Volutin Granules of Eimeria Parasites are Acidic Compartments and Have Physiological and Structural Characteristics Similar to Acidocalcisomes

LIA CAROLINA SOARES MEDEIROS,"'" FABIO GOMES," LUIS RENATO MAlA MACIEL,d SERGIO HENRI QUE SEABRA,"

ROBERTO DOCAMPO,r SILVIA MORENO,'" HELMUT PLATTNER,s JOACHTM HENTSCHEL,g URARA KA WAZOE,h HECTOR

BARRABIN," WANDERLEY DE SOUZA,"'" RENATO AUGUSTO DAMATTAJ and KILDARE MIRANDA"'c "Labortltr!rio de Ullraesiruiura Celular Herlh" Meye r. 1/l.,·lilLlto de Bio,,:" ica Carlos Chagas Filho and lnslilllio Nacional de C ilncia e

Tecnologia em Biologia Estrutural e Bioimagcns, Unillesidade Federal do Rio de Jan eiro, A II. Carlos Chaga.\· Filho, 373, bloco G subsolo, Cidade Ullil'ersilCfria, flha do Fundao, Rio de Janeiro, RJ 21941-902, Brazil, and

bLaboralario de Membranas Transporladoras, lnslillilo de l:JioqLIIllI.ica Mer/iea, Univesidade Federal do Rio de Janeiro, A 1'. Carlos Chagas Filho, 373, bloco H segulldo alldar, saia 25, Cidade Ullillersildria, llha do FlIndao, Rio de .Janeiro, RJ 21941-902, Brazil, and

cDireloria de Progral1la ,\', lnstilulo Naciollal de Metroiogia, Norlllaliza(,x{o e Qualidade Induslrial- INMETRO, RJ 25250-020, Brazil, and dLabOr£llrfrio de Biologia CelLilar e Tecidual, Cenlro de Biocilncias e Biolecnologia, Universidade ESladual do Norle Flumin.ense, Campos dos

GOYlacazes, RJ 280/3-602, Brazil, and cLaboraLO'rio de Teciloiogia elll CuilUra de Ce7u.las, Cenlro Universildrio ESladual da Zona Oesle, R.! 21070-200, Brazil, and

rDeparlmenl of Cellular Biology, Cenlerfor Tropical and Emerging Global Diseases, UniversilY ~f' Georgia. Alhens, Georgia 30602, USA, and gFachbereich Biology, Universilcic KOlIstall.Z, KOlI.slall.Z 7H457, Germany, and

hLaboratorio de Coccidiose A vicfrio, Departamento de Biologia Allimal, InSliluto de Biologia, Universidade Eslac/ual de Call1pil1as (UN/CAMP), Campinos, SP 13083-970, Brazil

ABSTRACT. The structural organization of parasites has been the subject of investigation by many groups and has lead to the identiflcation of structures and metabolic pathways that may represent targets for anti-parasitic drugs. A specific group of organelles named acidocaJci­somes has been identified in a number of organisms, including the apicomplexan parasites such as Toxoplasma and Plasmodium, whe re they have been shown to be involved in cation homeostasis, polyphosphate metabolism, and osmoregulation. Their structural counterparts in the ap icomplexan parasite Eimeria have not been fu ll y characterized. In this work, the ultrastructural and chemical properties of acidocalcisomes in Eimeria were characteri zed. Electron microscopy analysis of Eim.eria parasites showed the dense organelles called volutin granu les similar to acidocaJcisomes. Immunolocali zation of the vacuolar proton pyrophosphatase, considered as a marker for ac idocalcisomes, showed la­beling in vesicles of size and distribution similar to the dense organelles seen by electron microscopy. Spectrophotometric measurements of the kinetics of proton uptake showed a vacuolar proton pyrophosphatase activity. X-ray mapping revealed signi ficant amounts ofNa, Mg, P, K, Ca, and Zn in their matrix. The results suggest that volutin granules of Eimeria parasites are acidic, dense organelles, and possess structural and chemical properties analogous to those of other acidocalc isomes, suggesting a similar functional role in these parasites.

