Molecula & Biochemical Pa asi ology 241 (2021) 111347
A ailable online 18 Decembe 2020
0166-6851/© 2020 The Au ho s. Published by Else ie B.V. This is an open access a icle unde he CC BY-NC-ND license
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Sho communica ion
Gliding mo ili y p o ein LIMP p omo es op imal mosqui o midgu a e sal
and in ec ion by Plasmodium be ghei
Saskia Ega e
a
,
1
, Jo ge M. San os
b
,
1
,
2
, Jessica Keh e
a
, Julia Sa le
a
, F ied ich F ischknech
a
,
Gunna R. Mai
a
,
b
,
c
,
*
a
Pa asi ology, Depa men o In ec ious Diseases, Heidelbe g Uni e si y Medical School, Heidelbe g, Ge many
b
Ins i u o de Medicina Molecula Jo˜
ao Lobo An unes, Faculdade de Medicina da Uni e sidade de Lisboa, Edi ício Egas Moniz, A . P o . Egas Moniz, Lisbon, Po ugal
c
Iowa S a e Uni e si y, Biomedical Sciences, Ames, IA, Uni ed S a es
ARTICLE INFO
Keywo ds:
Gliding mo ili y
LIMP
Ookine e
T ansmission
T ansla ional ep ession
DOZI
ABSTRACT
Subs a e-dependen gliding mo ili y is key o mala ia ansmission. I media es hos cell a e sal, in asion and
in ec ion by Plasmodium and ela ed apicomplexan pa asi es. The 110 amino acid-long cell su ace p o ein LIMP
is essen ial o P. be ghei spo ozoi es whe e i is equi ed o he in asion o he mosqui o’s sali a y glands and
he li e cells o he oden hos . He e we de ine an addi ional ole o LIMP du ing mosqui o in asion by he
ookine e. limp mRNA is p o ided as a ansla ionally ep essed mRNP (messenge ibonucleop o ein) by he
emale game ocy e and he p o ein ansla ed in he ookine e. Pa asi es deple ed o limp (Δlimp) de elop ooki-
ne es wi h appa en no mal mo phology and no de ec du ing in i o gliding mo ili y, and ye display a p o-
nounced educ ion in oocys numbe s; compa ed o wild ype 82 % mo e Δlimp ookine es emain wi hin he
mosqui o blood meal explaining he dec ease in oocys s. As in he spo ozoi e, LIMP exe s a p o ound ole on
ookine e in ec ion o he mosqui o.
Gliding mo ili y o mala ia pa asi es is equi ed o mosqui o and
mammalian hos in ec ion. Despi e hei di e en mo phologies, all
in asi e li e cycle s ages o he mala ia pa asi e – me ozoi e, ookine e
and spo ozoi e – ely on a conse ed gliding mo ili y machine y o a-
e se di e se hos cell ba ie s in o de o es ablish hos cell in ec ions
and con inue he li e cycle [1]. Many o he mo ili y ac o s a e sha ed
wi h ela ed apicomplexan pa asi es such as Toxoplasma gondii. The
pea -shaped mala ia me ozoi e in ades ed blood cells. The ookine e is a
banana-shaped li e cycle s age ha is o med om a e ilised emale
game e (zygo e) in he mosqui o midgu and equi ed o es ablish an
in ec ion in he mosqui o; in o de o do so, he ookine e c osses he
blood meal sac as well as he mosqui o pe i ophic ma ix and midgu
epi helium o ans o m in o a sessile oocys . Spo ozoi es a e inally
o med in a pe iod o 2 weeks and a e dis inc ly slende -shaped; hey
mo e signi ican ly as e han ookine es and mig a e om he hemocoel
h ough he sali a y gland epi helium in o he gland lumen as well as
h ough he skin and ac oss he blood essel endo helium o ul ima ely
in ade hepa ocy es. The 110 amino acid-long cell su ace p o ein LIMP
is exp essed in ookine es and spo ozoi es, bu no me ozoi es. In he
spo ozoi e, LIMP de ines he pa asi e’s abili y o popula e he
mosqui o’s sali a y glands and is essen ial o he es ablishmen o an
in ec ion o he oden li e . In he ookine e, a dele ion o he LIMP gene
p oduces a obus , 50 %- educ ion in oocys numbe s [2]. The cause o
his de ec is howe e unknown. He e we sough o add ess he ole o
LIMP a his c ucial bo leneck in he mala ia pa asi e’s li e cycle
ocusing on key aspec s o ookine e biology: de elopmen , mo ili y and
in ec i i y.
