Running​ ​head:​ ​THE​ ​TSETSE​ ​FLY​ ​AND​ ​ITS​ ​SECONDARY​ ​ENDOSYMBIONT​ ​SODALIS​ ​GLOSSINIDIUS

Bacterial-Host​ ​Interactions:​ ​The​ ​Tsetse​ ​Fly​ ​and​ ​Its​ ​Secondary​ ​Endosymbiont​ ​Sodalis​ ​glossinidius

NS154​ ​-​ ​Life's​ ​Chemistry

Minerva​ ​Schools​ ​at​ ​KGI

THE​ ​TSETSE​ ​FLY​ ​AND​ ​ITS​ ​SECONDARY​ ​ENDOSYMBIONT​ ​SODALIS​ ​GLOSSINIDIUS ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​2

Bacterial-Host​ ​Interactions:​ ​The​ ​Tsetse​ ​Fly​ ​and​ ​Its​ ​Secondary​ ​Endosymbiont​ ​Sodalis​ ​glossinidius

Abstract

The​ ​tsetse​ ​fly​ ​(​Glossina​ ​spp.),​ ​a​ ​primary​ ​vector​ ​of​ ​African​ ​trypanosomes,​ ​has​ ​three​ ​known​ ​bacterial
endosymbionts​ ​which​ ​impact​ ​the​ ​physiology​ ​of​ ​their​ ​host.​ ​Sodalis​ ​glossinidius​ ​is​ ​its​ ​secondary​ ​endosymbiont,
of​ ​which​ ​knowledge​ ​of​ ​a​ ​clearly​ ​defined​ ​functional​ ​role​ ​within​ ​the​ ​host​ ​is​ ​still​ ​lacking.​ ​It​ ​has​ ​been​ ​shown​ ​that​ ​it
may​ ​influence​ ​its​ ​host's​ ​ability​ ​to​ ​serve​ ​as​ ​a​ ​vector​ ​of​ ​pathogenic​ ​trypanosomes​ ​and​ ​other​ ​findings​ ​show​ ​that​ ​its
removal​ ​from​ ​the​ ​host​ ​may​ ​decrease​ ​the​ ​lifespan​ ​of​ ​the​ ​tsetse​ ​fly.​ ​It​ ​is​ ​vertically​ ​transmitted​ ​from​ ​the​ ​milk
glands​ ​of​ ​the​ ​mother​ ​to​ ​the​ ​offspring​ ​but​ ​is​ ​not​ ​an​ ​obligate​ ​symbiont​ ​because​ ​it​ ​can​ ​be​ ​cultured​ ​outside​ ​its​ ​tsetse
host​ ​and​ ​symbiont–host​ ​coevolution​ ​studies​ ​have​ ​shown​ ​a​ ​lack​ ​of​ ​expected​ ​phylogenetic​ ​congruence​ ​in​ ​the
evolution​ ​of​ ​Sodalis​ ​and​ ​tsetse,​ ​suggesting​ ​that​ ​it​ ​has​ ​not​ ​undergone​ ​the​ ​high​ ​degree​ ​of​ ​reductive​ ​evolution​ ​that
is​ ​expected​ ​in​ ​long-standing​ ​obligate​ ​bacterial​ ​symbioses.​ ​They​ ​have​ ​maintained​ ​the​ ​genes​ ​implicated​ ​in
pathogenic​ ​cell​ ​invasion​ ​and​ ​iron​ ​acquisition​ ​systems,​ ​allowing​ ​them​ ​to​ ​exist​ ​with​ ​eukaryotic​ ​cells​ ​and​ ​obtain
biolimiting​ ​iron​ ​from​ ​the​ ​tsetse​ ​fly​ ​blood​ ​meal​ ​diet.
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Bacterial-Host​ ​Interactions:​ ​The​ ​Tsetse​ ​Fly​ ​and​ ​Its​ ​Secondary​ ​Endosymbiont​ ​Sodalis​ ​glossinidius

Most,​ ​if​ ​not​ ​all​ ​organisms,​ ​interact​ ​with​ ​microbes​ ​that​ ​influence​ ​critical​ ​aspects​ ​of​ ​their​ ​host's​ ​physiology.​ ​In

endosymbiosis,​ ​bacteria​ ​live​ ​within​ ​the​ ​body​ ​or​ ​cells​ ​of​ ​its​ ​hosts,​ ​providing​ ​mutual​ ​benefit​ ​to​ ​both​ ​species.​ ​They

also​ ​engage​ ​in​ ​pathogenic​ ​relationships​ ​with​ ​eukaryotic​ ​hosts​ ​(reviewed​ ​in​ ​Kobayashi​ ​&​ ​Crouch,​ ​2009)​ ​and

exert​ ​their​ ​effects​ ​by​ ​signalling​ ​within​ ​and​ ​across​ ​bacterial​ ​species,​ ​as​ ​well​ ​as​ ​with​ ​host​ ​cells,​ ​using​ ​quorum

sensing​ ​(Miller​ ​&​ ​Bassler,​ ​2001)​ ​The​ ​influence​ ​of​ ​endosymbionts​ ​have​ ​been​ ​characterised​ ​for​ ​various​ ​host

organisms.​ ​ ​(reviewed​ ​in​ ​McFall-Ngai​ ​et​ ​al.,​ ​2013)

