Cell, Vol. 119, 329–341, October 29, 2004, Copyright 2004 by Cell Press
A Role for Insect Galectins in Parasite Survival
phages of mammalian hosts and as extracellular pro-
mastigotes within the digestive tract of competent
insect vectors (Figure 1A). Amastigotes ingested by the
sand fly during blood feeding transform to dividing pro-
(Figure 1A). The bound promastigotes undergo numer-
ous divisions before differentiating into free-swimming
infective metacyclics (Kamhawi, 2002; Sacks, 2001). At-
tachment of promastigotes to the midgut lining of the
sand fly is crucial to the successful completion of their
life cycle in the fly, serving to prevent excretion of devel-
oping parasites along with the bloodmeal remnants.
Lipophosphoglycan (LPG), the major glycoconjugate
on the surface of Leishmania promastigotes, has been
implicated as the ligand-mediating midgut attachment
dylinositol lipid anchor (Figure 1B). The phosphoglycan
moieties of all LPG studied to date share a common
backbone consisting of repeating disaccharide units of
PO4-6Gal(?1-4)Man?1, where the C-3 position of the
Gal residue can either be unsubstituted (L. donovani,
Sudan), partially substituted with glucose side chains
(Indian L. donovani, L. mexicana, and L. chagasi), or
completely substituted with side chain sugars that ter-
minate primarily in galactose (L. major) or glucose and
arabinose (L. tropica) (McConville et al., 1995; Thomas
et al., 1992; Turco and Descoteaux, 1992). Figure 1B
shows the polymorphic nature of LPG in representative
parasite species. It is postulated that the composition,
length, and extent of sugar residues branching off the
disaccharide backbone repeats have developed in re-
sponse to a selection pressure applied by the nature and
diversity of sand fly midgut receptors. The involvement
of LPG side chain sugars in the species-specific attach-
ment of Leishmania parasites to their vectors has been
most clearly demonstrated for L. major and P. papatasi
(Pimenta et al., 1994). Oligosaccharide fragments corre-
spondingtotheterminalsidechainGal(?1-4)linked to the
galactosyl residue of the PO4-6Gal(?1-4)Man backbone
unit, and the tetrasaccharide formed by side chain substi-
tution of the backbone sequence with Gal(?1-3)Gal(?1-3),
inhibited binding of procyclic L. major promastigotes to
P. papatasi midguts in vitro (Pimenta et al., 1992). In
addition, L. major mutants deficient in ?1-3 galactosyl-
transferase activity and whose LPG lack galactose side
chains lost their ability to attach to the midgut of
P. papatasi (Butcher et al., 1996).
The nature of the LPG receptor in sand flies has been
of obvious interest but remains largely undefined. Sev-
eral publications have reported the presence of lectin-
like molecules within crude midgut lysates that are able
to agglutinate Leishmania parasites (Svobodova et al.,
1996; Volf et al., 1998, 2002; Wallbanks et al., 1986).
Dillon and Lane (1999) identified a microvillar protein
from the midgut of P. papatasi that binds the LPG of
L. major, though its final identity was not confirmed.
Shaden Kamhawi,1Marcelo Ramalho-Ortigao,1,2
Van M. Pham,2Sanjeev Kumar,3Phillip G. Lawyer,1
Salvatore J. Turco,4Carolina Barillas-Mury,3
David L. Sacks,1,5and Jesus G. Valenzuela2,5,*
1Laboratory of Parasitic Diseases
2Vector Molecular Biology Unit
Laboratory of Malaria and Vector Research
3Mosquito Immunity and Vector Competence Unit
Laboratory of Malaria and Vector Research
National Institutes of Allergy and Infectious
National Institutes of Health
Bethesda, Maryland 20892
4Department of Molecular and Cellular
University of Kentucky Medical Center
Lexington, Kentucky 40502
Insect galectins are associated with embryonic devel-
opment or immunity against pathogens. Here, we show
that they can be exploited by parasites for survival in
their insect hosts. PpGalec, a tandem repeat galectin
expressed in the midgut of the sand fly Phlebotomus
papatasi, is used by Leishmania major as a receptor
for mediating specific binding to the insect midgut, an
event crucial for parasite survival, and accounts for
species-specific vector competence. PpGalec is thus
identified as a keymolecule controlling vector compe-
tence for the most widely distributed form of cutane-
ous leishmaniasis in the Old World. In addition, these
studies demonstrate the feasibility of using midgut
receptors for parasite ligands as target antigens for
Leishmania produce a spectrum of diseases in humans,
including cutaneous, mucocutaneous, and visceral
forms determined in large part by the Leishmania spe-
ciestransmitted. Cutaneousleishmaniasisdueto L.ma-
jor is the most common leishmanial disease in the Old
World, widelydistributed from WestAfrica toSouth Asia
and particularly in the Middle East. The distribution of
of Leishmania and the diseases they produce are deter-
mined by the availability of competent vectors. Phlebot-
omine vectors of leishmaniases are also diverse and, in
the case of some sand fly species, will only permit the
complete development of the species of Leishmania
they transmit in nature. Thus, P. papatasi, the natural
vector of L. major, is refractory to the development of
other Leishmania species. Leishmania undergo a dige-
netic life cycle as intracellular amastigotes in macro-
5These authors contributed equally to this work.
