The symbiotic role of O-antigen of Burkholderia symbiont in association with host Riptortus pedestris

doi:10.1016/j.dci.2016.02.009

Developmental & Comparative Immunology

Volume 60, July 2016, Pages 202-208

https://doi.org/10.1016/j.dci.2016.02.009 Get rights and content

Highlights

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O-antigen defective mutant strains were generated for the insect gut symbiosis assay.
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LPS O-antigen is important for initial bacterial colonization in the host gut.
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O-antigen is not essential for bacterial persistence at later stage of symbiosis.

Abstract

Riptortus pedestris harboring Burkholderia symbiont is a useful symbiosis model to study the molecular interactions between insects and bacteria. We recently reported that the lipopolysaccharide O-antigen is absent in the Burkholderia symbionts isolated from Riptortus guts. Here, we investigated the symbiotic role of O-antigen comprehensively in the Riptortus-Burkholderia model. Firstly, Burkholderia mutant strains deficient of O-antigen biosynthesis genes were generated and confirmed for their different patterns of the lipopolysaccharide by electrophoretic analysis. The O-antigen-deficient mutant strains initially exhibited a reduction of infectivity, having significantly lower level of symbiont population at the second-instar stage. However, both the wild-type and O-antigen mutant symbionts exhibited a similar level of symbiont population from the third-instar stage, indicating that the O-antigen deficiency did not affect the bacterial persistence in the host midgut. Taken together, we showed that the lipopolysaccharide O-antigen of gut symbiont plays an exclusive role in the initial symbiotic association.

Introduction

Bacterial lipopolysaccharide (LPS) is the major outer membrane component of Gram-negative bacteria. It consists of three different regions: lipid A, core-oligosaccharide and O-antigen (Caroff and Karibian, 2003, Raetz and Whitfield, 2002). The innermost lipid A is a hydrophobic region anchored into the membrane and generally composed of a di-glucosamine backbone linked with four to seven fatty acids. A 2-keto-3-deoxyoctonate (Kdo) unit connects the lipid A to a core-oligosaccharide. The core-oligosaccharide is linked to the outermost region of LPS called O-antigen. The O-antigen consists of repeating oligosaccharide units. Bacteria with LPS lacking O-antigen is called rough-type bacteria, and bacteria harboring LPS O-antigen is called smooth-type bacteria.

LPS O-antigen provides a protective barrier against environmental and immunological factors for bacteria (Raetz and Whitfield, 2002). In pathogenesis, O-antigen is an important virulence factor that facilitates the interaction with host tissues and provides protection from membrane-active compounds of hosts (Trent et al., 2006). Although the roles of O-antigen may vary in different bacteria, many studies on pathogenic bacteria report that rough-type bacteria are susceptible to serum complement and antimicrobial peptides, resulting in much less efficiency to invade and survive in host (Burns and Hull, 1998, Gunn and Ernst, 2007, Murray et al., 2006, Nesper et al., 2001, VanDenBosch et al., 1997). Similarly, LPS O-antigen was reported to be essential for symbiotic association in some symbiotic model systems. In legume-Rhizobium symbiosis, O-antigen deficient mutant Rhizobium impairs root colonization probably due to susceptibility to antimicrobials (Ormeno-Orrillo et al., 2008). In squid-Vibrio symbiosis, O-antigen deficient mutant, waaL, shows a motility defect and significantly delayed colonization in light organ of squid (Post et al., 2012). Also pbgE mutant of Photorhabdus luminescens exhibiting rough-type LPS is unable to colonize the gut of the nematode (Bennett and Clarke, 2005). In case of leech-Aeromonas symbiosis, a high-molecular weight LPS is essential for bacterial colonization by providing the complement resistance to the symbionts onto digestive tract of leech (Braschler et al., 2003). In contrast to the general notion of the importance of LPS O-antigen in pathogenesis and symbiosis, we recently found that Burkholderia gut symbionts exist as the rough-type bacteria in an insect-bacteria symbiosis model (Kim et al., 2015).

