A new intracellular pathway of haem detoxification in the midgut of the cattle tick Boophilus microplus: aggregation inside a specialized organelle, the hemosome
1 Departamento de Bioquímica Médica, ICB, Universidade Federal
do Rio de Janeiro, Brazil
2 Departamento de Microbiologia Geral, IMPPG, Universidade Federal do Rio de
Janeiro, Brazil
3 Departamento de Parasitologia, ICB, Universidade de São Paulo,
Brazil
4 Divisão de Química, Petrobrás/CENPES, Rio de Janeiro,
Brazil
5 Centro de Biociências e Biotecnologia, Universidade Estadual do
Norte Fluminense, Brazil
* Author for correspondence (e-mail: marilvia{at}uenf.br)
Accepted 26 February 2003
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Summary |
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In the present study we followed the fate of the haem derived from haemoglobin hydrolysis in the digest cells of the midgut of fully engorged tick females. The tick does not synthesize haem, so during the initial phase of blood digestion, absorption is the major route taken by the haem, which is transferred from the digest cells to the tick haemocoel. After this absorptive period of a few days, most of the haem produced upon haemoglobin degradation is accumulated in the interior of a specialized, membrane-delimited, organelle of the digest cell, herein called hemosome. Haem accounts for 90% of the hemosome mass and is concentrated in the core of this structure, appearing as a compact, non-crystalline aggregate of iron protoporphyrin IX without covalent modifications. The unusual FTIR spectrum of this aggregate suggests that lateral propionate chains are involved in the association of haem molecules with other components of the hemosome, which it is proposed is a major haem detoxification mechanism in this blood-sucking arthropod.
Key words: tick, haemozoin, haem, cattle tick, Boophilus microplus
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Introduction |
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Organisms that feed on vertebrate haemoglobin face a very special situation
as digestion results in the production of remarkably high amounts of haem.
Biocrystallization of haem into an insoluble aggregate called haemozoin is a
major defence mechanism that makes it possible for several organisms to feed
on blood. The structure of haemozoin has recently been resolved
(Pagola et al., 2000) and its
presence, originally thought to be restricted to the malaria parasite, has now
been detected in Rhodnius prolixus, a blood-sucking insect
(Oliveira et al., 1999
), and
in the blood fluke Schistosoma mansoni
(Oliveira et al., 2000
). Haem
aggregation occurs either in an intracellular digestive vacuole where the
haemoglobin molecule is hydrolyzed, as in Plasmodium
(Goldberg et al., 1990
;
Slater et al., 1991
), or
extracellularly, as in Rhodnius and Schistosoma (Oliveira et
al., 1999
,
2000
).
After hatching, the Boophilus microplus larvae find their
vertebrate host. During the following 3 weeks, the tick larva feeds on small
amounts of blood, and after maturation, over a period slightly longer than 1
day, the adult female ingests blood equivalent to approximately 100 times its
own body mass. Being a single-host tick, the engorged female drops from the
bovine host and dies approximately 1 month later. Most of the digestion takes
place over a few days following the meal, in parallel with the development of
a large number of eggs. In insects, digestion takes place at the midgut lumen,
but ticks have developed a differentiated cell lineage that phagocytoses the
blood meal, so digestion is thought to be essentially intracellular,
accomplished by lysosomal hydrolytic enzymes in the interior of an acidic
vacuole (Gough and Kemp, 1995;
Mendiola et al., 1996
). In the
present work, we have studied the fate of haem in the midgut of Boophilus
microplus and show that, after hydrolysis of haemoglobin by the digest
cells, the major haem detoxification mechanism is its sequestration into a
non-crystalline aggregate that is different from haemozoin. This aggregate is
formed and stored in a particular type of intracellular vesicle that is
distinct from the digestive vesicle.
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Materials and methods |
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Histochemistry
Engorged females were dissected in modified Karnovsky's fixative (2.5%
glutaraldehyde, 4% paraformaldehyde, 100 mmol l1
CaCl2 and 0.1 mol l1 sodium cacodylate buffer, pH
7.3). Tissues were transferred to fresh fixative and kept at 4°C for 12 h.