In​ ​the​ ​invertebrate​ ​class,​​ ​Insecta​,​ ​endosymbionts​ ​are​ ​classified​ ​as​ ​either​ ​primary​ ​(obligate​ ​symbionts​ ​required

for​ ​host​ ​development​ ​and​ ​survival)​ ​or​ ​secondary​ ​(facultative​ ​symbionts​ ​that​ ​provide​ ​non-vital​ ​benefits​ ​to​ ​the

host).​ ​(White​ ​et​ ​al.,​ ​2013)​ ​The​ ​tsetse​ ​fly​ ​(​Glossina​ ​spp.)​ ​has​ ​three​ ​known​ ​bacterial​ ​endosymbionts:​ ​Wolbachia

spp​.​ ​which​ ​manipulates​ ​its​ ​host​ ​reproductive​ ​biology,​ ​(Alam​ ​et​ ​al.,​ ​2011)​ ​its​ ​obligate​ ​mutualist,​ ​Wigglesworthia,

which​ ​synthesises​ ​B​ ​vitamins​ ​that​ ​are​ ​absent​ ​from​ ​the​ ​fly's​ ​blood​ ​diet,​ ​(Pais​ ​et​ ​al.,​ ​2008)​ ​and​ ​its​ ​secondary

endosymbiont,​ ​Sodalis​ ​glossinidius​,​ ​whose​ ​exact​ ​function​ ​in​ ​the​ ​host​ ​has​ ​still​ ​not​ ​been​ ​discovered.

Consistent​ ​with​ ​the​ ​typical​ ​characterisation​ ​of​ ​secondary​ ​endosymbionts,​ ​S.​ ​glossinidius​ ​is​ ​localised​ ​in​ ​multiple

tsetse​ ​fly​ ​tissues.​ ​A​ ​small​ ​Gram-negative​ ​rickettsia-like​ ​micro-organism​ ​(now​ ​known​ ​to​ ​be​ ​Sodalis​ ​glossinidius​)

was​ ​observed​ ​in​ ​the​ ​midgut​ ​of​​ ​Glossina​ ​morsitans​ ​larvae,​ ​and​ ​it​ ​was​ ​hypothesised​ ​that​ ​they​ ​were​ ​residing​ ​on​ ​the

lining​ ​of​ ​the​ ​digestive​ ​tube.​ ​(Roberts​ ​&​ ​Pell,​ ​1976)​ ​Shaw​ ​and​ ​Moloo​ ​(1991)​ ​examined​ ​epithelial​ ​tissues​ ​from

the​ ​guts​ ​of​ ​Glossina​ ​species​ ​by​ ​electron​ ​microscopy​ ​and​ ​observed​ ​similar​ ​rickettsia-like​ ​organisms​ ​contained

within​ ​midgut​ ​epithelial​ ​cells.​ ​In​ ​1999,​ ​Cheng​ ​and​ ​Aksoy​ ​observed​ ​that​ ​localisation​ ​of​ ​secondary

endosymbionts​ ​(now​ ​identified​ ​to​ ​be​ ​S.​ ​glossinidius​)​ ​in​ ​Tsetse​ ​fly​ ​tissues​ ​increasingly​ ​diffused​ ​with​ ​age.​ ​At​ ​two

days​ ​old,​ ​the​ ​bacteria​ ​were​ ​observed​ ​only​ ​in​ ​the​ ​fly​ ​gut,​ ​while​ ​at​ ​four​ ​weeks​ ​old,​ ​it​ ​had​ ​colonised​ ​six​ ​other

tissues,​ ​including​ ​muscle​ ​and​ ​fat.​ ​(Fig.​ ​1)

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Fig. 1: Localisation of secondary symbionts within ​G. morsitans tissues with age – PCR-amplified S-symbiont DNA from (1) muscle,

(2)​ ​gut,​ ​(3)​ ​testis,​ ​(4)​ ​haemolymph,​ ​(5)​ ​fat,​ ​(6)​ ​milk​ ​gland,​ ​(7)​ ​salivary​ ​gland,​ ​(8)bacteriome,​ ​(9)​ ​ovary​ ​tissues

Vertical​ ​transmission​ ​of​ ​endosymbiotic​ ​bacteria​ ​from​ ​parent​ ​to​ ​offspring​ ​had​ ​been​ ​observed​ ​in​ ​other​ ​insect

species,​ ​specifically​ ​transovarial​ ​transmission​ ​but​ ​the​ ​mode​ ​of​ ​transformation​ ​of​ ​S.​ ​glossinidius​ ​gut​ ​bacteria​ ​in

tsetse​ ​flies​ ​remained​ ​unknown.​ ​Several​ ​hypothesised​ ​models​ ​of​ ​transmission​ ​were​ ​developed​ ​using​ ​observations

of​ ​ ​S.​ ​glossinidius​ ​localisation​ ​in​ ​tissues1.​ ​It​ ​was​ ​concluded​ ​that​ ​transovarial​ ​and​ ​paternal​ ​transmission​ ​of

bacteria​ ​was​ ​unlikely​ ​due​ ​to​ ​the​ ​absence​ ​of​ ​the​ ​bacteria​ ​in​ ​the​ ​ovary​ ​tissues2​ ​(Fig.​ ​1,​ ​lane​ ​9)​ ​ ​and​ ​mature​ ​Tsetse

fly​ ​sperm.​ ​(data​ ​not​ ​shown)​ ​The​ ​data​ ​showed​ ​that​ ​S.​ ​glossinidius​ ​was​ ​present​ ​in​ ​the​ ​milk​ ​glands​ ​of​ ​the​ ​mother

(Fig.​ ​1,​ ​lane​ ​6)​ ​and​ ​it​ ​was​ ​concluded​ ​that​ ​it​ ​was​ ​the​ ​most​ ​likely​ ​mode​ ​of​ ​bacterial​ ​transmission​ ​to​ ​offspring.

