- Email: [email protected]
Genus- and subgenus-specific oligonucleotide probes for Acanthamoeba
MOLECULAR
Molecular
and Biochemical
Parasitology
2&EMICAL PARASITOLOGY
71 (1995) 255-260
Short communication
Genus- and subgenus-specific oligonucleotide probes for Acanthamoeba Rebecca J. Gast ‘, Thomas J. Byers
*
Department of Molecular Genetics, The Ohio State Uniuersity, 484 W. 12th Avenue, Columbus, OH 43210-1292, Received 28 July 1994; accepted 23 February
Keywords: Acanthamoeba;
Oligonucleotide;
Ribosomal
DNA, Diagnostic
Acanthamoeba is a genus of opportunistic human pathogens. Infections include a chronic granulomatous amoebic encephalitis and Acanthamoeba keratitis, a painful and sight-threatening cornea1 infection [l]. In early stages of disease, misdiagnosis of amoebic keratitis as viral keratitis can result in inappropriate drug treatment [2]. Several diagnostic methods for Acanthamoeba are available [2,3], but are most useful at later stages of infection. Early detection and identification of amoebas in cornea1 infections are critical because they greatly increase the likelihood of rational and successful drug therapy [4]. In this report we describe very sensitive and specific oligonucleotide probes based on sequences of small ribosomal subunit RNA (srRNA) or DNA (srDNA). Our extensive analyses of srDNA target sequences and of probe hybridization to representative DNA slot blots demonstrates that these probes are very
Abbreviations: d2H,0, double distilled water; PCR, polymerase chain reaction; rcA, ribocluster A, srDNA, small subunit ribosomal DNA. 1 Current address: Department of Biology, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA. * Corresponding author. Tel.: (l-614) 292-4793; Fax: (l-614) 292-1538; e-mail: [email protected] 0166-6&X51/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0166-6851(94)00049-6
USA
1995
method
promising new diagnostic agents for Acanthamoeba in infections and environmental samples. Similar approaches also are promising for use with other protozoan pathogens [5-81. Our probes target all strains of the genus Acanthamoeba or subsets of strains identified by classical taxonomy or phylogenetic studies of srDNA sequences (Gast et al., data not shown; Ledee, Awwad and Byers, unpublished data). Probe specificity was tested by hybridization to slot blots of amoeba genomic DNA and by comparison of probe sequences with target sequences determined in this lab for 53 strains. The slot blots included genomic DNA from 28 isolates of Acanthamoeba representative of the 3 previously described morphological species groups [9], 8 other microorganisms and a virus that have been associated with CNS or ocular disease [1,3,10,11] and human cells (Table 1). Acanthamoeba and Hartmannella were grown as described previously [11,12]. Naegleria cell pellets were a gift from Dr. Takuro Endo. Bacteria and HeLa cells were grown using standard culture methods. Fungal cultures were gifts from the lab of P.E. Kolattukudy. Amoeba, human and bacterial DNAs were isolated by a scaled down UNSET procedure [13]. DNA from very dilute cell samples was co-precipitated with tRNA unless PCR was to be used. If
RJ. Gast, TJ. Byers / Molecular and Biochemical Parasitology
256 Table 1 Slot blot hybridization Group
results Strain a
Species
ATCC
No.
