Phylogenetic determinants of toxin gene distribution in genomes of Brevibacillus laterosporus.

Brevibacillus laterosporus is a globally ubiquitous, spore forming bacterium, strains of which have shown toxic activity against invertebrates and microbes and several have been patented due to their commercial potential. Relatively little is known about this bacterium. Here, we examined the genomes of six published and five newly determined genomes of B. laterosporus, with an emphasis on the relationships between known and putative toxin encoding genes, as well as the phylogenetic relationships between strains. Phylogenetically, strain relationships are similar using average nucleotide identity (ANI) values and multi-gene approaches, although PacBio sequencing revealed multiple copies of the 16S rDNA gene which lessened utility at the strain level. Based on ANI values, the New Zealand isolates were distant from other isolates and may represent a new species. While all of the genomes examined shared some putative toxicity or virulence related proteins, many specific genes were only present in a subset of strains.

Endophytic ability of the insecticidal bacterium Brevibacillus laterosporus in Brassica.

Brevibacillus laterosporus (Bl), is an insecticidal bacterium recorded as toxic to a range of invertebrates after ingestion. Isolates of Bl, which were initially recovered from surface-sterilised cabbage (Brassica oleracea var. capitata) seeds, were able to colonise brassica plants in the laboratory and field. The bacterium was recovered from surface-sterilised leaf, stem and root sections of seedlings after inoculation with Bl vegetative cells under laboratory conditions, and from mature cabbage plants sprayed with Bl in a field trial. The identity of the recovered bacterial isolates was confirmed by PCR through amplification of 16S rDNA and two strain-specific regions. The effect on diamondback moth (DBM) insect herbivory was tested with cabbage seedlings treated with one isolate (Bl 1951) as the strains are toxic to DBM after direct ingestion. While no effect on DBM larval herbivory was observed, there was a significant reduction of DBM pupation on the Bl 1951 colonised plants. The presence of Bl 1951 wild type cells within cabbage root tissue was confirmed by confocal microscopy, establishing the endophytic nature of the bacterium. The bacterium was also endophytic in three other brassica species tested, Chinese kale (Brassica oleracea var. alboglabra), oilseed rape (Brassica napus var. oleifera) and radish (Raphanus sativus).

Diet influences the bacterial and free fatty acid profiles of the cuticle of Galleria mellonella larvae.

The evolutionary success of insects is arguably due to their ability to build up a complex, highly-adaptable and very effective defense system against numerous pathogens, including entomopathogenic fungi. This system relies on the humoral immune system and cellular defense reactions. The first line of defense against biological pathogens is a cuticle formed of several layers. The cuticular lipids may contain hydrocarbons, free fatty acids (FFA), alcohols, waxes, glycerides, aldehydes and sterols. Cuticular fatty acids may also play a role in defending against fungal invasion. Our present findings show that the diet of insects can have a significant effect on their sensitivity and defense response to pathogens; for example, while G. mellonella larvae fed on beeswax had a similar appearance to those reared on a semi-artificial diet, they possessed a different cuticular free fatty acid (FFA) profile to those fed on a semi-artificial diet, and were less sensitive to Conidiobolus coronatus infection. It is possible that the presence of heneicosenoic acid (C21:1) and other long-chain free fatty acids (C22:0, C24:0, C26:0), as well as Brevibacillus laterosporus bacteria, on the cuticle of larvae fed on beeswax, plays a protective role against fungal invasion. Insect pests represent a global problem. An understanding of the basic mechanisms underlying the fungal infection of insects might provide a clearer insight into their defenses, thus allowing the design of more effective, and environmentally-friendly, means of controlling them. The greater wax moth is an excellent model for the study of immunology resistance. Knowledge of the influence of diet on pathogen resistance in insects can be also useful for creating a model of human diseases caused by pathogens, such as Candia albicans.

Survey of Brevibacillus laterosporus insecticidal protein genes and virulence factors.

The pathogenic action of the bacterium Brevibacillus laterosporus against invertebrates involves a toxin-mediated mechanism. Several studies, conducted with specific strains against diverse targets, suggested the implication of different toxins. Recent genome sequencing and annotation of some insecticidal strains revealed several putative virulence factors highly conserved in this species. After determining the pathogenicity of strain UNISS 18 against different Lepidopteran and Dipteran larvae, in this study we have investigated the actual expression of genes encoding for enzymes (i.e., chitinases, proteases), toxins, and other virulence factors, either in vitro and in vivo at the transcriptional level. Selected genes encode for two chitinases, a collagenase-like protease, a GlcNAc-binding protein, two protective antigen proteins, a bacillolysin, a thermophilic serine proteinase, two spore surface proteins, an insecticidal toxin homologous to Cry75Aa. All target genes were well expressed in pure bacterial cultures with significant differences between bacterial growth phases. Their expression level was generally enhanced in the bacterial population developing in the insect body cavity, compared with pure culture. The expression of certain genes increased substantially over time after insect inoculation. These results support a complex mechanism of action leveraging a variety of available virulence factors, and can also explain the ability of this bacterial species to act against diverse invertebrate targets.

Laboratory evaluation of Brevibacillus laterosporus strains as biocidal agents against Chrysomya megacephala (Fabricius, 1794) (Diptera: Calliphoridae) larvae.