Key Words. Acidocalcisome, Eimeria acervulina, Eimeria lenella, elemental mapping, vacuolar proton pyrophosphatase, volutin granu le.

E fMERIA species are intracellular protozoan parasites that in­fect poultry flocks all over the world, causing major parasitic

diseases of great economical importance. Infec tion by Eimeria species is responsible for considerable economical losses in poultry production, estimated to be 700 million dollars per year in the United States (Lillehoj et a l. 2007).

Several studies on the structural organ ization of parasitic pro­tists have shown that they conta in unique organell es and in trace l­lular structures th at are interesting e ither from the ce ll biology point of view, because new structures, interaction mechanisms, and metabolic reac tions are ide ntifi ed, or from the chemotherapy side. because the identificat ion of novel structu res and their physio­logical roles may reveal potential biochemical targets for the raLional developme nt of anti-parasitic drugs (De Souza 2002; Docampo 2008). In this regard, combined structural and bio­chemical studies on the mechanisms involved in the regulation of ion transport in protist parasites have led to the identification of an organel le named the acidocalc isome (Verces i, Moreno, and Docampo J 994; revie wed in Docampo et al. 2005).

The first microscopical observations of acidocalc isomes showed that they share some characterist ics w ith volut in g ran­ules, such as stain ing with basic dyes and a high calcium a nd phosphate content. For this reason, they have been considered to

Corresponding Author: K. Miranda, Laborat6rio de Ultraestrutura Celular Hertha Meyer, Instituto de Bioflsica Carlos Chagas Filho and Jnstituto Naciona l de Ciencia e Tecnologia em Biologia Estrutural e Bioimagens, Univesidade Federal do Rio de Janeiro, Av. Carl os Chagas Filho, 373. bloco G subsolo, Cidade Universit,;ria. Tlha do Fundao, Rio de Janeiro, RJ 2 1941 -902, Brazil- Telephone/FAX number: 55 2 1 2260 2364; e-mail: kl11iranda@biof.uflj.br

correspond to vo lutin or metachromatic granu les described previ­ously in some bacteria and protists (Kornberg 1995 ; Meyer 1904) . Acidocalcisomes have been characterized by their ac idi c nature, high electron de ns ity , and high concentration of e lements, such as Na, Mg, P, K, Ca, Fe. a nd Zn. which can be detected either by fluorim etric assays using appropriate fluorescent probes (Miranda et al. 2005; Verces i et al. 1994) or by analyti cal elec tron micro­scopy (LeFurgey et al. 2005; Miranda et al. 2000, 2004a, b, c, 2008; Scott e t al. 1997; Vickerman and Tetl ey 1977). Cat ion uptake and re lease and water transport across these organelles are £owered by a number of ion pumps (i.e. Ca2

-1--ATPases, V-H -ATPases,

V-H + -PPases) , exchangers (i.e. Na+/J-T+ and Ca2 +/H + exchang­ers), and channels (i.e . C I - channel, aquaporin) present in their enc losing membrane (reviewed in Docampo el al. 20(5). Because 01' the presence 01' these transport mechanisms, these organelles have been regarded to participate in several biological functions , such as control of intrace llul ar pH, ca lc ium homeostasis , ion stor­age, polyphosphate (po ly P) metabolism, and osmoregul ation (Besteiro e l a l. 2008; Fang et a l. 2007 ; Miranda el al. 2008; Rohloff and Docampo 2008; Schoijet et al. 2008). Little is known abou t acidocalcisomes in ap icomplexan parasites and most 01' the studies have been done in 7oxopiasI1la gondii. The biochemical characterization of acidocalc isomes was carried out mainly llsing permeab ili zed ce ll s and ce ll fractions of tachyzoites. and by e lec­tron microscopy. These studies have shown that the physiological and structural properties of T. gOlldii acidocalcisomes art: sim il ar to those that have been described previous ly for trypanosomes, where they are better characterized (Lu et al. 1998; Moreno and Zhong 1996; Rodri gues et al. 2000). The presence of acidocalci­somes in Plasmodium species has been reported (Bakar et al. 2009; Marchesini et al. 2000; Ruiz et al. 20(4), although littl e

416

informati on on their physiol ogical properties has been provided and the characteri zation of their fine structure in different developmental stages of the paras ite is lacking. The presence of acidocalcisomes in Eimeria parasites has for long been neglected and only a few images suggesting the presence of acidic organelles that resemble in shape acidocalcisomes of T Kondii have been shown in Eimeria acervulina (Miranda et a l. 2008). As with Plasl1/­odium spec ies, any detail ed structural and physiological character­izati on of these organe ll es in Eimeria parasites is lacking as well.

