Wolbachia

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1		Ann M Fallon	+ 1649 word(s)	1649	2021-08-09 10:03:53

This entry is adapted from 10.3390/insects12080706

Wolbachia is an intracellular bacterium that occurs in arthropods and in filarial worms. First described nearly a century ago in the reproductive tissues of Culex pipiens mosquitoes, Wolbachia is now known to occur in roughly 50% of insect species, and has been considered the most abundant intracellular bacterium on earth. In insect hosts, Wolbachia modifies reproduction in ways that facilitate spread of the microbe within the host population, but otherwise is relatively benign. In this “gene drive” capacity, Wolbachia provides a tool for manipulating mosquito populations. In mosquitoes, Wolbachia causes cytoplasmic incompatibility, in which the fusion of egg and sperm nuclei is disrupted, and eggs fail to hatch, depending on the presence/absence of Wolbachia in the parent insects. Recent findings demonstrate that Wolbachia from infected insects can be transferred into mosquito species that do not host a natural infection. When transinfected into Aedes aegypti, an important vector of dengue and Zika viruses, Wolbachia causes cytoplasmic incompatibility and, in addition, decreases the mosquito’s ability to transmit viruses to humans.

alpha-proteobacteria reproductive parasite symbiont mosquito insect cell lines genetic manipulation cell culture

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Subjects: Microbiology

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Ann Fallon

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Update Date: 31 Aug 2021

Table of Contents

1. Introduction

Wolbachia is an obligate intracellular microbe first described in reproductive tissues of Culex pipiens mosquitoes nearly a century ago ^[1]^[2]. Like Escherichia coli , Wolbachia is a Gram-negative bacterium in the phylum Proteobacteria: the purple bacteria and their relatives. Proteobacteria include nine monophyletic classes representing tremendous biodiversity. Among these, the genera Ehrlichia and Anaplasma , which can cause disease in humans, are classified with Wolbachia as members of the alpha-proteobacteria, in the order Rickettsiales, family Anaplasmataceae. Wolbachia is uniquely associated with invertebrates, does not infect vertebrate hosts, and replicates only within a eukaryotic host cell. In contrast, E. coli and many familiar Gram-negative pathogens of humans classified as gamma-proteobacteria can be cultured in liquid medium and plated on solid media as free-living microbes.

Knowledge of well-studied free-living bacteria provides an important framework for investigating the genetics and physiology of Wolbachia , now known to infect a high proportion of insect species, in addition to other arthropods and filarial worms, all members of the Ecdysozoa. Because of its widespread distribution among insects ^[3]^[4], Wolbachia provides a model system for exploring biological interactions between an intracellular microbe, the invertebrate host cells in which it resides, and the diversity of reproductive phenotypes with which it is associated ^[5]^[6]. In species that harbor Wolbachia, the bacterium is transmitted vertically, from mother to offspring, which retain the infection. In most arthropods, Wolbachia alters reproduction in diverse ways that favor its invasion of naive populations, and is sometimes considered a reproductive parasite. In contrast, Wolbachia is an essential symbiont in filarial worms ^[7]^[8]^[9]. In mosquitoes, Wolbachia causes a reproductive distortion called cytoplasmic incompatibility (CI), which has important applications in vector control ^[10].

2. Wolbachia in Insect Cell Lines

Wolbachia ’s obligate intracellular lifestyle complicates the biochemical and genetic analyses that could advance pest control and anti-filarial applications. Even with hosts amenable to laboratory rearing, maintenance of colonies, dissection of infected tissues, and embryonic microinjection are labor-intensive and time-consuming. Moreover, many existing laboratory colonies are highly inbred, complicating cage studies that address fitness. The utility of Wolbachia in control applications would be enhanced if the microbe could be experimentally manipulated by genetic engineering to express selectable markers, which in turn will be advanced by improving manipulation of Wolbachia in cell lines and expanding the diversity of Wolbachia strains that can be investigated in culture. A modest advance would be adaptation of a filarial strain of Wolbachia to a cell line; at present, Wolbachia -infected insect cell lines are used as a surrogate to identify new drugs that target Wolbachia for treatment of filarial diseases ^[11]^[12]^[13].