The o ma ion o he P. be ghei ookine e om he e ilized emale
game e (zygo e) depends on ma e nally p o ided gene p oduc s (mRNA)
ha a e p oduced in he emale game ocy e, bu a e only ansla ed a e
e ilisa ion. Hund eds o mRNAs – some 50 % o he de ec ed an-
sc ip ome – a e p o ided in ansla ionally silen mRNPs (messenge
ibonucleop o eins) [3]. When ansla ed in o p o ein hey a e ins u-
men al in many de elopmen al p ocesses o he ookine e and can e en
a ec he o ma ion o oocys -de i ed spo ozoi es [4–8]. E alua ing he
p o ein exp ession p o ile h ough C- e minal GFP- agging o LIMP in
* Co esponding au ho a : Iowa S a e Uni e si y, Biomedical Sciences, Ames, IA, Uni ed S a es
E-mail add ess: [email p o ec ed] (G.R. Mai ).
1
Au ho s con ibu ed equally.
2
P esen add ess: Depa men o Immunology and In ec ious Diseases, Ha a d T.H. Chan School o Public Heal h, Bos on, MA, Uni ed S a es.
Con en s lis s a ailable a ScienceDi ec
Molecula & Biochemical Pa asi ology
jou nal homepage: www.else ie .com/loca e/molbiopa a
h ps://doi.o g/10.1016/j.molbiopa a.2020.111347
Recei ed 29 June 2020; Recei ed in e ised o m 4 Decembe 2020; Accep ed 15 Decembe 2020
Molecula & Biochemical Pa asi ology 241 (2021) 111347
2
limp::g p mu an s by li e luo escence mic oscopy ound no ansla ion
in asexual s age pa asi es no game ocy es; limp::g p is ansla ed in he
ookine e s age and he p o ein is hen mainly isible nea o in he
c ys alloid, a ansien and pu a i e s o age o ganelle o he ookine e [9]
(Fig. 1A and p e iously shown in [2]). We obse ed no ansla ion o
limp in sexual p ecu so cells (game ocy es), despi e he ansc ip being
eadily de ec ed by RT-PCR in his li e cycle s age (see limp inpu lanes
labelled wi h ed a ows in Fig. 1B). Using pa asi e clones exp essing
GFP- agged ansla ional ep esso s DOZI (a homolog o yeas DHH1
DEAD-box RNA helicase) and CITH (a homolog o yeas ep esso o
ansla ion ini ia ion p o ein SCD6) [3,10] we iden i ied limp mRNA o
be associa ed wi h bo h p o eins by RNA-immunop ecipi a ion (RIP)
ollowed by RT-PCR de ec ion o he bound ansc ip . Monoclonal
an i-GFP an ibodies and a p o ein G sepha ose a ini y eagen we e
used o immunop ecipi a e DOZI::GFP and CITH::GFP and any bound
mRNA om en iched game ocy e popula ions (lanes IP-
α
GFP); con ols
included he use o an un ela ed an i-cmyc IgG (lanes IP-
α
cmyc) o
omission o IgG al oge he (lanes IP beads). Like p25 and p28 – bo h a e
hallma k ansla ionally ep essed ansc ip s encoding ookine e su ace
p o eins and hus impo an and p omising an imala ial ans-
mission-blocking accine a ge s [11–13] – limp mRNA is bound by
DOZI and CITH (Fig. 1B). As expec ed, mRNA encoding he