(Cheng​ ​&​ ​Aksoy,​ ​1999)

The​ ​observation​ ​of​ ​ ​S.​ ​glossinidius​ ​within​ ​diverse​ ​cells​ ​within​ ​the​ ​host​ ​flies​ ​raised​ ​questions​ ​about​ ​the

mechanisms​ ​of​ ​cell​ ​penetrance​ ​that​ ​were​ ​utilised​ ​by​ ​the​ ​bacteria.3The​ ​mechanisms​ ​of​ ​cell​ ​invasion​ ​by

1
​ ​#Plausibility:​ ​The​ ​models​ ​that​ ​were​ ​hypothesised​ ​for​ ​vertical​ ​transmission​ ​of​ ​S.​ ​glossinidius​ ​were​ ​based​ ​on​ ​the​ ​synthesis​ ​of​ ​various
experimental​ ​observations​ ​with​ ​scientific​ ​theory
2
​ ​#Deduction:​ ​The​ ​deductive​ ​argument​ ​is:​ ​"If​ ​vertical​ ​transmission​ ​occurs​ ​by​ ​this​ ​pathway,​ ​bacterial​ ​localisation​ ​should​ ​be​ ​observed
there.​ ​If​ ​no​ ​bacterial​ ​localisation​ ​is​ ​observed,​ ​then,​ ​vertical​ ​transmission​ ​does​ ​not​ ​occur​ ​by​ ​this​ ​pathway."
3
​ ​#GapAnalysis:​ ​They​ ​had​ ​observed​ ​that​ ​the​ ​bacteria​ ​existed​ ​intracellularly​ ​but​ ​they​ ​recognised​ ​that​ ​they​ ​were​ ​unaware​ ​of​ ​how​ ​cell
invasion​ ​occured
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pathogenic​ ​bacteria​ ​are​ ​well-understood​ ​while​ ​little​ ​is​ ​known​ ​about​ ​mechanisms​ ​for​ ​the​ ​host​ ​cell​ ​penetrance​ ​in

bacterial​ ​endosymbionts.​ ​To​ ​investigate​ ​this,​ ​Dale​ ​et​ ​al.​ ​(2001)​ ​hypothesised​ ​that​ ​these​ ​endosymbionts​ ​would

utilise​ ​the​ ​same​ ​mechanisms​ ​employed​ ​by​ ​pathogens​ ​to​ ​invade​ ​host​ ​cells.

Using​ ​the​ ​mutagenesis​ ​screening​ ​technique,​ ​they​ ​chemically​ ​induced​ ​random​ ​mutations​ ​in​ ​Sodalis​ ​colonies​ ​then

incubated​ ​the​ ​mutants​ ​with​ ​cultured​ ​insect​ ​cells​ ​to​ ​allow​ ​the​ ​bacteria​ ​to​ ​invade​ ​the​ ​cell.​ ​Invasion​ ​was​ ​observed

by​ ​microscopic​ ​examination​ ​and​ ​staining,​ ​and​ ​as​ ​expected,​ ​they​ ​noticed​ ​a​ ​time-dependent​ ​increase​ ​of

intracellular​ ​S.​ ​glossinidius​ ​density,​ ​indicating​ ​that​ ​the​ ​bacteria​ ​were​ ​dividing​ ​within​ ​the​ ​cell​ ​cytoplasm.​ ​ ​They

then​ ​selected​ ​the​ ​Sodalis​ ​mutants​ ​that​ ​were​ ​unable​ ​to​ ​invade​ ​the​ ​insect​ ​cells.​ ​The​ ​negative​ ​selection​ ​process

identified​ ​a​ ​single​ ​mutant​ ​clone​ ​that​ ​was​ ​able​ ​to​ ​attach​ ​but​ ​not​ ​invade​ ​the​ ​insect​ ​cell.​ ​Isolation​ ​of​ ​the​ ​mutated

DNA​ ​fragment​ ​and​ ​sequencing​ ​revealed​ ​high​ ​sequence​ ​similarity​ ​to​ ​the​ ​invC​ ​gene,​ ​which​ ​had​ ​previously​ ​been

identified​ ​as​ ​a​ ​key​ ​component​ ​of​ ​the​ ​type​ ​III​ ​secretion​ ​system​ ​employed​ ​by​ ​the​ ​pathogen,​ ​S.​ ​enterica​.​ ​They​ ​also

found​ ​that​ ​S.​ ​glossinidius​ ​with​ ​inactivating​ ​mutation​ ​in​ ​the​ ​invC​ ​gene​ ​were​ ​not​ ​transmitted​ ​vertically​ ​to

offspring.

The​ ​invC​ ​gene​ ​and​ ​its​ ​network​ ​of​ ​contributing​ ​genes​ ​allows​ ​S.​ ​glossinidius​ ​to​ ​invade​ ​nonphagocytic​ ​cells​ ​using

the​ ​trigger​ ​mechanism.​ ​Effector​ ​molecules​ ​that​ ​are​ ​a​ ​downstream​ ​product​ ​of​ ​the​ ​invC​ ​gene​ ​are​ ​injected​ ​into​ ​the

host​ ​cell​ ​cytoplasm​ ​through​ ​the​ ​implicated​ ​type​ ​III​ ​secretion​ ​system.​ ​These​ ​effector​ ​molecules​ ​activate​ ​a​ ​family

of​ ​Rho​ ​GTPases​ ​that​ ​stimulate​ ​actin​ ​polymerization.​ ​Actin​ ​filaments​ ​are​ ​components​ ​of​ ​the​ ​cytoskeleton​ ​that

function​ ​in​ ​determining​ ​cell​ ​shape​ ​and​ ​facilitating​ ​cell​ ​adhesion.​ ​These​ ​effector​ ​molecules​ ​cause​ ​actin,​ ​as​ ​well

as​ ​other​ ​cytoskeletal​ ​elements​ ​and​ ​proteins​ ​to​ ​be​ ​rearranged,​ ​causing​ ​the​ ​host-cell​ ​surface​ ​to​ ​ruffle,​ ​resulting​ ​in

large​ ​protrusions​ ​of​ ​the​ ​cell​ ​membrane​ ​ ​that​ ​fold​ ​over​ ​and​ ​trap​ ​the​ ​bacterium,​ ​inadvertently​ ​phagocytosing​ ​the

bacterium.4​ ​(Alberts​ ​et​ ​al.​ ​2002)