MATERIALS AND METHODS

PI'oduction of Eimeria acel"l'ulilla and Eimeria tellella 00-

cysts and purification of spol·ozoites. Hy- line chickens were reared from I d of age with ration free of anticoccidial drugs and water ad libitum in a pathogen-free room at the Avian Coccidiosis Laboratory , Department of Animal Biology, Institute of Biology, Universidade Estadual de Campinas (UNICAMP), Campinas, Sao Paulo , Brazil. Four-week-old chickens were inoculated with I x 104 sporulated oocysts of E. acervulina "Cu" strain (Kawa­zoe et a l. 2005) or 5 x 103 sporulated oocysts of E. ten ella "Pa" strain . Eimeria tenella "Pa" strain was isolated from chicken fe­ces at "Sitio Palmito" city of Frutal, state of Minas Gerais. in a backyard without contact with chickell food or anticoccidial drugs and kept in liquid nitrogen in the Laboratory of Avian Coccidi­osis, Biology Tnstitute at Universidade Estadual de Campinas, Sao Paulo, Brazi l. Oocysts were isolated from feces and sporulated after I wk of inoculation according to the procedure of either Long et al. (1976) and Chapman (1978) . Sporozoites, obtained frum oocysts after mec hanical rupture, enzymati c treatment, and purification by anion-exchan ge chromatography on D E-52 cel lu­lose columns (Schumatz, Crane, and Murray 1984), were col­lected and stored at 4 °C.

Visualization of acidic compartments. Sporozoites were in­cubated for 10 min with 25 ~lg/ml of acridine orange in phosphate­buffered sal ine (PBS), washed twice, and wet-mounted. Images were obtained in a Zeiss Axioplan epifluorescence microscope (Zeiss, Oberkochen, Baden-Wurttemberg) equipped with a 488-nm excitation filter set. Emi ssion signal (above 500 nm) was detected with a Hamamatsu digital CCD Model C5R 10 camera (Hamamatsu, Hamamatsu City, Shizuoka, Japan) and an image analysis sys tem attached to the microscope.

Vacuolar pl'oton pyrophosphatase (V-H + -PPase) activity. Acidification of internal compartments was followed by measuring changes in the absorbance of ac ridine orange at the wavelength pair 493- 530 nm in an SLM-Aminco DW2000 dual-wavelength spectrophotometer (SLM-Ami nco, Rochester, NY). Sporozoites of E. lene/ta (0.1 mg/ml of protein) were incubated at 28 "C in 2.5 ml standard reac ti on medium , containing 130 mM KC I, 2 mM MgCI2 , 111M oli gomycin , 10 mM H EPES buffer, pH 7.2, with 16 ~lM digitonin before addition of 3 ~lM acridine orange. PPi­driven proton uptake by the acidocalcisomes was measured with the addition of 100 pM sodium pyrophosphate to the reaction me­dium containing digitonin permeabilized ce ll s. The results shown are representati ve of at leas t three ex periments.

ImmunoftuOI'esccnce miCl"oscopy. Cells fixed in freshly pre­pared 4% (v/v) formaldehyde in PBS were allowed to adhere to poly l.-Iysine-coated covers lips, permeabilized with 0.3% (v/v) Triton X-J 00 in PBS , washed, incubated with 50 mM ammonium chl oride in PBS , and blocked with 3% (v/v) bovine serum albumin in PBS . Pol yclonal antibodies rai sed in rabbits against the putative hydrophobi c loop 111 of AraiJidopsis thalialla V-H + -PPase were kindly provided by Dr. Phillip Rea, University of Pennsylvani a (Sara fian et a l. 1992). Immuno fluorescence was carried out by incubating adhered permeabili zed cell s in a I: 100 dilution of the antibody for I h. After washing, a I : 100 dilution of lluorescein

417

isothiocyanate-coupled goat ant i-rabbit IgG was used as a sec­ondary antibody. Tmages were obtained in Zeiss Ax iopl an fluo­rescence microscope at the same conditi ons used for imaging acridine orange stained cells. Images were deconvolve d using a no neighbor method and a deblur algorithm.

