Molecular and Biochemical Parasitology, 49 (1991 ) 61-72 © 1991 Elsevier Science Publishers B.V. All rights reserved. / 0166-6851/91/$03.50 ADONIS 016668519100346S

61

MOLBIO 01603

Pathogenic and nonpathogenic Entamoeba histolytica: identification and molecular cloning of an iron-containing superoxide dismutase E g b e r t T a n n i c h , Iris B r u c h h a u s , R o l f D. W a l t e r a n d R o l f D. H o r s t m a n n Bernhard Nocht Institute for Tropical Medicine, Hamburg, F.R.G. (Received 22 February 1991; accepted 20 May 1991)

Superoxide dismutase (SOD) activity was determined in the cell lysate of the axenically cultured Entamoeba histolytiea isolate HM-I:IMSS. Under anaerobic culture conditions, 18.7 (_+ 4.9) units SOD activity (mg protein)-t were found. By inhibition studies the activity was attributed to an iron-containing type of SOD (FeSOD). Using degenerate oligonucleotide primers derived from regions highly conserved in prokaryotic FeSOD sequences, a genomic D N A fragment was amplified by the polymerase chain reaction. The fragment was used to isolate FeSOD specific cDNA clones from a pathogenic and a nonpathogenic E. histolytica isolate. A comparison of the 2 sequences revealed 5% nucleotide differences resulting in a single amino acid exchange. The primary structure showed the characteristics of an iron-containing type of SOD with a homology of approximately 55% with other FeSOD sequences. The enzyme was found to be encoded by single copy genes in both the pathogenic and the nonpathogenic E. histolytica, but restriction fragment lengths differed between the 2 groups. In 5 isolates studied, no correlation was found between pathogenic behavior of the amebae and the expression of FeSOD-related mRNA. Key words: Entamoeba histolytica; Iron-containing superoxide dismutase; cDNA sequence

Introduction

The protozoon Entamoeba histolytica is an intestinal parasite infecting 500 million people worldwide. It may cause life-threatening disease such as hemorrhagic colitis and extraintestinal abscesses [1]. Increasing evidence indicates that 2 genetically distinct forms of E. histolytica exist: nonpathogenic and pathogenic [2-5]. Studies are in progress to determine which factors enable pathogenic forms to destroy host tissues, i.e., to invade the Correspondence address." Egbert Tannich, Bernhard-NochtInstitut, Bernhard-Nocht-Str. 74, 2000 Hamburg 36, F.R.G. Abbreviations." SOD, superoxide dismutase; FeSOD, ironcontaining superoxide dismutase; MnSOD, manganese-containing superoxide dismutase; Cu/ZnSOD, copper/zinc-containing superoxide dismutase. Note." Nucleotide sequence data reported in this paper have been submitted to the EMBL, GenBank and DDBJ data bases with the accession numbers M63815 and M63816.

intestinal mucosa, enter the blood vessels, and disseminate to other organs.E, histolytica trophozoites normally exist under anaerobic or microaerobic conditions, but may respire aerobically. Under most culture conditions, however, more than 5% oxygen in the gas phase of the culture medium is deleterious for the amebae, possibly because of the accumulation of toxic oxygen metabolites such as superoxide radicals [6,7]. By invading aerobic tissues of the host, E. histolytica may become exposed to substantial amounts of superoxide radicals. The detoxification of superoxide radicals is generally mediated by the enzyme superoxide dismutase (SOD). Three different classes of SOD have been described which differ in their metal content and in their sensitivity to cyanide, azide and hydrogen peroxide (H202) [8]. Copper/zinc-containing SODs (Cu/ ZnSODs), which have been found in higher animals and plants, are inhibited by cyanide and H 2 0 2 . Manganese-containing SODs

62

(MnSODs), which have been found in prokaryotes, mitochondria, and chloroplasts, are insensitive to cyanide, azide, and H202. Ironcontaining SODs (FeSODs), which have been found in prokaryotes, plants, and protozoa, are inhibited by H202 and azide but not by cyanide [9-14]. Amino acid sequence data have shown that the 3 types of SOD segregate into 2 distinct phylogenetic families, the Cu/ZnSODs and the FeSODs/MnSODs. The FeSODs and MnSODs show a high degree of homology in primary and tertiary structure, while they appear to be unrelated to the Cu/ZnSODs [9]. Previously, evidence for the presence of SOD activity in E. histolytica trophozoites was presented in a preliminary report [15]. Here we describe the identification, structure, and expression of the enzyme.

