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Putative host-derived growth factors inducing colonization of Burkholderia gut symbiont in Riptortus pedestris insect
Developmental and Comparative Immunology 104 (2020) 103570
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Putative host-derived growth factors inducing colonization of Burkholderia gut symbiont in Riptortus pedestris insect
T
Junbeom Leea,1, Xinrui Maoa,1, You Seon Leea, Dong Jung Leea, Junghyun Kima, Jiyeun Kate Kimb, Bok Luel Leea,∗ a b
Global Research Laboratory, College of Pharmacy, Pusan National University, Busan, 46241, South Korea Department of Microbiology, College of Medicine, Kosin University, Busan, 49267, South Korea
A R T I C LE I N FO
A B S T R A C T
Keywords: Riptortus pedestris Burkholderia insecticola Host-derived growth factor Symbiont's proliferation
It is questionable that how gut symbiont can be proliferated in the host symbiotic organs, such as host midgut region, which are known to be highly stressful and nutritional depleted conditions. Since Riptortus-Burkholderia symbiosis system is a good model to study this question, we hypothesized that Burkholderia symbiont will use host-derived bacterial growth factor(s) to colonize persistently in the host midgut 4 (M4) region, which is known as symbiotic organ. In this study, we observed that although gut-colonized symbiotic Burkholderia cells did not grow in the nutrient-limited media conditions, these symbionts were able to grow dose-dependent manner by addition of host naïve M4 lysate, supporting that host-derived growth factor molecule(s) may exist in the host M4 lysate. By further experiments, a host-derived growth factor(s) did not lose its biological activity in the conditions of high temperature, treatment of phenol-chloroform or ethyl alcohol precipitation, indicating that a growth factor molecule(s) is neither a protein nor a DNA. Also, based on the biochemical analyses data, molecular weight of the host-derived bacterial growth factor(s) was turned out to be less than 3 kDa molecular mass and to give the positive chemical response to the ninhydrin reagent on thin layer chromatography. Finally, we found that one specific peak showing ninhydrin positive signal was separated by gel filtration column and induced proliferative activity for Burkholderia gut symbiont cells.
1. Introduction Microorganism-mediated symbiotic associations are known for a broad range of animals, plants, and other organisms (Margulis and Fester, 1991; Ruby et al., 2004). Especially, many insects are known to possess symbiotic bacteria within their cells, tissues, and guts (Douglas, 2014; Ferrari and Vavre, 2011). These insect-symbiont associations have been established for a long time and are known to affect the biology of host insects in various ways (Kim et al., 2013a). Some symbionts play indispensable roles, such as providing essential nutrients (Moran et al., 2008). Although there are numerous examples of the transfer of essential amino acids, vitamins and short chain fatty acids from microbial symbionts to their animal hosts (Douglas, 1994), only a few evidences were reported that how host supply a quantity of nutrients to support growth of diverse bacterial symbionts. Moreover, every microorganism requires certain basic nutrients for growth and maintenance of metabolic functions. The amounts and types of nutrients are widely different depending on the microorganism. These
nutrients include water, nitrogen source, vitamins, and minerals (Busta et al., 2003; Mossel et al., 1995). Also, according to previous studies, many symbiont accumulate granules of polyhydroxyalkanoate (PHA) within their cells, which protect bacteria from nutritional depletion and other environmental stresses (Anderson and Dawes, 1990; Kim et al., 2013b; Schwartzman and Ruby, 2016). In the case of Riptortus-Burkholderia symbiosis, Riptortus pedestris host insect possesses a specialized symbiotic organ, named midgut 4 (M4) region. M4 region of one nymph harbors numerous crypts with about 107 Burkholderia insecticola symbiont cells (Kim et al., 2014). In addition, our another report revealed that the symbiont titer at the pre-molt stage transiently was decreased in comparison with that of the preceding inter-molt stage during host insect's development (Kim et al., 2014). Based on these results, we supposed that symbiotic Burkholderia cells expelled from M4 region during molting period should be re-colonized in the M4 crypt during next developmental stage, suggesting that R. pedestris host insects must provide some nutrients to their symbionts to support proliferation and
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Corresponding author. E-mail address: [email protected] (B.L. Lee). 1 J.L. and X.M. contributed equally to this article. https://doi.org/10.1016/j.dci.2019.103570 Received 25 September 2019; Received in revised form 7 December 2019; Accepted 8 December 2019 Available online 10 December 2019 0145-305X/ © 2019 Elsevier Ltd. All rights reserved.
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Fig. 1. Identification of symbiotic Burkholderia growth activity in the Riptortus M4 lysates. (A) Examination of CFUs after incubation with non-heat-treated naïve M4 lysates of fifth-instar Apo- and Sym-nymphs. Negative control [(−) control] indicates the incubation with symbiotic Burkholderia cells only in M9 minimal media. BSA indicates bovine serum albumin with 400 μg of protein. Each sample was incubated at 30°C for 12 h. (B) The Burkholderia symbiont growths were estimated by the measurement of absorbance changes after incubation with gut-colonized symbiotic Burkholderia cells and naïve M4 lysate (left) or heat-treated M4 lysate (right) of fifth-instar Apo-nymphs. The absorbance at 600 nm was measured after every 2 h (unpaired t-test: *, P < 0.01; n = 5).
distilled water containing 0.05% ascorbic acid (DWA) with a final concentration of 107 cells/ml (Lee et al., 2019). After the insects had been fed with the bacterial inoculum solution for 2 days, fresh and sterile DWA was provided to the insects instead of the bacterial inoculum solution. Upon reaching adulthood, the insects were transferred to larger containers in which soybean plant pots were placed for feeding and cotton pads were attached to the cage walls for egg laying. Eggs were collected daily and transferred to new cages for hatching.
to maintain microbial community persistently. In this study, we aimed to identify growth factor(s) that is present in the host M4 region. Here we found that gut-colonized symbiotic Burkholderia cells suspended in M9 minimal medium were grown by addition of host-derived naïve M4 lysates collected from Burkholderiaabsent aposymbiotic insects (Apo-insect) and from Burkholderia-harboring symbiotic insects (Sym-insect). These data indicate that the naïve extract of host M4 region is able to induce growth of symbiotic Burkholderia cells. Because bacterial growth-inducing activity was more increased by heat-treatment of naïve Apo-M4 lysate, we prepared heattreated Apo-M4 lysate using naïve Apo-M4 lysate for the next purification steps. The heat-treated Apo-M4 lysates were separated on gel filtration column packed with polyvinyl-based resins and a host-derived growth factor(s) was separated with several peaks showing positive response to ninhydrin reagent. As the results, a specific peak purified by biochemical approaches showed symbiotic Burkholderia growth activity, suggesting that symbiotic Burkholderia cells may require the nutrient source or host-derived growth factor(s) for persistent colonization in the insect gut.