Sequence alignments. Multiple alignments between a con­served domain of Arabidapsis t/w/ial1a vacuolar proton pyro­phosphatase (326-pept ide sequence-Loop XII, 38 amino ac ids GPTSDNAGGIAEMAGMSHRIRERTDALDAAGNTTAATG), Tlypal/.osoma cru.zi, T. gonciii, and Plasmodium fatcipal"l/II/ (http://www.ncbi.n lm.nih .gov/blast), and sequences from Eimeria database deposited at the Sanger Instirute (http://www.san­ger.ac.uk) were conducted using the NCBT BLAST server (http://www.ncbi.nlm.nih.gov/blast) and the CLUSTALW2 pro­gram using defau lt parameters (Larkin e t a1. 2007).

Electl"On microscopy of ultrathin sections. Sporozoites of both species were washed in PBS , pH 7.2, fi xed in 2.5% (v/v) glutaraldehyde plus 4% fo rmaldehyde in 0 . 1 M sodium cacodylate buffer, postfixed in 1% (w/v) OS04, and embedded in Polybed812 epoxide resin. Sections were sta ined for 30 min in uranyl acetate, 5 min in lead citrate, and observed in a JEOL 1200EX elec tron microscope (.TEOL, Akishima. Tokyo, Japan) operat ing at 80 kV.

Imaging of unfixed whole cells. Cells were washed in PBS and resuspended droplets were applied to 100-mesh fonnvar­coated copper grids, allowed to adhere for 10 min , carefully blot­ted dry , and observed in an energy-filtering LEO EM 912 electron microscope (Zeiss) operating at 80 kV. E lectron spectroscopi c images were recorded at an energy loss of ~ 60eV with spec­trometer slit width of 20 eV.

Morphometric analysis. Volumetric density of the acidocalci­somes was determined in thin sections. Randomly selec ted cells were observed in a Zeiss EM 900 transmiss ion electron micro­scope (TEM) equipped with a Megaview III camera and an iTEM image analysi s system. Profiles of 73 cell s of each Eimeria species were acquired and measured. Number and absolute volume of acidocalcisomes were estimated using whole cells prepared as described above. Absolute volume of ac idocalcisomes was deter­mined by measuring the di ameters of ~ 50 organelles assuming they are spheres. Statistical s ignificance was determined by Stu­dent's I-test (P<0.05).

Electron pl"Obe X-my microanalysis and elemental mapping. Energy dispersive X-ray spectra were recorded from the dense ves­icles of whole cells dried onto Fonllvar-coated grids. Control spectra were collected from regions adjacent to the ac idocalcisomes and from the Formvar film. Specimens were analyzed in a LEO 912 Omega scanning transmission electron microscope (STEM; Zeiss). X-rays were collected for 200 s using aLi-drifted Si-detector (front area 30mm2

) equipped with an ATW atmospheric window. The microscope was operated at 80 kV using a tungsten filament, in the STEM imaging mode with spot size set to 40 nm. Analyses were performed using a Link multichannel energy analyzer and Link ISIS 3.00 software (Oxford Instruments, W iesbaden, Germany).

Localization of poly P. Sporocysts of E. acervulill(l and E. tmel/a were tlxed with 4% (v/v) formald ehyde in PBS for 10 min , permeabili zed with 0.3% (v/v) Triton X- IOO for 5 min . and incubated at room temperature with 50 ~lg/ml of the fluores­cent dye 4',6-diam idino-2-phenylindole (DAPT) for 30 min . Sam­ples were mounted on glass s lides and observed in a Zeiss Ax ioplan flu orescence microscope at an exc itati on wavelength of 370 nm and emission filtered lower than 500 nm. Images were deconvolved using a no neighbor method and a deblur algorithm .

