Burkholderia gut symbionts enhance the innate immunity of host Riptortus pedestris

Accepted Manuscript Burkholderia gut symbionts enhance the innate immunity of host Riptortus pedestris Jiyeun Kate Kim, Jun Beom Lee, Ye Rang Huh, Ho Am Jang, Chan Hee Kim, Jin Wook Yoo, Bok Luel Lee PII:

S0145-305X(15)30011-2

DOI:

10.1016/j.dci.2015.07.006

Reference:

DCI 2428

To appear in:

Developmental and Comparative Immunology

Received Date: 31 May 2015 Revised Date:

5 July 2015

Accepted Date: 6 July 2015

Please cite this article as: Kim, J.K., Lee, J.B., Huh, Y.R., Jang, H.A., Kim, C.H., Yoo, J.W., Lee, B.L., Burkholderia gut symbionts enhance the innate immunity of host Riptortus pedestris, Developmental and Comparative Immunology (2015), doi: 10.1016/j.dci.2015.07.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Developmental and Comparative Immunology: Short Communications

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Burkholderia gut symbionts enhance the innate immunity of host Riptortus

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pedestris

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Jiyeun Kate Kima, Jun Beom Leeb, Ye Rang Huhb, Ho Am Jangb, Chan Hee Kimb, Jin Wook Yoob,

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Bok Luel Leeb*

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Department of Department of Microbiology, Kosin University College of Medicine, Busan 602-703,

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South Korea; bGlobal Research Laboratory of Insect Symbiosis, College of Pharmacy, Pusan National

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University, Busan 609-735, South Korea

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*Corresponding author: Tel: +82-51-510-2809, FAX: +82-51-513-2801, E-mail address:

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[email protected] (B.L.Lee)

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ACCEPTED MANUSCRIPT Abstract

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The relation between gut symbiosis and immunity has been reported in various animal model studies.

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Here, we corroborate the effect of gut symbiont to host immunity using the bean bug model. The bean

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bug, Riptortus pedestris, is a useful gut symbiosis model due to the monospecific gut symbiont, genus

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Burkholderia. To examine the effect of gut symbiosis to host immunity, we generated the gut

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symbiont-harboring (symbiotic) insect line and the gut symbiont-lacking (aposymbiotic) insect line.

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Upon bacterial challenges, the symbiotic Riptortus exhibited better survival than aposymbiotic

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Riptortus. When cellular immunity was inhibited, the symbiotic Riptortus still survived better than

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aposymbioic Riptortus, suggesting stronger humoral immunity. The molecular basis of the strong

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humoral immunity was further confirmed by the increase of hemolymph antimicrobial activity and

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antimicrobial peptide expression in the symbiotic insects. Taken together, our data clearly demonstrate

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that Burkhoderia gut symbiont positively affect the Riptortus systemic immunity.

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Highlights (85 characters including spaces)

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Symbiotic insect survives a bacterial infection better than aposymbiotic insect.

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Symbiotic insect exhibits stronger humoral immunity than aposymbiotic insect.

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Hemolymph of symbiotic insect has a high level of antimicrobial activity.

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Gut symbiosis induces higher expression of antimicrobial peptides in host insect.

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Keywords

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Gut symbiosis, Innate immunity, Antimicrobial peptide, Riptortus pedestris, Burkholderia symbiont

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ACCEPTED MANUSCRIPT 1. Introduction

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Gut symbiosis has drawn a large amount of attention because the gut microbiota is known to be

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pivotal to human health (Sekirov et al., 2010). Gut symbionts are intimately involved in numerous

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aspects of host biology, from nutrient metabolism to brain and behavior (Cryan and Dinan, 2012;

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Sommer and Backhed, 2013). The intimate relationship between gut microbiota and human

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immunologic diseases implies gut microbial signals modulating host’s immune systems (Abt and Artis,

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2009). Using the germ-free murine model, the importance of the gut microbiota has been revealed in