2.2. Isolation of symbiotic Burkholderia cells from insect midgut The M4 regions from 10 fifth-instar Sym-nymphs were collected and placed in 200 μl of 10 mM phosphate buffer (PB; pH 7.0) as described (Byeon et al., 2015; Kim et al., 2013a). The M4 regions were cut with fine scissors to break the M4 crypts into small pieces. One milliliter of PB was added to the solution and symbiotic Burkholderia cells colonized in the crypt were re-suspended in the PB by gently pipetting. The solution was filtered through 5 μm pore filter to remove the midgut tissues. To remove other contaminants of host midgut, isolated symbiotic Burkholderia cells were sequentially washed with PB. Then, symbiotic Burkholderia cells collected by centrifugation at 20,000 × g for 10 min were rewashed with PB.
2. Materials and methods 2.1. Insect rearing and Burkholderia symbiont inoculation
2.3. Bioassay for measuring bacterial growth activity R. pedestris was maintained in our insect culturing room with 28°C under a long day cycle of 16 h light and 8 h dark as described (Lee et al., 2017). When the newborn nymphs were molted to second instar, in vitro cultured Burkholderia inoculated solution was provided with wet cotton balls in a petri dish. Burkholderia cells were suspended in
The M4 regions from 10 fifth-instar Apo- and Sym-nymphs were collected in 200 μl of PB and homogenized with plastic pestles. M4 lysates were centrifuged at 20,000 × g for 10 min. To separate midgut tissues and gut epithelial cells from symbiotic Burkholderia cells, this 2
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Fig. 2. Biochemical characterization of the Riptortus M4 lysates. (A) Symbiotic Burkholderia growth rates were estimated after treatment of fractions after performance of molecular weight 3 kDa cut off spin columns (Millipore). Negative control [(−) control] indicates the incubation of symbiotic Burkholderia cells only in M9 minimal media, and positive control [(+) control] means the addition of naïve Sym-M4 lysate to symbiotic Burkholderia cells suspended in M9 minimal media. 100°C sup means the supernatant of heat-treated Apo-M4 lysate. Less 3 kDa indicates the pass through of 3 kDa molecular weight cut off spin column (Millipore). More 3 kDa sample is the remaining solution on 3 kDa molecular weight cut off spin column. Each sample was incubated at 30°C for 6 h. (B) Symbiotic Burkholderia growth rates were estimated after treatment of proteinase K, ethyl alcohol precipitation or phenol-chloroform. Negative control [(−) control] indicates the incubation of symbiotic Burkholderia cells only in M9 minimal media. We normalized the growth rates to (−) control, which was set as 1. Five hundred of symbiotic Burkholderia cells were incubated with each treatment at 30°C for 6 h. All the values are means ± S.D. (n = 5). Different letters over bar indicates significant difference in treatment according to Duncan's multiple range test (P < 0.01).
100 μl of PB and homogenized with plastic pestles. The host gut-derived contaminant was removed by centrifugation at 20,000 × g for 10 min. Supernatant was collected as non-heat-treated naïve M4 lysate and used for measurement of bacterial growth activity.
procedure was repeated one more time and supernatant were finally collected. The starting bacterial solutions prepared in M9 minimal medium (0.6% Na2HPO4∙2H2O, 0.3% KH2PO4, 0.1% NH4Cl, 0.05% NaCl, 0.0003% CaCl2, 1 mM MgSO4, 0.2% glucose) by adjusting OD600nm to 0.1 (1.0 × 107 cells) using symbiotic Burkholderia cells isolated from Sym-M4 crypt were incubated with naïve Apo- and SymM4 lysates (400 μg). Each sample was incubated at 30°C for 12 h, and the samples were spread onto yeast-glucose (YG) agar plates (0.5% yeast extract, 0.4% glucose, and 0.1% NaCl) containing rifampicin (Rif; 30 μg/ml), cultured for 2 days, and subjected to colony counting. Colony forming units (CFUs) were calculated by colony numbers × dilution rate (Kim et al., 2013a).
The collected supernatant of naïve Apo-M4 lysate was heat-treated at 100°C again for 5 min and centrifuged at 20,000 × g for 10 min. Finally, clear and homogeneous supernatant was collected for further experiments.
2.4. Measurement of bacterial growth curves in liquid media
(3) Proteinase K treatment
Growth curves of symbiotic Burkholderia cells isolated from fifthinstar Sym-M4 crypt were examined by incubation with naïve Apo-M4 lysates or heat-treated Apo-M4 lysates in M9 minimal medium (Lee et al., 2015). The starting bacterial solutions prepared in M9 minimal medium by adjusting OD600nm to 0.1 using log-phased pre-cultured cells were incubated with M4 lysates on a rotator shaker at 180 rpm at 30°C for 16 h, whose OD600nm was monitored every 2 h using a spectrophotometer (UV-1700 PharmSpec, Shimadzu, Japan).
The heat-treated Apo-M4 lysate (50 μg) was incubated with 20 μg of proteinase K at 60°C for 1 h.
(2) Preparation of heat-treated Apo-M4 lysates from naïve Apo-M4 lysates
(4) Ethanol precipitation The heat-treated Apo-M4 lysates (50 μg) were incubated with 500 μl ethanol (2.5 volume of sample) at −20°C for 24 h and then was centrifuged at 20,000 × g at 4°C for 10 min. Recovered pellets were washed with 80% ethanol two times and then supernatant and washed solution were dried under vacuum at 60°C for 1 h.
2.5. Biochemical characterization of host-derived growth factors
(5) Phenol-chloroform extraction
(1) Preparation of naïve M4 lysate from Apo-insects
The mixture of acid phenol (400 μl) and chloroform (200 μl) were
The M4 lysates from fifth-instar Apo-nymphs were collected in 3
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Fig. 3. Purification of growth factor(s) derived from Apo-M4 lysate via two different gel filtration columns. (A) (i) shows the elution pattern of Apo-M4 lysate on HW55S size column and (ii) is the results of CFUs measurements of each peak of Fig. 3A (i). Five hundreds of symbiotic Burkholderia cells were treated with 50 μg of each peak. (B) (i) shows the elution pattern of HW-55S peak 5 on HW-40S column. (ii) is the results of CFUs measurements of each peak of Fig. 3B (i). The spots indicate the response by spreading of ninhydrin reagent on each peak of HW-55S column (Fig. 3A–iii) and on each peak of HW-40S column (Fig. 3B–iii) after thin layer chromatography. C1 and C2 indicate the fractions of heat-treated M4 lysate and peak 5 of HW-55S column. (unpaired t-test: *, P < 0.01; n = 5).
added into 50 μg heat-treated Apo-M4 lysate (100 μl) and vortexed at room temperature for 10 min. Then, aqueous layer was separated from phenol layer and dried under vacuum at 60°C for 1 h.
