Purification of high molecular-weight antibacterial proteins of insect pathogenic Brevibacillus laterosporus isolates

Brevibacillus laterosporus (Bl) is a Gram-positive and spore-forming bacterium belonging to the Brevibacillus brevis phylogenetic cluster. Globally, insect pathogenic strains of the bacterium have been isolated, characterised, and some activities patented. Two isolates, Bl 1821L and Bl 1951, exhibiting pathogenicity against the diamondback moth and mosquitoes, are under development as a biopesticide in New Zealand. However, due to the suspected activity of putative antibacterial proteins (ABPs), the endemic isolates often grow erratically. Various purification methods including size exclusion chromatography, sucrose density gradient centrifugation, polyethylene glycol precipitation, and ammonium sulphate precipitation employed in this study enabled the isolation of two putative antibacterial proteins of ~30 kD and ~48 kD from Bl 1821L and one putative antibacterial protein of ~30 kD from Bl 1951. Purification of the uninduced cultures of Bl 1821L and Bl 1951 also yielded the protein bands of ~30 kD and ~48 kD on SDS-PAGE which indicated their spontaneous induction. Disc diffusion assay was used to determine the antagonistic activities of the putative ABPs. Subsequent transmission electron microscope (TEM) examination of purified putative antibacterial protein-containing solution showed the presence of encapsulin (~30 kD) and polysheath (~48 kD) like structures. Although only the ~30 kD protein was purified from Bl 1951, both structures were seen in this strain under TEM. Furthermore, while assessing the antibacterial activity of some fractions of Bl 1951 against Bl 1821L in size exclusion chromatography method, population of Bl 1821L persister cells was noted. Overall, this work added a wealth of knowledge for the purification of the HMW proteins (bacteriocins) of the Gram-positive bacteria including Bl

Isolation, purification, and characterisation of a phage tail-like bacteriocin from the insect pathogenic bacterium Brevibacillus laterosporus

The Gram-positive and spore-forming bacterium Brevibacillus laterosporus (Bl) belongs to the Brevibacillus brevis phylogenetic cluster. Isolates of the species have demonstrated pesticidal potency against a wide range of invertebrate pests and plant diseases. Two New Zealand isolates, Bl 1821L and Bl 1951, are under development as biopesticides for control of diamondback moth and other pests. However, due to the often-restricted growth of these endemic isolates, production can be an issue. Based on the previous work, it was hypothesised that the putative phages might be involved. During investigations of the cause of the disrupted growth, electron micrographs of crude lysate of Bl 1821L showed the presence of phages’ tail-like structures. A soft agar overlay method with PEG 8000 precipitation was used to differentiate between the antagonistic activity of the putative phage and phage tail-like structures (bacteriocins). Assay tests authenticated the absence of putative phage activity. Using the same method, broad-spectrum antibacterial activity of Bl 1821L lysate against several Gram-positive bacteria was found. SDS-PAGE of sucrose density gradient purified and 10 kD MWCO concentrated lysate showed a prominent protein band of ∼48 kD, and transmission electron microscopy revealed the presence of polysheath-like structures. N-terminal sequencing of the ∼48 kD protein mapped to a gene with weak predicted amino acid homology to a Bacillus PBSX phage-like element xkdK, the translated product of which shared >90% amino acid similarity to the phage tail-sheath protein of another Bl published genome, LMG15441. Bioinformatic analysis also identified an xkdK homolog in the Bl 1951 genome. However, genome comparison of the region around the xkdK gene between Bl 1821L and Bl 1951 found differences including two glycine rich protein encoding genes which contain imperfect repeats (1700 bp) in Bl 1951, while a putative phage region resides in the analogous Bl 1821L region. Although comparative analysis of the genomic organisation of Bl 1821L and Bl 1951 PBSX-like region with the defective phages PBSX, PBSZ, and PBP 180 of Bacillus subtilis isolates 168 and W23, and Bacillus phage PBP180 revealed low amino acids similarity, the genes encode similar functional proteins in similar arrangements, including phage tail-sheath (XkdK), tail (XkdO), holin (XhlB), and N-acetylmuramoyl-L-alanine (XlyA). AMPA analysis identified a bactericidal stretch of 13 amino acids in the ∼48 kD sequenced protein of Bl 1821L. Antagonistic activity of the purified ∼48 kD phage tail-like protein in the assays differed remarkably from the crude lysate by causing a decrease of 34.2% in the number of viable cells of Bl 1951, 18 h after treatment as compared to the control. Overall, the identified inducible phage tail-like particle is likely to have implications for the in vitro growth of the insect pathogenic isolate Bl 1821L