DNA/RNA-binding p o ein ALBA-3, which is ansla ed in he game o-
cy e, does no co-IP wi h DOZI o CITH. The RIP da a explain he lack o
LIMP exp ession in he game ocy e; limp mRNA is a bona ide ma e nally
p o ided mRNA ha is kep ansla ionally quiescen in a complex
con aining DOZI and CITH in game ocy es. Once ansla ed, LIMP a -
ec s in i o ansmission om he in ec ed expe imen al mouse hos o
Anopheles emale mosqui oes; limp gene dele ion mu an s expe ience a
50 % educ ion in oocys numbe s [2]. This dec ease could esul om
de ec s in ookine e de elopmen , mo ili y, adhesion o in asion, bu he
unde lying cause is no clea . In o de o es whe he hei mo ili y was
a ec ed, we pe o med in i o ookine e cul u es om
game ocy e-in ec ed blood. Knock-ou pa asi es p oduced appa en ly
no mal ookine es as e alua ed by di e en ial in e e ence con as (DIC)
mic oscopy (Fig. 1C); hey showed no de ec in gliding mo ili y; a d op
o pu i ied ookine es in Roswell Pa k Memo ial Ins i u e (RPMI) 1640
medium was placed on a glass slide and imaged using an in e ed Zeiss
Axio e 200 M wide ield mic oscope wi h an XBO75 xenon lamp, he
AxioVision 4.7.2 so wa e, a 63X objec i e and a CoolSNAP™ HQ2 high
esolu ion CCD came a (Pho ome ics); images we e cap u ed e e y 20 s
o 15 min; he mo ing ookine es we e inally analysed using he
Fig. 1. LIMP is ma e nally p o ided o he ookine e whe e i ensu es op imal mosqui o in ec ion. (A) Li e luo escence o limp:g p blood s age game ocy e
shows he absence o LIMP::GFP exp ession, while imaging o blood meal- e ie ed ookine es highligh s LIMP::GFP localisa ion o disc e e oci (*) a and nea he
c ys alloid. Scale ba =5
μ
m. (B) RT-PCR analyses o DOZI::GFP and CITH::GFP RNA immunop ecipi a ion (RIP) elua es demons a e ha limp co-p ecipi a es wi h
bo h ansla ional ep esso s. Inpu : o al game ocy e mRNA; IP-
α
GFP: IP wi h an i-GFP an ibody; IP-
α
cmyc: IP wi h an i-c-myc an ibody; IP beads: no an ibody used
o IP. (C) Rep esen a i e images o wild ype and Δlimp ookine es isola ed om mosqui o midgu s. Scale ba =5
μ
m. (D) Quan i ica ion o pa asi e mo ili ies o
wild ype and Δlimp ookine es. Mann-Whi ney es ; means wi h SEM a e indica ed. 3 independen ookine e cul u es.(E) Oocys numbe s om wild ype and Δlimp-
in ec ed mosqui oes. Mann-Whi ney es ; means wi h SEM a e indica ed. 2 independen memb ane eeding assays. Midgu s wi h no pa asi es we e emo ed om he
g aph. Mean p e alence o in ec ion was 74 % o bo h g oups (Chi-squa e es , p =0.8877). (F) Numbe o wild ype and Δlimp ookine es e ained in he mosqui o
blood meal bolus 24 h a e in ec ion. Ra io pai ed es is indica ed. 4 independen memb ane eeding assays. Lines connec da a poin s om he same epli-
ca e expe imen .