Apo-nymphs and homogenized with plastic pestles. To prevent contamination from M4 oily substances and gut epithelial cells, the supernatant was collected by centrifugation at 20,000 × g for 10 min and then filtered with a 0.45 μm filter (referred as naïve M4 lysate). The naïve Apo-M4 lysates were heat-treated at 100°C and centrifuged. Then, the recovered supernatant was loaded on a Toyopearl HW series columns (HW–55S column ϕ 20 × 900 mm, HW-40S column ϕ 10 × 300 mm) equilibrated with 0.45 μm filtered double distilled water
2.6. Purification of host-derived growth factor molecule(s) from Apo-M4 lysates The M4 regions were collected in PB (500 μl) from 3,000 fifth-instar 4
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Table 1 Estimate of specific activity of host-derived growth factor molecule(s) during purification. Purification steps
Weight (mg) (A)
CFUs (B)
Units (C)
Total units (D)
Specific activities (E)
Heat treated Apo-M4 lysate Peak 5 on HW-55S Peak 5 on HW-40S
142.93 42.11 3.80
16500 33600 80800
33.0 67.2 161.6
94333.8 56595.8 12281.6
660 1344 3232
For calculating specific activities, one unit was defined as growth of 500 bacteria cells upon by using 50 μg of protein amounts. C = B/500. D = (A/0.05 mg) × C. E = D/A.
and eluted with the same water at a flow rate of 0.1 ml/min. To measure bacterial growth and specific activity, symbiotic Burkholderia cells isolated from fifth-instar Sym-M4 crypt were diluted with PB to 500–1000 CFUs, and then in naïve- and heat-treated M4 lysate or column-purified fractions (50 μg in 200 μl M9 minimal medium). Each sample was incubated at 30°C for 6 h, and the samples were spread onto YG-Rif (30 μg/ml) agar plates. CFUs were calculated by colony numbers × dilution rate.
Burkholderia growth until 10 h, Burkholderia growth was observed after 10 h incubation, suggesting that growth-inducing activity was induced slowly in the naïve Apo-M4 lysate (Fig. 1B, left). But, when supernatant of heat-treated Apo-M4 lysates were added to symbiotic Burkholderia in the M9 minimal medium and incubated at 30°C for different time points, symbiotic Burkholderia cells were grown after 6 h incubation (Fig. 1B, right), suggesting that Burkholderia growth-inducing activity was increased by heat-treatments and that the enrichment of active component(s) by heat treatment. At the same conditions, symbiotic Burkholderia cells were not grown by addition of BSA as a control protein, showing that BSA did not affect growth of gut-colonized symbiotic Burkholderia cells. Based on these results, we decided to use heat-treated Apo-M4 lysate as a source of growth factor purification.
2.7. Thin-layer chromatography (TLC) After lyophilization of purified fractions on Toyopearl HW series columns, each peak (10 μg sample) was loaded on TLC silica gel 60 F254 (aluminum sheet coated with adsorbent silica gel, 9 × 5 cm) (Merck). The TLCs were developed with mobile phase (solvent mixture of nbutanol: methanol: dichloromethane: water = 3 : 5: 2 : 2) and color of the plate was visualized by spraying of ninhydrin reagent (SigmaAldrich) followed by heating (95°C).
3.2. Biochemical characterization of symbiotic Burkholderia growthinducing factor(s) Because Apo-M4 lysate did not lose its biological activity by heattreatment, molecular weight of active molecule(s) was estimated by ultrafiltration method. Symbiotic Burkholderia growth activity was recovered at the fraction harboring less than 3 kDa molecular size (Fig. 2A). By further experiments, host-derived growth factor(s) showed the resistance against proteinase K and ethyl alcohol precipitation, and phenol-chloroform treatment (Fig. 2B). Taken together, these results suggested that the growth factor(s) derived from both of M4 lysates of Apo- and Sym-insects is neither a protein nor a DNA.
2.8. Statistical analysis The data were analyzed using one-way ANOVA with Tukey's HSD test or Duncan's multiple range test using SPSS Statistics 23. 3. Results 3.1. Bacterial growth inducing activity of the Riptortus M4 lysates against symbiotic Burkholderia cells
3.3. Purification of host-derived growth factor(s) from heat-treated Apo-M4 lysates
To examine the presence of a growth factor(s) in the host insect's midgut, we firstly prepared naïve M4 lysates from Apo-insect and Syminsect. Two naïve M4-lysates (400 μg protein amounts) were incubated with symbiotic Burkholderia cells in the M9 minimal medium at 30°C for 12 h. The reason of the use of M9 medium was attributed to contain the minimal nutrients with the absence of amino acids. When bovine serum albumin (BSA) was used as a control protein, symbiotic Burkholderia cells did not show any growth (Fig. 1A). But, naïve M4 lysate of Apoinsects induced growth of symbiotic Burkholderia cells (column 1). Also, naïve M4 lysate of Sym-insects induced symbiotic Burkholderia growth in M9 minimal medium (column 2), indicating a possibility of that both M4 lysates have some unidentified growth-induced factor(s). Therefore, to further confirm the presence of symbiotic Burkholderia growth-inducing activity of naïve M4 lysates, we decided to use Apo-M4 lysate. Although M4 lysates of Sym-insects also have shown symbiotic Burkholderia growth activities, it was supposed that Sym-M4 lysates may be inappropriate for a source of growth factor purification due to the contamination of unknown secretion factor of symbiotic Burkholderia cells and host derived cellular components. Next, Burkholderia growth curves were examined by measuring absorbance after incubation with different amount proteins of naïve Apo-M4 lysates (50, 100, 200, and 400 μg) and symbiotic Burkholderia cells (OD600nm = 0.1) in M9 minimal medium at the different time points. Although naïve Apo-M4 lysate did not induce symbiotic
When heat-treated Apo-M4 lysates derived from fifth-instar Aponymphs were loaded on Superdex or Sephacryl-series gel filtration columns, the host-derived growth factor(s) was not separated in spite of usage of several different buffer conditions (data not shown). However, when the same Apo-M4 lysate was separated on HW-55S gel filtration column, which is known to be packed with polyvinyl-based resins, a host-derived growth factor(s) was separated with eight peaks (Fig. 3A–i). After lyophilization of each peak, the fractions were incubated with symbiotic Burkholderia in order to find which peak has symbiotic Burkholderia growth-inducing activity. The growth activity of symbiotic Burkholderia was detected in peak 5 only (Fig. 3A–ii). When each peak was examined by treatment of ninhydrin reagent, several positive spots were shown in TLC analyses. The peak 5 showing growth activity also elicited similar pattern with the heat-treated Apo-M4 lysate (Fig. 3A–iii). However, when peak 6 shown a pattern similar to the heat-treated Apo-M4 lysate was treated with symbiotic Burkholderia cells, growth activity was not observed. For next purification step, peak 5 was separated on HW-40S gel filtration column. Peak 5 derived from HW-55S column was separated with seven peaks on HW-40S gel column (Fig. 3B–i). After lyophilization of each peak, when the same amount was incubated with symbiotic Burkholderia cells in order to find which peak has growth-inducing activity, symbiotic Burkholderia growth activity was only observed in peak 5 (Fig. 3B–ii). The peak 5 of 5