Not decided
result b
Fig. la 16s UNIV
Fig. lb RGPG
Fig. lc RGP2/3
Fig. Id RGPA
Seq. Dev. ’
Ray Comandon oc-15c
30137 30135 30867
Al + AZ+ A3+
Al + A2+ A3+
Al A2A3-
AlA2A3-
>8
A. castellanii
50374 50370 50493 50491 so373 50494 50492 30731 30730 50495 50498 B2 + 50372 50371 30487 50368 30134
Bl c2 D2 D4 c3 c7 C8 B6 B8 Dl D3 B2 Cl B4 c4 B7 C6
+ + + + + + f + + + + + + + + + +
Bl c2 D2 D4 c3 c7 C8 B6 B8 Dl D3 Cl Cl B4 c4 B7 C6
+ + + + + + + + + + + + + + + + +
Bl c2 D2 D4 c3 c7 C8 B5 B7 Dl D3 Cl B8 B4 c4 B6 C6
+ + + + + + + + + + + + + + + + +
Bl + c2 + D2 + D4 + C3C7CSB5 B7Dl + D3 + 0 B8 + B4 c4 B6 + C6 -
0 0 0 0 >8 >8 >8 6 >8 0 0
A. rhysodes A. terricola
Castellani * Ma e. * vo42 e.r * 180: 1 I,* Neff * VO06 b,* vo14 e, * s-7 * BH-2 * * V029 e, * V125 ‘I* Liu/El e,r * JAC/S2 * 73-1-16 es* Panola Mt ’ 85-6-116 e, * Pussard
A. A. A. A.
Diamond e, * Reich * Oak Ridge * PD,S * *
A4 + 30870 30884 30841
A4 A7 A8 A6
+ + + +
A4 A7 A8 A6
+ + + +
A4 A7 A8 A6
+ + + -
0 A7A8 A6-
82-12-324 e** 50497 88-2-27 e, * OC-3A * *
50496 c5 + 50379 30866
B5 + c5 + B3 + A5+
B5 + c5 + B3 + A5+
B3 + cs + B2 + A5+
83 + 0 B2A5-
El E2 E3 E4 E5 E6 D6 Fl D7 F2 F3 F4
El E2 E3 E4 E5 E6 D6 Fl D7 F2 F3 F4
El E2 E3 -
El E2 E3 -
E6 -
E6 +
E5 -
ES -
E4 -
IS-
F8 E7 F4 F3
F8 E7 F4 F3
culbertsoni palestinensis royreba lenticulata
A. species 88-2-37 cl * A. healyi
Controls
Slot location/Hybridization
A. astronyxis A. comandoni A. tubiashi
A. griffini A. hatchetti A. polyphaga
I11
71 (1995) 255-260
Aspergillus sp. Fusarium solani Helminthiosporium sp. Bacillus subtilis Pseudomonas aeruginosa Staphylococcus epidermis Hartmannella vermiformis Hartmannella vermiformis Naegleria lovaniensis Naegleria lovaniensis Herpes Simplex Virus Human HeLa cells Plasmid containing RGP2/3 target Plasmid containing RGPA target
a Isolates from human eye authors (* *). Liu/lE and Centers for Disease Control b Probe did (+) or did not ’ Sequence deviation, base
+ + + + + + + + + + + +
+ -
+ -
0 2 6 1
>8 4 >8 0 >8 >8
+
(e), brain (b) or lung (l), or the environment. Sequences currently available from GenBank (*) or from the Diamond were gifts from Drs. J.Y. Liu, East China Normal University (Shanghai, China) and G.S. Visvesvara, (Atlanta, GA, USA), respectively. (-) hybridize with the target. mismatches between RGPA and the best-tit srDNA target sequence.