Purification of high-molecular-weight antibacterial proteins of insect pathogenic Brevibacillus laterosporus isolates

Brevibacillus laterosporus (Bl) is a Gram-positive and spore-forming bacterium belonging to the Brevibacillus brevis phylogenetic cluster. Globally, insect pathogenic strains of the bacterium have been isolated, characterised, and some activities have been patented. Two isolates, Bl 1821L and Bl 1951, exhibiting pathogenicity against the diamondback moth and mosquitoes, are under development as a biopesticide in New Zealand. However, due to the suspected activity of putative antibacterial proteins (ABPs), the endemic isolates often grow erratically. Various purification methods, including size exclusion chromatography, sucrose density gradient centrifugation, polyethylene glycol precipitation, and ammonium sulphate precipitation employed in this study, enabled the isolation of two putative antibacterial proteins of ∼30 and ∼48 kD from Bl 1821L and one putative antibacterial protein of ∼30 kD from Bl 1951. Purification of the uninduced cultures of Bl 1821L and Bl 1951 also yielded protein bands of ∼30 and ∼48 kD on SDS-PAGE, which indicated their spontaneous induction. A disc diffusion assay was used to determine the antagonistic activities of the putative ABPs. Subsequent transmission electron microscope (TEM) examination of a purified putative antibacterial protein-containing solution showed the presence of encapsulin (∼30 kD) and polysheath (∼48 kD)-like structures. Although only the ∼30 kD protein was purified from Bl 1951, both structures were seen in this strain under TEM. Furthermore, while assessing the antibacterial activity of some fractions of Bl 1951 against Bl 1821L in the size exclusion chromatography method, the population of Bl 1821L persister cells was noted. Overall, this work added a wealth of knowledge about the purification of the high-molecular-weight (HMW) proteins (bacteriocins) of Gram-positive bacteria including Bl

Linocin M18 protein from the insect pathogenic bacterium Brevibacillus laterosporus isolates

Brevibacillus laterosporus (Bl) is a Gram-positive and spore-forming bacterium. Insect pathogenic strains have been characterised in New Zealand, and two isolates, Bl 1821L and Bl 1951, are under development for use in biopesticides. However, growth in culture is sometimes disrupted, affecting mass production. Based on previous work, it was hypothesised that Tectiviridae phages might be implicated. While investigating the cause of the disrupted growth, electron micrographs of crude lysates showed structural components of putative phages including capsid and tail-like structures. Sucrose density gradient purification yielded a putative self-killing protein of ∼30 kDa. N-terminal sequencing of the ∼30 kDa protein identified matches to a predicted 25 kDa hypothetical and a 31.4 kDa putative encapsulating protein homologs, with the genes encoding each protein adjacent in the genomes. BLASTp analysis of the homologs of 31.4 kDa amino acid sequences shared 98.6% amino acid identity to the Linocin M18 bacteriocin family protein of Brevibacterium sp. JNUCC-42. Bioinformatic tools including AMPA and CellPPD defined that the bactericidal potential originated from a putative encapsulating protein. Antagonistic activity of the ∼30 kDa encapsulating protein of Bl 1821L and Bl 1951 during growth in broth exhibited bacterial autolytic activity. LIVE/DEAD staining of Bl 1821L cells after treatment with the ∼30 kDa encapsulating protein of Bl 1821L substantiated the findings by showing 58.8% cells with the compromised cell membranes as compared to 37.5% cells in the control. Furthermore, antibacterial activity of the identified proteins of Bl 1821L was validated through gene expression in a Gram-positive bacterium Bacillus subtilis WB800N