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than 3 kDa (Fig. 2). Further experiments showed that a specific peak fraction induced symbiotic Burkholderia growth activity with contained several ninhydrin-positive spots on the TLC analyses (Fig. 3). The positive response to the ninhydrin reagent indicates that active growth factor(s) may harbor primary and secondary amino groups. However, recent study on the nutrient exchange between the R. pedestris host and the Burkholderia insecticola symbiont demonstrated that gut symbiotic bacteria provide nutrients to the host insects, as shown in other phytophagous insects (Ohbayashi et al., 2019). According to this report, the gut-colonized Burkholderia symbiont cells can produce all essential amino acids and vitamin B derivatives. These materials may be provided to the Riptortus host insects as essential nutrients, which are limited in the soybean food. Furthermore, transcriptomic analyses revealed that the Riptortus host insects likely provide ribose, rhamnose and myo-inositol, as well as fatty acids as carbon sources, allantoin and urea as nitrogen sources, and sulfate and taurine as sulfur sources to in vivo symbiont (gut-colonized Burkholderia cells). Therefore, we can assume that amino acids are not host-derived growth factor(s) because gut-colonized Burkholderia cells can produce essential amino acids by themselves. Although recent study suggested a possibility that fatty acids, urea and taurine are host-derived growth factor (s) (Ohbayashi et al., 2019), further research is required because their studies have been carried out at the gene level by transcriptomic analyses. Thus, to identify host-derived growth factor(s), further biochemical characterizations, such as determinations of molecular weight and whole structure of this growth factor(s) by NMR or LC-MS/MS, are definitely necessary. In conclusion, biochemical approaches for identifying the symbiotic association-triggering molecule(s) between R. pedestris and Burkholderia insecticola have provided an experimental evidence of how host insects regulates the gut symbiont population via environmental nutrient controlling.
HW-55S column was separated on the TLC analyses (Fig. 3B–iii). The peak 5 spots of HW-40S column showed similar pattern with those of peak 5 of HW-55S (Fig. 3B–iii). However, the uppermost spot of peak 5 of HW-55S was separated with the absence of growth activity. To examine whether specific activity was increased or not by further purification, specific activities were estimated. As shown in Table 1, initial specific activity was increased from 660 to 3232 (5 times increase) after two times gel filtration column chromatographies, supporting that hostderived growth factor(s) is now further purified by performance column chromatographies. 4. Discussion Our previous studies have shown that gut-colonized symbiotic Burkholderia produce PHA to adapt harsh environment of insect's gut (Jang et al., 2017; Kim et al., 2013b). In addition, when Burkholderia symbionts have established symbiotic relationship with host insect in M4 midgut region, water supplemented with food coloring dyes did not reach to M4 crypt region (Ohbayashi et al., 2015), indicating that some modulating mechanism is working in the vicinity of M4 region. Especially in M4 region, the symbiont titer at the pre-molt stage in the fifthinstar exhibited about 8-fold decrease in comparison with that of the preceding inter-molt stage (Kim et al., 2014). Furthermore, the examination of the susceptibility of cultured and symbiotic Burkholderia cells to antimicrobial peptides (AMPs) revealed that the gut-colonized symbiotic Burkholderia cells were highly susceptible to host AMPs, such as riptocin and rip-defensin (Kim et al., 2015). These observations suggested that environments of M4 midgut region may be quite stressful conditions and may be in the state of nutritional depletion for the symbiotic Burkholderia. Based on these results, we supposed that symbiotic Burkholderia cells expelled from M4 region during molting should be re-colonized in the M4 crypt during next developmental stage, suggesting that R. pedestris must provide some nutrients to their symbionts to support proliferation and to maintain microbial community persistently. The experimental evidence providing nutrient sources to symbiont was first provided in the squid-Vibrio symbiosis system (Graf and Ruby, 1998). Dr. Graf and Ruby have addressed the fundamental question of how the nature and dynamics of nutrient can be exchanged between cooperative bacterial symbionts and their animal hosts by examining the bioluminescent association between Vibrio fischeri and the squid Euprymna scolopes (Graf and Ruby, 1998). According to their report, the host squid E. scolopes provides at least 9 amino acids to the growing culture of symbiotic V. fischeri present in its light-emitting organ. In addition, when they collected and analyzed the extracellular fluid from this organ, in which the symbionts colonized, it was confirmed that this organ contained significant amounts of amino acids. The combined results suggested that host-derived free amino acids are a source of the amino acids supporting the growth of the symbiont cells. In the squidVibrio symbiotic association, other report also suggested that glucose could serve as the primary nutrient in some light organs on V. fischeri (Nealson, 1979). Moreover, V. fischeri cells have the unusual ability to utilize 3′:5′-cyclic adenosine monophosphate (cAMP) as a sole carbon, nitrogen, and phosphate source, leading to the hypothesis that the specificity of their symbiotic associations could be due to cAMP being the only host-derived nutrient released into the crypts of the light organ (Dunlap et al., 1992). We firstly assumed that the nutritional factor(s) provided by the host insects would be amino acids in the Riptortus-Burkholderia symbiotic association. To address this hypothesis, we tried to identify unidentified growth factor(s) that is present in the host M4 region. As results, host-derived growth factor(s) did not lose its biological activity in the conditions of high temperature, phenol-chloroform treatment or ethyl alcohol precipitation (Fig. 2), supporting that a growth factor molecule(s) is neither a protein nor a DNA. In addition, molecular weight of the host-derived growth factor(s) was turned out to be less
