signaling pathways which protect C. elegans against Cry5B toxin are also required for protection against Cry21Fa1. Thus, B. thuringiensis DB27 produces novel nematicidal protoxins Cry21Fa1 and Cry21Ha1 with synergistic action, which highlights the importance of naturally isolated strains as a source of novel toxins.     

Bioinformatic analysis of P granule-related proteins: insights into germ granule evolution in nematodes

Germ cells in many animals possess a specialized cytoplasm in the form of granules that contain RNA and protein complexes essential for the function and preservation of the germline. The mechanism for the formation of these granules is still poorly understood; however, the lack of conservation in their components across different species suggests evolutionary convergence in the assembly process. Germ granules are assumed to be present in all nematodes with a preformed germline. However, few studies have clearly identified these structures in species other than Caenorhabditis elegans and even less have carried functional analysis to provide a broader panorama of the granules composition in the phylum. We adopted a bioinformatics approach to investigate the extension of conservation in nematodes of some known C. elegans germ granule components, as a proxy to understand germ granules evolution in this phylum. Unexpectedly, we found that, in nematodes, the DEAD box RNA helicase Vasa, a conserved protein among different phyla, shows a complex history of clade-specific duplications and sequence divergence. Our analyses suggest that, in nematodes, Vasa’s function might be shared among proteins like LAF-1, VBH-1, and GLH-1/-2/-3 and GLH-4. Key components of P granules assembly in C. elegans, like the PGL protein family, are only preserved in Caenorhabditis species. Our analysis suggests that germ granules assembly may not be conserved in nematodes. Studies on these species could bring insight into the basic components required for this pathway.     

Discovery of Anthelmintic Drug Targets and Drugs Using Chokepoints in Nematode Metabolic Pathways

Parasitic roundworm infections plague more than 2 billion people (1/3 of humanity) and cause drastic losses in crops and livestock. New anthelmintic drugs are urgently needed as new drug resistance and environmental concerns arise. A “chokepoint reaction” is defined as a reaction that either consumes a unique substrate or produces a unique product. A chokepoint analysis provides a systematic method of identifying novel potential drug targets. Chokepoint enzymes were identified in the genomes of 10 nematode species, and the intersection and union of all chokepoint enzymes were found. By studying and experimentally testing available compounds known to target proteins orthologous to nematode chokepoint proteins in public databases, this study uncovers features of chokepoints that make them successful drug targets. Chemogenomic screening was performed on drug-like compounds from public drug databases to find existing compounds that target homologs of nematode chokepoints. The compounds were prioritized based on chemical properties frequently found in successful drugs and were experimentally tested using Caenorhabditis elegans. Several drugs that are already known anthelmintic drugs and novel candidate targets were identified. Seven of the compounds were tested in Caenorhabditis elegans and three yielded a detrimental phenotype. One of these three drug-like compounds, Perhexiline, also yielded a deleterious effect in Haemonchus contortus and Onchocerca lienalis, two nematodes with divergent forms of parasitism. Perhexiline, known to affect the fatty acid oxidation pathway in mammals, caused a reduction in oxygen consumption rates in C. elegans and genome-wide gene expression profiles provided an additional confirmation of its mode of action. Computational modeling of Perhexiline and its target provided structural insights regarding its binding mode and specificity. Our lists of prioritized drug targets and drug-like compounds have potential to expedite the discovery of new anthelmintic drugs with broad-spectrum efficacy.     

A Lover and a Fighter: The Genome Sequence of an Entomopathogenic Nematode Heterorhabditis bacteriophora

Heterorhabditis bacteriophora are entomopathogenic nematodes that have evolved a mutualism with Photorhabdus luminescens bacteria to function as highly virulent insect pathogens. The nematode provides a safe harbor for intestinal symbionts in soil and delivers the symbiotic bacteria into the insect blood. The symbiont provides virulence and toxins, metabolites essential for nematode reproduction, and antibiotic preservation of the insect cadaver. Approximately half of the 21,250 putative protein coding genes identified in the 77 Mbp high quality draft H. bacteriophora genome sequence were novel proteins of unknown function lacking homologs in Caenorhabditis elegans or any other sequenced organisms. Similarly, 317 of the 603 predicted secreted proteins are novel with unknown function in addition to 19 putative peptidases, 9 peptidase inhibitors and 7 C-type lectins that may function in interactions with insect hosts or bacterial symbionts. The 134 proteins contained mariner transposase domains, of which there are none in C. elegans, suggesting an invasion and expansion of mariner transposons in H. bacteriophora. Fewer Kyoto Encyclopedia of Genes and Genomes Orthologies in almost all metabolic categories were detected in the genome compared with 9 other sequenced nematode genomes, which may reflect dependence on the symbiont or insect host for these functions. The H. bacteriophora genome sequence will greatly facilitate genetics, genomics and evolutionary studies to gain fundamental knowledge of nematode parasitism and mutualism. It also elevates the utility of H. bacteriophora as a bridge species between vertebrate parasitic nematodes and the C. elegans model.     

A novel lipid binding site formed by the MAP kinase insert in p38 alpha

The p38 mitogen-activated protein (MAP) kinases function as signaling molecules essential for many cellular processes, particularly mediating stress response. The activity of p38 MAP kinases is meticulously regulated to reach the desired cellular phenotype. Several alternative activation and attenuation mechanisms have been characterized recently which include new phosphorylation sites. Here we present the crystal structure of p38α MAP kinase in complex with n-octyl-β-glucopyranoside detergent. The complex unveils a novel lipid-binding site formed by a local conformational change of the MAP kinase insert. This binding is the first attribution for a possible role of the MAP kinase insert in p38. The binding site can accommodate a large selection of lipidic molecules. In addition, we also show via biophysical methods that arachidonic acid and its derivatives bind p38α in vitro. Based on our analysis we propose that the binding of lipids could fine-tune p38α catalytic activity towards a preferred phenotype.     

Alcaligenes faecalis ZD02, a Novel Nematicidal Bacterium with an Extracellular Serine Protease Virulence Factor

Root knot nematodes (RKNs) are the world''s most damaging plant-parasitic nematodes (PPNs), and they can infect almost all crops. At present, harmful chemical nematicides are applied to control RKNs. Using microbial nematicides has been proposed as a better management strategy than chemical control. In this study, we describe a novel nematicidal bacterium named Alcaligenes faecalis ZD02. A. faecalis ZD02 was isolated from Caenorhabditis elegans cadavers and has nematostatic and nematicidal activity, as confirmed by C. elegans growth assay and life span assay. In addition, A. faecalis ZD02 fermentation broth showed toxicity against C. elegans and Meloidogyne incognita. To identify the nematicidal virulence factor, the genome of strain ZD02 was sequenced. By comparing all of the predicted proteins of strain ZD02 to reported nematicidal virulence factors, we determined that an extracellular serine protease (Esp) has potential to be a nematicidal virulence factor, which was confirmed by bioassay on C. elegans and M. incognita. Using C. elegans as the target model, we found that both A. faecalis ZD02 and the virulence factor Esp can damage the intestines of C. elegans. The discovery that A. faecalis ZD02 has nematicidal activity provides a novel bacterial resource for the control of RKNs.