S. Ega e e al.
Molecula & Biochemical Pa asi ology 241 (2021) 111347
3
manual acking plug-in o ImageJ [14] e ealing a speed o jus below
5
μ
m×min
−1
o mu an and wild ype pa asi es (Fig. 1D). Knock-ou
ookine es appea ed o show a sligh inc ease in gliding speed. Howe -
e , his inc ease is only ma ginal (p =0.0460) and we belie e i does no
bea any biological ele ance. We nex es ed he abili y o in i o-cul-
u ed ookine es o in ec he mosqui o ec o in a con olled s anda d
memb ane eeding assay (SMFA). To his end, wild ype and mu an
ookine es we e i s pu i ied, hen mixed wi h 500
μ
l naï e mouse blood
and inally o e ed o emale Anopheles s ephensi mosqui oes o 30 min
in wo independen eeding expe imen s (ei he o e ing 1.6 o 6 million
ookine es a a concen a ion o 3200 o 12,000 ookine es ×
μ
l
−1
). On
day 10 pos -in ec ion, oocys numbe s we e quan i ied ollowing
me cu och ome s aining. The gene dele ion mu an ep oduced he 50
% educ ion in oocys numbe s obse ed in ou p e ious publica ion
using di ec mosqui o eeds on in ec ed mice [2] (Fig. 1E). P e alence o
in ec ion was as ollows: eplica e 1–58% wild ype s 68 % knock-ou
(Chi-squa e es , p =0.4272) – and eplica e 2–89% wild ype s 79 %
knock-ou (Chi-squa e es , p =0.2898). Mean p e alence o in ec ion
calcula ed by combining bo h eplica es was 74 % o bo h wild ype and
knock-ou pa asi es (Chi-squa e es , p =0.8877). By bypassing he in
i o di e en ia ion o ookine es, ou esul s show ha he lowe numbe
o oocys s p oduced by knock-ou pa asi es canno be a ibu ed o an in
i o de ec in zygo e- o-ookine e ans o ma ion. To cha ac e ize he
knock-ou ’s ailu e o p oduce high oocys numbe s in mo e de ail, we
inally quan i ied ookine es in he blood meal a e 24 h o ou inde-
penden SMFA. The e o e, midgu s om in ec ed mosqui oes we e
collec ed in RPMI and he blood meal isola ed; he numbe o e ained
ookine es was quan i ied ia haemocy ome e . We ound he educ ion
in oocys numbe s o co ela e wi h an accumula ion o ookine es in he
mosqui o blood meal bolus. On a e age, we obse ed an 82% inc ease in
e ained knock-ou ookine es when compa ed o he wild ype con ols
(Table 1 and Fig. 1F), indica ing a ole o LIMP in a achmen /-
a e sal/in asion o he mosqui o midgu by he ookine e. Gi en he
obse ed 50 % educ ion in knock-ou oocys numbe s, one could expec
o ind wice as many ookine es e ained in he blood meal bolus (i.e.,
200 % o wild ype numbe s, o a 100 % inc ease). Ins ead, we obse e,
on a e age, an 82 % inc ease om wild ype in he numbe o e ained
ookine es. This is sligh ly lowe han he p edic ed 100 % inc ease.
Howe e , i is concei able ha some s ochas ic e en s migh ha e
esul ed in an unde es ima ion o e ained ookine es (e.g., loss o ooki-
ne es du ing sample p ocessing o pa ial exc e ion o he blood bolus
p io o gu collec ion). The e o e, i is plausible o an icipa e ha , wi h
u he eplica ion o his expe imen , he a e age pe cen inc ease
would p og essi ely ge close o he heo e ical alue o 100 %.In
summa y, ou da a show ha LIMP p o ein is p oduced om ansla-
ionally ep essed, ma e nally p o ided mRNA in he ookine e whe e i
helps o op imize mala ia pa asi e in ec ion o he mosqui o; limp-de-
ple ed zygo es de elop in o mo ile ookine es wi h appa en no mal
mo phology, and ye display a de ec in escaping he mosqui o blood
meal and eaching he midgu basal lamina whe e hey can ans o m
in o he spo ozoi e-p oducing oocys s age and con inue he li e cycle.