Segments of the anterior portion of the midgut were then washed in 0.1 mol
l1 sodium cacodylate buffer, pH 7.3. The tissues were
dehydrated in ethanol and embedded in Historesin (Leica, Wetzlar, Germany).
Semi-thin (5 µm) sections were observed by differential interference
contrast (DIC) microscopy (Axioplan, Zeiss, Esslingen, Germany).
Transmission electron microscopy
The tissues were fixed as above and post-fixed in buffered 1% osmium
tetroxide, 0.8% potassium ferrocyanide at room temperature for 1 h, dehydrated
in acetone and embedded in epoxide resin. Ultra-thin sections (70 nm) were
stained with uranyl acetate and lead citrate (Reynold's method, as described
in Glauert, 1974) and were
observed with a Zeiss 900 transmission electron microscope at 80 kV.
Cytochemistry
To locate areas of higher haem concentration, we used the methodology of
Graham and Karnovsky (1966),
which is based on the capacity of haem to promote oxidation of
3-3-diaminobenzidine (DAB). Midguts were dissected and fixed with 1%
glutaraldehyde in 0.1 mol l1 sodium cacodylate buffer, pH
6.5. After several washings in sodium cacodylate buffer, tissues were
incubated in 2.5 mmol l1 DAB for 1 h at 37°C, and then
transferred to 0.03% hydrogen peroxide for 1 h at 37°C. Controls were
performed in the absence of hydrogen peroxide.
Isolation of hemosomes
Engorged females were dissected on the day 10 after a blood meal (ABM) in
0.15 mol l1 NaCl, 20 mmol l1 sodium
phosphate buffer, pH 7.2, plus 10 mmol l1 CaCl2.
The gut content was centrifuged at 1000 g for 1 min and the
supernatant was discarded. The pellet was washed with the same solution
(3x) and further purified by means of a 4.5 ml Percoll0.2 mol
l1 sucrose (9:1 v/v), laid over a cushion of 0.5 ml 60%
sucrose and centrifuged at 53,000 g for 1 h in a Hitachi CB
ultracentrifuge (CA, USA).
Haem content and light absorption spectra
Alkaline pyridine-haemochrome derivatives were obtained as described by
Falk (1964), and visible
absorption spectra were obtained using a Zeiss spectrophotometer model
Spekord. Haem content was determined from the reduced minus oxidized spectra
of the pyridine alkaline derivative.
HPLC fractionation
High performance liquid chromatography (HPLC) was performed using a
Shimadzu LC-10AT (Maryland, USA) device equipped with a diode array detector
(SPD-M10A), and fractionation of hemosomes was performed using a Shimadzu
CLC-ODS column (15 mmx22 cm). Hemosomes were frozen, thawed, suspended
in deionised water and centrifuged at 12 000 g for 1 min. The
pellet, containing the hemosome core, was dissolved in 0.1 mol
l1 NaOH, and diluted 10x with solvent A [0.1 mol
l1 (NH4)H2PO4, pH 3.5, and
methanol (55:45, v/v)], and applied to the column. Solvent B was methanol. The
chromatography was performed using a 40 min gradient at a flow rate of 0.5 ml
min1, increasing the proportion of solvent B from 60% to
75%. Solvent B concentration was then raised to 100% and maintained for a
further 30 min.
Mass spectrometry
Mass spectra were acquired using a Finnigan LCQ-Duo ion trap mass
spectrometer (Finnigan, ThermoQuest Inc., San Jose, CA, USA). Samples from
HPLC fractionation of hemosomes, obtained as described above, were diluted
10x with HPLC-grade methanol (Carlo Erba, Milan, Italy), and introduced
into the electrospray source by injection of 10 µl samples through a 50
µm fused silica capillary at a flow rate of 510 µl
min1. Spectra were acquired at 3 s per scan over a
2002000 mass/charge (m/z) range. Source voltage was 4.5 kV,
and the electrospray ionisation (ESI) capillary voltage was set at 31.5 V.
Vaporizer and capillary temperatures were set at 32 and 200°C,
respectively. Spectra were collected in positive ion mode after instrument
mass calibration with an authentic haem standard (Sigma, St Louis, MI,
USA).