Riptortus pedestris (bean bug) harbors a single kind of gut symbiont, genus Burkholderia, in a specialized region of the posterior midgut (Kikuchi et al., 2005). This Burkholderia symbiont is not transmitted from mother to offspring, but it is orally acquired by Riptortus nymphs from environment. Possessing its free living ability, the symbionts isolated from the host midgut can be cultured in standard bacterial media and subjected to genetic modification (Kikuchi et al., 2007, Kikuchi et al., 2011a, Kikuchi et al., 2011b). Recently we used the genetically modified Burkholderia symbiont strains to understand molecular cross-talks between insect and bacteria (Kim et al., 2014a, Kim et al., 2014b, Kim et al., 2013a, Kim et al., 2013b). Furthermore, we attempted to understand molecular changes occurring in the Burkholderia cells as they become gut symbionts in the Riptortus host. The direct comparison between the symbiotic Burkholderia cells and the cultured Burkholderia cells revealed striking differences in the cell envelope structures. The symbiotic cells isolated from Riptortus midgut exhibited the rough-type LPS (Kim et al., 2015).

Because the O-antigen is important for the pathogenic bacteria to escape from the host immunological factors (Burns and Hull, 1998, Gunn and Ernst, 2007, Murray et al., 2006, Nesper et al., 2001, Trent et al., 2006, VanDenBosch et al., 1997) and for the several symbiotic bacteria to successfully colonize the host (Bennett and Clarke, 2005, Ormeno-Orrillo et al., 2008, Post et al., 2012), it was quite unexpected to find the loss of the O-antigen in the Burkholderia symbiont. Therefore, in this study, we investigated the role of the Burkholderia O-antigen in the symbiotic association with the host Riptortus host. By using the O-antigen mutant Burkholderia strains, we addressed the importance of the O-antigen in the initial stage as well as in the later stage of the symbiosis.

Section snippets

Bacteria and media

List of bacteria used in this study is shown in Table 1. Escherichia coli cells were cultured at 37 °C with LB medium (1% tryptone, 0.5% yeast extract, 0.5% NaCl). Burkholderia symbiont RPE75 cells were cultured at 30 °C with YG medium (0.4% glucose, 0.5% yeast extract, 0.1% NaCl) containing 30 μg/ml rifampicin (Kikuchi et al., 2011b).

Isolation of symbiotic Burkholderia from midgut

The symbiotic midguts, M4s, were dissected from fifth instar nymphs and placed in 50 μl of 10 mM phosphate buffer (PB, pH7.0). The M4 midguts were cut into pieces

Generation of the O-antigen mutant strains of Burkholderia symbiont

When the genome of Burkholderia symbiont strain RPE64 was searched for the candidate genes encoding enzymes for LPS O-antigen biosynthesis (Shibata et al., 2013), the gene clusters of the O-antigen biosynthesis genes were found in at least two locations in chromosome 1 (Fig. 1A). In order to generate mutant strains with different O-antigen structures, we targeted three glycosyltransferase genes (wbxA, wbxB and wbiF) and an epimerase gene (wbiG) involved in the LPS O-antigen synthesis. Although

Discussion

In this study, we investigated the role of the LPS O-antigen in the Riptortus-Burkholderia symbiosis using O-antigen biosynthesis mutant strains. Because LPS O-antigen is shown to be critical for bacterial colonization to host in other symbiotic model systems, we expected that O-antigen deficient mutant strains would exhibit impairment in the colonization to the midgut of R. pedestris. Our results demonstrated that the O-antigen deficient strains exhibit the lower level of colonization at the

Acknowledgments

We thank Naruo Kikoh (Open University, Japan) and Takema Fukatsu (AIST, Japan) for the resources and advice. This study was supported by the Global Research Laboratory Program (grant number 2011-0021535), Basic Science Research Program (grant number 2014R1A1A4A01007507) of the National Research Foundation of Korea, and a grant from Kosin University College of Medicine (2015).