4
​ ​#EmergentProperties:​ ​The​ ​cytoskeletal​ ​elements​ ​have​ ​their​ ​intended​ ​function​ ​but​ ​upon​ ​interaction​ ​with​ ​a​ ​novel​ ​molecule,
they​ ​produce​ ​an​ ​unexpected​ ​outcome​ ​that​ ​would​ ​otherwise​ ​not​ ​be​ ​observed.
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Recall​ ​that​ ​Cheng​ ​&​ ​Aksoy​ ​(1999)​ ​observed​ ​that​ ​the​ ​midgut​ ​tissues​ ​contained​ ​the​ ​highest​ ​density​ ​of​ ​S.

glossinidius​ ​across​ ​multiple​ ​tsetse​ ​fly​ ​species.​ ​The​ ​reduction​ ​of​ ​genome​ ​size​ ​has​ ​been​ ​characterised​ ​in​ ​bacterial

endosymbionts​ ​of​ ​insects,​ ​including​ ​S.​ ​glossinidius​.​ ​However,​ ​it​ ​was​ ​observed​ ​that​ ​the​ ​genes​ ​implicated​ ​in​ ​iron

acquisition​ ​systems​ ​are​ ​yet​ ​to​ ​be​ ​eliminated​ ​from​ ​the​ ​Sodalis​ ​genome.(Belda,​ ​Moya,​ ​Bentley​ ​&​ ​Silva,​ ​2010)

Multiple​ ​groups​ ​have​ ​attempted​ ​to​ ​investigate​ ​the​ ​action​ ​of​ ​iron-uptake​ ​systems​ ​in​ ​the​ ​midgut-residing​ ​S.

glossinidius​,​ ​given​ ​that​ ​iron-containing​ ​proteins​ ​would​ ​be​ ​available​ ​in​ ​its​ ​preferred​ ​locale​ ​after​ ​consumption​ ​of​ ​a

blood​ ​meal​ ​by​ ​its​ ​host​ ​tsetse​ ​fly.​ ​ ​(Runyen-Janecky,​ ​Brown,​ ​Ott,​ ​Tujuba​ ​&​ ​Rio,​ ​2010;​ ​Smith​ ​et​ ​al.​ ​2013;​ ​Hrusa

et​ ​al.​ ​2015)

Iron​ ​in​ ​the​ ​blood​ ​meal​ ​of​ ​tsetse​ ​flies​ ​is​ ​sequestered​ ​within​ ​molecules​ ​like​ ​heme​ ​and​ ​transferrin.​ ​(Zhou​ ​et​ ​al.,

2007)​ ​Most​ ​of​ ​the​ ​systems​ ​of​ ​iron​ ​acquisition​ ​in​ ​S.​ ​glossinidius​ ​involve​ ​the​ ​secretion​ ​of​ ​siderophores,​ ​e.g.

achromobactin,​ ​to​ ​capture​ ​iron-containing​ ​molecules.​ ​S.​ ​glossinidius​ ​is​ ​a​ ​Gram-negative​ ​bacterium​ ​with​ ​an

envelope​ ​consisting​ ​of​ ​an​ ​outer​ ​membrane,​ ​the​ ​periplasmic​ ​space​ ​and​ ​an​ ​inner​ ​membrane​ ​and​ ​because​ ​of​ ​its

complicated​ ​cellular​ ​architecture​ ​and​ ​the​ ​lack​ ​of​ ​energy​ ​sources​ ​within​ ​the​ ​outer​ ​membrane,​ ​the

iron-siderophore​ ​complexes​ ​are​ ​not​ ​able​ ​to​ ​simply​ ​diffuse​ ​across​ ​the​ ​outer​ ​membrane.​ ​Getting​ ​the​ ​iron​ ​into​ ​the

bacterial​ ​cell​ ​requires​ ​transporters​ ​and​ ​an​ ​energy​ ​source​ ​to​ ​drive​ ​transporter​ ​action.​ ​(Dale​ ​&​ ​Maudlin,​ ​1999;

Beveridge,​ ​1999)

In​ ​most​ ​gram-negative​ ​bacteria,​ ​this​ ​energy​ ​made​ ​available​ ​by​ ​the​ ​interactions​ ​between​ ​TonB​ ​and​ ​the

ExbB-ExbD​ ​complex​ ​in​ ​the​ ​inner​ ​membrane,​ ​which​ ​couples​ ​the​ ​proton​ ​motive​ ​force​ ​in​ ​the​ ​inner​ ​membrane​ ​to

the​ ​outer​ ​membrane.​ ​The​ ​TonB​ ​protein​ ​is​ ​able​ ​to​ ​span​ ​the​ ​periplasmic​ ​space​ ​such​ ​that​ ​its​ ​C-terminal​ ​domain

interacts​ ​with​ ​motifs​ ​at​ ​the​ ​N-terminal​ ​domain​ ​of​ ​the​ ​TonB-dependent​ ​outer​ ​membrane​ ​receptors​ ​while​ ​its

N-terminal​ ​domain​ ​connects​ ​it​ ​to​ ​the​ ​inner​ ​membrane,​ ​ultimately​ ​connecting​ ​the​ ​outer​ ​membrane​ ​transporters​ ​to

an​ ​energy​ ​source​ ​on​ ​the​ ​inner​ ​membrane​ ​surface.

