Identification of the most abundant secreted proteins from the salivary glands of the sand fly Lutzomyia longipalpis, vector of Leishmania chagasi
1 Vector Molecular Biology Unit, Laboratory of Malaria and Vector Research,
NIAID, National Institutes of Health, 12735 Twinbrook Parkway, Room 2E-22C,
Rockville, MD 20852, USA,
2 Structural Biology Unit, NIAID, Rockville, MD 20852, USA
3 Department of Entomology, Walter Reed Army Institute of Research,
Washington, DC 20307, USA
* Author for correspondence (e-mail: jvalenzuela{at}niaid.nih.gov)
Accepted 12 July 2004
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Summary |
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Key words: salivary gland transcript, salivary protein, New World sand fly, Lutzomyia longipalpis, saliva
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Introduction |
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Arthropod saliva modifies the physiology of the host at the site of the
bite, making it more permissive for pathogen invasion
(Titus and Ribeiro, 1988;
Ribeiro, 1989
;
Kamhawi, 2000
;
Nuttall et al., 2000
). In some
vector/parasite systems, immune response to arthropod feeding or arthropod
bites (probably to salivary proteins) precludes the establishment of the
pathogen in the vertebrate host (Bell et
al., 1979
; Jones and Nuttall,
1990
; Wikel et al.,
1997
; Nazario et al.,
1998
; Kamhawi et al.,
2000
). There are two hypotheses (not mutually exclusive) to
explain this protection. First, vertebrate immune response to insect salivary
proteins, particularly antibodies, may neutralize the effect of the salivary
component(s) responsible for pathogen establishment. There is evidence
supporting this hypothesis; Morris et al.
(2001
) demonstrated that
animals vaccinated with maxadilan, a potent vasodilator and immunomodulator
from Lu. longipalpis, raised antibodies against this salivary
protein, which resulted in animals protected against Le. major
infection. Second, vertebrate host immune response to salivary proteins
creates an inhospitable environment to the pathogen, which is either killed or
its future development negatively affected at the site of the bite. In this
case, the anti-salivary immune response may be mainly a cellular rather than
humoral response. Evidence to support this hypothesis comes from the work of
Kamhawi et al. (2000
), where
animals pre-exposed to Phlebotomus papatasi sand fly bites generated
a strong delayed-type hypersensitivity response at the site of the bite; this
response was related to protection against Le. major infection. Mice
vaccinated with a 15 kDa salivary protein (PpSP15) produced a strong
delayed-type hypersensitive response (DTH) and antibodies in black/6 mice,
resulting in protection against Le. major infection when parasites
were co-inoculates with salivary gland homogenate (SGH). Further evidence
suggested that DTH was responsible for the observed anti-Leishmania
protection and that antibodies were not necessary
(Valenzuela et al.,
2001a
).
In humans, we have reported a correlation between immune response to
Lu. longipalpis salivary proteins and cellular response to
Leishmania chagasi, a protective mechanism against leishmaniasis
(Barral et al., 2000).
Additionally, we have reported a number of salivary antigens recognized by
sera of humans, which may be related to protection against leishmaniasis
(Gomes et al., 2002
); however,
the identity of these proteins remains to be solved.
We have hypothesized that the protective effect of sand fly salivary
proteins is related to a cellular response to these molecules, probably from
CD4 T cells (Valenzuela et al.,
2001b). Accordingly, these responses depend on the immunogenetic
background of the vertebrate host; thus, targeting a single protein may not be
an adequate approach to identify the right vaccine for a population. A broader
approach or selection of multiple candidates may be required. High-throughput
approaches based on massive cDNA sequencing, proteomics and customized
computational biology approaches are helping us to reveal the proteins present
in the salivary glands of different blood-feeding arthropods, including the
mosquitoes Anopheles gambiae
(Francischetti et al., 2002
),
Anopheles stephensi (Valenzuela
et al., 2003
), Anopheles darlingi
(Calvo et al., 2004
) and
Aedes aegypti (Valenzuela et al.,
2002a
), the tick Ixodes scapularis
(Valenzuela et al., 2002b
) and
the bug Rhodnius prolixus
(Ribeiro et al., 2004
). In the
present work, we explored the proteins and transcripts encoded in the salivary
gland of Lu. longipalpis, targeting the isolation and full sequencing
of the secreted and putative secreted proteins, which are potential markers
for vector exposure and vaccine candidates to control Le. chagasi
infection.
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Materials and methods |
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Salivary gland cDNA library
Lu. longipalpis salivary gland mRNA was isolated from 80 salivary
gland pairs using the Micro-FastTrack mRNA isolation kit (Invitrogen, San
Diego, CA, USA). The PCR-based cDNA library was made following the
instructions for the SMART cDNA library construction kit (BD-Clontech, Palo
Alto, CA, USA) with some modifications (Valenzuela et al.,
2001a,
2002c
). The obtained cDNA
libraries (large, medium and small sizes) were plated by infecting log phase
XL1-blue cells (BD-Clontech), and the amount of recombinants was determined by
PCR using vector primers flanking the inserted cDNA and visualized on a 1.1%
agarose gel with ethidium bromide (1.5 µg ml1).
