The differential strain virulence of the candidate toxins of Photorhabdus akhurstii can be correlated with their inter-strain gene sequence diversity

Abstract

Photorhabdus akhurstii is an insect-parasitic bacterium that symbiotically associates with the nematode, Heterorhabditis indica. The bacterium possesses several pathogenicity islands that aids in conferring toxicity to different insects. Herein, we constructed the plasmid clones of coding sequences of four toxin genes (pirA, tcaA, tccA and tccC; each was isolated from four P. akhurstii strains IARI-SGMG3, IARI-SGGJ2, IARI-SGHR2 and IARI-SGMS1) in Escherichia coli and subsequently, their biological activity were investigated against the fourth-instar larvae of the model insect, Galleria mellonella via intra-hemocoel injection. Bioinformatics analyses indicated inter-strain amino acid sequence difference at several positions of the candidate toxins. In corroboration, differential insecticidal activity of the identical toxin protein (PirA, TcaA, TccA and TccC conferred 15-59, 27-100, 25-100 and 33-98% insect mortality, respectively, across the strains) derived from the different bacterial strains was observed, suggesting that the diverse gene pool in Indian strains of P. akhurstii leads to strain-specific virulence in this bacterium. These toxin candidates appear to be an attractive option to deploy them in biopesticide development for managing the insect pests globally.

Keywords: Bacterial strains; Galleria mellonella; PirA; RVA assay; TcaA; TccA; TccC.

Fig. 1

Sequence conservation of P. akhurstii PirA protein across the different bacterial genera of order Enterobacteriales. A maximum-likelihood phylogeny of PirA and its putative homologues is shown. Genbank accession numbers for different gene sequences are provided in the parentheses. Bootstrap values (corresponding to different cluster nodes) are shown as percentages. The bootstrap consensus tree was inferred from 1000 replicates to represent the evolutionary history and branches corresponding to partitions reproduced in less than 50% bootstrap replicates were collapsed. Sequence alignments were manually corrected by eliminating the gaps and missing data

Fig. 2

Multiple sequence alignment (MSA) of amino acid residues of PirA protein derived from P. akhurstii strain IARI-SGMG3, IARI-SGGJ2, IARI-SGHR2 and IARI-SGMS1 with P. luminescens PirA. Amino acid residues differed at 17 (neutral threonine changed to acidic glutamate), 31 (basic histidine altered to neutral tyrosine), 66 (neutral serine changed to basic lysine), 84 (nonpolar tyrosine altered to polar, sulphur-containing cysteine), 96 (nonpolar alanine changed to polar, hydroxyl-containing serine) and 113 (polar serine to nonpolar glycine)

Fig. 3

Sequence conservation of P. akhurstii TcaA protein across the different bacterial genera of order Enterobacteriales. A maximum-likelihood phylogeny of TcaA and its putative homologues is shown. Genbank accession numbers for different gene sequences are provided in the parentheses. Cluster nodes are strongly supported by a bootstrap analysis (1,000 replicates) value of more than 70%

Fig. 4

MSA of amino acid residues of TcaA protein derived from P. akhurstii strain IARI-SGMG3, IARI-SGGJ2, IARI-SGHR2 and IARI-SGMS1 with P. luminescens TcaA. Amino acid residues differed at numerous positions. When compared with other strains, insertion of two similar amino acids, such as asparagine and threonine (polar, neutral) at position 337 and 338 is evident in IARI-SGMG3 TcaA

Fig. 5

Sequence conservation of P. akhurstii TccA protein across the different bacterial genera of order Enterobacteriales. A maximum-likelihood phylogeny of TccA and its putative homologues is shown. Genbank accession numbers for different gene sequences are provided in the parentheses. Cluster nodes are strongly supported by a bootstrap analysis (1000 replicates) value of more than 70%