R.J. Gast. T.J. Byers /Molecular
and Biochemical Parasitology
PCR was used, DNA was isolated by a Chelex procedure [14] to avoid problems with PCR products resulting from contaminants in the tRNA preparations. PCR amplifications consisted of 40 cycles each of 1 min at 94°C 30 s at 57°C and 2 min at 72°C with RGPG and 892C as primers. Slot blots used approx. 1 pg of genomic DNA for amoeba or human cell samples and approx. 5 pg for fungal or bacterial samples. Four oligonucleotide probes synthesized by National Biosciences (NBI, Plymouth) were designed to hybridize to both nuclear srDNA and srRNA. The 16s UNIV probe (3’-ACGGTGCGg,CGGCGCCATTA”,G), corresponding to nt 630-650 of A. castel-
a
16s
UNIV
71 (1995) 255-260
257
Zanii Castellani (AcC; GenBank accession No. UO7413), was designed to recognize srRNA and srDNA from all prokaryotic and eukaryotic organisms [15] and was used as a positive control for hybridization. RGPG (3’-GGGGATCGTCGAACACTCTIT), designed as a probe for the entire genus Acanthamoeba, corresponds to AcC nt 1372-1392. RGP2/3 (T-ACCGTGGCCACI-I’ACTGAGGGG),
b
ABCDEF
RGPG A
BCDEF
1
RGPA
RGPA/3 ABCDEF
ABCDEF 1 2
1 2
3
3
4
4
5
5
6
6
7
7
8
8
Fig. 1. Slot blots of Acanthamoeba and control genomic DNAs. (a,b) Samples A-D, and E and F are two separate membranes probed with 16s UNIV, then stripped and reprobed with RGPG. (c,d) A single membrane probed with RGP2/3, then stripped and reprobed with RGPA. Sample positions (slots) differ in panels a and b compared to panels c and d (see Table 1). Membranes were prehybridized 30 min at 60°C with 100 /.rg denatured herring sperm DNA in 5 ml hybridization solution (7% SDS/S X SSC/OS M NaH,PO,/ 10 X Denhardt’s solution), then hybridized 4 h at 60°C for 16s UNIV, 50°C for RGP2/3 and RGPA, or 42°C for RGPG with 2 X 10’ cpm of oligonucleotide probe S-labelled with [y3’ P]ATP. Membranes were rinsed once with 2 X SSC at room temperature and then washed at 50°C (panels a,c,d) or 42°C (panel b) for 15 min in 2 X SSC/O.l% SDS and 15 min in 0.1 X SSC/O.l% SDS.
258
RJ. Gad, T.J. Byers/Molecular
and Biochemical Parasitology
&A) (Gast et al., data not shown), corresponded to AcC nt 275-291. RCA is a subgroup of strains, possibly all from group II, that are phylogenetically related by srDNA sequences differing by no more than 2.9% (Gast et al., data not shown). RCA includes all of the human isolates we have examined except Acanthamoeba species 88-2-27 and A. castellanii VOO6and VQ14. Results of hybridization reactions (Fig. 1) are summarized in Table 1. Probe 16s UNIV hybridized with all DNA samples (Fig. la). Human DNA contaminating the HSV sample probably explains hybridization with viral DNA (F3). RGPG hybridized to genomic DNA from all Acanthamoeba isolates (Fig. lb), but with a reduced signal for the three group-I species (Al-3). In an analysis of 53 strains, sequences exactly complementary to RGPG were found in 51 cases and sequences mismatching the probe at only 1 nt were found in A. tubiashi and A. astronyxis, two of the group-I species. The sequence of A. comandoni srDNA is unknown, but reduced stability due to single base mismatches probably explains the reduced signals observed for all three group-I strains. With the various probes used here, we found no cases in which positive signals were obtained when there was more than 1 base mismatch between the probe and the amoeba srDNA target. RGPG did not hybridize with any control genomic DNA except that from Pseudomonas aeruginosa (Fig. lb, E5) even though the hybridization temperature was lowered to detect any possible cross-hybridization with Hartmannella, the other amoeba genus. The nonspecific hybridization (the bacterial srDNA gene does not have the RGPG target) could be avoided by using amoeba srDNA sequences, obtained by PCR amplification with eukaryotic-specific primers, rather than genomic DNA in the slot blots (not illustrated, but see below). RGP2/3 hybridized with genomic DNA from all 21 group-II and -111 isolates except A. lenticulata and with all 4 unidentified strains (Fig. lc; Table 1, A6). It did not hybridize with group-I strains (Al-3) or any controls except the probe-containing plasmid (F4). In the 23 cases where a positive signal was obtained, RGP2/3 exactly matched the target in 22 cases and only mismatched at 1 base in the other case (VOO6). Sequence analysis of 12 A. Zenticulutu strains (Diedrich and Byers, unpublished data) and of