Figure 1. Leishmania Life Cycle and Representative LPG Structures of Several Parasite Species
(A) Macrophages containing amastigotes are ingested by sand flies during a bloodmeal. Amastigotes transform to promastigotes, which
attach to the insect midgut epithelium. Bound promastigotes multiply and differentiate into free-swimming infective metacyclics available
(B) Polymorphic structures of LPGs from Old World Leishmania species. Oligosaccharide core and lipid anchor domains are conserved
between species. Developmental modification of LPG expressed by L. major metacyclic promastigoes is also shown.
its function as the receptor of L. major parasites. Galec-
tins, a ?-galactoside binding family of lectins, have
gained wide recognition as multifunctional molecules
important in development, homeostasis, and immune
regulation (Hughes, 2001; Pace and Baum, 2004; Rabi-
sen, 2004).Recently, a rolefor galectins inthe establish-
ment of parasitic diseases in mammalian hosts was
was shown to bind to galectin-3 and to galectin-9, pro-
moting parasite interaction with its macrophage host
cell (Pelletier et al., 2003; Pelletier and Sato, 2002). Try-
itating its mobility through the extracellular matrix
(Moody et al., 2000). In insects, the number, nature, and
function of galectins are less known (Pace and Baum,
2004).To ourknowledge,onlyone tandemrepeatgalec-
tin, involved in embryogenesis and host defense, has
been fully characterized from Drosophila melanogaster
(Pace et al., 2002). Other reports describe the presence
of galactose-specific lectins that function in host de-
fense (Dimopoulos et al., 1998; Mello et al., 1999), with
one study suggesting a role for galactose-specific lec-
tins in the attachment of T. rangeli to the salivary glands
of its vector, Rhodnius prolixus (Basseri et al., 2002).
The present study, on the interaction of the protozoan
parasite L. major with its sand fly vector P. papatasi,
provides evidence for the role of insect galectins in the
establishment of parasites within disease vectors.
Identification of PpGalec as a Tandem
A nonamplified cDNA library from the midgut of female
P. papatasi sand flies was plated, and 700 plaques were
were clustered using BlastN with a cutoff of 10E?60ob-
taining 380 unique clusters of related sequences. All se-
quences within each cluster were compared with the
nonredundant (NR) protein database using the BlastX
program (Altschul et al., 1997). Sequences were analyzed
for signal secretory peptides and for transmembrane
domains. A cDNA with high similarities to a galactose
Bank accession number AY538600). This cDNA was rel-
atively abundant in the midgut library of P. papatasi,
representing cluster 19 (with four identical cDNA) of 380
clusters (total of 672 cDNA); only the first 24 clusters
had more than three cDNA per cluster. PpGalec cDNA
encodes a 311 amino acid protein with a predicted mo-
lecular weight of 35.4 kDa. This protein lacks a signal
PpGalec Mediates P. papatasi-L. major Binding
Figure 2. The Identification of PpGalec as a Tandem Repeat Galectin
(A) Schematic representation of the protein, showing the position of the two carbohydrate recognition domains (CRD) and the linker region.
(B) ClustalW alignment of tandem repeat galectins of Phlebotomus papatasi (Pp) (AY538600, present study), Rattus norvegicus galectin-4
(Rn4) (NP037107), Homo sapiens galectin-9 (Hs9) (NP033665), and Drosophila melanogaster (DmD) (AF338142). Identical and similar amino
acids are shaded in black and gray, respectively. Residues involved in ?-galactoside binding are marked with an asterisk.
(C) A phylogenetic tree comparing full-length PpGalec (AY538600) from P. papatasi (Pp) to selected tandem repeat galectins of different
vertebrate and invertebrate species. Hs4, H. sapiens galectin-4 (AAH03661); Hs8, H. sapiens galectin-8 (NP_006490.1); Hs9, H. sapiens
galectin-9 (NP_033665.1); Ss4, Susscrofa galectin-4 (Q29058); Oc4, Oryctolagus cuniculus galectin-4 (AF091738);Rn4, R. norvegicus galectin-4
(NP_037107); Rn9, R. norvegicus galectin-9 (NP_037109.1); Mm4, Mus musculus galectin-4 (AAH21632); Mm8, M. musculus galectin-8
(AAH40243); Mm9, M. musculus galectin-9 (NP_034838.1); Hc, Haemonchus contortus (AAD11972); Bm, Brugia malayi (AF237486); DmA,
D. melanogaster (AAL28440); DmB, D. melanogaster (NP_608553); DmC, D. melanogaster (CG11374); DmD, D. melanogaster (AF338142); AgA,
Anopheles gambiae(XP_310776); AgB,A. gambiae (XP_319586);Aa, Aedes aegypti(EST-7403, TIGRdatabase). Scale represents0.1 nucleotide
substitutions per site.
peptide and a transmembrane domain. A BLAST analy-
sis of PpGalec amino acid sequence defined it as a
tandem repeat galectin with two carbohydrate recogni-
tion domains (CRD) with affinity for ?-galactose-bearing
glycoconjugates, in which the CRDs occur within one
molecule, separated by a linker region (Figure 2A). Both
CRDs of PpGalec share consensus sequences with tan-
and human galectin-9 (Figure 2B). Most amino acids
considered to be involved in galactose binding (Sujatha
and Balaji, 2004) are highly conserved in PpGalec, in-
cluding H48, R52, V60, N62, and E73for CRD1; and H202, N204,
amino acid substitutions in galactose binding sites on
the PpGalec protein. These divergences occurred on
E227) on the CRD2 domain (Figure 2B). Analysis of the
phylogenetic relationship between representative tan-
dem repeat galectins shows that PpGalec separates
into a distinct cluster, supported by a high bootstrap
value, together with four other putative nonannotated
insect tandem repeat galectins (Figure 2C).