Massive sequencing of cDNA library
Lu. longipalpis salivary gland cDNA libraries were plated to a
number of 200 plaques per plate (150 mm Petri dish). The plaques were
randomly picked and transferred to a 96-well polypropylene plate (Novagen,
Madison, WI, USA) containing 75 µl of water per well. Four microliters of
the phage sample was used as a template for a PCR reaction to amplify random
cDNAs. The primers used for this reaction were sequences from the triplEX2
vector. PT2F1 (5'-AAG TAC TCT AGC AAT TGT GAG C-3') is positioned
upstream of the cDNA of interest (5' end), and PT2R1 (5'-CTC TTC
GCT ATT ACG CCA GCT G-3') is positioned downstream of the cDNA of
interest (3' end). Platinum Taq polymerase (Invitrogen) was used for
these reactions. Amplification conditions were: 1 hold of 75°C for 3 min,
1 hold of 94°C for 2 min and 30 cycles of 94°C for 1 min, 49°C for
1 min and 72°C for 1 min 20 s. Amplified products were visualized on a
1.1% agarose gel with ethidium bromide. PCR products were cleaned using the
PCR multiscreen filtration system (Millipore, Bedford, MA, USA). Three
microliters of the cleaned PCR product was used as a template for a
cycle-sequencing reaction using the DTCS labeling kit from Beckman Coulter
(Fullerton, CA, USA). The primer used for sequencing, PT2F3 (5'-TCT CGG
GAA GCG CGC CAT TGT-3'), is upstream of the inserted cDNA and downstream
of the primer PT2F1. Sequencing reaction was performed on a Perkin Elmer 9700
thermacycler (Foster City, CA, USA). Conditions were 75°C for 2 min,
94°C for 2 min and 30 cycles of 96°C for 20 s, 50°C for 10 s and
60°C for 4 min. After cycle-sequencing the samples, a cleaning step was
done using the multiscreen 96-well plate cleaning system from Millipore.
Samples were sequenced immediately on a CEQ 2000XL DNA sequencing instrument
(Beckman Coulter) or stored at 30°C.
Bioinformatics
Detailed description of the bioinformatic treatment of the data can be
found elsewhere (Valenzuela et al.,
2002c). Briefly, primer and vector sequences were removed from raw
sequences, compared against the GenBank non-redundant (NR) protein database
using the stand-alone 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. The
predicted translated proteins were searched for a secretory signal through the
SignalP server (Nielsen et al.,
1997
). Sequences were clustered using the BlastN program
(Altschul and Lipman, 1990
) as
detailed before (Valenzuela et al.,
2002c
), and the data presented in the format of
Table 1. The electronic version
of the table (available on request from
jvalenzuela{at}niaid.nih.gov)
has additional hyperlinks to ClustalX alignments
(Jeanmougin et al., 1998
) as
well as FASTA-formatted sequences for all clusters.
|
Full-length sequencing of selected cDNA clones
An aliquot (4 µl) of the -phage containing the cDNA of interest
was amplified using the PT2F1 and PT2R1 primers (same conditions as described
above). The PCR samples were cleaned using the multiscreen PCR 96-well
filtration system (Millipore). Cleaned samples were sequenced first with PT2F3
primer and subsequently with custom primers. Full-length sequences were again
compared with databases as indicated for the nucleotide sequences above, and
the data displayed as in Table
2.
|
SDS-PAGE
Tris-glycine gels (420%), 1 mm thick (Invitrogen), were used. Gels
were run with Tris-glycine SDS buffer according to manufacturer's
instructions. To estimate the molecular mass of the samples, SeeBlueTM
markers from Invitrogen (myosin, bovine serum albumin, glutamic dehydrogenase,
alcohol dehydrogenase, carbonic anhydrase, myoglobin, lysozyme, aprotinin, and
insulin, chain B) were used. SGH were treated with equal parts of 2xSDS
sample buffer (8% SDS in Tris-HCl buffer, 0.5 mol l1, pH
6.8, 10% glycerol and 1% bromophenol blue dye). Thirty pairs of homogenized
salivary glands per lane (approximately 30 µg protein) were applied when
visualization of the protein bands stained with Coomassie blue was desired.
For amino-terminal sequencing of the salivary proteins, 40 homogenized pairs
of salivary glands were electrophoresed and transferred to polyvinylidene
difluoride (PVDF) membrane using 10 mmol l1 CAPS, pH 11.0,
10% methanol as the transfer buffer on a Blot-Module for the XCell II
Mini-Cell (Invitrogen). The membrane was stained with Coomassie blue in the
absence of acetic acid. Stained bands were cut from the PVDF membrane and
subjected to Edman degradation using a Procise sequencer (Perkin-Elmer Corp.).
To find the cDNA sequences corresponding to the amino acid sequence obtained
by Edman degradation, we used a search program (written in Visual Basic by J.
M. C. Ribeiro) that checked these amino acid sequences against the three
possible protein translations of each cDNA sequence obtained in the DNA
sequencing project. A more detailed account of this program is found elsewhere
(Valenzuela et al.,
2002c).
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Results |
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Table 1 shows the most abundant clusters in descending order, from the most abundant sequences to the least. From these 40 clusters, 28 contain sequences with predicted secretory proteins, and those remaining represent clusters for housekeeping genes or unknown sequences without a clear secretory signal peptide. We found 65 sequences (out of 550) of probable housekeeping genes, arranged in 61 clusters, an average of 1.07 sequences per cluster. Examples of these housekeeping genes are: protein disulphide isomerase (cluster 35); 6-phosphogluconate dehydrogenase (cluster 36); ribosomal protein (cluster 39); actin (cluster 54); adenylate kinase (cluster 92); ATP-synthase (cluster 65) and cytochrome oxidase (cluster 57). These clusters represent only the clusters from the middle of Table 1 downwards, with only a few sequences per cluster (most are only one sequence per cluster) representing the low abundant messages.