71 (1995) 255-260
group-I species A. astronyxis and A. tubiashi revealed 7-nt mismatches between probe and target in each case. The basic sequences of A. lenticulata srDNAs are very different from those of other groupIII species and most strains of the species have unique srDNA introns (Ref. 16; and Diedrich, unpublished observations). The species probably should be reclassified into a different group. RGPA hybridized with DNA from 9 group II, 1 from group III and 2 unidentified species (Fig. Id; Table 1). Targets exactly matched the probe in all these strains except A. rhysodes where there was a single base mismatch. Based on comparisons of complete srDNA sequences, all these strains, including the group-III strain Diamond, can be placed in phylogenetically defined rcA, which appears to be a subset of group II. Thus, Diamond may belong in group II rather than III, a reassignment also supported by mitochondrial srDNA sequence comparisons (Awwad, Ledee and Byers, unpublished data). Where sequences are known, failure of hybridization by RGPA is due to mismatches at 2 or more positions (Table 1). RGPA, like RGPG, hybridized with P. aeruginosa genomic DNA (Fig. Id, E6). Again, the problem could be eliminated by hybridizing to PCR amplified srDNA rather than genomic DNA. It can take up to two weeks to culture amoebas from infections of the cornea for diagnosis. Thus, rapid and unequivocal direct detection of amoebas in tissue samples is very desirable. Tissue biopsies and epithelial scrapes used for microbiological evaluation of amoebic keratitis typically are very small samples and amoebas are greatly outnumbered by human cells. This suggests the need to develop an amoebas ecific PCR-based diagnostic technique. When Jr P-labelled RGP2/3 was hybridized to genomic DNA isolated from axenic amoeba cultures, lo’--lo3 amoebas could be detected (Fig. 2), but l-10 amoebas could be detected if PCR-amplified srDNA was the target. Detection sensitivity was reduced when RGP2/3 labelled at the 5’ end with digoxigenin was used for chemiluminescent detection (see the legend to Fig. 2). Detection sensitivity also was studied following PCR amplification of Acanthamoeba srDNA from mixtures containing l-lo4 amoebas plus lo5 HeLa cells plus or minus 1 pg of P. aeruginosa genomic DNA (not illustrated). The best results were obtained using RGPG and 892C
R.J. Gast, TJ. Byers/Molecular
1
2
and Biochemicaf Parasitology
3 IO4
103 102
10 1
0 g
is
g 2 Z
4
C Fig. 2. Minimum number of Ma strain amoebas detectable by hybridization of probe RGP2/3 to blots of Acanthamoeba DNA. Column 1, Genomic DNA from lo-fold dilutions of amoebas. Columns 2 and 3, PCR products obtained by amplifying a segment of amoeba srDNA using 892C and RGPG as primers and genomic DNA from IO-fold dilutions of amoebas as templates. Blots were probed as described in the legend to Fig. 1 with RGP2/3 5’-labelled with 32P in columns 1 and 2 or with digoxigenin (Genius System, Boehringer-Mannheim, Indianapolis, IN, uSA) in column-3. (C) Blot from control PCR reaction with no amoeba DNA added.
71 (1995) 255-260
2.59
oligonucleotide probes and genomic DNA, especially when axenic cultures are available. PCR amplified srDNAs, as used with Leishmania [9], would be the preferred probe targets if amoebas were mixed with other microorganisms or human cells. Lai et al. [17] developed a 125-bp nuclear large subunit ribosomal RNA gene probe that specifically hybridized to four Acanthamoeba isolates with a sensitivity similar to our probes. The advantage of our probes is that they have been designed to be more selective by basing their sequences on extensive knowledge of sequence variation and by making them smaller and more sensitive to target sequence differences.
Acknowledgements We thank many colleagues for cultures and helpful suggestions. This work was supported by NE1 Grant No. EY09073 to T.J.B. and P.A. Fuerst. R.J.G. submitted portions of this work to Ohio State University in partial fulfillment of requirements for the Ph.D.