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Experimental​ ​evidence​ ​gathered​ ​to​ ​date​ ​suggests​ ​an​ ​energy-transfer​ ​model​ ​involving​ ​interactions​ ​between

dimeric​ ​TonB​ ​and​ ​a​ ​complex​ ​formed​ ​by​ ​ExbB​ ​and​ ​ExbD.​ ​(Fig.​ ​2)​ ​While​ ​the​ ​exact​ ​mechanism​ ​remains​ ​to​ ​be

characterised,​ ​findings​ ​from​ ​Jordan​ ​et​ ​al.​ ​(2013)​ ​confirm​ ​that​ ​the​ ​ExbB–ExbD​ ​complex​ ​couples​ ​the​ ​ion

electrochemical​ ​gradient​ ​of​ ​the​ ​inner-membrane​ ​with​ ​a​ ​series​ ​of​ ​conformational​ ​changes​ ​in​ ​TonB.​ ​ ​In​ ​the​ ​first

step​ ​of​ ​the​ ​cycle,​ ​TonB​ ​is​ ​"charged"​ ​by​ ​the​ ​proton-motive​ ​force​ ​of​ ​the​ ​inner​ ​membrane,​ ​harnessed​ ​by​ ​the

ExbB–ExbD​ ​complex,​ ​changing​ ​its​ ​structural​ ​conformation.​ ​In​ ​the​ ​second​ ​step,​ ​the​ ​"energized"​ ​TonB​ ​spans

across​ ​the​ ​periplasmic​ ​space​ ​where​ ​upon​ ​interaction​ ​with​ ​an​ ​outer​ ​membrane​ ​receptor​ ​bearing​ ​an

iron-siderophore​ ​complex,​ ​TonB​ ​releases​ ​its​ ​stored​ ​energy,​ ​possibly​ ​as​ ​mechanical​ ​force​ ​due​ ​to​ ​conformational

changes​ ​into​ ​the​ ​discharged​ ​state.​ ​In​ ​the​ ​final​ ​step,​ ​TonB,​ ​in​ ​the​ ​discharged​ ​state,​ ​is​ ​reconciled​ ​with​ ​the

energy-harvesting​ ​ExbB–ExbD​ ​complex​ ​to​ ​repeat​ ​the​ ​cycle.​ ​(Ferguson​ ​&​ ​Deisenhofer,​ ​2002;​ ​Postle​ ​&​ ​Larsen,

2007)​ ​In​ ​correction​ ​of​ ​the​ ​proposed​ ​model,​ ​Jordan​ ​et​ ​al.​ ​(2013)​ ​found​ ​that​ ​TonB​ ​is​ ​actually​ ​restricted​ ​and

transfer​ ​energy​ ​to​ ​the​ ​transporters​ ​by​ ​rotation​ ​rather​ ​than​ ​migration​ ​across​ ​the​ ​periplasmic​ ​space.
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Fig.​ ​2:​​ ​Proposed​ ​model​ ​for​ ​energy​ ​transduction​ ​by​ ​TonB​ ​–​ ​TonB​ ​–​ ​Conformational​ ​changes​ ​in​ ​TonB​ ​enable​ ​storage​ ​and​ ​delivery​ ​of

energy​ ​harnessed​ ​from​ ​the​ ​ion​ ​electrochemical​ ​gradient​ ​of​ ​the​ ​inner-membrane​ ​by​ ​the​ ​interaction​ ​between​ ​dimeric​ ​TonB​ ​and​ ​the

ExbB–ExbD​ ​complex

All​ ​three​ ​studies​ ​investigating​ ​the​ ​function​ ​and​ ​regulation​ ​of​ ​various​ ​TonB-dependent​ ​iron​ ​uptake​ ​systems

observed​ ​that​ ​mutants​ ​with​ ​inactivated​ ​iron​ ​acquisition​ ​genes​ ​failed​ ​to​ ​proliferate,​ ​even​ ​in​ ​iron-rich

environments,​ ​confirming​ ​that​ ​iron​ ​is​ ​a​ ​limiting​ ​substrate​ ​in​ ​the​ ​survival​ ​of​ ​S.​ ​glossinidius​.​ ​They​ ​also​ ​found​ ​that

components​ ​of​ ​each​ ​of​ ​these​ ​systems​ ​are​ ​repressed​ ​by​ ​the​ ​iron-responsive​ ​transcriptional​ ​regulator​ ​Fur,​ ​in

iron-rich​ ​environments.​ ​Given​ ​the​ ​necessity​ ​of​ ​iron​ ​for​ ​bacterial​ ​function,​ ​the​ ​expression​ ​of​ ​genes​ ​involved​ ​in

iron​ ​uptake​ ​systems​ ​is​ ​tightly​ ​regulated.​ ​In​ ​E.​ ​coli,​ ​the​ ​Fur​ ​protein​ ​acts​ ​as​ ​a​ ​transcriptional​ ​repressor​ ​that​ ​is

allosterically​ ​inhibited​ ​by​ ​Fe​2+​.5​ ​Under​ ​iron-rich​ ​conditions,​ ​Fe​2+​binds​ ​to​ ​the​ ​Fur​ ​protein​ ​and​ ​the​ ​Fe-Fur​ ​complex

binds​ ​to​ ​regulatory​ ​elements​ ​located​ ​in​ ​the​ ​promoter​ ​region​ ​of​ ​the​ ​DNA​ ​sequences​ ​known​ ​as​ ​Fur​ ​boxes.​ ​The

binding​ ​of​ ​this​ ​complex​ ​prevents​ ​the​ ​transcription​ ​of​ ​these​ ​genes.​ ​Under​ ​iron-deficient​ ​conditions,​ ​when​ ​iron

levels​ ​are​ ​low,​ ​the​ ​Fe-Fur​ ​complex​ ​dissociates​ ​from​ ​the​ ​DNA​ ​and​ ​transcription​ ​is​ ​resumed.​ ​(Bagg​ ​&​ ​Neilands,