RESULTS

Volutin granules of Eimeria parasites al'e acidic. Sporo­zoites of E. acervulina and E. tellel/a incubated in the presence of

418

Fig. 1,2. Fluorescence detection of ac idic compartments of Eimeira tlcerl'lllil1lf and Eimeria t (, llella spo)"ozo ites . Several acidic organelles (stained in orange) are observed in the cytoplasm of E. ace rvuiil1 (1 (1) and E. leI/eli" (2). Note bindi ng of ac ridi ne orange to t.he nucleus and the refractil e body (green). Scale bars = 3 ~1I11 .

acrid ine orange showed orange labe ling in a number of ves icles and intracellul ar structures (Fig. I , 2, respective ly). A lthough green labe ling also occurred in the nucle us and in the refracti le

bod ies, due to intercalat ion of the dye w ith compo nen ts of these organe ll es, the num be r and di stribution of o ra nge-l a beled vesi­cles , presumably correspond ing to volutin granules, was similar to that obse rved for these organe lles us ing other techn iques.

Immunolocalization of the vacuolar proton pyrophospha­tase and pmton pyrophosphatase (V-H + -PPase) activity. An­tibod ies ra ised aga inst a conserved dom a in (326 p eptide) of A. thaliana vacuo lar proton pyrophosphatase (V-H + -PPase) have been shown to recogni ze ac idoca lcisomes in a number of paras ites (Miranda et al. 2004c; Soares M ede iros e t al. 2005). To confirm that the 326 domain was conserved in Eimeria parasites , sequence a lig nme nts o f the 326 peptide from A. thalialla V-H + -PPase w ith Eimeria seque nces were carri ed uul. Results shu wed that this du­m ain is h ighl y conserved in Eimeria paras ites, as occurs with other parasites suc h as Toxoplasma and Plasmodium and Try­I Ja IIO.\' 0 111(1, whe re the presence of ac idocalcisomes has been ex­tensively de monstrated (Fig. 3). Immuno fluoresce nce results revealed labe ling in intracellular organe ll es in both Eimeria species (Fig. 4A,B, SA,B) w ith size and distributi on similar to w hat was seen previously afte r ac ridine orange labeling (Fig. 1,2). M easure ment o f the pyrophosphate-driven H + uptake (i.e. vacuolar proton-pyrophosphatase act ivity) , ca rri ed o ut in per­meabili zec1 cells showed that acridine o ra nge uptake (ind icating ac idification) was tri ggered by add ition of 0 .1 mM sodium pyro­phosphate . Addit io n of 1 ~lM ni geri c in aboli shed ac ridine orange uptake (Fig. 6), suggesting an intrace llul ar PPi -dependent

3 T_90ndii Eimeria_sp P_£alciparum A thaliana T_cruzi

GPISDNAGGl AEMAGLGPEVRSRTDALDAAGNTTAAVG 39 GPI SDNAGGI~~LLGEEARSRTDALDAAGNTTAAVG 39 GPISDNAGGl AEMAGLFSEVReRTDILDAAGNTTAAIG 39 GFISDNAGGl AEMAGMSHRl RERTDALDAAGNTTAAIG 39 GPISDNAGGl AEMAHMGHEl REI TDALDAAGNTTAAIG 39 ************** * ** **** **** ** : *

4mio

Fig. 3-6. Antibodies raised aga inst a plant vacuolar proton pyrophosphatase (H+ -PPase) recognize Eillleritl sp. structures that are assoc iated with the Eimeria acidoca1ci somes. 3. Sequence ali gnment 01' the 326 peptide from Arabidupsis t/wlial/a with sequences from Eillleria and other parasites shows the conservation of the "326" domain . 4, 5. Immunofluorescence microscopy using ant ibodies rai sed agai nst A. I/W/illlw vacuolar proton pyro­phosphatase (Y-H+-PPase) shows labeling of large spheri ca l int race llular structures of Eilileria ace rvuliI/O (4A, B) and Eillleria lel/elia (5A, B), in a disposition sim il ar to the ac idoca lcisomes observed in whole cell images. Scale bars = 10 pm . 6. Pyrophosphate (PPiJ-drivcn proton uptake in digitonin­permeabi li zed sporozo ites of E. lenella. Traces are representative of three diFferent experiments. Trace before PPi add ition represents the control.