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Environ. Microbiol. 83 e00459-00417. Kim, J.K., Han, S.H., Kim, C.-H., Jo, Y.H., Futahashi, R., Kikuchi, Y., Fukatsu, T., Lee, B.L., 2014. Molting-associated suppression of symbiont population and up-regulation of antimicrobial activity in the midgut symbiotic organ of the Riptortus-Burkholderia symbiosis. Dev. Comp. Immunol. 43, 10–14. Kim, J.K., Kim, N.H., Jang, H.A., Kikuchi, Y., Kim, C.-H., Fukatsu, T., Lee, B.L., 2013a. Specific midgut region controlling the symbiont population in an insect-microbe gut symbiotic association. Appl. Environ. Microbiol. 79, 7229–7233. Kim, J.K., Son, D.W., Kim, C.-H., Cho, J.H., Marchetti, R., Silipo, A., Sturiale, L., Park, H.Y., Huh, Y.R., Nakayama, H., Fukatsu, T., Molinaro, A., Lee, B.L., 2015. Insect gut symbiont susceptibility to host antimicrobial peptides caused by alteration of the bacterial cell envelope. J. Biol. Chem. 290, 21042–21053. Kim, J.K., Won, Y.J., Nikoh, N., Nakayama, H., Han, S.H., Kikuchi, Y., Rhee, Y.H., Park, H.Y., Kwon, J.Y., Kurokawa, K., Dohmae, N., Fukatsu, T., Lee, B.L., 2013b. Polyester synthesis genes associated with stress resistance are involved in an insect-bacterium symbiosis. Proc. Natl. Acad. Sci. U.S.A. 110, E2381–E2389. Lee, J., Kim, C.-H., Jang, H.A., Kim, J.K., Kotaki, T., Shinoda, T., Shinada, T., Yoo, J.-W., Lee, B.L., 2019. Burkholderia gut symbiont modulates titer of specific juvenile hormone in the bean bug Riptortus pedestris. Dev. Comp. Immunol 103399. Lee, J.B., Byeon, J.H., Jang, H.A., Kim, J.K., Yoo, J.W., Kikuchi, Y., Lee, B.L., 2015. Bacterial cell motility of Burkholderia gut symbiont is required to colonize the insect gut. FEBS Lett. 589, 2784–2790. Lee, J.B., Park, K.-E., Lee, S.A., Jang, S.H., Eo, H.J., Jang, H.A., Kim, C.-H., Ohbayashi, T.,
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Putative host-derived growth factors inducing colonization of Burkholderia gut symbiont in Riptortus pedestris insect
T
Junbeom Leea,1, Xinrui Maoa,1, You Seon Leea, Dong Jung Leea, Junghyun Kima, Jiyeun Kate Kimb, Bok Luel Leea,∗ a b
Global Research Laboratory, College of Pharmacy, Pusan National University, Busan, 46241, South Korea Department of Microbiology, College of Medicine, Kosin University, Busan, 49267, South Korea
A R T I C LE I N FO
A B S T R A C T
Keywords: Riptortus pedestris Burkholderia insecticola Host-derived growth factor Symbiont's proliferation
It is questionable that how gut symbiont can be proliferated in the host symbiotic organs, such as host midgut region, which are known to be highly stressful and nutritional depleted conditions. Since Riptortus-Burkholderia symbiosis system is a good model to study this question, we hypothesized that Burkholderia symbiont will use host-derived bacterial growth factor(s) to colonize persistently in the host midgut 4 (M4) region, which is known as symbiotic organ. In this study, we observed that although gut-colonized symbiotic Burkholderia cells did not grow in the nutrient-limited media conditions, these symbionts were able to grow dose-dependent manner by addition of host naïve M4 lysate, supporting that host-derived growth factor molecule(s) may exist in the host M4 lysate. By further experiments, a host-derived growth factor(s) did not lose its biological activity in the conditions of high temperature, treatment of phenol-chloroform or ethyl alcohol precipitation, indicating that a growth factor molecule(s) is neither a protein nor a DNA. Also, based on the biochemical analyses data, molecular weight of the host-derived bacterial growth factor(s) was turned out to be less than 3 kDa molecular mass and to give the positive chemical response to the ninhydrin reagent on thin layer chromatography. Finally, we found that one specific peak showing ninhydrin positive signal was separated by gel filtration column and induced proliferative activity for Burkholderia gut symbiont cells.
1. Introduction Microorganism-mediated symbiotic associations are known for a broad range of animals, plants, and other organisms (Margulis and Fester, 1991; Ruby et al., 2004). Especially, many insects are known to possess symbiotic bacteria within their cells, tissues, and guts (Douglas, 2014; Ferrari and Vavre, 2011). These insect-symbiont associations have been established for a long time and are known to affect the biology of host insects in various ways (Kim et al., 2013a). Some symbionts play indispensable roles, such as providing essential nutrients (Moran et al., 2008). Although there are numerous examples of the transfer of essential amino acids, vitamins and short chain fatty acids from microbial symbionts to their animal hosts (Douglas, 1994), only a few evidences were reported that how host supply a quantity of nutrients to support growth of diverse bacterial symbionts. Moreover, every microorganism requires certain basic nutrients for growth and maintenance of metabolic functions. The amounts and types of nutrients are widely different depending on the microorganism. These
nutrients include water, nitrogen source, vitamins, and minerals (Busta et al., 2003; Mossel et al., 1995). Also, according to previous studies, many symbiont accumulate granules of polyhydroxyalkanoate (PHA) within their cells, which protect bacteria from nutritional depletion and other environmental stresses (Anderson and Dawes, 1990; Kim et al., 2013b; Schwartzman and Ruby, 2016). In the case of Riptortus-Burkholderia symbiosis, Riptortus pedestris host insect possesses a specialized symbiotic organ, named midgut 4 (M4) region. M4 region of one nymph harbors numerous crypts with about 107 Burkholderia insecticola symbiont cells (Kim et al., 2014). In addition, our another report revealed that the symbiont titer at the pre-molt stage transiently was decreased in comparison with that of the preceding inter-molt stage during host insect's development (Kim et al., 2014). Based on these results, we supposed that symbiotic Burkholderia cells expelled from M4 region during molting period should be re-colonized in the M4 crypt during next developmental stage, suggesting that R. pedestris host insects must provide some nutrients to their symbionts to support proliferation and
∗
Corresponding author. E-mail address: [email protected] (B.L. Lee). 1 J.L. and X.M. contributed equally to this article. https://doi.org/10.1016/j.dci.2019.103570 Received 25 September 2019; Received in revised form 7 December 2019; Accepted 8 December 2019 Available online 10 December 2019 0145-305X/ © 2019 Elsevier Ltd. All rights reserved.
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Fig. 1. Identification of symbiotic Burkholderia growth activity in the Riptortus M4 lysates. (A) Examination of CFUs after incubation with non-heat-treated naïve M4 lysates of fifth-instar Apo- and Sym-nymphs. Negative control [(−) control] indicates the incubation with symbiotic Burkholderia cells only in M9 minimal media. BSA indicates bovine serum albumin with 400 μg of protein. Each sample was incubated at 30°C for 12 h. (B) The Burkholderia symbiont growths were estimated by the measurement of absorbance changes after incubation with gut-colonized symbiotic Burkholderia cells and naïve M4 lysate (left) or heat-treated M4 lysate (right) of fifth-instar Apo-nymphs. The absorbance at 600 nm was measured after every 2 h (unpaired t-test: *, P < 0.01; n = 5).
distilled water containing 0.05% ascorbic acid (DWA) with a final concentration of 107 cells/ml (Lee et al., 2019). After the insects had been fed with the bacterial inoculum solution for 2 days, fresh and sterile DWA was provided to the insects instead of the bacterial inoculum solution. Upon reaching adulthood, the insects were transferred to larger containers in which soybean plant pots were placed for feeding and cotton pads were attached to the cage walls for egg laying. Eggs were collected daily and transferred to new cages for hatching.