Materials and Methods

Entamoeba histolytica isolates and culture conditions. The pathogenic E. histolytica isolate HM-I:IMSS was cultured in TYI-S-33 medium in the absence of bacteria (axenically). The pathogenic isolates SAW755, SAW891 and SAW1728 and the nonpathogenic isolates SAW142, SAW760, SAW1734, SAW1798 and SAW1799, provided by P. Sargeaunt (London), were grown in TYI-S-33 or TYSGM-9 in the presence of a mixed bacterial flora (xenically) [16,17]. All isolates were grown in sealed culture flasks under anaerobic conditions. They were characterized as being pathogenic or nonpathogenic using 3 criteria: (1) the clinical histories of the infected individuals; (2) isoenzyme classification [2,18]; and (3) genetic analysis [5,19].

Assay for superoxide dismutase activity. E. histolytica trophozoites of the pathogenic isolate HM-I:IMSS were harvested in early, mid-, or late logarithmic phase of growth by chilling on ice for 10 min and low-speed centrifugation at 4°C for 5 min, and washed twice in phosphate-buffered saline. The resulting pellet was freeze-thawed 5 times in solid CO2/acetone, and sedimented at 150000 x g

at 4°C for 40 min. The supernatant was dialyzed (cut-off 12-14 kDa) against a 50 mM phosphate buffer, pH 7.8, containing 0.1 mM EDTA. SOD activity was determined according to the method described by McCord and Fridovich [20] with the minor modification that the reaction volume was reduced to 900/~1. Bovine superoxide dismutase (Sigma) was used as a standard. To characterize the type of SOD present in E. histolytica, the assay was performed in the presence of potassium cyanide (1 mM), sodium azide (5 mM) or H202 (0.5 mM) [21].

Preparation of E. histolytica genomic DNA. E. histolytica trophozoites were harvested as described above. Nuclei were obtained by lysis of the cells in 1% Nonidet-P-40 and pelleted by centrifugation at 500 × g at 4°C for 5 rain. The pellet was resuspended, and DNA was released by treatment with proteinase K (1 mg m1-1) in a 10 mM Tris buffer, pH 8.3, containing 100 mM NaC1, 10 mM EDTA, and 0.5% N-lauroylsarcosine at 60°C for 2 h. The DNA was extracted twice with phenol/chloroform, 1:1 (v/v), once with chloroform, and precipitated with ethanol.

Amplification procedure. Two oligonucleotide primers FeSOD-S21 (5'-TGG ATC CAT ACT/A TTT TAT TGG) and FeSOD-AS23 (5'-TAA TAT/A GCA TGC TCC CAT/A ACA TC) were synthesized on an Applied Biosystems DNA synthesizer. The sequences of the primers were deduced from two regions found to be highly conserved in the amino acid sequences of all prokaryotic FeSODs analyzed so far [22-26]. FeSOD-S21 is a 21-mer sense oligonucleotide deduced from the residues flanking the histidine at position 73, and FeSOD-AS23 is a 23-mer antisense oligonucleotide deduced from the residues flanking the histidine at position 160 of the FeSOD of Escherichia coli [24]. Recognition sites for the restriction endonucleases BamHI and Sphl were introduced into the primer sequences to allow rapid subcloning in a predicted orientation (Fig. 1).

63 FeSOD-S21 (sense primer) I-- BamH I 5' TGG ATC CAT ACR TTT W N H T F 73

TAT TGG Y W

3'

FeSOD-AS23 (anti-sense primer) 5'

TA Y

I~SP hI ATA RGC ATG CTC CCA RAC Y A H E W V 160

ATC 3' D

R =AorT

Fig. 1. Oligonucleotide primers designed for the amplification of a fragment of the FeSOD gene by the polymerase chain reaction. Primer sequences were deduced from highly conserved amino acid sequences of FeSODs from various species. They comprise regions flanking the histidine sites at positions 73 and 160, respectively of the FeSOD of E. coli. Recognition sites for restriction endonucleases are indicated.

The polymerase chain reaction was performed as described [27]. Briefly, 2.5 U of Taq polymerase (Cetus Corp.) was used in 100 #1 of 10 mM Tris buffer, pH 8.3, containing 50 mM KC1, 1.5 mM MgCI, 200 #M deoxynucleotide triphosphates (Cetus Corp.), each oligonucleotide primer at 1.0 #M, 0.01% gelatin, and 0.1 #g of purified genomic DNA. The sample was amplified for 30 cycles in an automated Thermal Cycler (Perkin-ElmerCetus). Each cycle consisted of 1.5 min of denaturation at 94°C, 1.5 min of annealing at 40°C and 1.5 min of extension at 72°C. The final extension step was continued for additional 3 min.