419

Fig. 7-12. T hin secti ons of sporozo ites o f Eimeria acervulilla (7- JO) and Eimeria leI/ ella ( II, 12) aftc r conventi o nal glUlara ldehyde-osmium fi x­ati o n. Cell s display circul ar vacuoics with a dense mate ri a l assoc iated with th c inner face 01' the ir mem brancs s imil ar to th at observed in thc acid­ocaic isomes. AcidocaJci somes have s ingle membrane (9. whi te head arrow) with d iffe rent deg rees of preserva ti o n of th e dense materi al, from an a lmost to ta ll y em pty profile to vacuo les cOnLa ining a conside rable amount of dense mate ri a l ( 10, 12, white arrows). Appa re nL fus io n betwee n these organe ll es was seen (8) resulting in a polymorphic organi zati on. Sca le bars: 7 = I ,1m; 8 = 400 nl11 ; 9, 10 = 200 nl11 ; I I, 12 = I ,1111 .

420

10' p o

Ca

14

Ni

rr;-I LLlt.J

10

Fig. 13-1 5. Whole cell s show spheri cal acidocalc isomes that are the main s ite For cation storage in Eimeria parasites. ]3. Electron spec troscopic imag ing of who le unfi xed Eillleria (/ce rvulil1a (ilE betwee n 60 and 80 kV) . 14. Corresponding X- ray spectrum of th e acidocalcisomes in th e inset to Fig. 13 using energy- filtered scanning transmission electron mi croscopy (EFSTEM). Nickel peaks in the spec trum came fro m the grid and s il icon from th e detector itselF. Carbon and sulFur s ignal s were s imilar in the ac idoca lcisomes and control regions (cytoplasm, data not shown). Inset shows an increased sca le of the peak representing Z n. 15. An EFST EM electron spec troscopi c image of a porlion of th e cell di splayed in Fig. 13, inset (ilE between GO and 80 kY ). Ele menta l images o f the cell di splayed correspond to (P) phosphorus, (0) oxygen, (Ca) calc ium, (Mg) magnes ium . and (Zn) 7. inc . Scale bars: 13 = 3 flm; 15 (EFSTEM) = 500 nm ; P, 0 , Ca, Mg and Zn = 500 nm .

acidification process. Accordingly, ce lls incubated in the absence of Na-PPi , did not show acidificat ion (data not shown).

Ultrastructural analysis of acidocalcisome-Iike organelles. Tn thin sections, the volutin granules of both Eimeria species ap­peared as ves icles with a dense material apposed to the inner face of the membrane (Fig. 7- 12). Sporozoites showed volutin granu les w ith different degrees of preservation of the dense material , from an

almost totally empty profile to vacuoles containing a considerable amount of dense materi al (Fig. 10, 12, white arrows). Fusion between these organelles was occasionally observed (Fig. 8).

Elemental composition and morphometl'ic analysis of' volu­tin granules. The size, d istribution , and ultrastruc tural charac­te ri stics of the volutin granu les seen by e lec tron microscopy toge ther with their acidic nature and presence of a V-H +-PPase

activity strong ly indi cated that they mi ght correspond to ac id­ocalc isome- like structures. One e ffki ent technique that has been appli ed to the ob~ervation of acidoca lc isomes or organe ll es with s imilar e lectron scattering properties is the preparation or whole cells directly dried on Formvar-coated grids followed by ob~er­vation of contrast-tuned images in an ene rgy-fi ltered transmiss ion e lec tron mi croscopy (EFTEM). Tn these preparations, sphe rica l de nse organelles randomly spread throughout the cell body could be readily observed (Fig. 13). Approximately 40 ac idocalci somes, w ith diame ters between 100 and 400 nm , were observed per ce ll. Morphometri c a nalysi s showed that they occupied ~ 1.52% of the total volume of the ce ll (Tab le I ). X-ray mi croanalys is (Fig . 14) reveal ed considerabl e amounts of oxygen, magnesium , phos­phorus, ca lc ium , and z inc concentrated in these compartments. E leme ntal mapping showed that these e le me nts were mainly concentrated in the matrix of the de nse organell es (Fi g. 14, 15). A lthough very weak, the signal of z inc is present both in the spec trum and the map (Fig. 14, inset) .