(~‘-GTcAGAGGTGAAA’ITC~GG)
as primers to amplify a fragment of approx. 350 bp. These eukaryotic specific primers prevented amplification of bacterial srDNA and the amoeba-specific RGPG prevented amplification of human srDNA. The amount of amoeba PCR product was reduced, however, by the presence of relatively large amounts of either human or bacterial DNA possibly due to nonspecific competition for the primers. A minimum of l-10 amoebas could be detected when pure amoeba cultures were used, but the minimum was lo-100 when amoebas were mixed with human cells and 1000 when amoebas were mixed with human cells plus relatively large amounts of bacterial DNA. RGPG, RGP2/3 and RGPA recognize Acanthamoeba targets with high specificity and are most useful for identifying all members of the genus or of subgenus clusters of strains. Individual strains can be identified readily by srDNA sequencing since the sequences have been unique for each of the more than 50 strains that we have examined. We have shown that detection specificity and sensitivity can be achieved for Acanthamoeba using
References [l] John, D.T. (1993) Opportunistically pathogenic free-living amebae. In: Parasitic Protozoa (Kreier, J.P. and Baker, J.R.. eds), 2nd edn., pp. 143-246. Academic Press, San Diego, CA. [2] Palmer, M.L. and Hyndiuk, R.A. (1993) Contact lens-related infectious keratitis. Int. OphthaImoI. Clin. 33, 23-49. [3] Gordon, Y.J. (1993) Rapid diagnostic tests for infectious ocular disease. Int. Ophthalmol. Clin. 33, 153-161. [4] Bacon, AS., Dart, J.K., Ficker, L.A., Matheson, M.M. and Wright, P. (1993) Acanthamoeba keratitis. The value of early diagnosis. Opthalmology 100, 1238-1243. [5] MacPherson, J.M. and Gajadhar, A.A. (1993) Ribosomal RNA sequences for the specific detection of Toxoplasma gondii by hybridization assay. Mol. Cell. Probes 7, 97-103. [6] Holmdahl, O.J.M., Mattsson, J.G., Uggla, A. and Johansson, K.-E. (1993) Oligonucleotide probes complementary to variable regions of 18s rRNA from Sarcocystis species. Mol. Cell. Probes 7, 481-486. [7] Benavides, G.R., Sullivan, J.J., Steurer, F., McGraw, R.A. and Tarleton, R.L. (1993) Differentiation of trypanosomatid species by hybridization to selected rRNA probes. Mol. Cell. Probes 7, 89-96.
260
RJ. Gast, TJ. Byers / Molecular and Biochemical
[81 Uliana, S.R.B., Nelson, K., Beverley, S.M., Camargo, E.P. and Floerer-Winter, L.M. (1994) Discrimination amongst Ieishmania by polymerase chain reaction and hybridization with small subunit ribosomal DNA derived oligonucleotides. J. Euk. Microbial. 41, 324-330. [9] Visvesvara, G.S. (1991) Classification of Acanthamoeba. Rev. Infect. Dis. 13, S369-S372. [lo] O’Day, D.M. (1983) Fungal keratitis. In: The Cornea: Scientific foundation and clinical practice (O’Day, D.M., ed.), pp. 420-428. Little&Brown, Boston, MA. [ll] Weekers, P.H.H., Gast, R.J., Fuerst, P.A. and Byers, T.J. (19941 Sequence variations in small-subunit ribosomal RNAs of Hartmannella uermiformis and their phylogenetic implications. Mol. Biol. Evol. 11, 684-690. [121 Byers, T.J., Maynard, B.J., L&en, R.A., Martin, S.M. and Akins, R.A. (1980) Rapid growth of Acanthamoeba in defined media; induction of encystment by glucose-acetate starvation. J. Protozool. 27, 216-219.