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the development of the adaptive immune system, and the gut microbiota disturbances may lead to the

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dysregulation of adaptive immune cells, possibly resulting disorders such as inflammatory bowel

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disease. (Round and Mazmanian, 2009). Similarly, insect gut symbiosis models have showed the

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association of gut symbiosis and host’s innate immunity. Tsetse flies (Glossina spp.), the sole vector

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of protozoan African trypanosomes causing Human African trypanosomiasis (HAT, sleeping sickness),

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possess commensal Sodalis and obligate Wigglesworthia as their major gut microbes. The tsetse flies

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without Wigglesworthia exhibit an irregular expression of humoral and epithelial immunity-related

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genes and decreased numbers of phagocytic hemocytes (Weiss et al., 2012; Weiss et al., 2011). Some

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other insect studies researched on the effect of Wolbachia, which is not a gut symbiont, to host insect

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immunity. In fruit fly (Drosophila melanogaster) and mosquito (Aedes aegypti), Wolbachia symbiont

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increased the host survival against pathogen infections (Hedges et al., 2008; Moreira et al., 2009;

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Walker et al., 2011). One of mechanisms explaining this protection was proposed as an immune

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priming in which Wolbachia stimulates the basal immune responses and helps to prepare host insect

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against subsequent infections (Rances et al., 2012).

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Recently, the bean bug Riptortus pedestris, belonging to the stinkbug family Alydidae in the insect

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order Hemiptera, has been recognized as a useful gut symbiosis model because they harbor a specific

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gut symbiont of β-proteobacterial genus Burkholderia. This insect orally acquires their Burkholderia

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symbionts from the rhizosphere environment in every generation (Kikuchi et al., 2007). When the

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Burkholderia cells colonize the posterior midgut region (M4 region) of the Riptortus, the numerous

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crypts of the M4 region are filled with the Burkholderia cells, leading to the exclusive gut symbiotic

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ACCEPTED MANUSCRIPT association between Riptortus and Burkholderia symbiont (Kikuchi et al., 2005). Because newly

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hatched R. pedestris nymphs are aposymbiotic (not possessing symbionts), symbiotic and

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aposymbiotic insect lines are easily established by controlling the feeding of the cultured Burkholderia

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cells. Reflecting its free-living origin in the environment, Burkholderia symbiont is easily cultivable

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on standard microbiological media and consequently amenable to genetic manipulation. The

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genetically manipulated symbiont strains have been used to understand molecular mechanism of

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symbiotic association (Kim and Lee, 2015).

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Previous studies on the Riptortus-Burkholderia symbiosis show that Burkholderia gut symbiont

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positively affects the host fitness and gut development (Kikuchi et al., 2007; Kim et al., 2014b; Kim et

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al., 2014c; Kim et al., 2013c). In this study, we explored the effect of the Burkholderia gut symbiont

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to the Riptortus host systemic immunity. Using the symbiotic and aposymbiotic insects, survival rates

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against bacterial septic infection were measured to examine overall immune defense as well as

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humoral immune defense. Subsequently, the levels of antimicrobial activity of hemolymph and the

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expression of antimicrobial peptides were examined to determine the molecular basis of the difference

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in humoral immune responses between the symbiotic and aposymbiotic insects.

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2. Materials and methods

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2.1. Bacteria and media.

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Escherichia coli K12 and Staphylococcus aureus RN4220 cells were cultured at 37°C with LB

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medium (1% tryptone, 0.5% yeast extract, 0.5% NaCl). Burkholderia symbiont RPE75 strain, a

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spontaneous rifampicin resistant strain derived from RPE64 strain (Kikuchi et al., 2011), was cultured

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at 30°C with YG-RIF medium (0.4% glucose, 0.5% yeast extract, 0.1% NaCl, 30 µg/ml rifampicin).

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2.2. Insect rearing.