Poly P localization. Polyphosphate was locali zed by i1uores­cence microscopy in DAPT-stained samples according to the pro­ced ure described in " Materials and methods." F luorescence emi s~ i o n of DAP! shifts to a longer wavelength (to 525 nm) when it binds to poly P , g iving a green-ye llow emission color in po ly P containing structures. Poly P was present in s mall vesic les present ins ide the sporocysts of E. ac:ervulina and E. tenet/a with s ize and di stributi on similar to what was seen in sporozoites of both

42 1

Eilileria spec ies after ac ridine o range labe ling and immuno local­izat ion with anti bodies aga inst the H + -PPase (Fig. 16, 17).

DISCUSSION

Apicompl exans are natura l invaders with a compl ex li fe cycle , usually involving more than one host. T herefore, they must be prepared to face differe nt e nvironmental conditions, which re­quire ex treme changes in metaboli sm. Acidocalc isom es have been associated w ith a number of' functions in e nvironme nta l adapta­tion of severa l paras ites , s uch as osmoregu lation and pH homeo­stasis. In trypanosomatids, the participation of acidocalci somes in the regulatory volume decrease mechanism , poly P me tabolism , and calc ium storage has been de monstrated (Docampo e t a l. 2005). Tn T. gondii , acidoca lcisomes are the largest s tore or Ca2+ (Bouchot e t al. 1999; Luo e t a l. 200 I ; Moreno a nd Zhong 1996), indi cating a poss ible role of thi s organelle in cell signaling during host ce ll invasion and egress , processes that are crucial for maintaining the ir life cycle . Large amounts of short and long c hain poly P were detected in different blood stages of P. falcip ­arwl! whose concentrations increase dl11'ing differentiation of the blood stages, reaching maxim al concentrat ions in the trophozoite s tages and then decreasing in the schi zonts (Rui z e t al. 2004). This wou ld be compatible with a role fo r ac idocalc isomes in the adaptation of apicomplexan parasites to env ironmental stress

Fig. 16, 17. Loca li za ti on of po ly phosphate (po ly P) in sporocysts of Eim e ria ace lvltli l/" ( 16) and Eim eri" leI/ eli" ( 17). To localize poly P, spo rocysts of both Eim er;a spec ies were incuhated wi th 4 ',6-d iamidino-2-pheny li ndo le (DA PI ). Differential interferentia l contrast microscopy of sporocyst, of E. acerIlU/;1/{1 ( 16A) and E. teoella ( 17A); 168, 178. Fluorescence images o f DAPI staining , showing several organelles containing poly P. sim ilar in s ize and di stribution to what corresponds to th e ac idocalc isol11es in sporllzo ites . ( 16C, 17C) Merge. Scale bars = 3 ;1111 .

422

Table 1. Morphometri c analys is of ac idocalc isomes of Eimeria lellella.

Acidoca lci somes Refracti Ie body

Number per cell

40 ± 8 I ± O

Stat isti cal signifi cance was determined by Student's I-test (P < O.OS). Values are expressed as means :1:: SEM (n = 73). NO, not determined.

(Miranda et al. 20 I 0) , a nd suggest that the same function s may be present in Eimeria paras ites . Fusion between these organelle s was occas io nal (Fig . 8), a s obse rved before in T gOlldii (Miranda e t al. 2008) a nd Leishmall.ia paras ites (Miranda e t a l. 2004c), whe re polymorphic acidoca lc isomes have been seen. These fus ion a nd budding events indi cate that the ir parti c ipa tion in the physio lo gy of the parasite is dy nami c.