(rPpGalec), strong agglutination was observed above
300 ng/?l of protein (data not shown). For flow cytomet-
ric analyses of binding, the subagglutination concentra-
tion of 300 ng/ul of rPpGalec was used. L. major V1
procyclics whose LPG bears typical side chains con-
geneously bound His-tagged rPpGalec (Figure 4A) with
a significant (p ? 0.05) 4-fold increased binding over
control (anti-His stained parasites not incubated with
rPpGalec) (Figure 4B). As an additional negative control,
L. major V1 did not bind a His-tagged recombinant 35
kDa salivary protein of Lu. longipalpis (data not shown).
Promastigotes of two other Leishmania species, L. don-
ovani “1S”, whose LPG is unsubstituted, and L. tropica
“KK27”, which has an extensively branched LPG with
side chains terminating mainly with glucose and arabi-
nose, showed no significant binding of rPpGalec (Figure
4B). Moreover, L. major NIH/SD, a West African geo-
side chains (Joshi et al., 1998), as well as “Spock,” a
mutant derived from L. major V1 and selected for ab-
sence of side chains (Butcher et al., 1996), both failed
to bind rPpGalec (Figure 4B). L. major “LV39,” which
bears LPG side chains consisting of elongated galactose
son et al., 2003), also did not bind PpGalec above back-
ground. Importantly, L. major V1 metacyclics, which ex-
pess twice the number of repeat units as procyclic LPG
and which replace many of their side chain terminal
sugars with arabinose (McConville et al., 1992; Sacks
et al., 1990), lost their capacity to bind rPpGalec (Figure
4B). When parasites were stained with the monoclonal
anti-LPG WIC79.3,which specificallyrecognizes thega-
lactose-containing sidechains ofL. majorLPG (Kelleher
et al., 1994), we observed a correlation between anti-
LPG staining of parasites and binding of rPpGalec (Fig-
Developmental Expression and Tissue
Specificity of PpGalec
PpGalec was expressed at low levels throughout the
upregulated in adult females (Figure 3A). Adult males
showed a low level of gene expression. In adult females,
remaining constant up to 72 hr postbloodmeal (Figure
3B). Moreover, its expression appears to be specific to
midgut tissues, as shown by its absence from the car-
cass (Figure 3B).
Restricted Distribution of PpGalec
in Phlebotomine Sand Flies
ent in P. papatasi and P. duboscqi, a sister species of
P. papatasi, both belonging to the subgenus Phleboto-
mus. It was absent from two other species of the genus
Phlebotomus: P. sergenti (subgenus Paraphlebotomus)
and P. argentipes (subgenus Euphlebotomus). It was
species belonging to the New World genus Lutzomyia
tasi cell line). It was absent from LL5 (a Lu. longipalpis
cell line) (Figure 3C) and genomic DNA preparations of
D. melanogaster and A. gambiae (data not shown). As a
with a Lu. longipalpis S6 ribosomal protein cDNA probe
tested (Figure 3D).
A mouse antiserum raised against purified recombi-
nant PpGalec was found to specifically recognize a na-
tive protein of approximately 35 kDa in P. papatasi and
P. perniciosus, or Lu. longipalpis midguts (Figure 3E).
Distribution and Surface Expression of Native
PpGalec in the Midgut of P. papatasi
Unfed P. papatasi midguts were opened and incubated
cific antibody. Compared to controls, anti-PpGalec
brightly stained the entire insect midgut (Figure 5A).
Anti-PpGalec did not stain Lu. longipalpis midguts (Fig-
ure 5B). Examination of luminal surface sections by
confocal microscopy revealed that the expression of
PpGalec varies among midgut cells (Figure 5C). To in-
vestigate the distribution of PpGalec across the midgut
epithelium, multiple confocal sections were obtained
and merged into four groups representing the luminal,
middle, basal, and muscle planes (Figure 5C). P. papa-
tasi midgut cells expressed PpGalec on their luminal
surface (Figure 5C, lumen). These cells have regular
geometrical shapes (usually pentagons or hexagonal),
and their nuclei are centered and roughly equidistant
from eachother (Figure 5C,middle). A fineactin network
can be observed in the basal plane (Figure 5C, basal)
immediately above the muscle layer. In a lateral view, it
is clear that PpGalec is expressed in the cytoplasm
but accumulates along the luminal surface of the cell
In Vitro Binding of Leishmania Species
to Recombinant PpGalec
tigotes were incubated with recombinant PpGalec
PpGalec Mediates P. papatasi-L. major Binding
Figure 3. Developmental Expression and Tissue Specificity of PpGalec in Phlebotomus papatasi
(A) RT-PCR of larval stages (L2–L4), early pupae (EP), late pupae (LP), adult females (F), and adult males (M). RNA was isolated from 20
specimens of each stage. One of two representative experiments.
(B) Midguts of 5–6 females were dissected at 0, 14, 30, 48, and 72 hr post-blood feeding. RNA was isolated from both the midgut and carcasses
for RT-PCR. G, gut; C, carcass. One of two representative experiments.