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The bean bugs R. pedestris were reared in our insect laboratory at 28°C under a long day regime of 16

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h light and 8 h dark as described (Kim et al., 2013b). Nymphal insects were reared in clean plastic

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containers (34 cm long, 19.5 cm wide and 27.5 cm high) supplied with soybean seeds and DWA

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ACCEPTED MANUSCRIPT (distilled water containing 0.05% ascorbic acid). Upon reaching adulthood, the insects were

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transferred to bigger containers (35 cm long, 35 cm wide and 40 cm high) in which soybean plant pots

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were placed for feeding and cotton pads were attached to the walls for egg laying. Eggs were collected

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daily and transferred to new cages for hatching.

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2.3. Burkholderia symbiont infection.

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Burkholderia symbiont strain RPE75 was cultured at 30°C in YG-RIF medium. An inoculum solution

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was prepared by suspending mid-log phase cultured Burkholderia cells in DWA at a concentration of

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107 cells/ml. Newly-molted second instar nymphs were provided with wet cotton balls soaked with the

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inoculum solution for two days and then reared on sterile DWA and soybean seeds. To generate the

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aposymbiotic insect not harboring Burkholderia cells, we did not provide Burkholderia inoculum

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solution but only sterile DWA.

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2.4. Bacterial septic infection and measurement of survival rate.

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Bacterial cells were washed with PBS (phosphate buffered saline, Gibco) and suspended with Grace’s

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Insect Medium (Gibco) to have 1010 cells/ml bacterial solutions. Two µl of the bacterial cell solutions

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were injected to male adult R. pedestris three days after the molting into adult. The survival rate was

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monitored every 24 h after bacteria septic injection.

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To inhibit phagocytosis activity of hemocytes, we injected latex bead solution (FluoSpheres®

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Fluorescent Microspheres; Invitrogen) prior to the bacterial injection. Beads were washed with PBS

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and suspended with Grace’s Insect Medium to have 3.6×1013 beads/ml solution. One µl of the bead

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solution was injected to adult R. pedestris one day prior to the bacterial challenge. The insect’s

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survival was monitored every 24 h.

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2.5. Hemolymph preparation.

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Hemolymph was collected from the insects that were injected with E. coli as described above. Around

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17 h post injections, the hemolymph were collected in decoaggulation buffer (30 mM trisodium citrate,

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26 mM citric acid, 15 mM sodium chloride, 20 mM EDTA, pH 4.6) by cutting legs. The ratio of

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ACCEPTED MANUSCRIPT hemolymph to decoaggulation buffer was 1:9. The collected hemolymph solution was centrifuged at

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20,000 × g for 15 min and the supernatants were boiled at 95°C for 5 min. After cooling the solution,

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the hemolymph solution was centrifuged once more at 20,000 × g for 15 min and the final

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supernatants were collected.

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2.6. Hemolymph susceptibility assay.

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The mid-log phase E. coli and S. aureus cells were washed with PBS (phosphate buffered saline,

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Gibco) and suspended with PBS to have approximately 105 cells per 50 µl. To fifty µl of cell solution,

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350 µl of different concentrations of hemolymph solutions were added. The different concentrations of

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hemolymph solution were prepared by containing 0.5, 0.75, 1 or 1.25 µl of the hemolymph

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supernatant prepared as mentioned above. The mixture of bacteria cells and hemolymph was

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incubated at 37°C for 2 h. After incubation, the cell and hemolymph solutions were diluted and mixed

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with 16 ml of 1% LB agar which was previously autoclaved and kept unsolidified at 50°C, and poured

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onto a Petri dish. Colony forming unit (CFU) were counted after 10 h incubation at 37°C.

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2.6. Measurement of expression of AMP genes.

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The expression of AMP genes were measured in the fat body of insects at 17 h post septic injection.