to maintain microbial community persistently. In this study, we aimed to identify growth factor(s) that is present in the host M4 region. Here we found that gut-colonized symbiotic Burkholderia cells suspended in M9 minimal medium were grown by addition of host-derived naïve M4 lysates collected from Burkholderiaabsent aposymbiotic insects (Apo-insect) and from Burkholderia-harboring symbiotic insects (Sym-insect). These data indicate that the naïve extract of host M4 region is able to induce growth of symbiotic Burkholderia cells. Because bacterial growth-inducing activity was more increased by heat-treatment of naïve Apo-M4 lysate, we prepared heattreated Apo-M4 lysate using naïve Apo-M4 lysate for the next purification steps. The heat-treated Apo-M4 lysates were separated on gel filtration column packed with polyvinyl-based resins and a host-derived growth factor(s) was separated with several peaks showing positive response to ninhydrin reagent. As the results, a specific peak purified by biochemical approaches showed symbiotic Burkholderia growth activity, suggesting that symbiotic Burkholderia cells may require the nutrient source or host-derived growth factor(s) for persistent colonization in the insect gut.
2.2. Isolation of symbiotic Burkholderia cells from insect midgut The M4 regions from 10 fifth-instar Sym-nymphs were collected and placed in 200 μl of 10 mM phosphate buffer (PB; pH 7.0) as described (Byeon et al., 2015; Kim et al., 2013a). The M4 regions were cut with fine scissors to break the M4 crypts into small pieces. One milliliter of PB was added to the solution and symbiotic Burkholderia cells colonized in the crypt were re-suspended in the PB by gently pipetting. The solution was filtered through 5 μm pore filter to remove the midgut tissues. To remove other contaminants of host midgut, isolated symbiotic Burkholderia cells were sequentially washed with PB. Then, symbiotic Burkholderia cells collected by centrifugation at 20,000 × g for 10 min were rewashed with PB.
2. Materials and methods 2.1. Insect rearing and Burkholderia symbiont inoculation
2.3. Bioassay for measuring bacterial growth activity R. pedestris was maintained in our insect culturing room with 28°C under a long day cycle of 16 h light and 8 h dark as described (Lee et al., 2017). When the newborn nymphs were molted to second instar, in vitro cultured Burkholderia inoculated solution was provided with wet cotton balls in a petri dish. Burkholderia cells were suspended in
The M4 regions from 10 fifth-instar Apo- and Sym-nymphs were collected in 200 μl of PB and homogenized with plastic pestles. M4 lysates were centrifuged at 20,000 × g for 10 min. To separate midgut tissues and gut epithelial cells from symbiotic Burkholderia cells, this 2
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Fig. 2. Biochemical characterization of the Riptortus M4 lysates. (A) Symbiotic Burkholderia growth rates were estimated after treatment of fractions after performance of molecular weight 3 kDa cut off spin columns (Millipore). Negative control [(−) control] indicates the incubation of symbiotic Burkholderia cells only in M9 minimal media, and positive control [(+) control] means the addition of naïve Sym-M4 lysate to symbiotic Burkholderia cells suspended in M9 minimal media. 100°C sup means the supernatant of heat-treated Apo-M4 lysate. Less 3 kDa indicates the pass through of 3 kDa molecular weight cut off spin column (Millipore). More 3 kDa sample is the remaining solution on 3 kDa molecular weight cut off spin column. Each sample was incubated at 30°C for 6 h. (B) Symbiotic Burkholderia growth rates were estimated after treatment of proteinase K, ethyl alcohol precipitation or phenol-chloroform. Negative control [(−) control] indicates the incubation of symbiotic Burkholderia cells only in M9 minimal media. We normalized the growth rates to (−) control, which was set as 1. Five hundred of symbiotic Burkholderia cells were incubated with each treatment at 30°C for 6 h. All the values are means ± S.D. (n = 5). Different letters over bar indicates significant difference in treatment according to Duncan's multiple range test (P < 0.01).
100 μl of PB and homogenized with plastic pestles. The host gut-derived contaminant was removed by centrifugation at 20,000 × g for 10 min. Supernatant was collected as non-heat-treated naïve M4 lysate and used for measurement of bacterial growth activity.
procedure was repeated one more time and supernatant were finally collected. The starting bacterial solutions prepared in M9 minimal medium (0.6% Na2HPO4∙2H2O, 0.3% KH2PO4, 0.1% NH4Cl, 0.05% NaCl, 0.0003% CaCl2, 1 mM MgSO4, 0.2% glucose) by adjusting OD600nm to 0.1 (1.0 × 107 cells) using symbiotic Burkholderia cells isolated from Sym-M4 crypt were incubated with naïve Apo- and SymM4 lysates (400 μg). Each sample was incubated at 30°C for 12 h, and the samples were spread onto yeast-glucose (YG) agar plates (0.5% yeast extract, 0.4% glucose, and 0.1% NaCl) containing rifampicin (Rif; 30 μg/ml), cultured for 2 days, and subjected to colony counting. Colony forming units (CFUs) were calculated by colony numbers × dilution rate (Kim et al., 2013a).
The collected supernatant of naïve Apo-M4 lysate was heat-treated at 100°C again for 5 min and centrifuged at 20,000 × g for 10 min. Finally, clear and homogeneous supernatant was collected for further experiments.
2.4. Measurement of bacterial growth curves in liquid media
(3) Proteinase K treatment
Growth curves of symbiotic Burkholderia cells isolated from fifthinstar Sym-M4 crypt were examined by incubation with naïve Apo-M4 lysates or heat-treated Apo-M4 lysates in M9 minimal medium (Lee et al., 2015). The starting bacterial solutions prepared in M9 minimal medium by adjusting OD600nm to 0.1 using log-phased pre-cultured cells were incubated with M4 lysates on a rotator shaker at 180 rpm at 30°C for 16 h, whose OD600nm was monitored every 2 h using a spectrophotometer (UV-1700 PharmSpec, Shimadzu, Japan).
The heat-treated Apo-M4 lysate (50 μg) was incubated with 20 μg of proteinase K at 60°C for 1 h.
(2) Preparation of heat-treated Apo-M4 lysates from naïve Apo-M4 lysates
(4) Ethanol precipitation The heat-treated Apo-M4 lysates (50 μg) were incubated with 500 μl ethanol (2.5 volume of sample) at −20°C for 24 h and then was centrifuged at 20,000 × g at 4°C for 10 min. Recovered pellets were washed with 80% ethanol two times and then supernatant and washed solution were dried under vacuum at 60°C for 1 h.