(C) Dot blot of genomic DNA isolated from various sand fly species and probed with DIG-labeled PpGalec cDNA: P. papatasi Israeli strain
(PPIS), P. papatasi North Sinai strain (PPNS), P. duboscqi (PDKY), P. sergenti (PSSS), P. argentipes (PAIN), Lutzomyia longipalpis (LLJB),
Lu. Verrucarum (LVER). DNA of sand fly cell lines LL5 and PP9 were also tested (1, 1?g; 2, 2 ?g DNA). As controls, ribosomal protein S3
(PPS3), tubulin (Tub), actin and midgut chitinase (Chit) from P. papatasi, and Lu. longipalpis ribosomal protein S6 (LLS6) were included. One
of three representative experiments.
(D) Dot blot of genomic DNA in (C) stripped and reprobed with DIG-labeled control DNA (LLS6).
(E) Expression of PpGalec in various sand fly species: P. papatasi (1), P. duboscqi (2), P. sergenti (3), P. argentipes (4), P. perniciosus (5), and
Lu. longipalpis (6). Equivalent of one midgut was loaded per well. One of three representative experiments.
Ex Vivo and In Vivo Inhibition of L. major
Parasite Binding to P. papatasi Midgut
by Anti-PpGalec Antibody
Anti-PpGalec antibodies were used to test the role of
PpGalec in LPG and parasite binding to the midgut
ex vivo. Midguts that were preincubated with anti-
PpGalec followed by incubation with procyclic L. major
V1 PG (delipidated LPG) and detection with monoclonal
anti-LPG showed an almost complete inhibition of PG
binding (Figure 6A, panel 1). In contrast, midguts incu-
Figure 4. In Vitro Binding of Leishmania Spe-
cies to His-Tagged Recombinant PpGalec
(A) Mean fluorescence of L. major (V1) incu-
bated with FITC anti-His alone (black) or with
recombinant PpGalec (rPpGalec) followed by
FITC anti-His (gray).
(B) Mean fluorescence of various Leishmania
strains bound to His-tagged rPpGalec shown
as fold-over controls incubated with FITC
anti-His alone. Strains used were the follow-
ing: V1, L. major V1; LV39, L. major “LV39”;
NIH/SD, L. major “Seidman”; 1S, L. donovani
“1S”; KK27, L. tropica “KK27”; Spock, L. ma-
jor “Spock”; V1 met, L. major metacyclics.
On average, 50,000 cells were acquired for
each sample. Mean of four experiments ?
(C) Mean fluorescence of the parasites noted
above stained with monoclonal anti-LPG
WIC79.3, which specifically recognizes the
galactose-containing side chains of L. ma-
bated with preimmune serum stained brightly with anti-
LPG (Figure 6A, panel 2). Recognition of native PpGalec
in the midgut of P. papatasi by anti-PpGalec is shown
inpanel 3of Figure6A.These resultsindicate thatnative
PpGalec is available to LPG ligands in the lumen and
binding of L. major promastigotes. This was confirmed
by exvivobindingstudies usingtheparasites themselves,
a 72% inhibition in the number of bound parasites (p ?
0.0006) compared with midguts preincubated with pre-
immune serum (Figure 6B).
A series of studies were conducted on P. papatasi
fed on bloodmeals containing L. major amastigotes and
reconstituted with serum from PpGalec-immunized
mice. At day 3 postinfection, prior to excretion of the
digested bloodmeal, there was no significant difference
in the number of parasites in sand flies fed on anti-
PpGalec or preimmune serum (Figures 6C and 6D). At
day 6 postinfection, however, when the bloodmeal was
lost, there was an 86% decrease (p ? 0.001) in the
number of parasites retained in the midgut of sand flies
fed on anti-PpGalec compared with controls, and infec-
tions appeared to be completely lost in half of the anti-
PpGalec fed flies (Figure 6C). The inhibitory effect of
anti-PpGalec on parasite survival was clearly timed with
bloodmeal excretion, as indicated by the comparison
of parasite loads in day 5-infected flies that had passed
PpGalec Mediates P. papatasi-L. major Binding
Figure 5. Distribution and Surface Expression of PpGalec on Phlebotomus papatasi Midgut
(A) Midguts incubated with preimmune serum or with anti-PpGalec. One of three representative experiments.
(B) Midguts of P. papatasi and Lu. longipalpis incubated with anti-PpGalec. A Dapi staining of Lu. longipalpis midgut is shown. One of three
(C) Midguts were incubated with either preimmune or anti-PpGalec sera and detected with Cy-3-conjugated anti-mouse (red). Alexa 488-
conjugated phalloidin (green) and Dapi were used to stain actin and nuclei, respectively. Multiple confocal sections were obtained and merged
into four groups, representing the lumen, middle, basal, and muscle planes of the midgut epithelium. Scale bars, 5 ?m. Arrow indicates the
position of the horizontal section shown as a side view in the bottom panel.
(D) Side view of merged sections from the luminal surface (L) to the basal layer (B) of the midgut epithelium. Scale bar, 5 ?m.
or not yet passed their bloodmeal remnants (Figure 6D).
The massive loss of infection at this early stage pre-
rior midgut, such that only three of 27 of the flies (11%)
examined at day 14 harbored metacyclic promastigotes
capable of initiating infection in the vertebrate host. By
Figure 6. Effect of Blocking PpGalec on Parasite Binding and Survival
(A) Inhibition of Leishmania major procyclic PG binding by preincubation of midguts with anti-PpGalec. As controls, midguts were preincubated
with either anti-PpGalec or preimmune serum and detected with FITC-anti-mouse antibody. One of two representative experiments.
(B) Ex vivo binding of L. major parasites to the midgut of Phlebotomus papatasi following preincubation with either preimmune (?) or anti-
PpGalec (?) sera. One of three representative experiments.