Economic evaluation of pharmacist-led medication reviews in residential aged care facilities

Introduction: Medication reviews is a widely accepted approach known to have a substantial impact on patients’ pharmacotherapy and safety. Numerous options to optimise pharmacotherapy in older people have been reported in literature and they include medication reviews, computerised decision support systems, management teams, and educational approaches. Pharmacist-led medication reviews are increasingly being conducted, aimed at attaining patient safety and medication optimisation. Cost effectiveness is an essential aspect of a medication review evaluation. Areas covered: A systematic searching of articles that examined the cost-effectiveness of medication reviews conducted in aged care facilities was performed using the relevant databases. Pharmacist-led medication reviews confer many benefits such as attainment of biomarker targets for improved clinical outcomes, and other clinical parameters, as well as depict concrete financial advantages in terms of decrement in total medication costs and associated cost savings. Expert commentary: The cost-effectiveness of medication reviews are more consequential than ever before. A critical evaluation of pharmacist-led medication reviews in residential aged care facilities from an economical aspect is crucial in determining if the time, effort, and direct and indirect costs involved in the review rationalise the significance of conducting medication reviews for older people in aged care facilities

Trap cropping in South Asia: Concepts, limitations, and future strategy

Cultural methods are some of the most widely adopted approaches in integrated pest management. Trap cropping is based on the principle of using a relatively more preferred crop species to keep the pest away from the main crop and reduce pest damage. This technique has tremendous potential to keep the pest below the economic damage threshold and can be used for pest management in organic farming. Furthermore, trap crops can be linked to habitat management and conservation biological control to improve multiple ecosystem services in an agroecosystem. While trap cropping is one of the most common cultural pest management control methods in subsistence farming in South Asia, it has not yet become common in conventional agriculture, nor has this practice been well documented in this region. This work broadly reviews the most relevant literature related to trap cropping used in pest management in this region. Regional cooperation for knowledge-sharing and research collaborations, motivating farmers to promote organic farming, along with increased research and policy interventions to favor sustainable agriculture have been done to promote this pest management practice in South Asia

Comparison of Phenacoccus solenopsis specimens from different regions of Pakistan using COI molecular barcoding (Hemiptera: Pseudococcidae)

Because correct identification of insects is crucial for pest management involving chemical or biological control agents, we have used a molecular approach to identify and characterize specimens of the cotton pest Phenacoccus solenopsis Tinsley (Sternorrhyncha: Pseudococcidae) present in different regions of Pakistan. The specimens were analyzed through DNA sequence analysis of their mitochondrial COI (mtCOI) gene using an improved procedure that could distinguish between the pest and its associated parasitoid. Our analysis showed no variation among the mealybug specimens from different geographical locations of Pakistan and confirmed that this is the same species and haplotype that is infesting cotton plants in other parts of Asia. This information will assist in the development of biological control programs against P. solenopsis in Pakistan and other Asian countries

OVX836 a recombinant nucleoprotein vaccine inducing cellular responses and protective efficacy against multiple influenza A subtypes

Influenza vaccine: engineered nucleoprotein with improved protective efficacy against multiple strains Circulating influenza A virus (IAV) strains differ in their surface proteins each year, and vaccines eliciting an immune response to these proteins are often only partially protective. Internal viral proteins, such as the nucleoprotein (NP), are highly conserved, and cellular immunity to NP has been correlated with protection from diverse strains. However, current IAV vaccines induce a poor immune response to NP. In this study, led by Fergal Hill from Osivax, researchers develop an oligomeric version of NP with improved immunogenicity. Vaccination of mice with oligomeric NP results in an improved NP-specific T-cell response, including CD8+ tissue memory T cells in the lung, and protects mice against three different IAV subtypes. Co-administration with the currently used inactivated influenza vaccine further improves protection against virus infection in mice. These results encourage further pre-clinical and clinical development for this vaccine candidate