1987)​ ​They​ ​hypothesised​ ​similar​ ​action​ ​of​ ​a​ ​Fur​ ​protein​ ​in​ ​the​ ​iron​ ​acquisition​ ​systems​ ​of​ ​Sodalis​.​ ​Iron​ ​levels

were​ ​manipulated​ ​using​ ​the​ ​iron​ ​chelator,​ ​EDDA,​ ​which​ ​binds​ ​to​ ​the​ ​iron​ ​ions​ ​to​ ​prevent​ ​further​ ​reactions​ ​and

limit​ ​their​ ​bioavailability.​ ​Expression​ ​levels​ ​of​ ​the​ ​genes​ ​were​ ​measured​ ​and​ ​they​ ​observed​ ​higher​ ​expression

under​ ​low-iron​ ​conditions​ ​than​ ​iron-rich​ ​conditions.​ ​To​ ​confirm​ ​transcriptional​ ​regulation​ ​by​ ​Fur​,​ ​they​ ​cultured

the​ ​inactivated-Fur​ ​mutant​ ​ ​S.​ ​glossinidius​ ​line​ ​(​URSOD6)​ ​under​ ​both​ ​conditions​ ​and​ ​saw​ ​very​ ​high​ ​expression

of​ ​uptake​ ​genes,​ ​suggesting​ ​that​ ​transcription​ ​was​ ​no​ ​longer​ ​repressed,​ ​compared​ ​to​ ​wild-type​ ​ ​S.​ ​glossinidius.

While​ ​these​ ​studies​ ​showed​ ​how​ ​S.​ ​glossinidius​ ​are​ ​able​ ​to​ ​take​ ​advantage​ ​of​ ​their​ ​host's​ ​blood​ ​meal​ ​diet,

colonising​ ​the​ ​midgut​ ​epithelial​ ​cells,​ ​its​ ​exact​ ​effects​ ​on​ ​and​ ​functions​ ​within​ ​the​ ​tsetse​ ​fly​ ​have​ ​still​ ​not​ ​been

5
​ ​#SystemDynamics:​ ​The​ ​Fur​ ​transcriptional​ ​protein​ ​is​ ​very​ ​sensitive​ ​to​ ​the​ ​concentration​ ​of​ ​iron​ ​and​ ​acts​ ​accordingly​ ​to
regulate​ ​transcription.
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discovered.​ ​ ​Welburn​ ​and​ ​Maudlin​ ​(1991,​ ​1999)​ ​showed​ ​that​ ​Sodalis​ ​glossinidius​ ​might​ ​influence​ ​the

susceptibility​ ​of​ ​tsetse​ ​flies​ ​to​ ​trypanosome​ ​infection​ ​by​ ​manipulating​ ​the​ ​efficacy​ ​of​ ​the​ ​tsetse​ ​fly's​ ​immune

system.​ ​Trypanosomes,​ ​the​ ​ ​trypanosomiasis-causing​ ​parasites​ ​that​ ​are​ ​transferred​ ​to​ ​humans​ ​and​ ​other​ ​animals

by​ ​tsetse​ ​flies,​ ​are​ ​only​ ​able​ ​to​ ​infect​ ​the​ ​host​ ​fly's​ ​gut​ ​if​ ​trypanocidal​ ​lectin​ ​is​ ​avoided.​ ​Sodalis​ ​is​ ​thought​ ​to

produce​ ​a​ ​lectin-inhibitory​ ​sugar​ ​through​ ​another​ ​of​ ​its​ ​metabolic​ ​pathways​ ​so,​ ​if​ ​the​ ​concentration​ ​of​ ​the

inhibitory​ ​sugar​ ​is​ ​high​ ​such​ ​that​ ​it​ ​is​ ​sufficient​ ​to​ ​cause​ ​lectin​ ​trypanocidal​ ​inhibition,​ ​trypanosome​ ​infection

rates​ ​would​ ​be​ ​increased.​ ​(Welburn​ ​and​ ​Maudlin,​ ​1991,​ ​1999)​ ​Dale​ ​and​ ​Welburn​ ​(2001)​ ​reported​ ​that

eradication​ ​of​ ​Sodalis​ ​by​ ​antibiotic​ ​manipulation​ ​significantly​ ​reduced​ ​the​ ​susceptibility​ ​of​ ​tsetse​ ​fly​ ​host's​ ​to

trypanosome​ ​but​ ​also​ ​decreased​ ​the​ ​lifespan​ ​of​ ​the​ ​tsetse​ ​fly​ ​compared​ ​to​ ​the​ ​wild-type.​ ​They​ ​used​ ​a​ ​novel

antibiotic,​ ​streptozotocin,​ ​which​ ​was​ ​able​ ​to​ ​selectively​ ​eliminate​ ​Sodalis​ ​from​ ​tsetse​ ​fly​ ​without​ ​impacting