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The dissected fat bodies were subjected to RNA extraction using RiboEx (GeneAll, South Korea). The

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RNA samples were reverse transcribed using TOPscript RT DryMIX containing oligo-dT primers

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(enzynomics, South Korea) to synthesize cDNA. cDNA was subjected to real-time quantitative PCR

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after mixed with a TOPreal qPCR 2x PreMIX with SYBR green (enzynomics, South Korea) and 0.25

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µM each of the primers: rip-defensin (accession number AK416895) primer set, 5’-TCG GTC GGA

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CTG AGA CTG AA-3’, 5’-TTG CCG CCT TTG TAT CCC TT-3’; riptocin (AB842297) primer set,

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5’- TCC GAA GCT GAG GGT CTT CCC G-3’, 5’-TCC GCA TCC AAG TTC GCG TCC-3’; rip-

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thanatins (AB842298) primer set, 5’- GTC TGC CTT CGT TGA AGA CG-3’, 5’- ATT CGC TTG

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CAA ACG CCG-3’; elongation factor 1 alpha (AB591382) primer set, 5’- CCT GCA TCC GTT GCT

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TTT GT-3’, 5’-GGC ATC GAG GGC TTC AAT AA-3’. The PCR temperature profile was set to

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95°C for 10 min followed by 40 cycles of 95°C for 10 sec, 60°C for 15 sec and 72°C for 20 sec using

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ACCEPTED MANUSCRIPT CFX96 real time PCR system (Bio-Rad). The comparative CT (∆∆CT) method was used to calculate

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relative gene expression levels with elongation factor 1 alpha (EF1α) gene of R. pedestris as an

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endogenous control gene.

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2.7. Statistical analyses.

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The statistical significance of data was determined using a Gehan-Breslow-Wilcoxon survival test or

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two-way ANOVA with Sidak’s post-hoc test, provided in the Prism GraphPad software.

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3.1. Insect harboring gut symbiont survives better against bacterial septic infection than insects lacking

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gut symbiont.

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To address the question whether the host’s systemic immunity can be affected by gut symbiosis, the

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survival rates of Riptortus hosts harboring Burkholderia symbiont (symbiotic insects) and lacking

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Burkholderia symbiont (aposymbiotic insects) were monitored after bacterial challenges. When the

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Gram-negative E. coli cells were injected into hemocoele of adult Riptortus, the symbiotic insects

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exhibited significantly better survival than the aposymbiotic insects (Gehan-Breslow-Wilcoxon test, P

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= 0.0018) (Fig. 1A, i). The symbiotic insects also survived the Gram-positive S. aureus septic

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infection better than the aposymbiotic insects (Gehan-Breslow-Wilcoxon test, P < 0.0001) (Fig. 1A, ii).

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These results indicate that gut symbiosis with Burkholderia symbiont positively affect the Riptortus

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host’s defense against systemic bacterial infection.

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3.2. The symbiotic insects have better humoral immune defense than the aposymbiotic insects.

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The insect immunity comprises cellular immunity and humoral immunity, in which the phagocytosis

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of hemocytes is a main cellular immune response, and the action of antimicrobial peptides (AMPs) is a

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major part of humoral immune responses. To examine the effect of gut symbiosis to humoral 7

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(Supplementary Fig. S1). These beads are taken up, but not degraded, by hemocytes, and thus

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effectively block the cellular immune response (Elrod-Erickson et al., 2000). Even though the cellular

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immune responses were inhibited by the beads, the symbiotic insects still exhibited significantly better

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survival than the aposymbiotic insects against E. coli infection (Gehan-Breslow-Wilcoxon test, P =

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0.035) (Fig. 1B, i) and against S. aureus infection (Gehan-Breslow-Wilcoxon test, P = 0.031) (Fig. 1B,

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ii), indicating stronger humoral immune responses in insects harboring gut symbionts.

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3.3. The antimicrobial activity of hemolymph of the symbiotic insect is stronger than that of the

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aposymbiotic insects.