(C) In vivo infection of P. papatasi with 3 ? 106L. major parasites/ml mixed with blood containing either preimmune (?) or anti-PpGalec (?)
sera. Parasite load was determined at day 3 and day 6 postinfection. One of two representative experiments.
(D) In vivo infection of P. papatasi with 3 ? 106L. major parasites/ml mixed with blood containing either preimmune (?) or anti-PpGalec (?)
sera. Parasite loads were determined at days 3, 5, and 14 postinfection. Data are pooled from two independent experiments.
PpGalec Mediates P. papatasi-L. major Binding
contrast, 18 of 33 (55%) sand flies fed on preimmune
serum developed metacyclics in numbers typical of ma-
ture, transmissible infections.
Binding of rPpGalec was restricted to L. major V1
bearing poly-Gal (?1-3) side chains on its LPG (Figures
4A and 4B), emulating the species-restricted vector
competence of P. papatasi to L. major infections. The
absence of binding of rPpGalec to L. major strains NIH/
SD and “Spock,” lacking poly-Gal (?1-3) side chains,
reinforces the role of LPG-mediated binding to PpGalec
in parasite survival, as these strains failed to maintain
infection in P. papatasi (Joshi et al., 1998; Butcher et
above background despite its polygalactosylation. This
may be explained by the fact that the side chains of
LV39 LPG are not typical of L. major but contain ?1-3-
linked galactose residues up to eight units in length,
some of which are arabinose capped (Dobson et al.,
2003). More critically, “LV39” promastigotes survive
poorly in P. papatasi compared with V1 promastigotes,
and a massive initial inoculum is required to establish
post-bloodmeal infection in these flies (Sacks et al.,
2000). It is possible that this structural variant arose
of sand fly selection, or else it reflects the structures of
the natural isolates, transmitted by P. papatasi bearing
a variant of PpGalec that can better accommodate their
particular polygalactose epitopes, or expressing a core-
ceptor for parasite attachment.
It is pertinent to note that metacyclic promastigotes
of L. major did not bind rPpGalec (Figure 4B). This is
entirely consistent with the fact that the downregulation
of polygalactose epitopes during metacyclogenesis re-
sults in the loss of LPG-mediated parasite binding to
the midgut. This structural modification is thought to be
crucial to the development of transmissible infections,
since free-swimming infective metacyclics are neces-
sary for successful transmission from the sand fly to
the mammalian host (Pimenta et al., 1992; Sacks and
A prerequisite to the function of PpGalec as a midgut
receptor of L. major is its abundance and surface ex-
distribution of PpGalec throughout the midgut of P. pa-
patasi. In addition, analysis of midgut sections using
confocal microscopy showed that PpGalec is present
on the luminal surface of epithelial midgut cells (Figures
5C and 5D). This is consistent with the cell surface local-
ization of other galectins, which, despite the absence
sical pathways to the cell surface, where they bind to
appropriately glycosylated surface molecules (Cooper,
2002; Hughes, 2001; Rabinovich et al., 2002). Dillon and
Lane (1999) reported the specific binding of L. major
LPG to a microvillar peptide in the gut of P. papatasi
receptor. Danielsen and van Deurs (1997) demonstrated
peptide, is externalized via nonclassical pathways, where
it directly associates and coimmunoprecipitates with
aminopeptidase N, a transmembrane brush border en-
zyme in the small intestine of the pig. We propose that
the digestive enzyme reported by Dillon and Lane (1999)
may have been coprecipitated with PpGalec, and the
latter was in fact responsible for the observed specific
binding to L. major LPG.
Here, we report on the characterization of a galectin
gene homolog from a cDNA library of the midgut of
P. papatasi and demonstrate the function of the protein,
named PpGalec, as a specific receptor for L. major pro-
cyclic LPG. The observed homology of PpGalec to ga-
lactose binding proteins, together with previous studies
indicating that poly-Gal(?1-3) side chains on the LPG of
L. major are responsible for specific binding to P. papa-
tasi midguts, suggested that PpGalec might be the mid-
gut receptor for L. major in this sand fly species. This
is supportedby a numberof lines ofevidence presented
in this report: (1) expression of PpGalec in P. papatasi
is strongly upregulated in adult females (Figure 3A) and
is restricted to midgut tissue (Figure 3B); (2) expres-
sion of PpGalec is restricted to P. papatasi and P. du-
boscqi (Figures 3C and 3E), sister species belonging to
the subgenus Phlebotomus, each of which transmits
L. major in nature; (3) the binding specificity of rPpGalec
is restricted to Leishmania promastigotes bearing poly-
is distributed throughout the abdominal and thoracic
midgut and localized on the luminal surface of midgut
cells (Figure 5); (5) antibodies against PpGalec inhibited
ex vivo midgut binding of L. major PG and parasites
(Figures 6A and 6B); and (6) PpGalec antibodies fed to
survival in the insect midgut (Figures 6C and 6D).
nonidentical carbohydrate recognition domains (CRD)
separated by a linker region (Figure 2A) (Bianchet et
al., 2000; Cooper, 2002). Galectins, a widely distributed
family of lectins reported from fungi to mammals, are
implicated in cell-cell and cell-matrix interactions,
immunity (Hughes, 2001; Pace and Baum, 2004; Rabi-
sen, 2004). They share evolutionarily conserved se-
quences in CRD that have a cation-independent affinity
to ?-galactosides (Cooper, 2002; Hughes, 2001; Rabi-
novich et al., 2002; Yang and Liu, 2003) and typically
bind to type I Gal?1,3GlcNAc or type II Gal?1,4GlcNAc
of PpGalec are homologs to CRDs of other galectins;
however, five amino acids involved in galactose bind-
ing (Sujatha and Balaji, 2004) exhibit some divergence
(Figure 2B), possibly a reflection of the binding specific-
ity of the molecule (Arata et al., 2001; Bianchet et al.,
2000). Phylogenetic analysis (Figure 2C) suggests that
PpGalec, together with putative nonannotated galectins
from D. melanogaster, Aedes aegypti, and An. gambiae,
may represent a novel family of galectins.