Construction and screening of the cDNA libraries. Using poly(A) + RNA purified from the pathogenic isolate HM-I:IMSS and the nonpathogenic isolate SAW142, 2ZAP libraries were constructed using the ZAPeDNA Synthesis Kit following the instructions of the manufacturer (Stratagene). Each library contained 107 independent recombinant phages. The 280-bp amplified genomic DNA fragment was radioactively labeled (specific activity 1 × 108 cpm #g-~) and used to screen the two eDNA libraries by hybridization under

low-stringency conditions (2 × SSC at 50°C; 1 x SSC = 0.15 M NaC1/0.015 M sodium citrate, pH 7). Hybridizing phages were isolated, and the plasmids were released according to the instructions of the manufacturer (Stratagene).

Standard DNA and RNA technologies. Nucleotide sequencing, Southern blotting, Northern blotting, and primer extension experiments were performed according to published procedures [28]. For primer extension studies, an oligonucleotide primer, Eh-FeSOD-AS19 (5'GCT TAT CAT GAT GGA ATT C), complementary to nucleotides 85-103 of the cDNA sequences (Fig. 2) was used. The nucleotide sequence of the extension product in the pathogenic E. histolytica isolate HMI:IMSS was determined using the procedure described by Frohman et al. [29]. Briefly, after reverse transcription of poly(A) + RNA with the primer Eh-FeSOD-AS19, the complementary DNA was tailed in a terminal deoxynucleotidyl transferase reaction with dATP. Using a 17-mer oligo(dT) primer and EhFeSOD-AS19, the tailed cDNA was amplified by the polymerase chain reaction, subcloned into the plasmid vector pBS (Stratagene), and sequenced by the dideoxy chain-termination method [30]. Computer analysis, eDNA and protein sequences were analyzed using the DNASIS and PROSIS programs, respectively (Pharmacia LKB). Comparisons to published protein sequences (National Biomedical Research Foundation Database, release 6/90) were performed using the FASTA searching program [31]. Results

Determination of superoxide dismutase activity in the lysate of E. histolytica trophozoites. A cell lysate of the axenically cultured E. histolytica isolate HM-I:IMSS was analyzed for superoxide dismutase activity in 8 independent experiments. It was found to contain 18.7

64

a a nos. Eh-FeSOD cEh-FeSODp cEh-FeSODnp

GATTCGATAATAATGTCTTTcCAATTACCAcAATTACCTTATGcTTATAATGcTCTTGAGcCTCATATTAGTAAAGAGAcTcTTGAATTc ? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . o ..... ,A,.o,, .......

Eh-FeSOD cEh-FeSODp cEh-FeSODnp

30 40 50 H H D K H H A T Y V N K L N G L V K G T E Q E H K T L E E CATcATGATAAGcATcACGCTACTTATGTTAATAAGTTAAATGGTCTTGTAAAAGGAAcTGAACAAGAACATAAAACTCTTGAAGAATTA • .C ..... C . . A . . C . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

M

S

F

Q

L

P

Q

L

P

¥

10 A

Y

N

A

L

E

P

H

I

S

20 K

E

T

L

E

F

L

Eh-FeSOD cEh-FeSODp cEh-FeSODnp

60 70 80 I K Q K P T Q A I Y N N A A Q A W N H A F Y W K C M C G C G ATTAAACAAAAGC CAACTCAAGCAATTTATAATAATGCAGCCCAAGCATGGAATCATGC ATTCTATTGGAAATGTATGTGTGGATGTGGA ........... A .......................... T..T ........... C .................................... 5 '- T G G A T C C A T A C R T T T T A T T G G 3' FeSOD-S21 90 i00 ii0 V K P S E Q L I A K L T A A F G G L E E F K K K F T E K A V GTTAAACCATC TGAAC AATTAATTGCTAAATTAACTGC TGCTTTTGGAGGATTAGAAGAATTTAAGAAGAAGTTTACTGAGAAAGCTGTT ...................... C ............................................................... A...

Eh-FeSOD cEh-FeSODp cEh-FeSODnp

120 130 140 II[ G H F G S G W C W L V E H D G K L E I I D T H D A IVI N P M T ~ACATTTT~GT~AT~TGTT~TT~TTGAACA~ATGGTAAGTTAGAGATTATTGATACTCA~ATGCTGTT~TCC~TGACC ........ C ....................................................... C ..... C . . . A . . . . . . . . . . . . . .