Wigglesworthia​ ​populations.​ ​The​ ​mechanism​ ​by​ ​which​ ​Sodalis​ ​removal​ ​reduced​ ​host​ ​lifespan​ ​remains

unknown.
THE​ ​TSETSE​ ​FLY​ ​AND​ ​ITS​ ​SECONDARY​ ​ENDOSYMBIONT​ ​SODALIS​ ​GLOSSINIDIUS
10

References
Alam,​ ​U.,​ ​Medlock,​ ​J.,​ ​Brelsfoard,​ ​C.,​ ​Pais,​ ​R.,​ ​Lohs,​ ​C.,​ ​&​ ​Balmand,​ ​S.​ ​et​ ​al.​ ​(2011).​ ​Wolbachia​ ​Symbiont​ ​Infections​ ​Induce
Strong​ ​Cytoplasmic​ ​Incompatibility​ ​in​ ​the​ ​Tsetse​ ​Fly​ ​Glossina​ ​morsitans.​ ​Plos​ ​Pathogens​,​ ​7​(12),​ ​e1002415.
http://dx.doi.org/10.1371/journal.ppat.1002415

Alberts​ ​B,​ ​Johnson​ ​A,​ ​Lewis​ ​J,​ ​et​ ​al.​ ​Molecular​ ​Biology​ ​of​ ​the​ ​Cell.​ ​4th​ ​edition.​ ​New​ ​York:​ ​Garland​ ​Science;​ ​2002.​ ​Cell
Biology​ ​of​ ​Infection.​ ​Available​ ​from:​ ​https://www.ncbi.nlm.nih.gov/books/NBK26833/

Belda,​ ​E.,​ ​Moya,​ ​A.,​ ​Bentley,​ ​S.,​ ​&​ ​Silva,​ ​F.​ ​(2010).​ ​Mobile​ ​genetic​ ​element​ ​proliferation​ ​and​ ​gene​ ​inactivation​ ​impact​ ​over​ ​the
genome​ ​structure​ ​and​ ​metabolic​ ​capabilities​ ​of​ ​Sodalis​ ​glossinidius,​ ​the​ ​secondary​ ​endosymbiont​ ​of​ ​tsetse​ ​flies.​ ​BMC
Genomics​,​ ​11​(1),​ ​449.​ ​http://dx.doi.org/10.1186/1471-2164-11-449

Beveridge,​ ​T.​ ​(1999).​ ​Structures​ ​of​ ​Gram-Negative​ ​Cell​ ​Walls​ ​and​ ​Their​ ​Derived​ ​Membrane​ ​Vesicles.​ ​Journal​ ​Of​ ​Bacteriology​,
181​(16),​ ​4725–4733.​ ​Retrieved​ ​from​ ​https://www.ncbi.nlm.nih.gov/pmc/articles/PMC93954/

Cheng,​ ​Q.,​ ​&​ ​Aksoy,​ ​S.​ ​(1999).​ ​Tissue​ ​tropism,​ ​transmission​ ​and​ ​expression​ ​of​ ​foreign​ ​genes​ ​in​ ​vivo​ ​in​ ​midgut​ ​symbionts​ ​of
tsetse​ ​flies.​ ​Insect​ ​Molecular​ ​Biology​,​ ​8​(1),​ ​125-132.​ ​http://dx.doi.org/10.1046/j.1365-2583.1999.810125.x

Dale,​ ​C.,​ ​&​ ​Maudlin,​ ​I.​ ​(1999).​ ​Sodalis​ ​gen.​ ​nov.​ ​and​ ​Sodalis​ ​glossinidius​ ​sp.​ ​nov.,​ ​a​ ​microaerophilic​ ​secondary​ ​endosymbiont
of​ ​the​ ​tsetse​ ​fly​ ​Glossina​ ​morsitans​ ​morsitans.​ ​International​ ​Journal​ ​Of​ ​Systematic​ ​Bacteriology​,​ ​49​(1),​ ​267-275.
http://dx.doi.org/10.1099/00207713-49-1-267

Dale,​ ​C.,​ ​Young,​ ​S.,​ ​Haydon,​ ​D.,​ ​&​ ​Welburn,​ ​S.​ ​(2001).​ ​The​ ​insect​ ​endosymbiont​ ​Sodalis​ ​glossinidius​ ​utilizes​ ​a​ ​type​ ​III
secretion​ ​system​ ​for​ ​cell​ ​invasion.​ ​Proceedings​ ​Of​ ​The​ ​National​ ​Academy​ ​Of​ ​Sciences​,​ ​98​(4),​ ​1883-1888.
http://dx.doi.org/10.1073/pnas.98.4.1883

Ferguson,​ ​A.,​ ​&​ ​Deisenhofer,​ ​J.​ ​(2002).​ ​TonB-dependent​ ​receptors—structural​ ​perspectives.​ ​Biochimica​ ​Et​ ​Biophysica​ ​Acta
(BBA)​ ​-​ ​Biomembranes​,​ ​1565​(2),​ ​318-332.​ ​http://dx.doi.org/10.1016/s0005-2736(02)00578-3

Hrusa,​ ​G.,​ ​Farmer,​ ​W.,​ ​Weiss,​ ​B.,​ ​Applebaum,​ ​T.,​ ​Roma,​ ​J.,​ ​&​ ​Szeto,​ ​L.​ ​et​ ​al.​ ​(2015).​ ​TonB-Dependent​ ​Heme​ ​Iron​ ​Acquisition
in​ ​the​ ​Tsetse​ ​Fly​ ​Symbiont​ ​Sodalis​ ​glossinidius.​ ​Applied​ ​And​ ​Environmental​ ​Microbiology​,​ ​81​(8),​ ​2900-2909.
http://dx.doi.org/10.1128/aem.04166-14

Jordan,​ ​L.,​ ​Zhou,​ ​Y.,​ ​Smallwood,​ ​C.,​ ​Lill,​ ​Y.,​ ​Ritchie,​ ​K.,​ ​&​ ​Yip,​ ​W.​ ​et​ ​al.​ ​(2013).​ ​Energy-dependent​ ​motion​ ​of​ ​TonB​ ​in​ ​the
Gram-negative​ ​bacterial​ ​inner​ ​membrane.​ ​Proceedings​ ​Of​ ​The​ ​National​ ​Academy​ ​Of​ ​Sciences​,​ ​110​(28),​ ​11553-11558.
http://dx.doi.org/10.1073/pnas.1304243110