H e re, we have shown that the ultrastructural , c he mi cal, and phys iologica l prope rti es of volu tin g ra nules of E. ten ella and E. acervulina resemble in severa l as pects acidocalc isomes o f Toxo­plasma , Plasmodium., and trypanosomatid species. F luo rescence mic roscopy of acridine o range-sta ined cell s , spectrophotometri c measurements of the ki ne tics of pyrophosphate-driven proton uptake, and poly P locali zation suggest that both Eimeria spec ies have org ane lles w ith phys io logical propert ies a na logous to those of acidocaJcisomes so far c haracteri zed (Docampo e t al. 2005 ; Miranda e t al. 2008) . Tn add ition , thin sections and EFTEM im­ages of w ho le intact sporozoites showed the presence of a number of de nse organe ll es, 100-300 nm in diamete r. The advantage of thi s type of preparation is the obse rvatio n of the whole paras ite (and whole organe ll es) without the poss ible s ide effec ts of fi xa­tives a nd other c he micals used in the rout ine processing necessary for TEM. This reduces s ig nificantl y the ex trac tion of the mine ra l content from the ac idocalcisomes and, therefore, allows the qual­itative d etec ti o n of several cat ions within the organelle by analyt­ica l T EM (Miranda e t al. 2000, 2004a, b , c, 2008 ; M o re ira e t a l. 2005; Soares M edeiros e t al. 2005) . Tn additi o n, as cell s are imaged as a whole m ount, seve ra l mo rphom e tri c parame te rs suc h as the numbe r o f acidocalcisomes pe r ce ll , the ir shape and mean vo lume is carri ed out with hi ghe r prec ision . Furthe rmore, X-ray micro­a nalys is revealed conside rable amounts of oxygen, m agnes ium, phosphorus , calc ium. and z inc concentrated in these compa rt­me nts, show ing that the c he mi cal prope rties of Eil71e ira de nse g ranules are s imil a rly to what has been re ported previous ly in the ac idocalc isom es of trypanosoma tid and ap icomplexan paras ites (Miranda e t a l. 2000 , 2004a, 2010; Ru iz e t a l. 2004; SCOll el al. 1997 ; Soares M ede iros e t al. 2 (05).

Because of the ir e nzyme re pertoire, wh ic h in some aspec ts diffe r from mammalian (;e ll s, and due to the ir ac idi c c haracteri s­ti cs , which a ll ow the m to accumulate bas ic d rugs, it has bee n postul a ted that ac idocalc isom es are pote ntial targets for anti­paras iti c drugs in a numbe r 01' protozoa, inc luding ap icomple xan parasites (rev iewed in Docampo a nd More no 2008). Anti -cocc i­di a l compo unds suc h as m o ne ns in (Na + /H + exchanger) have been suggested to inte rfe re w ith ac idoca lc isom e phys iology (Docampo and More no 200 I), w he reas b isphospho nates, pyrophosphate an ­a logs used to treat a vari e ty of bo ne resorpti o n d iseases (R odan 1998), have al so been show n to have activ ity aga ins t diffe re nt protozoan paras ites in v itro and in v ivo. Accumulat ion of these compo unds in the acidoca lc isom es has also bee n s uggested (Do­campo a nd More no 2008).

T he ide ntifi cation of ac idocalc isomes in diffe re nt s pec ies of Eimeria strong ly s uggests th at these organe lles a re conserved a mo ng the different m e mbe rs o f the Eimeria genus. The ide n­tifi cation of acidocalci so mes in Eillleria parasites, the re fore ,

% Volume occupied

I.S2 ± 0.3 30.S ± 3.7

Mean diameter (nm)

364.07 ± 14 NO

re presents a step toward further understanding and contro lling the bio logy of Eimeria paras itism, and may also provide inte rest­ing targets for the future de velopment of anti-cocc id ia l drugs against pa ras ites of ve ter.ina ry importa nce.

ACKNOWLEDGMENTS

This work was supported by g rants to the authors from the fo l­lowing Braz ilian agencies: Conselho Nacional d e Desenvolv i­me nto Cientffico e Tecnologico (CNPq), Programa Jovens Pesqui sadores CNPq/Braz il , and Funda~ao Carlos C hagas Filho de Amparo a Pesqu isa do Es tado do Ri o d e Jane iro (FAPERJ). S.N.J.M. was suppo rted by the U S N ati o na l Tnstitutes of Health (AI-07965) , and R.D. was supported by the Barbara and Sanfo rd Orkin/Georg ia R esearch A lliance Chai r.

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