The accumula ion o Δlimp mu an s in he midgu lumen is ep oducible
and s ong, bu no absolu e, allowing he es ablishmen o oocys s in a
labo a o y en i onmen whe e P. be ghei ypically can p oduce hund eds
o oocys s in an in ec ed mosqui o. In he ield on he o he hand,
absence o he conse ed P. alcipa um LIMP p o ein migh well cause a
comple e ansmission blockade o human mala ia pa asi es. The much
smalle numbe o P. alcipa um game ocy es p esen in mala ia-posi i e
indi iduals – 450 a he han 10,000 ound in he labo a o y o he
oden model P. be ghei – ansla es in o ield mosqui oes ypically
hos ing, on a e age, a ound 6 ookine es, and jus 2 oocys s pe in ec ed
mosqui o; in addi ion, only
1
/
3
o mosqui oes ac ually do become
in ec ed [15]. The game ocy emia numbe s unde labo a o y condi ions
can each 10 % o he o al pa asi e popula ion in he oden model; i is
hus ha we obse e la ge numbe s o oocys s p oduced in wild ype
condi ions. The absence o LIMP causes a 50 % educ ion in oocys
numbe s. In he con ex o he high baseline in ec ion his is no enough
o block he li e cycle; his occu s ul ima ely du ing sali a y gland and
li e cell in ec ion by he spo ozoi e [2]. The accumula ion o
mo phologically no mal ookine es in he mosqui o blood meal is also
documen ed o mu an s deple ed o he pe o in-like p o eins
PPLP3/MAOP, PPLP4 and PPLP5 [16–19], o he
cell- a e sal-p o ein- o -ookine es-and-spo ozoi es (CelTOS) [20,21];
he e, he block in P. be ghei ansmission is almos absolu e. Ookine e
a e sal o he mosqui o midgu epi helium is no comple ely unde -
s ood and i has no been p o en unequi ocally whe he he pa asi e
p edominan ly pene a es he cells o he epi helial issue o he midgu –
pe haps wi h he help o po e- o ming PPLPs and CelTOS – and/o
mo es be ween hese cells in o de o each he basal lamina o he dis al
side o he midgu whe e hey es ablish oocys s ha go on o p oduce
spo ozoi es [22]. The midgu in asion de ec o Δlimp pa asi es could
esul om a de ec in adhesion o he pe i ophic ma ix, he luminal
side o he midgu epi helium o a ailu e o a e se he epi helial cell
laye . These scena ios would be consis en wi h ou p e ious indings o
he spo ozoi e, whe e bo h adhesion and a e sal p ocesses ely on
LIMP [2]. Pe haps his dual ole o LIMP could be used o in e e e wi h
bo h mala ia pa asi e ansmission s ages: ookine e and spo ozoi e.
Decla a ions o Compe ing In e es
None.
Acknowledgemen s
We hank Mi iam Reinig (Uni e si y o Heidelbe g Medical School)
as well as Ana Pa ei a and Leono Pinho (iMM Jo˜
ao Lobo An unes) o
mosqui o ea ing. This s udy was suppo ed by Fundaç˜
ao pa a a Ciˆ
encia
e a Tecnologia g an s o GRM (PTDC/BIA-BCM/105610/2008 and
PTDC/SAU-MIC/122082/2010) and JMS (SFRH/BD/63849/2009); he
Na ional Ins i u es o Alle gy and In ec ious Diseases o GRM
(1R21AI139579-01A1); he Ho izon 2020 F amewo k P og amme
Ma ie Sklodowska-Cu ie g an ag eemen No 660211 o SE; as well as a
Human F on ie Science P og am G an (RGY/0071/2011) o FF. The
unde s had no ole in s udy design, da a collec ion and in e p e a ion,
o he decision o submi he wo k o publica ion. The au ho s ha e no
compe ing in e es s o decla e.
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Table 1
Da a summa y o ookine e e en ion expe imen s. Numbe o e ained ooki-
ne es is plo ed in Fig. 1F.
Expe imen
#
To al #
ookine es
used (x10
6
)
Ookine es/
μ
L
o blood a
eed (x10
3
)
# e ained
ookine es
% inc ease
om
wild ype
wild ype Δlimp
1 1.6 3.2 1163 2038 75
2 6.0 12.0 1300 2500 92
3 4.0 8.0 200 350 75
4 2.5 5.0 700 1300 86
a e age 3.525 7.05 841 1547 82
S. Ega e e al.
Molecula & Biochemical Pa asi ology 241 (2021) 111347
4
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