Another set of cDNA found in this library contains clones that do not have similarities to other genes in the NCBI databank, do not have an assigned function (by CDD analysis) and do not have a secretory signal peptide. We found 34 sequences (out of 550) of these types of genes arranged in 23 clusters (1.48 sequences per cluster).
The most abundant clones found in this cDNA library are the ones coding for
secretory proteins. We found 451 cDNA (out of 550 sequences) of potentially
secreted proteins arranged in 40 clusters (an average of 11.27 sequences per
cluster). Although four of these clusters (cluster 33 with two sequences,
cluster 84 with one sequence, cluster 88 with one sequence and cluster 110
with one sequence) resulted in no signal peptide in our analysis, we included
these sequences as secretory proteins because they are truncated versions of
the salivary adenosine deaminase cDNA and the salivary hyaluronidase cDNA,
previously analyzed and reported to be secretory proteins
(Charlab et al., 1999).
Therefore, the number of cDNAs coding for secretory proteins is 10.53 times
greater than the cDNA coding for housekeeping genes and 7.61 times greater
than the cDNA coding for proteins with unknown function; overall, cDNA coding
for secretory proteins is 4.4 times greater than cDNA coding for non-secreted
proteins. The cDNAs coding for secretory proteins represent 82% of the cDNAs
sequenced in this sand fly library. In fact, out of the first 40 clusters, 28
belong to cDNA coding for secretory proteins
(Table 1).
Proteome analysis of Lu. longipalpis salivary proteins
The supernatant of Lu. longipalpis SGH was separated by
one-dimension SDS-PAGE, the proteins transferred to PVDF membrane, and Edman
degradation performed on the Coomassie blue-stained bands.
Fig. 1 (left side) shows the
resulting N-terminal sequence of 17 proteins. By searching our database using
an in-house program (written by J. M. C. Ribeiro) similar to the BLOCK
program, we were able to identify all the coding cDNA, which correspond to the
14 N-terminal sequences (right side of Fig.
1). We identify by Edman degradation the N-terminus of the cDNA
corresponding to clusters 2, 3, 5, 7, 8, 8b, 9, 11, 12, 13, 14, 17, 18, 19,
22, 27 and 115. With the exception of cluster 115, all clusters were within
the first 40 clusters. Not all the attempted Edman degradation experiments
resulted in a sequence, probably because of the small amount of protein or
because these proteins were blocked at the N-terminal end.
|
Full-length sequence of clones coding for secreted proteins
Because we are primarily interested in the identification and
characterization of secreted proteins from this cDNA library, we selected the
35 clusters containing sequences with a clear signal peptide and obtained full
sequence on all of them. Table
2 shows the analysis of these sequences, including the signal
peptide cleavage site, molecular mass, isoelectric point and best match to
NCBI database. The last column of Table
2 indicates whether the clone has been found in the proteome
analysis. Extensive analysis is presented in the electronic version of
Table 2 (available upon request
from
jvalenzuela{at}niaid.nih.gov).
The selected cDNAs in Table 2 are arranged in descending order from the cluster containing the largest number of cDNA to the clusters containing the least number (from the most abundant transcripts to the least abundant transcripts in this cDNA library).
Description of cDNAs coding for secreted proteins
Cluster 1 (LJL35; GenBank acc. no. AF132516) RGD peptide
The cDNA from this cluster codes for a secreted 6 kDa peptide with no
similarities to other proteins in databanks. We previously isolated this
transcript by PCR subtraction technique
(Charlab et al., 1999). The
function of this protein remains unknown, although it may inhibit platelet
aggregation by interfering with the binding of platelet receptor to fibrinogen
by the RGD motif present on it. We did not find this protein in our proteomic
analysis, probably because of its small size or because the N-terminal amino
acid is blocked.
Clusters 2, 9 and 22 family of yellow-related proteins
Proteins from this family were found in cluster 2 (GenBank acc. no.
AF132518), cluster 9 (GenBank acc. no. AY445935) and cluster 22 on this cDNA
library.
Cluster 2 codes for a secreted protein of 45 kDa previously characterized
by PCR subtraction (Charlab et al.,
1999). The gene coding for this protein was first described in
Drosophila melanogaster (Geyer et
al., 1986
). When comparing with other Diptera, this protein was
not found in the salivary glands of Ae. aegypti
(Valenzuela et al., 2002a
),
An. gambiae (Francischetti et
al., 2002
) or An. stephensi
(Valenzuela et al., 2003
). It
was, however, described in Ae. aegypti whole-larvae extract and
purified as a dopachrome-converting enzyme
(Johnson et al., 2001
). The
homologue of Lu. longipalpis yellow protein was also described in the
salivary glands of the sand fly P. papatasi
(Valenzuela et al., 2001b
).
The function of this protein in the saliva remains to be elucidated. In the
proteomic analysis (Fig. 1),
this cluster codes for one of the most abundant proteins (AYVEIGYSLRNIT) in
the saliva of this sand fly. Interestingly, a protein with similar molecular
mass was the most recognized antigen by the sera of individuals who were
exposed to sand flies and developed a cellular immunity to Le.
chagasi (Gomes et al.,
2002
). Therefore, this protein is a candidate marker for
epidemiological studies on vector exposure and may be a potential vaccine
candidate to control Le. chagasi infection.