The abundant expression of PpGalec in adult female
midgut tissue (Figures 3A and 3B) fulfills basic require-
ments of a receptor for L. major. Moreover, among the
seven vector species examined, its expression is re-
stricted to the known sand fly vectors for L. major,
P. papatasi, and P. duboscqi (Figures 3C and 3E).
g for 1 hr. The pellet was washed several times over 2 days in TE50/
20/PI. The pellet was solubilized in 6 M guanidine-HCl, 100 mM
Tris-HCl (pH 8.0), and 2 mM EDTA; incubated for 1 hr at RT; and
centrifuged at 20,000 rpm for 1 hr at 4?C. DTT (10 mg/ml) was added
to the supernatant and incubated at RT for 2 hr. The solution was
added to a refolding buffer (0.1 M Tris-HCl [pH 9.0], 0.9 mM GSSG,
2 mM EDTA, 0.5 M NaCl, and 200 mM lactose, protease inhibitor
mix) and incubated for 48–66 hr at 4?C. The recombinant protein
was purified by reverse phase HPLC using a 40 min gradient of
10%–80% acetonitrile with 0.1% trifluoroacetic acid, at 2 ml/min
using a 1 ? 25 cm octadecyl silica column (Model 218TP510 from
Vydac). Eluent was collected at 1 min intervals. Fractions were spot-
ted on a PVDF membrane, and positive anti-His fractions were
pooled. An aliquot of the positive anti-His fractions was dried and
run in a SDS-PAGE 4%–20% to confirm the molecular weight and
in batches of 250 ?g in the presence of 40 ?g of bovine serum al-
Because anti-PpGalec recognized and could block
the native protein on the luminal surface of midgut cells,
the role of PpGalec in parasite attachment could be
directly tested. Preincubation of P. papatasi midguts
with anti-PpGalec resulted in a significant decrease in
both L. major PG and promastigote binding (Figures 6A
and 6B). This is strong evidence that PpGalec provides
an essential recognition site for parasite attachment. Its
potential as a transmission-blocking vaccine was con-
firmed in studies showing that flies fed on infective
bloodmeals reconstituted with sera from PpGalec-
immunized mice lost such a substantial number of para-
sites during bloodmeal excretion (Figure 6D) that they
failed to recover mature, transmissible infections.
Identifying and targeting midgut molecules essential
for parasite survival represents a novel strategy for de-
velopment of transmission-blocking vaccines against
Parasites were grown as previously described (Kamhawi et al.,
2000). Procyclic promastigotes were harvested from 1–2 day loga-
rithmic phase cultures. The following strains of Leishmania were
used in this study: the NIH Friedlin strain of L. major, clone V1 (NIH/
V1) (MHOM/IL/80/FN); L. donovani (MHOM/SD/00/1S-2D); the West
African NIH Seidman (NIH/SD) strain of L. major (MHOM/SN/74/
Seidman); L. tropica (MHOM/AF/88/KK27); and L. major “Spock”
mutant (Butcher et al., 1996). L. major (V1) metacyclics were purified
by negative selection with 50 ?g/ml peanut agglutinin from 5–6 day
Phlebotomus papatasi cDNA Library Construction
A midgut library of P. papatasi was constructed from ten adult
females using the Micro Fast Track mRNA isolation kit (Invitrogen)
tech) as described previously (Valenzuela et al., 2002a). Detailed
description of the bioinformatic treatment of the data can be found
elsewhere (Valenzuela et al., 2002b). Briefly, primer and vector se-
quences were removed from raw sequences, compared against the
GenBank nonredundant (NR) protein database using the standalone
BlastX program found in the executable package at (ftp://ftp.ncbi.
nlm.nih.gov/blast/executables/) (Altschul et al., 1997), and searched
against the Conserved Domains Database (CDD) (ftp://ftp.ncbi.nlm.
nih.gov/pub/mmdb/cdd/), which includes all Pfam (Bateman et al.,
2000) and SMART (Schultz et al., 1998, 2000) protein domains. Se-
quences were clustered using the BlastN program (Altschul et al.,
1990) as detailed before (Valenzuela et al., 2002b). Cluster designa-
tions were assigned in order, cluster 1 having the largest number
of representative sequences. Sequences were submitted to the Sig-
nalP server (http://www.cbs.dtu.dk/services/SignalP-2.0/) for verifi-
cation of secretory signal peptide and to the TMHMM Server v. 2.0
brane domains (Krogh et al. 2001).