Eh-FeSOD cEh-FeSODp cEh-FeSODnp

Eh-FeSOD cEh-FeSODp cEh-FeSODnp

Eh-FeSOD cEh-FeSODp cEh-FeSODnp

150 160 170 N G M K P L L T C D V W E H A Y Y I D T R N N R A A Y L E H AAT~AATGA~CCATTATTAACTTGTGATGTTTGGG~cA~cTTATTAcATTGACAcTAGAAAC~CAGAGCTGCTTACTTAG~cAT ......................................... C ..... C ........ T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3'-CTACARACCCTCGTACG~T~T-5' FeSOD-AS23 180 W W N V V N W K F V E E Q L * T~TGG~TGTAGTCAACT~AAGTTCGTTG~GAAC~CTCT~GTG~GTTTCACTTTTCCCCTCIA)n . . . . . . . . C ..... T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A ...... T T T T C T ( A ) n

Fig. 2. Sequence di~rences between FeSODs ~om pathogenic and nonpathogenic E. h~to@tica. Shown are the eDNA sequence and cDNA-deduced amino acid sequence of the enzyme derived ~om the pathogenic E. h~to@tica isolate HM-I :IMSS (cEH-FeSODp and Eh-FeSOD, resp.). The stop codon is marked by an asterix. For the enzyme ~om the nonpathogenic E. h~to@tica isolate SAW142, only di~ring nucleotides are presented (cEh-FeSODnp); conserved nucleotides are indicated by dots. The single amino acid exchange (valine vs. isoleucine at position 141) between the sequences of the pathogenic and the nonpathogenic isolate is marked by a box. The nucleotide sequence obtained by primer extension only is underlined. Primer sequences used ~ r genomic amplification are marked FeSOD-S21 and FeSOD-AS23, respectively.

(_+ 4.9) units SOD activity (rag protein)-~. There were no significant differences in activity with regard to the growth phase of the cells being early, mid-, or late logarithmic at the time of harvest. The activity appeared not to be due to contamination by metals since it was not reduced by dialyzing the lysate against an excess amount of buffer and it was sensitive to boiling. E. histolytica SOD activity was insensitive to inhibition by potassium cyanide (1 mM) but sensitive to hydrogen peroxide (0.5 mM) and sodium azide (5 mM) (Table I). These results suggested that the SOD activity detected in the E. histolytica cell lysate was due to the presence of an iron-containing type of SOD. The SOD activity and the inhibition values were comparable to those found in other protozoa (Table I).

Amplification of a genornic DNA fragment of E. histolytica representing part of an FeSOD gene. At present, amino acid sequence data of FeSODs are available from various prokaryotic organisms. Comparing the sequences, 2 regions were identified that are highly conserved in the enzymes of various species. These regions flank either one of the histidine residues (positions 73 and 160, respectively) that are responsible for metal binding. Deduced from these conserved regions, 2 oligonucleotide primers were constructed (Fig. 1). According to the high content of adenosine and thymidine within coding sequences of E. histolytica, the degeneracy of the oligonucleotide was reduced by using adenosine and thymidine at variable positions. These oligonucleotide primers and purified genomic D N A

C

65 TABLE I Superoxide dismutase activity in crude extracts of various protozoa, and effects of inhibitors on enzyme activity Organism

Entamoeba histolytica Tritrichomonas foetus Monocercomonas Leishmania tropica Trypanosoma brucei Crithidiafasciculata

Activity

Percent inhibition by

(Units (mg protein)-I)a

KCN (1 mM) NaN3 (5 mM) H:O2 (0.5 mM)

18.7 66.4 5.5 1.5 23.0 4.8 14.2 9.2

0 0 n.d. b n.d. 0 0 n.d. 0

+ 4.9 + 5.4 _+ 1.0 __+ 0.5 __+ 2.3 + 0.5 ± 2.6

(n=8) (n = 4) (n = 5) (n = 5) (n=4)

65 23 n.d. n.d. 55 56 n.d. 30

Ref.

86 82 n.d. n.d. 88 74 n.d. 76

11 10 10 12 12/14 12 14

aValues are means _+ standard deviation. bNot determined. TABLE II

of the axenically cultured E. histolytica isolate HM-l:IMSS were used in a polymerase chain reaction. Analysis of the amplified material by electrophoresis in an ethidium bromide stained agarose gel revealed a single fragment of approximately 280 bp. The nucleotide sequence of this fragment contained a single open reading frame. The deduced amino acid sequence showed substantial homology to those of other FeSODs.