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How host organisms evolved and maintain specific mutualisms with microorganisms is a fundamental question that is subject to intensive research. In the large majority of insect mutualisms, the host-microbe specificity is maintained by a “partner fidelity” mechanism, mainly through direct symbiont transmission from mother to offspring. Such vertical symbiont transmission is remarkably diverse in insects, including ovarial transmission, milk-gland transmission, coprophagy, egg-smearing, and capsule transmission. In contrast to the insect-microbe symbioses, many animals and plants do not vertically transmit their symbionts but acquire symbionts from ambient environments every generation. Sophisticated “partner choice” mechanisms are at play to establish these mutualisms and maintain them over evolutionary timescales. This symbiont transmission mode, called horizontal transmission or environmental acquisition, is rarely found in insects that maintain specific associations, but recent studies have described this type of symbiosis in a few insect groups. The symbiosis between the bean bug Riptortus pedestris and its gut symbiont Burkholderia insecticola is one of the model systems that is intensively studied to understand how host-symbiont specificity and mutualistic interactions are maintained in insects with horizontal symbiont transmission. Phylogenetic analyses of symbionts in natural insect populations and bacterial inoculation tests in the laboratory revealed a high degree of specificity in this symbiosis while mutant screening of the symbiotic bacterium, genomics and transcriptomics, and histological observations have identified underpinning morphological, genetic and molecular bases. In this chapter, we focus on symbiont transmission modes and mechanisms observed in the amazing diversity of microbial symbioses in insects, highlighting the ways in which they have likely evolved.
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The lipopolysaccharide core oligosaccharide of Burkholderia plays a critical role in maintaining a proper gut symbiosis with the bean bug Riptortus pedestris
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Lipopolysaccharide, the outer cell-wall component of Gram-negative bacteria, has been shown to be important for symbiotic associations. We recently reported that the lipopolysaccharide O-antigen of Burkholderia enhances the initial colonization of the midgut of the bean bug, Riptortus pedestris. However, the midgut-colonizing Burkholderia symbionts lack the O-antigen but display the core oligosaccharide on the cell surface. In this study, we investigated the role of the core oligosaccharide, which directly interacts with the host midgut, in the Riptortus–Burkholderia symbiosis. To this end, we generated the core oligosaccharide mutant strains, ΔwabS, ΔwabO, ΔwaaF, and ΔwaaC, and determined the chemical structures of their oligosaccharides, which exhibited different compositions. The symbiotic properties of these mutant strains were compared with those of the wild-type and O-antigen–deficient ΔwbiG strains. Upon introduction into Riptortus via the oral route, the core oligosaccharide mutant strains exhibited different rates of colonization of the insect midgut. The symbiont titers in fifth-instar insects revealed significantly reduced population sizes of the inner core oligosaccharide mutant strains ΔwaaF and ΔwaaC. These two strains also negatively affected host growth rate and fitness. Furthermore, R. pedestris individuals colonized with the ΔwaaF and ΔwaaC strains were vulnerable to septic bacterial challenge, similar to insects without a Burkholderia symbiont. Taken together, these results suggest that the core oligosaccharide from Burkholderia symbionts plays a critical role in maintaining a proper symbiont population and in supporting the beneficial effects of the symbiont on its host in the Riptortus–Burkholderia symbiosis.
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In vitro assays showed that the mutant was much less resistant to osmotic and centrifugal pressures and more susceptible to lysozyme, suggesting that cell wall integrity is important for the symbiotic association [95]. Subsequently, other enzymes involved in cell wall biosynthesis and turnover, N-acetylmuramyl-l-alanine amidase (AmiC), and in O-antigen biosynthesis (WbxA, WbiF, and WbiG), were also found to play key roles in the symbiotic association [96,97]. By comparative proteome analysis between free-living (i.e. in vitro) cells and symbiotic (isolated from the midgut crypts: i.e. in vivo) cells of the Burkholderia symbiont, it has been demonstrated that polyhydroxyalkanoate (PHA) biosynthesis genes phaB and phaC are colonization factors of the symbiont [98].
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To further confirm the effects of the M1 lysate, the combined mixtures of midgut lysates, except for that with the M1 lysate, were treated with Burkholderia as described in Fig. 2A. Although cultured Burkholderia and ΔwbiG mutants were not susceptible to midgut lysates except for the M1 lysate (Fig. 2B, lanes 2–5 and 12–15), native symbiotic Burkholderia were susceptible to the M4B lysate (lanes 9 and 10), which confirmed that symbiotic Burkholderia are susceptible to the M4B lysate and ΔwbiG mutants are susceptible to only the M1 lysate. Since we used an epimerase-deficient ΔwbiG mutant as an LPS O-antigen-deficient mutant, we prepared two additional LPS O-antigen glycosyltransferase-deficient ΔwbiF and ΔwbxA mutants (Kim et al., 2016; Shibata et al., 2013). Another LPS O-antigen glycosyltransferase-deficient ΔwbxB mutant was excluded from this study due to the presence of LPS O-antigen as shown by our previous study (Kim et al., 2016).
Riptortus pedestris, a common pest in soybean fields, harbors a symbiont Burkholderia in a specialized posterior midgut region of insects. Every generation of second nymphs acquires new Burkholderia cells from the environment. We compared in vitro cultured Burkholderia with newly in vivo colonized Burkholderia in the host midgut using biochemical approaches. The bacterial cell envelope of in vitro cultured and in vivo Burkholderia differed in structure, as in vivo bacteria lacked lipopolysaccharide (LPS) O-antigen. The LPS O-antigen deficient bacteria had a reduced colonization rate in the host midgut compared with that of the wild-type Burkholderia. To determine why LPS O-antigen-deficient bacteria are less able to colonize the host midgut, we examined in vitro survival rates of three LPS O-antigen-deficient Burkholderia mutants and lysates of five different midgut regions. The LPS O-antigen-deficient mutants were highly susceptible when cultured with the lysate of a specific first midgut region (M1), indicating that the M1 lysate contains unidentified substance(s) capable of killing LPS O-antigen-deficient mutants. We identified a 17 kDa protein from the M1 lysate, which was enriched in the active fractions. The N-terminal sequence of the protein was determined to be a soybean Kunitz-type trypsin inhibitor. These data suggest that the 17 kDa protein, which was originated from a main soybean source of the R. pedestris host, has antibacterial activity against the LPS O-antigen deficient (rough-type) Burkholderia.