2.5. Biochemical characterization of host-derived growth factors
(5) Phenol-chloroform extraction
(1) Preparation of naïve M4 lysate from Apo-insects
The mixture of acid phenol (400 μl) and chloroform (200 μl) were
The M4 lysates from fifth-instar Apo-nymphs were collected in 3
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Fig. 3. Purification of growth factor(s) derived from Apo-M4 lysate via two different gel filtration columns. (A) (i) shows the elution pattern of Apo-M4 lysate on HW55S size column and (ii) is the results of CFUs measurements of each peak of Fig. 3A (i). Five hundreds of symbiotic Burkholderia cells were treated with 50 μg of each peak. (B) (i) shows the elution pattern of HW-55S peak 5 on HW-40S column. (ii) is the results of CFUs measurements of each peak of Fig. 3B (i). The spots indicate the response by spreading of ninhydrin reagent on each peak of HW-55S column (Fig. 3A–iii) and on each peak of HW-40S column (Fig. 3B–iii) after thin layer chromatography. C1 and C2 indicate the fractions of heat-treated M4 lysate and peak 5 of HW-55S column. (unpaired t-test: *, P < 0.01; n = 5).
added into 50 μg heat-treated Apo-M4 lysate (100 μl) and vortexed at room temperature for 10 min. Then, aqueous layer was separated from phenol layer and dried under vacuum at 60°C for 1 h.
Apo-nymphs and homogenized with plastic pestles. To prevent contamination from M4 oily substances and gut epithelial cells, the supernatant was collected by centrifugation at 20,000 × g for 10 min and then filtered with a 0.45 μm filter (referred as naïve M4 lysate). The naïve Apo-M4 lysates were heat-treated at 100°C and centrifuged. Then, the recovered supernatant was loaded on a Toyopearl HW series columns (HW–55S column ϕ 20 × 900 mm, HW-40S column ϕ 10 × 300 mm) equilibrated with 0.45 μm filtered double distilled water
2.6. Purification of host-derived growth factor molecule(s) from Apo-M4 lysates The M4 regions were collected in PB (500 μl) from 3,000 fifth-instar 4
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Table 1 Estimate of specific activity of host-derived growth factor molecule(s) during purification. Purification steps
Weight (mg) (A)
CFUs (B)
Units (C)
Total units (D)
Specific activities (E)
Heat treated Apo-M4 lysate Peak 5 on HW-55S Peak 5 on HW-40S
142.93 42.11 3.80
16500 33600 80800
33.0 67.2 161.6
94333.8 56595.8 12281.6
660 1344 3232
For calculating specific activities, one unit was defined as growth of 500 bacteria cells upon by using 50 μg of protein amounts. C = B/500. D = (A/0.05 mg) × C. E = D/A.
and eluted with the same water at a flow rate of 0.1 ml/min. To measure bacterial growth and specific activity, symbiotic Burkholderia cells isolated from fifth-instar Sym-M4 crypt were diluted with PB to 500–1000 CFUs, and then in naïve- and heat-treated M4 lysate or column-purified fractions (50 μg in 200 μl M9 minimal medium). Each sample was incubated at 30°C for 6 h, and the samples were spread onto YG-Rif (30 μg/ml) agar plates. CFUs were calculated by colony numbers × dilution rate.
Burkholderia growth until 10 h, Burkholderia growth was observed after 10 h incubation, suggesting that growth-inducing activity was induced slowly in the naïve Apo-M4 lysate (Fig. 1B, left). But, when supernatant of heat-treated Apo-M4 lysates were added to symbiotic Burkholderia in the M9 minimal medium and incubated at 30°C for different time points, symbiotic Burkholderia cells were grown after 6 h incubation (Fig. 1B, right), suggesting that Burkholderia growth-inducing activity was increased by heat-treatments and that the enrichment of active component(s) by heat treatment. At the same conditions, symbiotic Burkholderia cells were not grown by addition of BSA as a control protein, showing that BSA did not affect growth of gut-colonized symbiotic Burkholderia cells. Based on these results, we decided to use heat-treated Apo-M4 lysate as a source of growth factor purification.
2.7. Thin-layer chromatography (TLC) After lyophilization of purified fractions on Toyopearl HW series columns, each peak (10 μg sample) was loaded on TLC silica gel 60 F254 (aluminum sheet coated with adsorbent silica gel, 9 × 5 cm) (Merck). The TLCs were developed with mobile phase (solvent mixture of nbutanol: methanol: dichloromethane: water = 3 : 5: 2 : 2) and color of the plate was visualized by spraying of ninhydrin reagent (SigmaAldrich) followed by heating (95°C).
3.2. Biochemical characterization of symbiotic Burkholderia growthinducing factor(s) Because Apo-M4 lysate did not lose its biological activity by heattreatment, molecular weight of active molecule(s) was estimated by ultrafiltration method. Symbiotic Burkholderia growth activity was recovered at the fraction harboring less than 3 kDa molecular size (Fig. 2A). By further experiments, host-derived growth factor(s) showed the resistance against proteinase K and ethyl alcohol precipitation, and phenol-chloroform treatment (Fig. 2B). Taken together, these results suggested that the growth factor(s) derived from both of M4 lysates of Apo- and Sym-insects is neither a protein nor a DNA.