Sand fly colonies were reared at Walter Reed Army Institute of
Medical Research and at The National Institutes of Health. The fol-
lowing sand flies species were used in this study: Phlebotomus
Egypt (PPNS); P. duboscqi from Kenya (PDKY); P. sergenti from
South Sinai, Egypt (PSSS); P. argentipes from India (PAIN); P. perni-
ciosus from Italy; Lutzomyia longipalpis from Brazil (LLJB); and Lu.
verrucarum from Peru (LVER). Anopheles gambiae were reared in
the Laboratory of Malaria and Vector Research at NIH. Drosophila
melanogaster flies were kindly provided by Dr. Denise Montell,
Johns Hopkins School of Medicine.
Sand Fly Cell Lines
Embryonic cell lines developed from eggs of P. papatasi (PP9) and
L. longipalpis (LL5) (Tesh and Modi, 1983) were grown at 26?C in
Mitsuhashi-Maramorosch medium (MM) supplemented with 20%
heat-inactivated fetal bovine serum, 100 U/ml of penicillin, 100 ?g/
ml streptomycin, 200 mM L-glutamine, and 2 mM myo-inositol.
Sequence Analysis of PpGalec
A BLAST search of PpGalec-predicted amino acid sequence was
carried out against several databases including the National Center
for Biotechnology Information (NCBI) databases, the Berkeley Dro-
sophila Genome Project, and the Sanger Center Anopheles genome
database. Sequence alignments and phylogenetic tree analysis
used the ClustalW package (Thompson et al., 1997). Phylogenetic
trees were constructed by the neighbor-joining method (Saitou and
Nei, 1987). Bootstrapping of phylogenetic trees, corrected for multi-
ple substitutions and excluding positions with gaps, was done with
the Clustal package for 1000 trials. Phylogenetic trees were format-
ted with TreeView (Page, 1996) using the ClustalW output.
RT-PCR was performed on total RNA extracted from pools of 20
L2, L3, and L4 larval stages, early pupae, late pupae, adult females
and males (less than 5 hr old) using RNA STAT-60 solution (Tel-Test
Inc.). For tissue specificity and response to blood feeding, total RNA
was extracted from carcasses and midguts of six adult females at
0, 14, 30, 48, and 72 hr post-bloodmeal using the RNeasy Mini Kit
(Qiagen). For RT-PCR, 0.5 ?g of total RNA and 0.6 ?M of PpGalec
forward and reverse primers were added to a ready-to-use RT-PCR
bead (Amersham Pharmacia Biotech). The reaction mixture was
incubated at 42?C for 30 min and 95?C for 5 min. PCR conditions
were 25 cycles of 94?C for 45 s, 50?C for 2 min, 72?C for 2 min,
followed by 10 min extension at 72?C. PCR products were visualized
in 1.2% ethidium bromide-stained agarose gels.
Expression of Recombinant PpGalec
PpGalec full-length cDNA was PCR amplified using the forward and
reverse primers 5?-ATGACTACCTCATTCTCTGGTCAA-3? and 5?-
CAGCGTGTGATATCAGATACGAGA-3?, respectively. The PCR prod-
uct was cloned into pCRT7NT TOPO expression vector (Invitrogen)
according to manufacturer’s instructions. rPpGalec was expressed
as a His-tagged protein using the RTS proteomaster system follow-
ing manufacturer’s instructions (Roche Laboratories).
Genomic DNA Dot Blot
Genomic DNA was isolated from PP9 and LL5 cell lines, from 2- to
5-day-old female sand flies of various species, and from D. melano-
gaster and A. gambiae using the Cells and Tissue DNA Isolation Kit
(Amersham Pharmacia Biotech). DNA was resuspended into 10 ?l
hydration buffer and sonicated. The DNA concentration was deter-
mined spectrophotometrically, and 1–2 ug of DNA was blotted onto
Purification of Recombinant PpGalec
Expression reaction (1 ml) was mixed with 1 ml of 50 mM Tris (pH
8.0), 20 mM EDTA, and Complete Protease Inhibitor Mix (Roche
Laboratories)(TE 50/20/PI).Thesample wascentrifugedat 20,000?
PpGalec Mediates P. papatasi-L. major Binding
a nylon membrane. A PCR product of PpGalec was used as positive
control. Negative controls used were cDNA of ribosomal protein 3
(PPS3), tubulin, actin, and chitinase from P. papatasi as well as
cDNA of ribosomal protein S6 from Lu. longipalpis (LLS6). DNA was
denatured in 1.5 M NaCl/0.5N NaOH, neutralized in 0.5 M Tris/1.5
M NaCl, and crosslinked to the blots. Hybridization was performed
overnight at 50?C with a digoxigenin-labeled PpGalec cDNA probe
and detected by chemiluminescence according to manufacturer’s
protocols (Roche Molecular Biochemicals). For control reactions,
the blot was stripped and hybridized to a digoxigenin-labeled LLS6
cDNA probe from Lu. longipalpis.
Samples were incubated with either anti-PpGalec or preimmune
serum (1:300) overnight at 4?C, followed by 4 hr at RT with Cy3-
anti-mouse-conjugated antibody (1:500) (Amersham). Actin was
stained by incubating the samples for 20 min at RT with 6.6 ?M
Alexa488-conjugated phalloidin in methanol (Molecular Probes, Eu-
gene, OR) diluted 1:40 in 1% BSA/PBS. Midguts were washed and
mounted in Vectashield (Vector Laboratories, Inc.) containing Dapi.
Finalimages wereobtainedusing theconfocalmicroscope DMIRE2
from Leica Microsystems, Inc. (Exton, PA).