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The symbiotic role of O-antigen of Burkholderia symbiont in association with host Riptortus pedestris

Highlights

Abstract

Introduction

Section snippets

Bacteria and media

Isolation of symbiotic Burkholderia from midgut

Generation of the O-antigen mutant strains of Burkholderia symbiont

Discussion

Acknowledgments

Carbohydr. Res.

J. Biol. Chem.

J. Biol. Chem.

Methods Enzymol.

The pbgPE operon in Photorhabdus luminescens is required for pathogenicity and symbiosis

J. Bacteriol.

Complement resistance is essential for colonization of the digestive tract of Hirudo medicinalis by Aeromonas strains

Appl. Environ. Microbiol.

Comparison of loss of serum resistance by defined lipopolysaccharide mutants and an acapsular mutant of uropathogenic Escherichia coli O75:K5

Infect. Immun.

Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia

Microbiol. Rev.

The structure and function of Francisella lipopolysaccharide

Ann. N. Y. Acad. Sci.

Mechanisms of cyclic-di-GMP signaling in bacteria

Annu. Rev. Genet.

The multiple signaling systems regulating virulence in Pseudomonas aeruginosa

Microbiol. Mol. Biol. Rev.

Insect-microbe mutualism without vertical transmission: a stinkbug acquires a beneficial gut symbiont from the environment every generation

Appl. Environ. Microbiol.

An ancient but promiscuous host-symbiont association between Burkholderia gut symbionts and their heteropteran hosts

ISME J.

Specific developmental window for establishment of an insect-microbe gut symbiosis

Appl. Environ. Microbiol.

Gut symbiotic bacteria of the genus Burkholderia in the broad-headed bugs Riptortus clavatus and Leptocorisa chinensis (Heteroptera : Alydidae)

Appl. Environ. Microbiol.

Efficient colonization of the bean bug Riptortus pedestris by an environmentally transmitted Burkholderia symbiont

Appl. Environ. Microbiol.

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

ISME J.

Purine biosynthesis, biofilm formation, and persistence of an insect-microbe gut symbiosis

Appl. Environ. Microbiol.