2.8. Statistical analysis The data were analyzed using one-way ANOVA with Tukey's HSD test or Duncan's multiple range test using SPSS Statistics 23. 3. Results 3.1. Bacterial growth inducing activity of the Riptortus M4 lysates against symbiotic Burkholderia cells
3.3. Purification of host-derived growth factor(s) from heat-treated Apo-M4 lysates
To examine the presence of a growth factor(s) in the host insect's midgut, we firstly prepared naïve M4 lysates from Apo-insect and Syminsect. Two naïve M4-lysates (400 μg protein amounts) were incubated with symbiotic Burkholderia cells in the M9 minimal medium at 30°C for 12 h. The reason of the use of M9 medium was attributed to contain the minimal nutrients with the absence of amino acids. When bovine serum albumin (BSA) was used as a control protein, symbiotic Burkholderia cells did not show any growth (Fig. 1A). But, naïve M4 lysate of Apoinsects induced growth of symbiotic Burkholderia cells (column 1). Also, naïve M4 lysate of Sym-insects induced symbiotic Burkholderia growth in M9 minimal medium (column 2), indicating a possibility of that both M4 lysates have some unidentified growth-induced factor(s). Therefore, to further confirm the presence of symbiotic Burkholderia growth-inducing activity of naïve M4 lysates, we decided to use Apo-M4 lysate. Although M4 lysates of Sym-insects also have shown symbiotic Burkholderia growth activities, it was supposed that Sym-M4 lysates may be inappropriate for a source of growth factor purification due to the contamination of unknown secretion factor of symbiotic Burkholderia cells and host derived cellular components. Next, Burkholderia growth curves were examined by measuring absorbance after incubation with different amount proteins of naïve Apo-M4 lysates (50, 100, 200, and 400 μg) and symbiotic Burkholderia cells (OD600nm = 0.1) in M9 minimal medium at the different time points. Although naïve Apo-M4 lysate did not induce symbiotic
When heat-treated Apo-M4 lysates derived from fifth-instar Aponymphs were loaded on Superdex or Sephacryl-series gel filtration columns, the host-derived growth factor(s) was not separated in spite of usage of several different buffer conditions (data not shown). However, when the same Apo-M4 lysate was separated on HW-55S gel filtration column, which is known to be packed with polyvinyl-based resins, a host-derived growth factor(s) was separated with eight peaks (Fig. 3A–i). After lyophilization of each peak, the fractions were incubated with symbiotic Burkholderia in order to find which peak has symbiotic Burkholderia growth-inducing activity. The growth activity of symbiotic Burkholderia was detected in peak 5 only (Fig. 3A–ii). When each peak was examined by treatment of ninhydrin reagent, several positive spots were shown in TLC analyses. The peak 5 showing growth activity also elicited similar pattern with the heat-treated Apo-M4 lysate (Fig. 3A–iii). However, when peak 6 shown a pattern similar to the heat-treated Apo-M4 lysate was treated with symbiotic Burkholderia cells, growth activity was not observed. For next purification step, peak 5 was separated on HW-40S gel filtration column. Peak 5 derived from HW-55S column was separated with seven peaks on HW-40S gel column (Fig. 3B–i). After lyophilization of each peak, when the same amount was incubated with symbiotic Burkholderia cells in order to find which peak has growth-inducing activity, symbiotic Burkholderia growth activity was only observed in peak 5 (Fig. 3B–ii). The peak 5 of 5
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than 3 kDa (Fig. 2). Further experiments showed that a specific peak fraction induced symbiotic Burkholderia growth activity with contained several ninhydrin-positive spots on the TLC analyses (Fig. 3). The positive response to the ninhydrin reagent indicates that active growth factor(s) may harbor primary and secondary amino groups. However, recent study on the nutrient exchange between the R. pedestris host and the Burkholderia insecticola symbiont demonstrated that gut symbiotic bacteria provide nutrients to the host insects, as shown in other phytophagous insects (Ohbayashi et al., 2019). According to this report, the gut-colonized Burkholderia symbiont cells can produce all essential amino acids and vitamin B derivatives. These materials may be provided to the Riptortus host insects as essential nutrients, which are limited in the soybean food. Furthermore, transcriptomic analyses revealed that the Riptortus host insects likely provide ribose, rhamnose and myo-inositol, as well as fatty acids as carbon sources, allantoin and urea as nitrogen sources, and sulfate and taurine as sulfur sources to in vivo symbiont (gut-colonized Burkholderia cells). Therefore, we can assume that amino acids are not host-derived growth factor(s) because gut-colonized Burkholderia cells can produce essential amino acids by themselves. Although recent study suggested a possibility that fatty acids, urea and taurine are host-derived growth factor (s) (Ohbayashi et al., 2019), further research is required because their studies have been carried out at the gene level by transcriptomic analyses. Thus, to identify host-derived growth factor(s), further biochemical characterizations, such as determinations of molecular weight and whole structure of this growth factor(s) by NMR or LC-MS/MS, are definitely necessary. In conclusion, biochemical approaches for identifying the symbiotic association-triggering molecule(s) between R. pedestris and Burkholderia insecticola have provided an experimental evidence of how host insects regulates the gut symbiont population via environmental nutrient controlling.
HW-55S column was separated on the TLC analyses (Fig. 3B–iii). The peak 5 spots of HW-40S column showed similar pattern with those of peak 5 of HW-55S (Fig. 3B–iii). However, the uppermost spot of peak 5 of HW-55S was separated with the absence of growth activity. To examine whether specific activity was increased or not by further purification, specific activities were estimated. As shown in Table 1, initial specific activity was increased from 660 to 3232 (5 times increase) after two times gel filtration column chromatographies, supporting that hostderived growth factor(s) is now further purified by performance column chromatographies. 4. Discussion Our previous studies have shown that gut-colonized symbiotic Burkholderia produce PHA to adapt harsh environment of insect's gut (Jang et al., 2017; Kim et al., 2013b). In addition, when Burkholderia symbionts have established symbiotic relationship with host insect in M4 midgut region, water supplemented with food coloring dyes did not reach to M4 crypt region (Ohbayashi et al., 2015), indicating that some modulating mechanism is working in the vicinity of M4 region. Especially in M4 region, the symbiont titer at the pre-molt stage in the fifthinstar exhibited about 8-fold decrease in comparison with that of the preceding inter-molt stage (Kim et al., 2014). Furthermore, the examination of the susceptibility of cultured and symbiotic Burkholderia cells to antimicrobial peptides (AMPs) revealed that the gut-colonized symbiotic Burkholderia cells were highly susceptible to host AMPs, such as riptocin and rip-defensin (Kim et al., 2015). These observations suggested that environments of M4 midgut region may be quite stressful conditions and may be in the state of nutritional depletion for the symbiotic Burkholderia. Based on these results, we supposed that symbiotic Burkholderia cells expelled from M4 region during molting should be re-colonized in the M4 crypt during next developmental stage, suggesting that R. pedestris must provide some nutrients to their symbionts to support proliferation and to maintain microbial community persistently. The experimental evidence providing nutrient sources to symbiont was first provided in the squid-Vibrio symbiosis system (Graf and Ruby, 1998). Dr. Graf and Ruby have addressed the fundamental question of how the nature and dynamics of nutrient can be exchanged between cooperative bacterial symbionts and their animal hosts by examining the bioluminescent association between Vibrio fischeri and the squid Euprymna scolopes (Graf and Ruby, 1998). According to their report, the host squid E. scolopes provides at least 9 amino acids to the growing culture of symbiotic V. fischeri present in its light-emitting organ. In addition, when they collected and analyzed the extracellular fluid from this organ, in which the symbionts colonized, it was confirmed that this organ contained significant amounts of amino acids. The combined results suggested that host-derived free amino acids are a source of the amino acids supporting the growth of the symbiont cells. In the squidVibrio symbiotic association, other report also suggested that glucose could serve as the primary nutrient in some light organs on V. fischeri (Nealson, 1979). Moreover, V. fischeri cells have the unusual ability to utilize 3′:5′-cyclic adenosine monophosphate (cAMP) as a sole carbon, nitrogen, and phosphate source, leading to the hypothesis that the specificity of their symbiotic associations could be due to cAMP being the only host-derived nutrient released into the crypts of the light organ (Dunlap et al., 1992). We firstly assumed that the nutritional factor(s) provided by the host insects would be amino acids in the Riptortus-Burkholderia symbiotic association. To address this hypothesis, we tried to identify unidentified growth factor(s) that is present in the host M4 region. As results, host-derived growth factor(s) did not lose its biological activity in the conditions of high temperature, phenol-chloroform treatment or ethyl alcohol precipitation (Fig. 2), supporting that a growth factor molecule(s) is neither a protein nor a DNA. In addition, molecular weight of the host-derived growth factor(s) was turned out to be less
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