This yellow-related protein is highly similar to proteins found in cluster
9 (GenBank acc. no. AY445935) and cluster 22.
Fig. 2 shows the ClustalW
alignment of this protein with related proteins, including yellow-related
salivary proteins from P. papatasi
(Valenzuela et al., 2001b),
Ae. aegypti midgut dopachrome conversion enzyme
(Johnson et al., 2001
) and
D. melanogaster yellow protein. Similarities among the phlebotomine's
yellow proteins are greater than non-salivary yellow from other Diptera and
are marked as gray-shaded amino acids (Fig.
2A). Bootstrap neighbor-joining analysis
(Fig. 2B) resulted in grouping
insect yellow proteins in three distinct clades: the first group contains the
Aedes, Anopheles and Drosophila yellow-related proteins
(non-salivary); the second group contains the two P. papatasi
yellow-related salivary proteins and the Lu. longipalpis yellow
salivary protein from cluster 2; and the third group contains the Lu.
longipalpis yellow salivary proteins from clusters 9 and 22.
|
Cluster 3 (LJL08; GenBank acc. no. M77090) maxadilan
This cluster codes for the most studied salivary protein from a sand fly.
It was discovered to be the most potent vasodilator from any organism
(Ribeiro et al., 1989) and
later as an immunomodulatory molecule
(Soares et al., 1998
).
Recently, Morris et al. reported that mice vaccinated with this 6 kDa peptide
produced antibodies against this molecule, and these animals were protected
against Le. major infection
(Morris et al., 2001
). The
N-terminal of this protein, XDATXQFRKAIEDDK, was found in the proteomic
analysis. Surprisingly, the cDNA we found in this library is only 69%
identical to the previously reported maxadilan. Differences may be due to
strain differences or the polymorphism reported for this molecule
(Lanzaro et al., 1999
).
Clusters 4, 6, 7, 18, 25, 26, 34, 67 and 115 novel peptides from the saliva of Lu. longipalpis
With our approach, we identified a number of small peptides in this cDNA
library. Only two peptides were previously reported from the salivary glands
of Lu. longipalpis: maxadilan
(Ribeiro et al., 1989) and an
RGD-containing peptide (Charlab et al.,
1999
). Following is the description of the nine clusters
containing these novel peptides. Sequences of these peptides are shown in
Fig. 3.
|
Cluster 5 (LJM04; GenBank acc. no. AF132517) PpSP15-like proteins
This cDNA was previously isolated by PCR subtraction and named SL1. This
cDNA codes for a protein of 14 kDa and is similar to the protein of 15 kDa
from P. papatasi (PpSP15) that conferred protection against Le.
major infection. The N-terminus predicted from this cDNA, EHPEEKXIRELAR,
was present in our proteomic analysis (Fig.
1). Previously, we reported three similar proteins (three
different clusters) on the salivary glands of P. papatasi (PpSP12,
PpSP14 and PpSP15). We found only one cluster of this family of proteins in
the Lu. longipalpis cDNA library.
Clusters 8, 8b, 11, 14, 17 and 19 family of putative anticoagulants (C-type lectin)
One of the most abundant families of protein found in this cDNA library is
the family of putative anticoagulants with homology to C-type lectins. Six
clusters (8, 8b, 11, 14, 17 and 19) belong to this family.
Cluster 8 (LJL91; GenBank acc. no. AY445934). This cDNA codes for
a secreted protein of 16 kDa and is similar to the previously described
anticoagulant from Lu. longipalpis
(Charlab et al., 1999). These
proteins contain a C-type lectin or C-type lectin-like domain. This domain
functions as a calcium-dependent carbohydrate-binding pocket involved in
extracellular matrix organization, pathogen recognition and cell-to-cell
interactions. Factor IX/X and Von Willebrand factor binding proteins contain
these domains, suggesting that the anticoagulant(s) of Lu.
longipalpis may be binding some of the targets of these coagulation
factors and inhibiting the blood coagulation cascade. An alignment showing all
the C-type lectin proteins found in this cDNA library is shown in
Fig. 4A. A ClustalW alignment
of this protein and other proteins containing lectin-like domains is shown in
Fig. 4B. Phylogenetic tree
analysis of C-type lectin-like proteins from Lu. longipalpis and
other organisms is shown in Fig.
4C. C-type lectin-like salivary proteins have only been described
in Lu. longipalpis sand flies.
|
Cluster 10 (LJL34; GenBank acc. no. AF132511) antigen 5-related proteins
This cluster codes for a secreted protein of 29 kDa similar to antigen
5-related protein from vespid venom (Lu et
al., 1993). Similar proteins have been isolated from the salivary
glands of Ae. aegypti (Valenzuela
et al., 2002a
) and An. gambiae
(Francischetti et al., 2002
).
We previously isolated this cDNA from the salivary glands of Lu.
longipalpis by PCR subtraction
(Charlab et al., 1999
).
Cluster 12 (LJL13; GenBank acc. no. AF420274) D7-related protein
This cluster codes for a secreted protein of 26 kDa and belongs to the D7
family of proteins (Valenzuela et al.,
2002c). One salivary D7 protein from An. stephensi was
shown to be an inhibitor of the blood coagulation Factor XII
(Isawa et al., 2002
). The
N-terminus of the predicted salivary protein, WQDVRNADQTL, was found in the
proteomic analysis (Fig.
1).