Inhibition of L. major PG Binding by Anti-PpGalec
for 20 min at 4?C, washed twice in PBS, and incubated with anti-
PpGalec or preimmune (1:20) for 1 hr at RT. After washing with PBS,
the guts were incubated with 50 ?g/ml L. major phosphoglycan (PG)
(Orlandi and Turco, 1987; Pimenta et al., 1992) for 1 hr at RT. After
washing, the guts were stained with Alexa Flour 488 (BD Biosci-
ences)-conjugated anti-LPG WIC79.3 (1:100) for 40 min at RT. The
guts were washed another five times in PBS and mounted on slides
for fluorescence microscopy.
Mouse Anti-PpGalec Antibodies
Polyclonal anti-PpGalec antibodies were prepared by repeated im-
munization of five BALB/c mice with purified rPpGalec protein
(SpringValleyLaboratories Inc.).Preimmuneserawere usedascon-
The midguts of two 5-day-old female sand flies from each species
were dissected and placed in 30 ?l PBS in groups of two. One gut
equivalent of homogenates was separated on 4%–12% NuPAGE
Bis-Tris gels using MES SDS running buffer (Invitrogen). Proteins
were transferred onto a nitrocellulose membrane for 1 hr at 30V and
with polyclonal anti-PpGalec (1:200) for 2 hr at RT followed by incu-
bation with anti-mouse IgG-HRP (1:5000) (Santa Cruz Biotechnol-
ogy)for40 minatRT.Theblotswere detectedusingtheSuperSignal
West Pico Chemiluminescent Substrate (Pierce) and exposed to
BioMax ML film (Eastman-Kodak).
Inhibition of Parasite Binding Ex Vivo by Anti-PpGalec
P. papatasi midguts were opened and fixed as mentioned above
and incubated with either anti-PpGalec or preimmune sera (1:10)
for 1 hr at RT. The guts were washed three times with PBS and
incubated with 2 ? 106parasites for 1 hr at RT. Individual midguts
were washed four times in PBS and placed in 30 ?l of 1% BSA/
Inhibition of Parasite Binding In Vivo by Anti-PpGalec
P. papatasi females were membrane fed on a bloodmeal containing
3 ? 106amastigotes/ml. The blood consisted of red blood cells
reconstituted with anti-PpGalec or preimmune sera. At each time
and dissected in PBS. Individual midguts were placed in 30 ?l of
1% BSA/PBS and homogenized. The number of promastigotes per
midgut was counted using a hemocytometer.
Immunostaining and Flow Cytometry
Lyophilized His-tagged rPpGalec was resuspended in PBS to a
concentration of 300 ?g/ml. Round-bottom 96-well plates (Corning)
were blocked with 1% BSA, and 500,000 Leishmania promastigotes
of various species were added to each well in triplicate and centri-
fuged at 3500 rpm for 10 min. One set of wells was incubated with
50 ?l of PpGalec, the other two with PBS containing 0.1% bovine
serum albumin (PBS/BSA), overnight at 4?C. The plates were main-
tained at 4?C during two washes with PBS/BSA and until they were
PpGalec complexes and one of the two sets of wells incubated
with PBS/BSA were stained with anti-His (C-term)-FITC antibody
(Invitrogen) (1:500). The final set of wells was stained with Alexa
Flour 488 (BD Biosciences)-conjugated anti-LPG WIC79.3 (1:400).
Both antibodies were incubated with parasites for one hr/RT,
washed three times with PBS/BSA, and resuspended in 150 ?l PBS/
BSA for FACS analysis. On average, 50,000 cells were acquired
for each sample, and the data were collected and analyzed using
TheKruskal-Wallistest wasusedtoanalyzeinvitro bindingofLeish-
to compare binding and in vivo survival of parasites. Values were
considered significant at the 95% confidence interval.
Thanks to Dr. Jose ´ M.C. Ribeiro for bioinformatics analysis and
manuscript review, Drs. John Andersen and L. Fabiano Oliveira for
their critical comments, Amy Seitz and David Reynoso for technical
assistance, Jurak Kabat and Owen Schwartz for confocal micros-
copy support, Dr. Robert W. Gwadz for his encouragement, and
Nancy Shulman for editorial assistance. S.J.T. is supported by NIH
Ex Vivo Staining of Sand Fly Midguts with Anti-PpGalec
Two- to five-day-old female sand flies, maintained on 50% sucrose,
were dissected in PBS. For each fly, the crop, malpighian tubules,
and hindgut were removed, leaving only the midgut. The midgut
was opened along its dorsal surface to expose the inner side. The
opened midguts were placed in the concave well of a microscope
chamber slide in groups of eight to ten, fixed in 2% paraformalde-
hyde for 20 min at 4?C, washed twice in PBS, and blocked with 1%
BSA for 1 hr at RT. The guts were incubated with anti-PpGalec or
preimmune serum (1:20) for 1 hr at RT, washed four times in PBS,
and incubated with FITC goat anti-mouse IgG (BD Pharmingen)
onto slides using VectaShield with Dapi (Vector Laboratories) and
viewed under a fluorescent microscope.
Received: May 21, 2004
Revised: September 2, 2004
Accepted: September 10, 2004
Published: October 28, 2004
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The GenBank accession numbers for the PpGalec (Pp), Brugia ma-
layi (Bm), Drosophila melanogaster (DmA), Anopheles gambiae
A. gambiae (AgB) sequences reported in this paper
are AY538600, AF237486, AAL28440, XP_310776, and XP_319586,
respectively. The name given to the Aedes aegypti sequence re-
ported in this paper according to the TIGR database is EST-7403.