The author’s research focuses on systematic exploration of Wolbachia propagation in cultured cells as a substitute for the differentiated host tissues, such as ovaries and testes, in which Wolbachia is most abundant. Cell lines used to propagate Wolbachia are listed in Table 1 , wherein supergroup designations are noted after the strain name; for example, w Pip_B indicates that w Pip is classified in supergroup B. With the exception of a single member of supergroup F from the cat flea ^[14], only members of supergroups A and B, sometimes called the “pandemic” supergroups, have been maintained in insect cell lines. The reader should note that, in some cases, an infected cell line may have been sub-cultured only a limited number of times and/or has a very long doubling time, and that the same cell line may have been infected with the same strain of Wolbachia by different investigators, and given a different name. An important incentive for employing cell lines was the possibility that preadaption to cultured cells might improve the likelihood that Wolbachia would establish in novel hosts infected by embryonic microinjection, and towards this end, a few lines have been maintained for several years ^[15]. In other cases, which are not reviewed in detail here, infected cell lines have been used to test effects of Wolbachia on viral replication in efforts that generally validate the anti-pathogen responses seen in transinfected mosquitoes. Finally, as with Wolbachia itself, a uniform descriptive label for infected cell lines remains to be developed.

Table 1. Cell lines in which Wolbachia strains have been propagated.

Cell Line Designation	Wolbachia Strain_Supergroup	Source of Wolbachia	Reference	Comments
Dipteran cell lines
Aedes albopictus (mosquito)
Aa23	wAlbB_B	Aedes albopictus embryos	^[16]	First infected cell line; established from naturally infected Ae. albopictus; one of two Wolbachia strains
Aa23(T)	wMel_A	infected RML-12 cells	^[17]	12 passages
Aa23(T)	wRi_A wCof_A wAlbB_B wPip_B wCauA_A wCauB_B	D. simulans eggs D. simulans eggs infected Aa23 cells Cx. pipiens eggs Cadra cautella eggs Cadra cautella eggs	^[18]	Demonstration of shell vial technique; details focus on wRi
Aa23(T)	wMelPop	w¹¹¹⁸ embryos	^[15]	Generated wMelPop-CLA
NIAS-AeAl-2	wStri_B wKue_A wCauA_A	L. striatellus ovary Ephestia kuehniella eggs Cadra cautella eggs	^[19]	Infected from small inoculum; one ovary, or 80–100 eggs; Infected AeAl-2 cells form aggregates; occasional addition of uninfected cells to infected cultures
NIAS-AeAl-2	wCau_A wCauB_B wKue_A	Ephestia kuehniella eggs Ephestia kuehniella eggs Ephestia kuehniella eggs	^[20]	Two stages: infection and maintenance
RML-12	wMelPop-CLA_A	infected Aa23 cells	^[15]	wMelPop transferred to cells; serial passage; reintroduction into original host by microinjection; some loss of virulence; “genetic adaptation” to improve transfer to new hosts
RML-12	wMel_A	O’Neill et al.; cited in ^[17] personal communication	^[17]	Maintained for 3 years
C6/36	wRi_A	D. simulans eggs	^[18]
C6/36	wMel_A	infected RML-12 cells	^[17]	Stable; higher density than RML-12 cells
C6/36	wAlbB_B	infected Aa23 cells	^[21]
C6/36	wAlbB_B	infected Aa23 cells	^[22]
C6/36	wMelPop-CLA_A	RML-12-CLA	^[23]	C6/36.wMelPop-CLA
C6/36	wAlbB_B	infected Aa23 cells	^[24]	Virus screen
C7-10	wStri_B	NIAS-AeAl-2	^[25]	Called C/wStri1 line
C7-10	wAlbB_B	infected Aa23 cells	^[26]	Infected line: C7-10B
C7-10	wRi_A	D. simulans eggs	^[26]	Infected line: C7-10R C7-10R more stable, uniform than C7-10B
TK-6 (C7-10)	wAlb_B	infected Aa23 cells	^[27]	Stable 5 months
Mtx-5011-256	wStri_B	C/wStri1 cells	^[28]	Lower MOI than C7-10; aneuploidy a factor?
Aedes aegypti mosquito
Aag2	wAlbB_B	infected Aa23 cells	^[29]	Line called Aag2.wAlbB
Aag2	wAlbB_B	infected Aa23 cells	^[30]	Line called w-Aag2
Aag2	wMel_A	D. melanogaster embryos	^[31]^[32]	Line called Aag-2wMel
Aag2	wMel_A wMelPop-CLA_A	Infected RML-12 cells Infected RML-12 cells	^[33]	^[15]
Aa-20	wMelPop-CLA_A	Not stated	^[34]	Mos 20; CVCL_Z353; ^[35]
Anopheles gambiae mosquito
Mos-55	wMelPop-CLA_A	infected Aa23 cells	^[15]
Sua5B	wAlbB_B wRi_A	infected Aa23 cells D. simulans eggs	^[36]	Best was 1/10³ cells infected
Drosophila melanogaster
S2	wRi_A	D. simulans eggs	^[18]
S2	strain from Dm2008Wb1cells	infected, D. melanogaster	^[37]	(from abstract; Russian)
Dm2008Wb1	primary cell culture	infected, D. melanogaster	^[37]	(from abstract; Russian)
JW-18	wMel-Pop_A	infected, D. melanogaster	^[13]	Albendazole sulfone inhibits
1182-48	wMelPop_A	infected JW-18 cells	^[38]	Acentriolar haploid line
S2R+	wMelPop_A	infected JW-18 cells	^[38]	Tetraploid male cells; higher Wolbachia titers
Lutzomyia longipalpis (sandfly)
LL5	wMelPop-CLA_A wMel_A	infected RML-12 cells infected RML-12 cells	^[39]	Immune activation unstable; no effect on Leishmania
Lulo	wMelPop-CLA_A wMel_A (unstable)	infected RML-12 cells infected RML-12 cells	^[39]
Culicoides sonorensis (Biting midge)
W3	wAlbB_B	infected Aa23 cells	^[40]	Line W3
W8	wAlbB_B	infected Aa23 cells	^[40]	Higher density than W3
Hematobia irritans (Horn fly)
HIE-18	wAlbB_B wMel_A wMelPop_A	infected Aa23 cells infected Aag2 cells infected Aag2 cells	^[41]	50 passages
Lepidopteran
BCIRL-HZ-AM1-G5 Heliothis zea	wStri_B	L. striatellus ovary	^[19]
Sf9 Spodoptera frugiperda	wRi_A	D. simulans eggs	^[18]
Sf9 Spodoptera frugiperda	wCauB_B	Ephestia kuehniella eggs	^[20]
Tick
Ixodes scapularis	wAlbB_B, wStri_B wCfe_F	infected mosquito cells cat fleas	^[14]	wStri_B, 29 passages wCfe_F, 2 passages
Ixodes ricinus	wAlbB_B, wStri_B	infected mosquito cells	^[14]
Riphicephalus microplus	wAlbB_B, wStri_B	infected mosquito cells	^[14]
Mammal
L929 (mouse)	wStri_B	L. striatellus ovary	^[19]	Cells maintained at 28 °C
Filarial screening
Aa23	wAlbB_B		^[11]	Anti-filarial screen
C6/36	wAlbB_B	infected Aa23 cells	^[12]	Macrofilaricides
JW-18	wMelPop_A	D. melanogaster w¹¹¹⁸	^[13]	Anti-filarial screen