Cluster 13 (LJL23; GenBank acc. no. AF131933) apyrase
This cluster codes for a secreted protein of 35 kDapreviously characterized
by PCR subtraction (Charlab et al.,
1999). This protein belongs to a family first described in
Cimex lectularius and shown to be a salivary apyrase
(Valenzuela et al., 1998
).
Later, the clone coding for an homologous cDNA from P. papatasi was
expressed and demonstrated to be a calcium-dependent apyrase
(Valenzuela et al., 2001b
).
Recently, a Cimex homologue from humans was demonstrated to have ATPase and
low ADPase activity (Murphy et al.,
2003
), while the insect apyrases have high ADPase activities. The
N-terminus of the predicted salivary protein, APPGVEWYHFGL, was found in the
proteomic analysis (Fig. 1). A
protein with similar molecular mass to the cDNA prediction for this cluster
(35 kDa) was recognized with high frequency among serum of individuals exposed
naturally to sand flies who developed a cellular immunity to Le.
chagasi (Gomes et al.,
2002
). Therefore, this protein may be a good marker for vector
exposure and potential vaccine candidate to control Le. chagasi
infection.
Clusters 16, 21, 23, 58 novel sequences
Cluster 16 (LJL143; GenBank acc. no. AY445936). This cluster codes
for a secreted protein of 32 kDa. It has no significant homology to known
proteins from GenBank. This protein may only be present in phlebotomines,
because we did not find similar sequences in either the Drosophila or
Anopheles genome or any other databases.
Cluster 21 (LJM114; GenBank acc. no. AY455907). This cluster codes for a secreted protein of 14 kDa, with no similarities to other proteins in the databank. It is a relatively abundant cDNA in this library and is therefore a novel protein in sand fly saliva.
Cluster 23 (LJM78; GenBank acc. no. AY455908). This cluster codes for a secreted protein of 37 kDa, with no similarities to other proteins on the databases searched. This cDNA represents a novel protein from the saliva of a sand fly.
Cluster 58 (LJS03; GenBank acc. no. AY455914). This cDNA codes for a secreted protein of 15 kDa, with no significant similarities to other proteins in the searched databases.
Cluster 20 (LJL04; GenBank acc. no. AY455906) collagen binding-like proteins
This cluster codes for a secreted protein of 29 kDa, with similarities to
PpSP32 salivary protein from P. papatasi and to the collagen adhesion
protein from Bacillus cereus (acc. no. NP_830673). This 29 kDa
protein is rich in glycine (51 amino acids), arginine (25 amino acids),
proline (25 amino acids) and lysine (23 amino acids). These four amino acids
represent 44% of the amino acids in this protein. Additionally, the pI of this
protein is 10.2, making it a very basic protein. Different short repeats were
identified in this protein, particularly the repeats GQG and GTRP
(Fig. 5). Because of its
similarities to the collagen-binding protein from B. cereus, the high
pI, the richness in glycine and the repeated amino acid motifs, this salivary
protein may bind to the extracellular matrix proteins of the vertebrate
host.
|
Cluster 27 (LJL11; GenBank acc. no. AF132510) 5'-nucleotidase
This cluster codes for a secreted protein of 61 kDa. It is the secreted
5'-nucleotidase from Lu. longipalpis, previously isolated by
PCR subtraction (Charlab et al.,
1999). The N-terminus of the predicted salivary protein,
EDGSYEIIILHTN, was found in the proteomic analysis
(Fig. 1), suggesting that this
protein is very abundant in the saliva of this insect.
Cluster 38 (LJL09; GenBank acc. no. AY455911) angiotensin converting enzyme
This cDNA codes for a secreted protein of 71 kDa, with high similarities to
angiotensin converting enzyme (ACE) from An. gambiae, D.
melanogaster, chicken and human (Fig.
6). The function as a peptidase remains to be elucidated. The
activity may be similar to the kininase activity observed in the tick I.
scapularis (Ribeiro and Mather,
1998).
|
Cluster 56 (LJM26; GenBank acc. no. AY455913) serine protease inhibitor
This cDNA codes for a secreted protein of 49 kDa, with high similarities to
the serpin family of protease inhibitors
(Fig. 7). This protein may
function as an anticoagulant in the saliva or may be responsible for
regulation of the insect immune response. Anticomplement activity in Lu.
longipalpis has been recently described
(Cavalcante et al., 2003);
because of its putative anti-protease activity, this cDNA may code for the
salivary anti-complement activity.
|
Cluster 71 (LJL138; GenBank acc. no. AY455916) endonuclease
This cDNA codes for a protein of 44 kDa, with high similarities to
non-specific RNA/DNA endonucleases from different organisms. A similar cDNA
was isolated from the salivary glands of the tsetse fly (Glossina
morsitans) and Culex quinquefasciatus
(Ribeiro et al., 2004) and the
protein was named TsaI (Li et al.,
2001
). The function as an endonuclease remains to be elucidated.
The presence of endonuclease in the saliva of an insect is puzzling and it is
interesting to note that enzymes directed to nucleotide metabolism, such as
5'-nucleotidase and adenosine deaminase, are present in this sand fly
(Charlab et al., 2000
).
Cluster 97 (LJS138; GenBank acc. no. AY455917) translocon-associated protein
This cDNA codes for a secreted protein of 16 kDa, with high similarities to
translocon-associated protein from different organisms and may be associated
with an ER protein export function.
Cluster 113 (LJS193; GenBank acc. no. AY455918) palmitoyl thioesterase
This cDNA codes for a protein of 32 kDa, with high similarities to
palmitoyl-(protein) thioesterase from different organisms.
