Ho Am Jang

Purine biosynthesis-deficient Burkholderia mutants are incapable of symbiotic accommodation in the stinkbug

The Riptortus–Burkholderia symbiotic system represents a promising experimental model to study the molecular mechanisms involved in insect–bacterium symbiosis due to the availability of genetically manipulated Burkholderia symbiont. Using transposon mutagenesis screening, we found a symbiosis-deficient mutant that was able to colonize the host insect but failed to induce normal development of host’s symbiotic organ. The disrupted gene was identified as purL involved in purine biosynthesis. In vitro growth impairment of the purL mutant and its growth dependency on adenine and adenosine confirmed the functional disruption of the purine synthesis gene. The purL mutant also showed defects in biofilm formation, and this defect was not rescued by supplementation of purine derivatives. When inoculated to host insects, the purL mutant was initially able to colonize the symbiotic organ but failed to attain a normal infection density. The low level of infection density of the purL mutant attenuated the development of the host’s symbiotic organ at early instar stages and reduced the host’s fitness throughout the nymphal stages. Another symbiont mutant-deficient in a purine biosynthesis gene, purM, showed phenotypes similar to those of the purL mutant both in vitro and in vivo, confirming that the purL phenotypes are due to disrupted purine biosynthesis. These results demonstrate that the purine biosynthesis genes of the Burkholderia symbiont are critical for the successful accommodation of symbiont within the host, thereby facilitating the development of the host’s symbiotic organ and enhancing the host’s fitness values.

Specific midgut region controlling the symbiont population in an insect-microbe gut symbiotic association.

ABSTRACT Many insects possess symbiotic bacteria that affect the biology of the host. The level of the symbiont population in the host is a pivotal factor that modulates the biological outcome of the symbiotic association. Hence, the symbiont population should be maintained at a proper level by the hosts control mechanisms. Several mechanisms for controlling intracellular symbionts of insects have been reported, while mechanisms for controlling extracellular gut symbionts of insects are poorly understood. The bean bug Riptortus pedestris harbors a betaproteobacterial extracellular symbiont of the genus Burkholderia in the midgut symbiotic organ designated the M4 region. We found that the M4B region, which is directly connected to the M4 region, also harbors Burkholderia symbiont cells, but the symbionts therein are mostly dead. A series of experiments demonstrated that the M4B region exhibits antimicrobial activity, and the antimicrobial activity is specifically potent against the Burkholderia symbiont but not the cultured Burkholderia and other bacteria. The antimicrobial activity of the M4B region was detected in symbiotic host insects, reaching its highest point at the fifth instar, but not in aposymbiotic host insects, which suggests the possibility of symbiont-mediated induction of the antimicrobial activity. This antimicrobial activity was not associated with upregulation of antimicrobial peptides of the host. Based on these results, we propose that the M4B region is a specialized gut region of R. pedestris that plays a critical role in controlling the population of the Burkholderia gut symbiont. The molecular basis of the antimicrobial activity is of great interest and deserves future study.

Burkholderia gut symbionts enhance the innate immunity of host Riptortus pedestris

The relation between gut symbiosis and immunity has been reported in various animal model studies. Here, we corroborate the effect of gut symbiont to host immunity using the bean bug model. The bean bug, Riptortus pedestris, is a useful gut symbiosis model due to the monospecific gut symbiont, genus Burkholderia. To examine the effect of gut symbiosis to host immunity, we generated the gut symbiont-harboring (symbiotic) insect line and the gut symbiont-lacking (aposymbiotic) insect line. Upon bacterial challenges, the symbiotic Riptortus exhibited better survival than aposymbiotic Riptortus. When cellular immunity was inhibited, the symbiotic Riptortus still survived better than aposymbioic Riptortus, suggesting stronger humoral immunity. The molecular basis of the strong humoral immunity was further confirmed by the increase of hemolymph antimicrobial activity and antimicrobial peptide expression in the symbiotic insects. Taken together, our data clearly demonstrate that Burkhoderia gut symbiont positively affect the Riptortus systemic immunity.

Bacterial cell motility of Burkholderia gut symbiont is required to colonize the insect gut.