Isolation and characterization of cDNA clones coding for an FeSOD from a pathogenic and a nonpathogenic E. histolytica isolate. Approximately 150 000 recombinant phage each from the cDNA libraries derived from the pathogenic isolate HM-I:IMSS and the nonpathogenic isolate SAW142 were screened under low stringency with the 280-bp genomic fragment obtained by in vitro amplification. Twentyeight and 10 phages, respectively, reacted with the probe. Five of each of the hybridizing phages were purified, and the cDNA inserts, designated cEh-FeSODp/1-5 and cEh-FeSODnp/1-5, were sequenced. All 5 inserts derived from the pathogenic isolate contained identical 3'-ends plus poly(A) tails, but differed in length at their Y-ends, suggesting that they were independent cDNA clones derived from the same m R N A species. The same result was obtained for the 5 cDNA clones derived from the nonpathogenic isolate. The longest cDNA clones of each group differed in length by one nucleotide only.

Both sequences had a single open reading frame but lacked an in-frame initiation codon. Primer extension experiments revealed extension products of 16 and 17 nucleotides, respectively. For the pathogenic isolate HMI:IMSS, the extension product was amplified by anchored PCR, subcloned and sequenced. The additional sequence contained an in-frame initiation codon and 2 nucleotides that were missing from the coding sequence (Fig. 2). A comparison of the two sequences derived from the pathogenic and the nonpathogenic isolate showed an exchange of 5% of the nucleotides, which was distributed over the entire sequences. These nucleotide differences resulted in a single amino acid exchange (valine vs. isoleucine at position 141) (Fig. 2). The amino acid sequences were aligned to those of FeSODs and MnSODs from various species. The degree of homology was found to be 53-56% to other FeSODs and 32-41% to MnSODs (Table II).

Southern and Northern blot analyses. Genomic DNA from trophozoites of the pathogenic E. histolytica isolate HM-I:IMSS and the nonpathogenic isolate SAW142 was digested with the restriction enzymes EcoRI and XmnI and hybridized to cEh-FeSODp under low-stringency conditions. In both digests, only one hybridizing fragment was found, suggesting that a single copy gene is coding for the FeSOD in each of the 2 forms of E. histolytica. The size of the hybridizing fragment in the EcoRI-digested DNA corresponded to 1.0 kb

66

Similarity between the amino acid sequences of FeSODs and MnSODs from various species, expressed as the percentage of identical amino acids

FeSOD E. h&tol, a FeSOD P. leiog, b FeSOD Ps. ovalis c FeSOD E. coh~ MnSOD E. coli MnSOD T. thermo, e MnSOD B. stearo, f MnSOD S. cerev, g MnSOD Human

FeSOD E.histol.

FeSOD P.leiog.

FeSOD Ps.ovalis

FeSOD E.coli

MnSOD E.coli

MnSOD MnSOD T.thermo. B.stearo.

MnSOD S.cerev.

** 53 56 53 37 38 41 32 36

** 65 74 40 37 49 34 37

** 65 43 41 52 34 42

** 42 40 49 34 40

** 52 60 39 43

62 42 49

** 44

** 39 50

MnSOD Human

aEntamoeba histolytica; bphotobacterium leiognathi; CPseudomonas ovalis; dEscherichia coli; eThermus thermophilus; rBacillus stearothermophilus," gSaccharomyces cerevisiae.

and appeared identical in both samples. In contrast, the size of the hybridizing fragment in the XmnI digested DNA differed between the pathogenic and the nonpathogenic isolate; in the former it was 1.8 kb and in the latter 1.7 kb

l

p

(Fig. 3A). When genomic DNA from 7 additional isolates, 3 pathogenic and 4 nonpathogenic, was digested with XmnI and hybridized under the same conditions, it was evident that the restriction fragment length

i

np

EcoR ]

t

p

m

I

u~

m

~

~

u~

m

m

u~

np

p

np

p

np

p

p

np

np

np

Xmn I

Xmn

I

Fig. 3. Southern blots of pathogenic (p) and nonpathogenic (np) isolates of E. histolytica hybridized with the cDNA corresponding to the FeSOD of the pathogenic isolate HM-1 :IMSS (cEH-FeSODp). Genomic DNA was digested with restriction enzymes as indicated and hybridized under low stringency conditions, and washed in 2 × SSC at 50°C. (A) Hybridization of DNA from the pathogenic isolate HM-I:IMSS (p) and the nonpathogenic isolate SAW142 (np), both digested with 2 different enzymes, as indicated. (B) Hybridization of DNA from 9 different isolates, 4 pathogenic (p) and five nonpathogenic (np), all digested with Xmnl. Size markers are indicated in kb.

67 B

:~ ~ ~ o ~ '

Pathogenic and nonpathogenic Entamoeba histolytica: identification and molecular cloning of an iron-containing superoxide dismutase.

Superoxide dismutase (SOD) activity was determined in the cell lysate of the axenically cultured Entamoeba histolytica isolate HM-1:IMSS. Under anaero...
920KB Sizes 0 Downloads 0 Views