The biocidal activity of three strains of Brevibacillus laterosporus upon the post-embryonic developmental stages of Chrysomya megacephala was evaluated. Bioassays were performed to verify lethal and sub-lethal effects including ultra-structural changes in the midgut. Among the strains assayed, Shi3 presented the highest larval mortality rates, achieving 70% at a concentration of 1×108 spores/g of diet. Transmission electron microscopy revealed intestinal alterations caused by all strains tested. The findings of this study indicate that Shi3 represents a promising tool for use in the biocontrol of C. megacephala.

Spore surface proteins of Brevibacillus laterosporus are involved in insect pathogenesis.

Outer spore envelope proteins of pathogenic bacteria often present specific virulence factors and tools to evade the defence system of their hosts. Brevibacillus laterosporus, a pathogen of invertebrates and an antimicrobial-producing species, is characterised by a unique spore coat and canoe-shaped parasporal body (SC-CSPB) complex surrounding the core spore. In the present study, we identified and characterised major proteins of the SC-CSPB complex of B. laterosporus, and we investigated their entomopathogenic role. Employing a proteomic approach and a B. laterosporus-house fly study model, we found four highly conserved proteins (ExsC, CHRD, CpbA and CpbB) that function as insect virulence factors. CpbA was associated with a significantly higher mortality of flies and greater relative gene expression levels during sporulation, compared to the other SC-CSPB proteins. Taken together, we suggest that spore surface proteins are a part of a complex set of toxins and virulence factors that B. laterosporus employs in its pathogenicity against flies.

Brevibacillus laterosporus pathogenesis and local immune response regulation in the house fly midgut.

The insect midgut represents the primary site of action of the entompathogenic bacterium Brevibacillus laterosporus. While most studies on this microorganism focus on the identification and characterization of possible virulence factors and toxins, little is known about the insect immune defense mechanisms that are activated against this pathogen. In this study we have investigated the local immune response of different house fly stages to B. laterosporus at the transcriptional level, and we tested the hypothesis that an improvement in entomopathogenicity can be achieved by impairing host innate immunity. Gene expression analyses showed that immediately after spore ingestion (6-12h) both larvae and adults increased the transcription rate of immune related genes in the midgut tissues, with special regard to those encoding for the main house fly antimicrobial peptides (AMPs) (i.e., attacin, cecropin, defensin, diptericin, domesticin, muscin) and for prophenoloxydase that is normally involved in the cascade of events leading to the generation of reactive oxygen species (ROS) and other factors with antibacterial properties. In experiments evaluating the use of an immunosuppressive agent to enhance the virulence of B. laterosporus against adult house flies, a significant downregulation of the same genes was observed 12-24h after the administration of sub-lethal doses of the botanical compound azadirachtin. Consequently, a significant increase in B. laterosporus entomopathogenic action was observed when flies were preliminarily or simultaneously exposed to a sub-lethal dose of azadirachtin. These results provide an important contribution to the prospect of employing immune-impairing tools to implement pest management strategies.

Transformation of Brevibacillus, a soil microbe to an uropathogen with hemagglutination trait.

An urinary tract infection (UTI) with Brevibacillus agri, an aerobic soil bacteria is discussed. The preliminary urine analysis tested negative for UTI, while the patient was diagnosed with focal pyelonephritis. The urine sample was analyzed for the presence of possible micro-organisms. The isolated micro-organism was phenotypically characterized and compared with a standard B. agri strain and an established uropathogen Eschericia coli, CFT073. Each experimental mouse was trans-urethrally infected using 2.5 × 10(8) c.f.u. for the generation of an UTI model. The kidney tissues were stored in buffered 10 % formaldehyde solution for histopathological analysis. The destruction of the glomerular and tubular morphology with prominent hemagglutination in the tubular region in the mouse kidneys were confirmed by light microscopic examination of the H&E stained sections. Hence, the identity of pathogen was confirmed using the 16S rRNA gene sequencing. The phylogenetic tree constructed using the 16S rRNA sequence obtained from the isolated microbial strain showed 99 % similarity with the strain, B. agri AB112716. Finally this study concludes based on the phenotypic characteristics, production of lipopolysaccharide, the ability to aggregate in the presence of ammonium sulphate, agglutinate erythrocytes in the presence of mannose, and the potential to resist the interactions of serum, the possibility that the soil microbe could have undergone genotypic modification to cause UTI. However, further detailed and in-depth genetic analysis are required to point out exactly how the soil bacterium has adapted itself to cause infection in a human subject.

Production of an in vitro-derived deletion mutation of Brevibacillus laterosporus by constructing a homology-driven integration vector.

In this study, a homology-driven integration vector and electroporation system was developed to delete a protease gene in the pathogenic bacterium Brevibacillus laterosporus strain G4. Furthermore, an in vitro protease-deficient mutation was generated by introducing the integration vector with a 445-bp protease BLG4 fragment into B. laterosporus chromosomal target via homologous recombination. The BLG4-deficient mutant showed a significant drop in protease activity as compared to the wild-type G4 strain, but had a slight effect on bacterial growth and sporulation. The results revealed that the developed method can become an important tool for studying the molecular pathogenesis mechanisms of B. laterosporus.