Kobayashi,​ ​D.,​ ​&​ ​Crouch,​ ​J.​ ​(2009).​ ​Bacterial/Fungal​ ​Interactions:​ ​From​ ​Pathogens​ ​to​ ​Mutualistic​ ​Endosymbionts.​ ​Annual
Review​ ​Of​ ​Phytopathology​,​ ​47​(1),​ ​63-82.​ ​http://dx.doi.org/10.1146/annurev-phyto-080508-081729

McFall-Ngai,​ ​M.,​ ​Hadfield,​ ​M.,​ ​Bosch,​ ​T.,​ ​Carey,​ ​H.,​ ​Domazet-Lošo,​ ​T.,​ ​&​ ​Douglas,​ ​A.​ ​et​ ​al.​ ​(2013).​ ​Animals​ ​in​ ​a​ ​bacterial
world,​ ​a​ ​new​ ​imperative​ ​for​ ​the​ ​life​ ​sciences.​ ​Proceedings​ ​Of​ ​The​ ​National​ ​Academy​ ​Of​ ​Sciences​,​ ​110​(9),​ ​3229-3236.
http://dx.doi.org/10.1073/pnas.1218525110

Miller,​ ​M.,​ ​&​ ​Bassler,​ ​B.​ ​(2001).​ ​Quorum​ ​Sensing​ ​in​ ​Bacteria.​ ​Annual​ ​Review​ ​Of​ ​Microbiology​,​ ​55​(1),​ ​165-199.
http://dx.doi.org/10.1146/annurev.micro.55.1.165

Pais,​ ​R.,​ ​Lohs,​ ​C.,​ ​Wu,​ ​Y.,​ ​Wang,​ ​J.,​ ​&​ ​Aksoy,​ ​S.​ ​(2008).​ ​The​ ​Obligate​ ​Mutualist​ ​Wigglesworthia​ ​glossinidia​ ​Influences
Reproduction,​ ​Digestion,​ ​and​ ​Immunity​ ​Processes​ ​of​ ​Its​ ​Host,​ ​the​ ​Tsetse​ ​Fly.​ ​Applied​ ​And​ ​Environmental​ ​Microbiology​,
74​(19),​ ​5965-5974.​ ​http://dx.doi.org/10.1128/aem.00741-08

Postle,​ ​K.,​ ​&​ ​Larsen,​ ​R.​ ​(2007).​ ​TonB-dependent​ ​energy​ ​transduction​ ​between​ ​outer​ ​and​ ​cytoplasmic​ ​membranes.​ ​Biometals​,
20​(3-4),​ ​453-465.​ ​http://dx.doi.org/10.1007/s10534-006-9071-6

Roberts,​ ​M.,​ ​&​ ​Pell,​ ​P.​ ​(1976).​ ​Micro-organisms​ ​in​ ​the​ ​midgut​ ​of​ ​tsetse​ ​fly​ ​larvae.​ ​Microbios​,​ ​17​(70),​ ​213-220.
THE​ ​TSETSE​ ​FLY​ ​AND​ ​ITS​ ​SECONDARY​ ​ENDOSYMBIONT​ ​SODALIS​ ​GLOSSINIDIUS
11
Runyen-Janecky,​ ​L.,​ ​Brown,​ ​A.,​ ​Ott,​ ​B.,​ ​Tujuba,​ ​H.,​ ​&​ ​Rio,​ ​R.​ ​(2010).​ ​Regulation​ ​of​ ​High-Affinity​ ​Iron​ ​Acquisition
Homologues​ ​in​ ​the​ ​Tsetse​ ​Fly​ ​Symbiont​ ​Sodalis​ ​glossinidius.​ ​Journal​ ​Of​ ​Bacteriology​,​ ​192​(14),​ ​3780-3787.
http://dx.doi.org/10.1128/jb.00161-10

Shaw,​ ​M.,​ ​&​ ​Moloo,​ ​S.​ ​(1991).​ ​Comparative​ ​study​ ​on​ ​Rickettsia-like​ ​organisms​ ​in​ ​the​ ​midgut​ ​epithelial​ ​cells​ ​of​ ​different
Glossina​ ​species.​ ​Parasitology​,​ ​102​(02),​ ​193.​ ​http://dx.doi.org/10.1017/s003118200006248x

White​ ​J.A.,​ ​Giorgini​ ​M.,​ ​Strand​ ​M.R.,​ ​Pennacchio​ ​F.​ ​(2013)​ ​Arthropod​ ​Endosymbiosis​ ​and​ ​Evolution.​ ​In:​ ​Minelli​ ​A.,
Boxshall​ ​G.,​ ​Fusco​ ​G.​ ​(eds)​ ​Arthropod​ ​Biology​ ​and​ ​Evolution.​ ​Springer,​ ​Berlin,​ ​Heidelberg

Zhou,​ ​G.,​ ​Kohlhepp,​ ​P.,​ ​Geiser,​ ​D.,​ ​Frasquillo,​ ​M.,​ ​Vazquez-Moreno,​ ​L.,​ ​&​ ​Winzerling,​ ​J.​ ​(2007).​ ​Fate​ ​of​ ​blood​ ​meal​ ​iron​ ​in
mosquitoes.​ ​Journal​ ​Of​ ​Insect​ ​Physiology​,​ ​53​(11),​ ​1169-1178.​ ​http://dx.doi.org/10.1016/j.jinsphys.2007.06.009