Insect cell lines in which Wolbachia has been maintained. Columns from left to right show: (1) cell lines, arranged in groups according to species from which the cell line was derived; (2) Wolbachia strain_supergroup; (3) source of the material introduced into the cell line; (4) reference; (5) brief comments.

3. Why Cultured Cells?

If the streamlined Wolbachia genome can be genetically engineered in the future, propagation of the altered genome will require efficient reintroduction into a host cell to allow replication and expansion of transformant populations. Use of cell lines offers a practical means of producing the large quantities of Wolbachia that will be needed to develop transformation protocols that are sufficiently robust for use in basic research and pest control applications. Although isolated examples of successful transformation of intracellular microorganisms such as Coxiella burnetti, the pathogen that causes Q fever, have been achieved, these remain labor intensive and have low frequencies of success ^[42]. Nevertheless, over the past two decades, remarkable progress towards cell-free culture of Coxiella has been achieved, despite its streamlined 2 Mb genome ^[43]^[44]. These successes underscore the importance of detailed attention to culture conditions and metabolic activities of obligate intracellular microbes. Wolbachia lacks pathogenicity to humans, and its genome is more extensively streamlined, relative to that of Coxiella. Nevertheless, the long evolutionary history of Wolbachia’s interaction with invertebrate hosts and its adaptations for germline transmission contribute to the value of Wolbachia as a model system for understanding the biology of obligate intracellular bacteria in invertebrate cells and manipulating their biology for control of insect pests.

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