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Discussion |
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It is interesting to note that we needed to sequence only 550 cDNA to identify the majority of secretory salivary proteins for this sand fly. The most abundant transcripts in this Lu. longipalpis salivary gland cDNA library correspond to putative secreted proteins, which indicates that most transcripts in this organ are directed to secretion. From these 550 cDNA, we obtained 143 different clusters of related sequences, suggesting that the cDNA library was diverse enough to have a good representation of the transcripts present in the salivary gland of this sand fly.
From the 143 different clusters or families of proteins, we identified 35 clusters of proteins containing a secretory signal peptide. Interestingly, the majority of the cDNAs sequenced (451 out of 550) were in these 35 clusters. We confirmed by Edman degradation the presence of 17 secreted proteins in the salivary gland of this sand fly. These 17 proteins are included in these 35 clusters. In fact, all the proteins that resulted with N-terminus data had a corresponding cDNA in these 35 clusters, and the predicted signal cleavage site (SignalP program for these 35 clusters) matched perfectly with the N-terminus obtained, with the exception of two proteins. These two proteins probably underwent further processing by a protease.
When comparing these 35 clusters with sequences deposited in existing databases, we found that nine of these cDNAs correspond with already described Lu. longipalpis proteins. We found eight cDNAs with low homology to already described Lu. longipalpis proteins, suggesting that they belong to related families of proteins but are different enough to be clustered in different groups. We found 10 cDNAs with homology to other proteins in databases, including an angiotensin converting enzyme, a protease and a palmitoyl-hydrolase; the rest of the cDNAs (seven) did not match to any protein in the existing databases. These are probably novel proteins only present thus far in this sand fly.
The sand fly Lu. longipalpis is the main vector of Le.
chagasi, the causal agent of visceral leishmaniasis. The relationship
between Lu. longipalpis saliva and human visceral leishmaniasis was
recently undertaken. Barral et al.
(2000) studied serum from
children living in an endemic area of visceral leishmaniasis and found a
positive correlation between children producing antibodies against Lu.
longipalpis and delayed-type hypersensitive response (DTH) to Le.
chagasi. DTH to Leishmania is a marker for protection against
this parasite, while positive serology is a sign of poor prognosis. On the
other hand, no correlation was found between children producing antibodies to
Lu. longipalpis and positive serology to Le. chagasi. In an
extension of this work, we recently reported that children from an endemic
area of leishmaniasis, who in a 6-month period developed antibodies to Le.
chagasi, did not produce detectable antibodies to Lu.
longipalpis salivary proteins. On the other hand, individuals that
developed a cellular response to Le. chagasi (DTH) produced IgG, IgG1
and IgE antibodies to Lu. longipalpis salivary proteins
(Gomes et al., 2002
). These
data support the hypothesis that induction of an immune response to salivary
proteins from Lu. longipalpis may facilitate a protective immune
response to Le. chagasi.
The salivary proteins, as well as the cDNA coding for secreted proteins
identified in this work, are good candidates as markers for vector exposure to
continue performing epidemiological studies with single recombinant proteins
instead of whole SGH. In previous work, we identified five salivary antigens
by western blot that resulted in molecular masses of 45, 44, 43, 35 and 17 kDa
(Gomes et al., 2002). In the
present study, we have identified a limited number of cDNAs that code for a
molecular mass similar to these antigens; these are the yellow-related
proteins (clusters 2, 9 and 22), an apyrase (cluster 13) and an anticoagulant
protein (clusters 8, 14, 19). Recombinant expression of these proteins will
determine whether these are the antigens recognized by individuals exposed to
sand flies that showed protection to Le. chagasi infection.
Ultimately, these salivary proteins represent good vaccine candidates to
control Leishmania infection. We reported that a delayed-type
hypersensitivity response to a sand fly salivary protein was responsible for
the protective effect against Le. major infection in mice
(Valenzuela et al., 2001a).
Because this type of protection is T cell dependent and the response may
depend on the immunogenetic background of the host, focusing on a single
protein may not be a proper vaccine strategy in an outbred population. This
high-throughput approach is providing a larger candidate repertoire to select
the best vaccine candidate or the best cocktail of protective salivary
components.
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Altschul, S. F. and Lipman, D. J. (1990). Protein database searches for multiple alignments. Proc. Natl. Acad. Sci. USA 87,5509 -5513.[Abstract]
Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J.,
Zhang, Z., Miller, W. and Lipman, D. J. (1997). Gapped BLAST
and PSI-BLAST: a new generation of protein database search programs.
Nucleic Acids Res. 25,3389
-3402.
Barral, A., Honda, E., Caldas, A., Costa, J., Vinhas, V.,
Rowton, E. D., Valenzuela, J. G., Charlab, R., Barral-Netto, M. and Ribeiro,
J. M. (2000). Human immune response to sand fly salivary
gland antigens: a useful epidemiological marker? Am. J. Trop. Med.
Hyg. 62,740
-745.
Bateman, A., Birney, E., Durbin, R., Eddy, S. R., Howe, K. L.
and Sonnhammer, E. L. (2000). The Pfam protein families
database. Nucleic Acids Res.
28,263
-266.