We generated aBurkholderia mutant, which is deficient of anN‐acetylmuramyl‐l‐alanine amidase, AmiC, involved in peptidoglycan degradation. When non‐motile ΔamiC mutantBurkholderia cells harboring chain form were orally administered toRiptortus insects, ΔamiC mutant cells were unable to establish symbiotic association. But, ΔamiC mutant complemented withamiC gene restored in vivo symbiotic association. ΔamiC mutant cultured in minimal medium restored their motility with single‐celled morphology. When ΔamiC mutant cells harboring single‐celled morphology were administered to the host insect, this mutant established normal symbiotic association, suggesting that bacterial motility is essential for the successful symbiosis between host insect andBurkholderia symbiont.

Understanding regulation of the host-mediated gut symbiont population and the symbiont-mediated host immunity in the Riptortus-Burkholderia symbiosis system.

Valuable insect models have tremendously contributed to our understanding of innate immunity and symbiosis. Bean bug, Riptortus pedestris, is a useful insect symbiosis model due to harboring cultivable monospecific gut symbiont, genus Burkholderia. Bean bug is a hemimetabolous insect whose immunity is not well-understood. However, we recently identified three major antimicrobial peptides of Riptortus and examined the relationship between gut symbiosis and host immunity. We found that the presence of Burkholderia gut symbiont positively affects Riptortus immunity. From studying host regulation mechanisms of symbiont population, we revealed that the symbiotic Burkholderia cells are much more susceptible to Riptortus immune responses than the cultured cells. We further elucidated that the immune-susceptibility of the Burkholderia gut symbionts is due to the drastic change of bacterial cell envelope. Finally, we show that the immune-susceptible Burkholderia symbionts are able to prosper in host owing to the suppression of immune responses of the symbiotic midgut.

An antimicrobial protein of the Riptortus pedestris salivary gland was cleaved by a virulence factor of Serratia marcescens

ABSTRACT Recently, our group demonstrated that the bean bug, Riptortus pedestris, is a good experimental symbiosis model to study the molecular cross‐talk between the host insect and the gut symbiont. The Burkholderia symbiont is orally acquired by host nymphs from the environment in every generation. However, it is still unclear how Riptortus specifically interacts with entomopathogens that are abundant in the environmental soil. In preliminary experiments, we observed that a potent entomopathogen, Serratia marcescens, can colonize the midgut of Riptortus insects and was recovered from the midgut when Serratia cells were orally administered, suggesting that this pathogenic bacterium can escape host immune defenses in the salivary fluid. We examined how orally fed Serratia cells can survive in the presence of antimicrobial substances of the Riptortus salivary fluid. In this study, a 15 kDa trialysin‐like protein from the salivary gland of R. pedestris and a potent virulence factor of Serratia cells, a serralysin metalloprotease, from the culture medium of S. marcescens were successfully purified to homogeneity. When the purified Riptortus trialysin (rip‐trialysin) was incubated with purified serralysin, rip‐trialysin was specifically hydrolyzed by serralysin, leading to the loss of antimicrobial activity. These results clearly demonstrated that a potent virulent metalloprotease of S. marcescens functions as a key player in the escape from the salivary fluid‐mediated host immune response, resulting in successful colonization of S. marcescens in the host midgut. HighlightsSalivary glands of insects contain some antimicrobial substances to protect the host.Two proteins, host antimicrobial peptides and pathogen virulence factor were purified.Antimicrobial peptides were specifically hydrolyzed by virulence factor.virulent factor can escape from the salivary fluid‐mediated host immune response.

Gut symbiotic bacteria stimulate insect growth and egg production by modulating hexamerin and vitellogenin gene expression

ABSTRACT Recent studies have suggested that gut symbionts modulate insect development and reproduction. However, the mechanisms by which gut symbionts modulate host physiologies and the molecules involved in these changes are unclear. To address these questions, we prepared three different groups of the insect Riptortus pedestris: Burkholderia gut symbiont‐colonized (Sym) insects, Burkholderia‐non‐colonized (Apo) insects, and Burkholderia‐depleted (SymBurk‐) insects, which were fed tetracycline. When the hemolymph proteins of three insects were analyzed by SDS‐PAGE, the hexamerin‐&agr;, hexamerin‐&bgr; and vitellogenin‐1 proteins of Sym‐adults were highly expressed compared to those of Apo‐ and SymBurk‐‐insects. To investigate the expression patterns of these three genes during insect development, we measured the transcriptional levels of these genes. The hexamerin‐&bgr; gene was specifically expressed at all nymphal stages, and its expression was detected 4–5 days earlier in Sym‐insect nymphs than that in Apo‐ and SymBurk‐‐insects. However, the hexamerin‐&agr; and vitellogenin‐1 genes were only expressed in adult females, and they were also detected 6–7 days earlier and were 2‐fold higher in Sym‐adult females than those in the other insects. Depletion of hexamerin‐&bgr; by RNA interference in 2nd instar Sym‐nymphs delayed adult emergence, whereas hexamerin‐&agr; and vitellogenin‐1 RNA interference in 5th instar nymphs caused loss of color of the eggs of Sym‐insects. These results demonstrate that the Burkholderia gut symbiont modulates host development and egg production by regulating production of these three hemolymph storage proteins. HighlightsGut symbionts are known to modulate host development and egg production.Expressions of hemolymph proteins are specifically changed by presence of gut symbiont.Burkholderia symbiont promotes the hosts development and fecundity via symbiosis‐affected host molecule.This study describes molecular cross‐talk between insects and gut symbionts.