FTIR spectrometry
Dried samples of standard haemin (Sigma) or hemosome core (isolated as
described above) were used to obtain reflectance spectra, acquired for 60
cycles with a Fourier Transform Infrared (FTIR) spectrometer (Nicolet, Magna
550).
Elemental mapping
To evaluate iron and nitrogen concentrations in different compartments of
the digest cell, samples were processed as described for transmission electron
microscopy, without post-fixation, and analysed using a Zeiss 912 Omega
transmission electron microscope with a LaB6 filament. For
elemental mapping, unstained ultra-thin sections (nominal thickness
3050 nm) were analysed at 120 kV with an objective aperture of 12.5
mrad and an energy-selecting aperture of 20 eV. Images were digitised with a
14-bit slow scan CCD camera (Proscan, Germany) controlled by an image analysis
system (ESI Pro, SIS GmbH, Germany). Elemental maps were calculated with the
three-window power-law method for iron L edge (pre-edge images at 660 and 690
eV, post-edge at 720 eV) and the two-window method was used for nitrogen K
edge (pre-edge images at 390 eV, post-edge image at 410 eV).
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Results |
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On days 13 after the blood meal (ABM), digest cells are attached to the gut wall and have areas of direct contact with the basal lamina, which are reduced by day 5 and are no longer observed by the days 15 and 20 (Fig. 1). On the first day, the haem-peroxidase label was strongly concentrated on the basal surface of the digest cells, near to the basal lamina, suggesting that these cells were delivering haem to the haemolymph (Fig. 1A). By the third day the digest cells began to show heavily stained dark granules (Fig. 1B), hereafter called hemosomes, which progressively increased both in size and number and eventually occupied most of the cell cytoplasm 2 weeks ABM (Fig. 1D,E). The development of hemosomes occurred in parallel to the progressive detachment of the digest cells from the gut wall, which would presumably result in reduction of metabolite transfer to the haemocoel. These results imply a role for hemosomes as a site of haem sequestration.
Digest cells from the third day ABM were isolated by opening the midgut and gently washing the luminal content with phosphate-buffered saline. Observation of intact cells by light microscopy (Fig. 2A) revealed that blood digestion is clearly polarized in these large (>90 µm) cells. Typically, one side of their cytoplasm is occupied by large digestive vacuoles displaying an intense red colour, suggesting the presence of undigested haemoglobin, whereas hemosomes are concentrated on the other side of the cell (Fig. 2A). When squeezed between the lamina and the coverslip, the perinuclear location of the hemosomes in the digest cells is clearly shown during this initial phase of blood digestion (Fig. 2B).
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When the midgut was stained with DAB and observed by transmission electron microscopy, the fully grown, mature hemosomes appear as strongly labelled, very electron-dense structures (Fig. 3A). Hemosomes are delimited by a membrane and have a very compact core, which is surrounded by a cortical region encompassing several distinct layers. Fig. 3B shows a growing hemosome not yet fully developed, displaying strong peroxidase activity near the surface of the vesicle. The core of the hemosome comprises an assembly of homogeneous particles of haem aggregates (mean size 40±3 nm), which seem to detach from the hemosome membrane, suggesting a model mechanism for the formation of this structure (Fig. 3C).
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Hemosomes isolated by Percoll gradient ultracentrifugation were suspended in deionised water, frozen, thawed and centrifuged again. The pellet obtained, when observed by transmission electron microscopy, shows the core of the hemosome displaying almost its original appearance, only the more external layers being removed by this treatment (Fig. 4A). The hemosome pellet was dried, weighed and the haem content measured by the alkaline pyridine derivative, revealing that haem alone accounts for 90±3% of the composition of this organelle. The haem aggregate from the hemosome preparation was dissolved in NaOH, isolated by reverse phase chromatography and analysed by electrospray ionisation mass-spectrometry (ESI-MS) (Fig. 4BI). A major singly charged ([M+H]+) ion species was observed at m/z 616.4 (Fig. 4C). When submitted to sequential fragmentation by tandem ESI-MS/MS, this ion species gave rise to daughter ions of m/z 557.4, 498.5, 483.5 and 468.6 (Fig. 4DF). The same ion fragments were obtained from an authentic haem standard (Fig. 4GI). Therefore, we can conclude that hemosome-derived fractions showing absorption at the Soret band (398 nm) are actually iron-protoporphyrin IX (Fig. 4J). Furthermore, ESI-MS data show that there is no covalent modification of the haem molecule upon haemoglobin digestion and accumulation into the organelle. However, the FTIR spectrum of hemosome is clearly distinct from that of standard haem (Fig. 5). Some peaks found in the hemosome core could be assigned to other non-haem components of the aggregate (indicated by asterisks in the figure). A remarkable feature, however, is the absence of the characteristic 1704 cm1 carboxylate stretch (arrow), which suggests that these groups are interacting with other components of the aggregate.