Bell, J. F., Stewart, S. J. and Wikel, S. K. (1979). Resistance to tick-borne Francisella tularensis by tick-sensitized rabbits: allergic klendusity. Am. J. Trop. Med. Hyg. 28,876 -880.[Medline]
Calvo, E., Andersen, J., Francischetti, I. M., del Capurro, M., deBianchi, A. G., James, A. A., Ribeiro, J. M. and Marinotti, O. (2004). The transcriptome of adult female Anopheles darlingi salivary glands. Insect Mol. Biol. 13, 73-88.[Medline]
Cavalcante, R. R., Pereira, M. H. and Gontijo, N. F. (2003). Anti-complement activity in the saliva of phlebotomine sand flies and other haematophagous insects. Parasitology 127,87 -93.[CrossRef][Medline]
Charlab, R., Valenzuela, J. G., Rowton, E. D. and Ribeiro, J.
M. (1999). Toward an understanding of the biochemical and
pharmacological complexity of the saliva of a hematophagous sand fly
Lutzomyia longipalpis. Proc. Natl. Acad. Sci. USA
96,15155
-15160.
Charlab, R., Rowton, E. D. and Ribeiro, J. M. (2000). The salivary adenosine deaminase from the sand fly Lutzomyia longipalpis. Exp. Parasitol. 95, 45-53.[CrossRef][Medline]
Francischetti, I. M., Valenzuela, J. G., Pham, V. M., Garfield, M. K. and Ribeiro, J. M. (2002). Toward a catalog for the transcripts and proteins (sialome) from the salivary gland of the malaria vector Anopheles gambiae. J. Exp. Biol. 205,2429 -2451.[Medline]
Geyer, P. K., Spana, C. and Corces, V. G. (1986). On the molecular mechanism of gypsy-induced mutations at the yellow locus of Drosophila melanogaster. EMBO J. 5,2657 -2662.[Abstract]
Gillespie, R. D., Mbow, M. L. and Titus, R. G. (2000). The immunomodulatory factors of blood-feeding arthropod saliva. Parasite Immunol. 22,319 -331.[CrossRef][Medline]
Gomes, R. B., Brodskyn, C., de Oliveira, C. I., Costa, J., Miranda, J. C., Caldas, A., Valenzuela, J. G., Barral-Netto, M. and Barral, A. (2002). Seroconversion against Lutzomyia longipalpis saliva concurrent with the development of anti-Leishmania chagasi delayed-type hypersensitivity. J. Infect. Dis. 186,1530 -1534.[CrossRef][Medline]
Isawa, H., Yuda, M., Orito, Y. and Chinzei, Y.
(2002). A mosquito salivary protein inhibits activation of the
plasma contact system by binding to factor XII and high molecular weight
kininogen. J. Biol. Chem.
277,27651
-27658.
Jeanmougin, F., Thompson, J. D., Gouy, M., Higgins, D. G. and Gibson, T. J. (1998). Multiple sequence alignment with Clustal X. Trends Biochem. Sci. 23,403 -405.[CrossRef][Medline]
Johnson, J. K., Li, J. and Christensen, B. M. (2001). Cloning and characterization of a dopachrome conversion enzyme from the yellow fever mosquito, Aedes aegypti. Insect Biochem. Mol. Biol. 31,1125 -1135.[CrossRef][Medline]
Jones, L. D. and Nuttall, P. A. (1990). The effect of host resistance to tick infestation on the transmission of Thogoto virus by ticks. J. Gen. Virol. 71,1039 -1043.[Abstract]
Kamhawi, S. (2000). The biological and immunomodulatory properties of sand fly saliva and its role in the establishment of Leishmania infections. Microbes Infect. 2,1765 -1773.[CrossRef][Medline]
Kamhawi, S., Belkaid, Y., Modi, G., Rowton, E. and Sacks, D.
(2000). Protection against cutaneous leishmaniasis resulting from
bites of uninfected sand flies. Science
290,1351
-1354.
Lanzaro, G. C., Lopes, A. H., Ribeiro, J. M., Shoemaker, C. B., Warburg, A., Soares, M. and Titus, R. G. (1999). Variation in the salivary peptide, maxadilan, from species in the Lutzomyia longipalpis complex. Insect Mol. Biol. 8, 267-275.[CrossRef][Medline]
Lerner, E. A., Ribeiro, J. M., Nelson, R. J. and Lerner, M.
R. (1991). Isolation of maxadilan, a potent vasodilatory
peptide from the salivary glands of the sand fly Lutzomyia longipalpis.J. Biol. Chem. 266,11234
-11236.
Li, S., Kwon, J. and Aksoy, S. (2001). Characterization of genes expressed in the salivary glands of the tsetse fly, Glossina morsitans morsitans. Insect Mol. Biol. 10, 69-76.[CrossRef][Medline]
Lu, G., Villalba, M., Coscia, M. R., Hoffman, D. R. and King, T.
P. (1993). Sequence analysis and antigenic cross-reactivity
of a venom allergen, antigen 5, from hornets, wasps, and yellow jackets.
J. Immunol. 150,2823
-2830.
Morris, R. V., Shoemaker, C. B., David, J. R., Lanzaro, G. C.
and Titus, R. G. (2001). Sandfly maxadilan exacerbates
infection with Leishmania major and vaccinating against it protects
against L. major infection. J. Immunol.
167,5226
-5230.
Murphy, D. M., Ivanenkov, V. V. and Kirley, T. L. (2003). Bacterial expression and characterization of a novel, soluble, calcium-binding, and calcium-activated human nucleotidase. Biochemistry 42,2412 -2421.[CrossRef][Medline]
Nazario, S., Das, S., de Silva, A. M., Deponte, K., Marcantonio,
N., Anderson, J. F., Fish, D., Fikrig, E. and Kantor, F. S.