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We further addressed the molecular basis of the strong humoral immune defense of the symbiotic

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insects. The activated hemolymph of the symbiotic and aposymbiotic adult insects were collected at

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17 h post E. coli injection and subjected to boiling and centrifugation to remove large proteins. The

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antimicrobial activity of the hemolymph solution was accessed by their killing effect to the E. coli

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cells. When the same volume of hemolymph of the symbiotic and aposymbiotic insects was compared,

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the hemolymph of symbiotic insects exhibit much stronger antimicrobial activity than that of

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aposymbiotic insects (two-way ANOVA, P < 0.0001) (Fig. 2A).

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3.4. The symbiotic insect exhibits higher AMP expression than the aposymbiotic insect.

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Riptortus is a hemimetabolous insect whose innate immune mechanisms are much less studied than

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holometabolous insects such as Drosophila, Manduca and Tenebrio (Lemaitre and Hoffmann, 2007).

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However, we have identified three major AMPs of Riptortus by protein purification (Kim et al., 2015)

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confirmed by the data of expressed-sequence tags (EST) (Futahashi et al., 2013) and RNA sequencing

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(Futahashi et al., unpublished data). In searching for the molecular basis of the strong humoral

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immunity and the increased antimicrobial activity of hemolymph of the symbiotic insect, we examined

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ACCEPTED MANUSCRIPT the expression of the Riptortus AMPs in the fat body of the symbiotic and aposymbiotic adult insects.

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While the basal expressions of riptocin, rip-defensin and rip-thanatin were similar between the

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symbiotic and aposymbiotic insects, the expression of AMPs were significantly more increased in the

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symbiotic insects than in the aposymbiotic insects upon bacterial infection (Fig. 2B). These results

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indicate that gut symbiosis can enhance the induction of AMP expression in the fat body in response

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to the bacterial systemic challenge.

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Here, we report that gut symbiosis enhances the host immunity in the bean bug Riptortus model

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system. The gut symbiont-harboring bean bug survives better in bacterial septic challenges and

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exhibits a strong humoral immune response by expressing significantly higher level of antimicrobial

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peptides than the gut symbiont-free bean bug. Along with the previous reports from other animal

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studies, our study again reinforces the intimate association between gut symbiosis and host immunity.

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By utilizing the simplicity of the Riptortus-Burkholdera gut symbiosis, we hope to further extend our

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understanding how the gut symbionts affect the host systemic immunity in molecular level.

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Through this study, we additionally acquired information about the characteristics of Riptortus

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immune responses. Data in Figure 1B, in which the latex bead injection distinguished the effect of

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humoral and cellular immune responses, suggest that different immune responses of the Riptortus are

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critical to E. coli and S. aureus infections. In E. coli infection, we did not observe a significantly

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different survival rate between bead-injected and none-injected insects, indicating little protective

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effect of the cellular immune response against E. coli. However, the survival rate after S. aureus

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infection was significantly different between bead-injected and none-injected insect (aposymbiotic

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insect, P = 0.036; symbiotic insect, P = 0.049 by Gehan-Breslow-Wilcoxon test), suggesting that the

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cellular immune response contributes significantly to the host defense against S. aureus infection.

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These results correlate with our recent data about the hemolymph antimicrobial activity. The activated

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hemolymph exhibited a dose dependent antimicrobial activity against E. coli, but not against S. aureus

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ACCEPTED MANUSCRIPT (Kim et al., unpublished data). Therefore, it seems that the cellular immunity is important for the S.

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aureus clearance in the hemocoele of Riptortus, and the humoral immunity is more effective to the

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defense against E. coli.