Parasitology
71 (1995) 255-260
[13] Hugo, E.R., Gast, R.J., Byers, T.J. and Stewart, V.J. (19921 Purification of amoeba mtDNA using the UNSET procedure. In: Protocols in Protozoology (Lee, J.J. and Soldo, A.T., eds.), pp. D7.1-7.2. Allen Press, Lawrence. 1141 Walsh, P.S., Metzger, D.A. and Higuchi, R. (1991) Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material. BioTechniques 10, 506-513. [15] Giovannoni, S.J., DeLong, E.R., Olsen, G.J. and Pace, N.R. (1988) Phylogenetic group specific oligonucleotide probes for identification of single microbial cells. J. Bacterial. 170, 720-26. [16] Gast, R.J., Fuerst, P.A. and Byers, T.J. (1994) Discovery of group I introns in the nuclear small subunit ribosomal RNA genes of Acanthamoeba. Nucleic Acids Res. 22, 592-596. [17] Lai, S., Asgari, M. and Henney Jr., H.R. (1994) Non-radioactive DNA probe and polymerase chain reaction procedures for the specific detection of Acanthamoeba. Mol Cell. Probes 8, 81-89.
Molecular
and Biochemical
Parasitology
2&EMICAL PARASITOLOGY
71 (1995) 255-260
Short communication
Genus- and subgenus-specific oligonucleotide probes for Acanthamoeba Rebecca J. Gast ‘, Thomas J. Byers
*
Department of Molecular Genetics, The Ohio State Uniuersity, 484 W. 12th Avenue, Columbus, OH 43210-1292, Received 28 July 1994; accepted 23 February
Keywords: Acanthamoeba;
Oligonucleotide;
Ribosomal
DNA, Diagnostic
Acanthamoeba is a genus of opportunistic human pathogens. Infections include a chronic granulomatous amoebic encephalitis and Acanthamoeba keratitis, a painful and sight-threatening cornea1 infection [l]. In early stages of disease, misdiagnosis of amoebic keratitis as viral keratitis can result in inappropriate drug treatment [2]. Several diagnostic methods for Acanthamoeba are available [2,3], but are most useful at later stages of infection. Early detection and identification of amoebas in cornea1 infections are critical because they greatly increase the likelihood of rational and successful drug therapy [4]. In this report we describe very sensitive and specific oligonucleotide probes based on sequences of small ribosomal subunit RNA (srRNA) or DNA (srDNA). Our extensive analyses of srDNA target sequences and of probe hybridization to representative DNA slot blots demonstrates that these probes are very
Abbreviations: d2H,0, double distilled water; PCR, polymerase chain reaction; rcA, ribocluster A, srDNA, small subunit ribosomal DNA. 1 Current address: Department of Biology, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA. * Corresponding author. Tel.: (l-614) 292-4793; Fax: (l-614) 292-1538; e-mail: [email protected] 0166-6&X51/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0166-6851(94)00049-6
USA
1995
method
promising new diagnostic agents for Acanthamoeba in infections and environmental samples. Similar approaches also are promising for use with other protozoan pathogens [5-81. Our probes target all strains of the genus Acanthamoeba or subsets of strains identified by classical taxonomy or phylogenetic studies of srDNA sequences (Gast et al., data not shown; Ledee, Awwad and Byers, unpublished data). Probe specificity was tested by hybridization to slot blots of amoeba genomic DNA and by comparison of probe sequences with target sequences determined in this lab for 53 strains. The slot blots included genomic DNA from 28 isolates of Acanthamoeba representative of the 3 previously described morphological species groups [9], 8 other microorganisms and a virus that have been associated with CNS or ocular disease [1,3,10,11] and human cells (Table 1). Acanthamoeba and Hartmannella were grown as described previously [11,12]. Naegleria cell pellets were a gift from Dr. Takuro Endo. Bacteria and HeLa cells were grown using standard culture methods. Fungal cultures were gifts from the lab of P.E. Kolattukudy. Amoeba, human and bacterial DNAs were isolated by a scaled down UNSET procedure [13]. DNA from very dilute cell samples was co-precipitated with tRNA unless PCR was to be used. If
RJ. Gast, TJ. Byers / Molecular and Biochemical Parasitology
256 Table 1 Slot blot hybridization Group
results Strain a
Species
ATCC
No.
