Variations of Indole Metabolites and NRPS-PKS Loci in Two Different Virulent Strains of Xenorhabdus hominickii.

Xenorhabdus hominickii ANU1 is known to be an entomopathogenic bacterium symbiotic to nematode Steinernema monticolum. Another bacterial strain X. hominickii DY1 was isolated from a local population of S. monticolum. This bacterial strain X. hominickii DY1 was found to exhibit high insecticidal activities against lepidopteran and coleopteran species after hemocoelic injection. However, these two X. hominickii strains exhibited significant variations in insecticidal activities, with ANU1 strain being more potent than DY1 strain. To clarify their virulence difference, bacterial culture broths of these two strains were compared for secondary metabolite compositions. GC-MS analysis revealed that these two strains had different compositions, including pyrrolopyrazines, piperazines, cyclopeptides, and indoles. Some of these compounds exhibited inhibitory activities against phospholipase A2 to block eicosanoid biosynthesis and induce significant immunosuppression. They also exhibited significant insecticidal activities after oral feeding, with indole derivatives being the most potent. More kinds of indole derivatives were detected in the culture broth of ANU1 strain. To investigate variations in regulation of secondary metabolite production, expression level of leucine-responsive regulatory protein (Lrp), a global transcription factor, was compared. ANU1 strain exhibited significantly lower Lrp expression level than DY1 strain. To assess genetic variations associated with secondary metabolite synthesis, bacterial loci encoding non-ribosomal protein synthase and polyketide synthase (NRPS-PKS) were compared. Three NRPS and four PKS loci were predicted from the genome of X. hominickii. The two bacterial strains exhibited genetic variations (0.12∼0.67%) in amino acid sequences of these NRPS-PKS. Most NRPS-PKS genes exhibited high expression peaks at stationary phase of bacterial growth. However, their expression levels were significantly different between the two strains. These results suggest that differential virulence of the two bacterial strains is caused by the difference in Lrp expression level, leading to difference in the production of indole compounds and other NRPS-PKS-associated secondary metabolites.

Variation in pathogenicity of different strains of Xenorhabdus nematophila; Differential immunosuppressive activities and secondary metabolite production.

Xenorhabdus nematophila, an entomopathogenic bacterium, is mutualistic with the nematode Steinernema carpocapsae. The bacterium produces secondary metabolites to inhibit target insect phospholipase A2 (PLA2) and induce immunosuppression, which is required for the pathogenicity of this bacterium-nematode complex. However, it was unclear if immunosuppressive intensity of the bacteria was correlated with their insecticidal potency. We compared six different X. nematophila strains inhibiting the immune responses of the beet armyworm (Spodoptera exigua) to explain their virulence variations. In addition to four known strains obtained from the Korean Agricultural Culture Collection, we identified two new strains (SK1 and SK2) of X. nematophila from two different isolates of S. carpocapsae. Although all six strains were virulent, they showed significant variation in median lethal bacterial dosage (LD50). The LD50 of most strains was 15-30 CFU/larva, however, the LD50 of the SK1 strain was more than two-fold higher against S. exigua larvae. Immunosuppressive activities of the six strains were measured by comparing hemocyte-spreading behavior and nodule formation; the SK1 strain was significantly less potent than other bacterial strains. These suppressed hemocyte behaviors were recovered by adding arachidonic acid (a catalytic product of PLA2) into all six strains. Bacterial culture broth was fractionated with different organic solvents and the ability to inhibit immune response and PLA2 activity were assessed. All organic extracts had immunosuppressive activities and PLA2-inhibitory activities. GC-MS analysis showed that these organic extracts possessed a total of 87 different compounds. There were variations in chemical components among the six bacterial strains. Organic extracts of SK1 strain, which exhibited the lowest virulence, contained the least number of secondary metabolites.

Biosynthetic pathway of arachidonic acid in Spodoptera exigua in response to bacterial challenge.

Eicosanoids play crucial roles in mediating insect immune responses. In vertebrates, phospholipase A2 (PLA2) releases arachidonic acid (AA) from phospholipids (PLs) for biosynthesis of various eicosanoids. However, little AA is found in PLs of lepidopteran insects. Spodoptera exigua, a lepidopteran insect, is known to use eicosanoids to mediate immunity. Although AA was not detected in PLs of hemocytes and fat body (two immune tissues) of naïve larvae, it was detected at small but significant level after bacterial infection, suggesting induction of AA biosynthesis for immunity. Based on a mammalian AA biosynthetic pathway, this study hypothesizes that AA is synthesized from C18 polyunsaturated fatty acid (PUFA) precursor by subsequent desaturation and elongation reactions because PLs of S. exigua larvae are rich in linoleic acid. After inhibiting PLA2 activity to prevent release of free fatty acids, different PUFA precursors were injected to S. exigua larvae followed by assessment of eicosanoid-mediated cellular immune response. ω-6 PUFAs were effective in inducing immune response whereas α-linolenic acid (an ω-3 PUFA) was not. Several fatty acyl desaturases (SeDESs) have been predicted from S. exigua transcriptomes. Specific inhibitors against Δ5 or Δ6 DESs inhibited eicosanoid-mediated immune responses. Furthermore, RNA interference (RNAi) specific to Δ5 or Δ6 DES genes significantly suppressed eicosanoid-mediated immune responses. Four very long chain fatty acid elongase genes (SeEloV-A ∼ SeEloV-D) were predicted. Among respective RNAi treatments of these genes, only one RNAi treatment specific to type 5 elongase (SeEloV-B) suppressed eicosanoid-mediated immune response. These results suggest that S. exigua larvae can synthesize AA from linoleic acid via Δ5- and Δ6-desaturations by SeDESs along with chain elongation by SeEloV-B. Finally, this study showed significant fitness cost of uncontrolled AA biosynthesis. AA injection alone without bacterial challenge significantly induced both cellular and humoral immune responses. This unnecessary energy expense due to free AA resulted in reduced pupal size and decreased adult egg production. The detrimental effect of free AA explains physiological significance of little AA content in lepidopteran insects except for life-or-death situation such as pathogen infection.

Theileria annulata seroprevalence among different cattle breeds in Rajshahi Division, Bangladesh.

An epidemiological survey of Theileria annulata infection was undertaken in a cattle population in Rajshahi Division, Bangladesh. The local cattle breeds from the area (North Bengal Gray and Deshi) and crosses between the local breeds and Holstein cattle were predominantly screened. In total, 192 cattle serum samples were collected in two areas of Rajshahi Division, the Rajshahi District (n=147) and Natore District (n=45). The samples were screened with an enzyme-linked immunosorbent assay using T. annulata surface protein (TaSP) as the antigen. The seroprevalence was 80.0% (36/45) in Natore and 20.4% (30/147) in Rajshahi. A logistic regression analysis showed that the sampling location was significantly associated with seropositivity, whereas age, sex and breed were not. Although the logistic regression analysis did not show a linear dependence on age, we considered age-specific seroprevalence separately in the two districts. Seroprevalence did not differ significantly among age categories in the Natore District. In contrast, all the cattle <1 year old in the Rajshahi District were seronegative (11/11). Seroprevalence in the 1- and 2-year-old cattle was significantly lower in the Rajshahi District than in the Natore District. In the older age categories (3, 4 and >5 years), seroprevalence did not differ significantly between the Natore and Rajshahi Districts. These results suggest that the cattle in the Rajshahi District were sporadically exposed to T. annulata, whereas most cattle in the Natore District became infected during an early phase of life.