Specialized digestive mechanism for an insect-bacterium gut symbiosis

Abstract

In Burkholderia-Riptortus symbiosis, the host bean bug Riptortus pedestris harbors Burkholderia symbionts in its symbiotic organ, M4 midgut, for use as a nutrient source. After occupying M4, excess Burkholderia symbionts are moved to the M4B region, wherein they are effectively digested and absorbed. Previous studies have shown that M4B has strong symbiont-specific antibacterial activity, which is not because of the expression of antimicrobial peptides but rather because of the expression of digestive enzymes, mainly cathepsin L protease. However, in this study, inhibition of cathepsin L activity did not reduce the bactericidal activity of M4B, indicating that there is an unknown digestive mechanism that renders specifically potent bactericidal activity against Burkholderia symbionts. Transmission electron microscopy revealed that the lumen of symbiotic M4B was filled with a fibrillar matter in contrast to the empty lumen of aposymbiotic M4B. Using chromatographic and electrophoretic analyses, we found that the bactericidal substances in M4B existed as high-molecular-weight (HMW) complexes that were resistant to protease degradation. The bactericidal HMW complexes were visualized on non-denaturing gels using protein- and polysaccharide-staining reagents, thereby indicating that the HMW complexes are composed of proteins and polysaccharides. Strongly stained M4B lumen with Periodic acid-Schiff (PAS) reagent in M4B paraffin sections confirmed HMW complexes with polysaccharide components. Furthermore, M4B smears stained with Periodic acid-Schiff revealed the presence of polysaccharide fibers. Therefore, we propose a key digestive mechanism of M4B: bacteriolytic fibers, polysaccharide fibers associated with digestive enzymes such as cathepsin L, specialized for Burkholderia symbionts in Riptortus gut symbiosis.

Keywords: Burkholderia; Riptortus pedestris; fibers; midgut; symbiosis.

Figure 1

(A) Midgut regions of bean bug. Burkholderia cells uptaken by mouth move through midgut regions in the order of M1, M2, M3, M4B, and M4. M4 midgut region is wherein they colonize and proliferate. After filling up the M4, excess Burkholderia symbionts flow back to M4B where they are digested and absorbed by the host. (B) TEM image of M4B lumen of symbiotic bean bug. TEM image was obtained from the M4B specimen of a bean bug harboring Burkholderia symbiont in M4. The fibrillar matter is indicated with green arrowheads. (C) TEM image of M4B lumen of aposymbiotic bean bug. The M4B specimen was prepared from a bean bug without Burkholderia symbionts. (D) TEM images of M4B lumen located close to M4 of symbiotic bean bug. Intact Burkholderia cells coming from M4 (blue arrows in the third panel) are lysed as they move toward the fibrillar matter of M4B. Remnants of dissolved Burkholderia cells in the fibrillar matters are indicated by red arrows in the first panel. The fibrillar matter is indicated with green arrowheads. Scale bars are shown on the images.

Figure 2

(A) Size-exclusion chromatography of M4B lysate. The M4B lysate prepared from 200 bean bugs was separated by a Superdex 200 (1 × 30 cm) column according to their size and collected 1 mL of eluate per fraction. The protein contents of eluates were detected by UV absorbance at 280 nm (OD₂₈₀, red line). Bactericidal activity of each fraction was determined by optical density at 600 nm of a 12-h culture of Burkholderia symbiont after treating with fractions. The bactericidal rate (light blue column) was calculated using the following formula: {1 – (OD₆₀₀ of sample / OD₆₀₀ of control)} × 100. (B) Size-exclusion chromatography of M4B fractions with protease K treatment. Eluates with bactericidal activity from the first size exclusion chromatography (5 mL) were concentrated. Prior to loading onto a Superdex 200 column, half of the concentrates were non-treated for use as a control (i), and the other half was treated with protease K (100 μg/mL) for 1 h (ii). (C) SDS-PAGE analysis of M4B fractions with protease K treatment. BSA was treated with protease K to verify the proteolytic activity (Lanes 1 and 2). Proteins of M4B fractions without treatment (Lane 3), protease K-treatment (Lane 4), and heat-denaturation and protease K treatment (Lane 5) were separated on SDS-PAGE and visualized by Coomassie Brilliant Blue staining. Protein bands of protease K are shown at 29 kDa in Lanes 2, 4, and 5. (D) Denaturing and non-denaturing SDS-PAGE analyses of M4B fractions with protein or polysaccharide staining. Proteins and polysaccharides on the denaturing and non-denaturing SDS-PAGE gels were visualized by Coomassie Brilliant Blue staining and ProQ emerald 300 staining, respectively. The HMW complexes are shown in non-denaturing gels (red arrowheads). (E) H&E and PAS staining of M4B paraffin sections. The paraffin-embedded M4B section was stained with H&E (i) or hematoxylin and PAS (ii). M4B tissues were stained with hematoxylin, showing blue-purple color. However, the M4B lumen was specifically stained with PAS, showing magenta color. (F) PAS staining of M4B smear. The M4B lumen contents were smeared on slide glasses and stained with PAS. Polysaccharide fibers are visualized in magenta color (i–iii). (G) Schematic illustration of the lysis of Burkholderia symbiont by bacteriolytic fibers. As coming from M4 to M4B, Burkholderia symbionts move toward polysaccharide fibers tightly associated with digestive enzymes, named bacteriolytic fibers. The contact with the bacteriolytic fibers induces lysis and digestion of Burkholderia symbionts, and consequently absorption by M4B. Scale bars are shown on the images.