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Digestive vacuoles are protein-rich structures, and therefore a high local concentration of nitrogen was expected. Haem accumulation, on the other hand, should result in a local increase of iron concentrations. Elemental mapping of the hemosome by energy-filtering transmission electron microscopy was used to evaluate nitrogen and iron concentrations. Based on the results shown in Fig. 6, we propose a maturation cycle for this organelle. Typically, hemosomes are smaller on day 4 ABM and present the iron-rich core surrounded by a boundary, with lower density of both iron and nitrogen. They were frequently found in close association with the much larger digestive vacuoles, which showed a strong nitrogen signal, together with a lower concentration of iron. On day 8 ABM, hemosomes larger in size and of compact aspect were more frequent and the clear, low-iron boundary between the core and the surface was no longer observed in these vesicles. By day 20 ABM, no more digestive vacuoles were found and the hemosomes had become less dense structures, with several distinct internal layers on both the iron and nitrogen maps.
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Discussion |
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We have shown previously that during the initial days after a blood meal
(ABM) there is a huge accumulation of haem in the oocytes
(Braz et al., 1999), and also
have presented evidence that this haem originates from the haemolymph and is
delivered by a haemolipoprotein called HeLp
(Maya-Monteiro et al., 2000
).
These results are in agreement with the observed intense concentration of haem
at the basal surface of the digest cell during the initial days ABM, which
coincide with the period of most intense vitellogenic oocyte growth,
suggesting that by this time the absorption pathway seems to predominate over
hemosome formation (Fig. 1A,B).
This phenomenon is dependent on the accumulation of vitellin, the major yolk
protein of oviparous animals (Sappington
and Raikhel, 1998
), which in ticks is a haemoprotein
(Rosell and Coons, 1991
) that
functions as a haem reservoir to support embryo development
(Logullo et al., 2002
).
One important question concerns the cytoplasmic route taken by haem on its way from the digestive vesicles to the haemocoel. The haem might be exocytated by the digest cells at its basal surface or, alternatively, might be transported through the membrane of the digestive vesicles to the cytoplasm and then transferred to the haemocoel via the cellular membrane. Transmission electron microscopy, using 3-3-diaminobenzidine, did not provide a definitive answer to this question, but, based on haem-peroxidase activity, we observed that it is not only the digestive vesicles that are stained by DAB, but also the cytoplasm itself, which seems to have a higher haem content compared to the cytoplasm of the neighboring basophilic cells (F. A. Lara, data not shown). We were not, however, able to find DAB-positive exocytic vesicles at the basal surface of digest cells. Taken together these data would favor the hypothesis of a haem transport pathway from the digestive vesicles through the cytosol to reach the haemocoel at the basal surface.
After the first week ABM, the intensity of haem absorption seems to
decrease, with a decline in the concentration of haem close to the haemocoel
and reduction of contact of digest cells with the basal lamina
(Fig. 1D,E), when compared to
the previous days. At this time, haem released from haemoglobin in midgut
cells becomes directed to hemosomes, which are the most prominent organelle of
senescent digest cells (Fig.
1D,E). Almost 10% of the haem present in the meal of an adult
fully engorged female is found in its ovary
(O'Hagan, 1974) and by the end
of digestion (2 weeks ABM) >98% of the haem in the gut is associated with
hemosomes (F. A. Lara, data not shown). These structures have been described
by other authors as residual bodies, and are usually excreted along with the
faeces at the end of digestion (Tarnowski
and Coons, 1989
). Iron was suggested to be their main constituent
and therefore these structures were named siderosomes in earlier literature
(Walker and Fletcher,
1987
).