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Immune priming is one of the proposed mechanisms in enhanced insect immunity by Wolbachia

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symbiont (Moreira et al., 2009; Rances et al., 2012). It suggests that harboring the Wolbachia

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symbiont increases the basal immune responses in host insects. In our data, the AMP expression in the

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Burkholderia-harboring bean bugs was similar to that of aposymbiotic bean bugs after buffer-injection

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(Fig. 2B) and none-injection (data not shown). These suggest that the basal immune responses of the

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symbiotic bean bugs were not significantly different from the aposymbiotic bean bugs. However, upon

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bacterial infection, the extent of the activated immune responses was much greater in the gut

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symbiont-harboring bean bug (Fig. 2B).

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The molecular mechanism of how gut symbionts enhance the host systemic immunity, through general

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health improvement by nutritional support or specific immune enhancement by gut symbiont-derived

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signals, is a big question to answer. In order to answer this question, the hemimetabolous Riptortus

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innate immunity should be understood in great detail as well as the mechanisms of the gut symbiosis.

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Currently, RNA sequencing analysis on the immune genes of the R. pedestris has been done

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(Futahashi et al., unpublished data) and expected to provide important information about

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hemimetabolous insect immunity. We also have been investigating molecular mechanisms of

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establishing the Riptortus gut symbiosis (Kim et al., 2014a; Kim et al., 2013a). Therefore, by studying

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various aspects of the Riptortus-Burkholderia model system and interconnecting the results, we hope

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to unveil the molecular cross-talks between Burkholderia gut symbiont and Riptortus innate immunity

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in near future.

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ACCEPTED MANUSCRIPT Acknowledgements

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This study was supported by the Global Research Laboratory Program (grant number 2011-0021535)

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and Basic Science Research Program (grant number 2014R1A1A4A01007507) of the National

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Research Foundation of Korea.

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Figure legends

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Fig. 1. (A) Bean bug survival rate after bacterial challenges. The symbiotic (SYM) insects (blue line)

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and the aposymbiotic (APO) insects (red line) were injected with E. coli cells (i) or S. aureus cells (ii)

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and monitored for their survival. For control insect groups, the same volume of buffer was injected

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(mock injection). For each sample group, thirty insects were monitored. (B) Bean bug survival rate

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after bead injection and bacterial challenges. The latex bead solutions were injected to insects one day

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prior to the E. coli (i) or S. aureus (ii) challenge to inhibit the cellular immune responses. The control

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groups of insects were prepared by either injected bead only or bacteria only. For each sample group,

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thirty three insects were monitored.

326 Fig. 2. (A) Antimicrobial activity of the activated hemolymphs. The E. coli-injected hemolymphs of

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the symbiotic (SYM) and aposymbiotic (APO) insects were compared for their antimicrobial activities

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to the E. coli cells. CFUs were normalized by hemolymph-untreated CFUs set to 100%. Means and

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SDs are shown (n=4). (B) Expression level of AMPs. The fat bodies of the symbiotic and

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aposymbiotic insects were collected to measure the expression of riptocin (i), rip-defensin (ii) and rip-

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thanatins (iii) at 17 h post injection with buffer, E. coli cells or S. aureus cells. The expression level

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was normalized by the comparative CT (∆∆CT) method using EF1α as a reference gene. Columns and

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bars indicate means and SDs (n = 3). Statistically significant differences between the symbiotic and

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aposymbitotic insects were analyzed by two-way ANOVA with Sidak’s correction (NS, not

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significant; ***, P < 0.0001).

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Supplementary Fig. S1. Bacterial phagocytosis of hemocytes of bead-injected or noneinjected insects. Adult Riptortus was either none-injected or bead-injected (FluoSpheres® Fluorescent Microspheres, 3.6×1010 beads per insect). After 12 h initial bead-injection, FITClabelled bacteria (approximately, 2 x 107 bacteria cells per insect) were injected to the insects. In order to examine the bacterial uptake of hemocytes, hemolymph was collected at the 3 h post injection of FITC-labelled bacteria. In contrary to the hemocytes from no-bead injected insects, the hemocytes from bead-injected insects were saturated with beads without FITClabelled bacteria.