Not decided
result b
Fig. la 16s UNIV
Fig. lb RGPG
Fig. lc RGP2/3
Fig. Id RGPA
Seq. Dev. ’
Ray Comandon oc-15c
30137 30135 30867
Al + AZ+ A3+
Al + A2+ A3+
Al A2A3-
AlA2A3-
>8
A. castellanii
50374 50370 50493 50491 so373 50494 50492 30731 30730 50495 50498 B2 + 50372 50371 30487 50368 30134
Bl c2 D2 D4 c3 c7 C8 B6 B8 Dl D3 B2 Cl B4 c4 B7 C6
+ + + + + + f + + + + + + + + + +
Bl c2 D2 D4 c3 c7 C8 B6 B8 Dl D3 Cl Cl B4 c4 B7 C6
+ + + + + + + + + + + + + + + + +
Bl c2 D2 D4 c3 c7 C8 B5 B7 Dl D3 Cl B8 B4 c4 B6 C6
+ + + + + + + + + + + + + + + + +
Bl + c2 + D2 + D4 + C3C7CSB5 B7Dl + D3 + 0 B8 + B4 c4 B6 + C6 -
0 0 0 0 >8 >8 >8 6 >8 0 0
A. rhysodes A. terricola
Castellani * Ma e. * vo42 e.r * 180: 1 I,* Neff * VO06 b,* vo14 e, * s-7 * BH-2 * * V029 e, * V125 ‘I* Liu/El e,r * JAC/S2 * 73-1-16 es* Panola Mt ’ 85-6-116 e, * Pussard
A. A. A. A.
Diamond e, * Reich * Oak Ridge * PD,S * *
A4 + 30870 30884 30841
A4 A7 A8 A6
+ + + +
A4 A7 A8 A6
+ + + +
A4 A7 A8 A6
+ + + -
0 A7A8 A6-
82-12-324 e** 50497 88-2-27 e, * OC-3A * *
50496 c5 + 50379 30866
B5 + c5 + B3 + A5+
B5 + c5 + B3 + A5+
B3 + cs + B2 + A5+
83 + 0 B2A5-
El E2 E3 E4 E5 E6 D6 Fl D7 F2 F3 F4
El E2 E3 E4 E5 E6 D6 Fl D7 F2 F3 F4
El E2 E3 -
El E2 E3 -
E6 -
E6 +
E5 -
ES -
E4 -
IS-
F8 E7 F4 F3
F8 E7 F4 F3
culbertsoni palestinensis royreba lenticulata
A. species 88-2-37 cl * A. healyi
Controls
Slot location/Hybridization
A. astronyxis A. comandoni A. tubiashi
A. griffini A. hatchetti A. polyphaga
I11
71 (1995) 255-260
Aspergillus sp. Fusarium solani Helminthiosporium sp. Bacillus subtilis Pseudomonas aeruginosa Staphylococcus epidermis Hartmannella vermiformis Hartmannella vermiformis Naegleria lovaniensis Naegleria lovaniensis Herpes Simplex Virus Human HeLa cells Plasmid containing RGP2/3 target Plasmid containing RGPA target
a Isolates from human eye authors (* *). Liu/lE and Centers for Disease Control b Probe did (+) or did not ’ Sequence deviation, base
+ + + + + + + + + + + +
+ -
+ -
0 2 6 1
>8 4 >8 0 >8 >8
+
(e), brain (b) or lung (l), or the environment. Sequences currently available from GenBank (*) or from the Diamond were gifts from Drs. J.Y. Liu, East China Normal University (Shanghai, China) and G.S. Visvesvara, (Atlanta, GA, USA), respectively. (-) hybridize with the target. mismatches between RGPA and the best-tit srDNA target sequence.