Here we show that haem, and not iron, is the major component of these
vesicles, accounting for 90% of its mass. By means of sequestering most of the
haem in excess of its physiological demand inside the hemosome, the tick
counteracts any haem toxicity that could otherwise result in severe tissue
damage. Thus hemosome formation should be regarded as a detoxification
mechanism, functionally equivalent to the formation of haemozoin, a
crystalline haem aggregate that occurs in other haemoglobin-eating parasites
such as Plasmodium (Slater et
al., 1991), Rhodnius prolixus
(Oliveira et al., 1999
) and
Schistosoma mansoni (Oliveira et
al., 2000
). The haem aggregate found in the tick hemosome is not
haemozoin, however, as revealed by its distinct FTIR spectra
(Fig. 5) and by the absence of
birefringence observed by polarizing microscopy and the lack of X-ray
diffraction (M. F. Oliveira, data not shown).
With respect to hemosome structure, the absence of a 1704
cm1 carboxylate stretch in the FTIR spectrum
(Fig. 5) suggests that haem
association to non-haem components of the hemosome involves the lateral
propionate chains of the porphyrin ring. Haem iron-carboxylate bonding
as found in haemozoin can be excluded by the absence of the 1660
cm1 peak, suggesting that the hemosome core comprises a new
type of haem-based supramolecular structure. Characterization of the non-haem
components of the hemosome and of the molecular structure of this haem
aggregate are underway in our laboratory. Another essential feature is the
presence of a delimiting membrane (Fig.
4), indicating that the hemosome and digestive vacuoles are
distinct structures and pointing to the existence of a haem transport system
between both cellular compartments. Blood-feeding organisms are found in very
different taxonomic groups and all have haem as the main end product of
haemoglobin digestion, frequently being described under the generic
denomination of haematin in the previous literature. Structural
characterization of a haem aggregate is only available, however, for
haemozoin. The presence of haemozoin has been investigated in several species
of haematophagous animals but with negative results in most cases, except for
Plasmodium, Rhodnius and Schistosoma, as mentioned above
(Oliveira et al., 1999,
2000
). To our knowledge this
is the first characterization of a non-haemozoin haem aggregate. A goal for
future research is to compare the structural organization of haem aggregates
in different blood-feeding organisms.
In most organisms, during turnover of haem proteins the protoporphyrin ring
is cleaved by haem oxygenase and the iron atom is eventually reused for de
novo synthesis of haem (White and
Granick, 1963; Ponka,
1997
). The only exceptions to this are some pathogenic protozoa
and bacteria, in which a complex array of proteins has evolved to take up haem
efficiently from the vertebrate host
(Wandersman and Stojiljkovic,
2000
). Among higher organisms, the tick Boophilus
microplus (Braz et al.,
1999
) and the hemipteran Rhodnius prolixus
(Braz et al., 2002
), both
blood-sucking arthropods, are the only exceptions reported to date. We have
recently shown that HeLp (Haem Lipoprotein), the major lipoprotein of
Boophilus microplus haemolymph, is capable of binding haem and
delivering it to the tick tissues
(Maya-Monteiro et al., 2000
).
The same protein has also been described in two other tick species and
therefore its presence may be a common feature of ticks
(Gudderra et al., 2002
).
During the first week ABM, haem absorption is more evident and digest cells
that attach to the basal lamina may have direct contact with the haemocoel, so
it is conceivable that direct transfer of haem from digest cells to HeLp may
occur. Haem is never found free in its unbound form, however, due to its
potential toxic effects previously mentioned, and is always associated to
proteins. The transfer of haem to HeLp must therefore be preceded by transfer
from the digestive vacuole to the surface of the digest cell. The precise
pathway may include the hemosomes, which also concentrate close to the basal
lamina at the same time, as an intermediary compartment. Alternatively, the
hemosome may represent a divergent route taken by haem when its levels exceed
the tick's needs. Haem-binding proteins participating in intracellular
trafficking may be the targets for the development of new methods of acari
control, as they participate in a detoxification pathway that is specific for
this group of animals.
Taken all together, our data describe a new process for haem detoxification by means of sequestration inside the hemosome. The ability to form this structure is proposed to be an important adaptation of this organism in order to use vertebrate blood as its sole food source.
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Acknowledgments |
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