Fig. 6

MSA of amino acid residues of TccA protein derived from P. akhurstii strain IARI-SGMG3, IARI-SGGJ2, IARI-SGHR2 and IARI-SGMS1 with P. luminescens TccA. Amino acid residues differed at several positions. For example—polar, hydroxyl threonine changed to nonpolar, aliphatic alanine at position 253, 415, 779, 858 and 914; polar, hydroxyl serine altered to nonpolar, aliphatic glycine at position 83, 256 and 656; polar, hydroxyl threonine changed to nonpolar, aliphatic valine at position 277 and 868; polar, hydroxyl threonine changed to nonpolar, aliphatic isoleucine at position 340 and 630; neutral alanine altered to acidic glutamate at position 369

Fig. 7

Sequence conservation of P. akhurstii TccC protein across the different bacterial genera of order Enterobacteriales. A maximum-likelihood phylogeny of TccC and its putative homologues is shown. Genbank accession numbers for gene different sequences are provided in the parentheses. Cluster nodes are strongly supported by a bootstrap analysis (1,000 replicates) value of more than 70%

Fig. 8

MSA of amino acid residues of TccC protein derived from P. akhurstii strain IARI-SGMG3, IARI-SGGJ2, IARI-SGHR2 and IARI-SGMS1 with P. luminescens TccC. Amino acid residues differed at a number of positions. For example—polar, hydroxyl threonine changed to nonpolar, aliphatic alanine at position 65, 86 and 405; neutral glycine altered to acidic glutamate at position 37; polar, amide-containing glutamine changed to nonpolar, aromatic tyrosine at position 56; neutral glycine altered to acidic aspartate at position 81; polar, hydroxyl serine changed to nonpolar, cyclic proline at position 107; polar serine altered to nonpolar, aromatic phenylalanine at position 139; polar, amide-containing asparagine changed to nonpolar, cyclic proline at position 140; neutral, amide-containing glutamine altered to basic lysine at position 145; polar, hydroxyl serine changed to nonpolar, aliphatic alanine at position 158; neutral glutamine altered to basic arginine at position 452; polar, hydroxyl serine changed to nonpolar, aliphatic glycine at position 491

Fig. 9

The biological activity of PirA (a), TcaA (b), TccA (c) and TccC (d) toxins derived from P. akhurstii strain IARI-SGMG3 on fourth-instar larvae of G. mellonella. Different forms of the toxin (whole E. coli cells (10³ CFU) harbouring recombinant plasmids, sonicated cells (10 µg protein) harbouring recombinant plasmids, heat-inactivated sonicated cells (10 µg) and Proteinase K-digested sonicated cells (10 µg)) were intra-hemocoel injected into the insect larvae and incubated for 3 days. Concentrated cell supernatants (1 µg) and E. coli cells (10 µg) harbouring the empty plasmid were used as the control. X- and Y-axis in time–response curves indicate days after toxin injection and percent insect mortality, respectively. Error bars represent the standard error of the mean of three experiments. Asterisks indicate the significant difference in treatment (*P < 0.05; **P < 0.01) when compared with the control

Fig. 10

Strain-specific biological activity of PirA (a), TcaA (b), TccA (c) and TccC (d) toxins derived from P. akhurstii strain IARI-SGMG3, IARI-SGGJ2, IARI-SGHR2 and IARI-SGMS1 on fourth-instar larvae of G. mellonella. Sonicated E. coli cells (10 µg protein) harbouring recombinant plasmids were intra-hemocoel injected into the insect larvae and incubated for 3 days. E. coli cells (10 µg) harbouring the empty plasmid were used as the control. X- and Y-axis in time–response curves indicate days after toxin injection and percent insect mortality, respectively. Error bars represent the standard error of the mean of three experiments. Treatments with different letters are significantly different at P < 0.01, Tukey’s multiple comparisons test. Images (e) depict the hemolymph discoloration in TcaA toxin-challenged larvae as compared to healthy insect at 72 h after injection