The lipopolysaccharide core oligosaccharide of Burkholderia plays a critical role in maintaining a proper gut symbiosis with the bean bug Riptortus pedestris

Lipopolysaccharide, the outer cell-wall component of Gram-negative bacteria, has been shown to be important for symbiotic associations. We recently reported that the lipopolysaccharide O-antigen of Burkholderia enhances the initial colonization of the midgut of the bean bug, Riptortus pedestris. However, the midgut-colonizing Burkholderia symbionts lack the O-antigen but display the core oligosaccharide on the cell surface. In this study, we investigated the role of the core oligosaccharide, which directly interacts with the host midgut, in the Riptortus–Burkholderia symbiosis. To this end, we generated the core oligosaccharide mutant strains, ΔwabS, ΔwabO, ΔwaaF, and ΔwaaC, and determined the chemical structures of their oligosaccharides, which exhibited different compositions. The symbiotic properties of these mutant strains were compared with those of the wild-type and O-antigen–deficient ΔwbiG strains. Upon introduction into Riptortus via the oral route, the core oligosaccharide mutant strains exhibited different rates of colonization of the insect midgut. The symbiont titers in fifth-instar insects revealed significantly reduced population sizes of the inner core oligosaccharide mutant strains ΔwaaF and ΔwaaC. These two strains also negatively affected host growth rate and fitness. Furthermore, R. pedestris individuals colonized with the ΔwaaF and ΔwaaC strains were vulnerable to septic bacterial challenge, similar to insects without a Burkholderia symbiont. Taken together, these results suggest that the core oligosaccharide from Burkholderia symbionts plays a critical role in maintaining a proper symbiont population and in supporting the beneficial effects of the symbiont on its host in the Riptortus–Burkholderia symbiosis.

A midgut lysate of the Riptortus pedestris has antibacterial activity against LPS O-antigen-deficient Burkholderia mutants.

ABSTRACT Riptortus pedestris, a common pest in soybean fields, harbors a symbiont Burkholderia in a specialized posterior midgut region of insects. Every generation of second nymphs acquires new Burkholderia cells from the environment. We compared in vitro cultured Burkholderia with newly in vivo colonized Burkholderia in the host midgut using biochemical approaches. The bacterial cell envelope of in vitro cultured and in vivo Burkholderia differed in structure, as in vivo bacteria lacked lipopolysaccharide (LPS) O‐antigen. The LPS O‐antigen deficient bacteria had a reduced colonization rate in the host midgut compared with that of the wild‐type Burkholderia. To determine why LPS O‐antigen‐deficient bacteria are less able to colonize the host midgut, we examined in vitro survival rates of three LPS O‐antigen‐deficient Burkholderia mutants and lysates of five different midgut regions. The LPS O‐antigen‐deficient mutants were highly susceptible when cultured with the lysate of a specific first midgut region (M1), indicating that the M1 lysate contains unidentified substance(s) capable of killing LPS O‐antigen‐deficient mutants. We identified a 17 kDa protein from the M1 lysate, which was enriched in the active fractions. The N‐terminal sequence of the protein was determined to be a soybean Kunitz‐type trypsin inhibitor. These data suggest that the 17 kDa protein, which was originated from a main soybean source of the R. pedestris host, has antibacterial activity against the LPS O‐antigen deficient (rough‐type) Burkholderia. HighlightsBurkholderia LPS O‐antigen‐deficient bacteria had a reduced colonization rate in the host midgut.In vitro survival rates of three LPS O‐antigen‐deficient mutants were examined.A specific midgut contains substance killing LPS O‐antigen‐deficient mutants.17 kDa trypsin inhibitor has antibacterial activity against the LPS O‐antigen‐deficient mutant.