(1998). Prevention of Borrelia burgdorferi transmission
in guinea pigs by tick immunity. Am. J. Trop. Med.
Hyg. 58,780
-785.
Nielsen, H., Engelbrecht, J., Brunak, S. and von Heijne, G. (1997). A neural network method for identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Int. J. Neural Syst. 8,581 -599.[Medline]
Nuttall, P. A., Paesen, G. C., Lawrie, C. H. and Wang, H. (2000). Vectorhost interactions in disease transmission. J. Mol. Microbiol. Biotechnol. 2, 381-386.[Medline]
Ribeiro, J. M. (1987). Vector salivation and parasite transmission. Mem. Inst. Oswaldo Cruz 82(Suppl. 3),1 -3.
Ribeiro, J. M. (1989). Vector saliva and its role in parasite transmission. Exp. Parasitol. 69,104 -106.[CrossRef][Medline]
Ribeiro, J. M. (1995). Blood-feeding arthropods: live syringes or invertebrate pharmacologists? Infect. Agents Dis. 4,143 -152.[Medline]
Ribeiro, J. M. and Mather, T. N. (1998). Ixodes scapularis: salivary kininase activity is a metallo dipeptidyl carboxypeptidase. Exp. Parasitol. 89,213 -221.[CrossRef][Medline]
Ribeiro, J. M., Vachereau, A., Modi, G. B. and Tesh, R. B. (1989). A novel vasodilatory peptide from the salivary glands of the sand fly Lutzomyia longipalpis. Science 243,212 -214.[Medline]
Ribeiro, J. M., Andersen, J., Silva-Neto, M. A., Pham, V. M., Garfield, M. K. and Valenzuela, J. G. (2004). Exploring the sialome of the blood-sucking bug Rhodnius prolixus. Insect Biochem. Mol. Biol. 34,61 -79.[CrossRef][Medline]
Schultz, J., Milpetz, F., Bork, P. and Ponting, C. P.
(1998). SMART, a simple modular architecture research tool:
identification of signaling domains. Proc. Natl. Acad. Sci.
USA 95,5857
-5864.
Schultz, J., Copley, R. R., Doerks, T., Ponting, C. P. and Bork,
P. (2000). SMART: a web-based tool for the study of
genetically mobile domains. Nucleic Acids Res.
28,231
-234.
Soares, M. B., Titus, R. G., Shoemaker, C. B., David, J. R. and
Bozza, M. (1998). The vasoactive peptide maxadilan from sand
fly saliva inhibits TNF-alpha and induces IL-6 by mouse macrophages through
interaction with the pituitary adenylate cyclase-activating polypeptide
(PACAP) receptor. J. Immunol.
160,1811
-1816.
Titus, R. G. and Ribeiro, J. M. (1988). Salivary gland lysates from the sand fly Lutzomyia longipalpis enhance Leishmania infectivity. Science 239,1306 -1308.[Medline]
Valenzuela, J. G., Charlab, R., Galperin, M. Y. and Ribeiro, J.
M. (1998). Purification, cloning, and expression of an
apyrase from the bed bug Cimex lectularius. A new type of
nucleotide-binding enzyme. J. Biol. Chem.
273,30583
-30590.
Valenzuela, J. G., Belkaid, Y., Garfield, M. K., Mendez, S.,
Kamhawi, S., Rowton, E. D., Sacks, D. L. and Ribeiro, J. M.
(2001a). Toward a defined anti-Leishmania vaccine targeting
vector antigens: characterization of a protective salivary protein.
J. Exp. Med. 194,331
-342.
Valenzuela, J. G., Belkaid, Y., Rowton, E. and Ribeiro, J.
M. (2001b). The salivary apyrase of the blood-sucking sand
fly Phlebotomus papatasi belongs to the novel Cimex family of
apyrases. J. Exp. Biol.
204,229
-237.
Valenzuela, J. G., Pham, V. M., Garfield, M. K., Francischetti, I. M. and Ribeiro, J. M. (2002a). Toward a description of the sialome of the adult female mosquito Aedes aegypti. Insect Biochem. Mol. Biol. 32,1101 -1122.[CrossRef][Medline]
Valenzuela, J. G., Francischetti, I. M., Pham, V. M., Garfield, M. K., Mather, T. N. and Ribeiro, J. M. (2002b). Exploring the sialome of the tick Ixodes scapularis. J. Exp. Biol. 205,2843 -2864.[Medline]
Valenzuela, J. G., Charlab, R., Gonzalez, E. C., de Miranda-Santos, I. K., Marinotti, O., Francischetti, I. M. and Ribeiro, J. M. (2002c). The D7 family of salivary proteins in blood-sucking Diptera. Insect Mol. Biol. 11,149 -155.[CrossRef][Medline]
Valenzuela, J. G., Francischetti, I. M., Pham, V. M., Garfield, M. K. and Ribeiro, J. M. (2003). Exploring the salivary gland transcriptome and proteome of the Anopheles stephensi mosquito. Insect Biochem. Mol. Biol. 33,717 -732.[CrossRef][Medline]
Wikel, S. K., Ramachandra, R. N., Bergman, D. K., Burkot, T. R. and Piesman, J. (1997). Infestation with pathogen-free nymphs of the tick Ixodes scapularis induces host resistance to transmission of Borrelia burgdorferi by ticks. Infect. Immun. 65,335 -338.[Abstract]