R.J. Gast. T.J. Byers /Molecular
and Biochemical Parasitology
PCR was used, DNA was isolated by a Chelex procedure [14] to avoid problems with PCR products resulting from contaminants in the tRNA preparations. PCR amplifications consisted of 40 cycles each of 1 min at 94°C 30 s at 57°C and 2 min at 72°C with RGPG and 892C as primers. Slot blots used approx. 1 pg of genomic DNA for amoeba or human cell samples and approx. 5 pg for fungal or bacterial samples. Four oligonucleotide probes synthesized by National Biosciences (NBI, Plymouth) were designed to hybridize to both nuclear srDNA and srRNA. The 16s UNIV probe (3’-ACGGTGCGg,CGGCGCCATTA”,G), corresponding to nt 630-650 of A. castel-
a
16s
UNIV
71 (1995) 255-260
257
Zanii Castellani (AcC; GenBank accession No. UO7413), was designed to recognize srRNA and srDNA from all prokaryotic and eukaryotic organisms [15] and was used as a positive control for hybridization. RGPG (3’-GGGGATCGTCGAACACTCTIT), designed as a probe for the entire genus Acanthamoeba, corresponds to AcC nt 1372-1392. RGP2/3 (T-ACCGTGGCCACI-I’ACTGAGGGG),
b
ABCDEF
RGPG A
BCDEF
1
RGPA
RGPA/3 ABCDEF
ABCDEF 1 2
1 2
3
3
4
4
5
5
6
6
7
7
8
8
Fig. 1. Slot blots of Acanthamoeba and control genomic DNAs. (a,b) Samples A-D, and E and F are two separate membranes probed with 16s UNIV, then stripped and reprobed with RGPG. (c,d) A single membrane probed with RGP2/3, then stripped and reprobed with RGPA. Sample positions (slots) differ in panels a and b compared to panels c and d (see Table 1). Membranes were prehybridized 30 min at 60°C with 100 /.rg denatured herring sperm DNA in 5 ml hybridization solution (7% SDS/S X SSC/OS M NaH,PO,/ 10 X Denhardt’s solution), then hybridized 4 h at 60°C for 16s UNIV, 50°C for RGP2/3 and RGPA, or 42°C for RGPG with 2 X 10’ cpm of oligonucleotide probe S-labelled with [y3’ P]ATP. Membranes were rinsed once with 2 X SSC at room temperature and then washed at 50°C (panels a,c,d) or 42°C (panel b) for 15 min in 2 X SSC/O.l% SDS and 15 min in 0.1 X SSC/O.l% SDS.
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and Biochemical Parasitology
&A) (Gast et al., data not shown), corresponded to AcC nt 275-291. RCA is a subgroup of strains, possibly all from group II, that are phylogenetically related by srDNA sequences differing by no more than 2.9% (Gast et al., data not shown). RCA includes all of the human isolates we have examined except Acanthamoeba species 88-2-27 and A. castellanii VOO6and VQ14. Results of hybridization reactions (Fig. 1) are summarized in Table 1. Probe 16s UNIV hybridized with all DNA samples (Fig. la). Human DNA contaminating the HSV sample probably explains hybridization with viral DNA (F3). RGPG hybridized to genomic DNA from all Acanthamoeba isolates (Fig. lb), but with a reduced signal for the three group-I species (Al-3). In an analysis of 53 strains, sequences exactly complementary to RGPG were found in 51 cases and sequences mismatching the probe at only 1 nt were found in A. tubiashi and A. astronyxis, two of the group-I species. The sequence of A. comandoni srDNA is unknown, but reduced stability due to single base mismatches probably explains the reduced signals observed for all three group-I strains. With the various probes used here, we found no cases in which positive signals were obtained when there was more than 1 base mismatch between the probe and the amoeba srDNA target. RGPG did not hybridize with any control genomic DNA except that from Pseudomonas aeruginosa (Fig. lb, E5) even though the hybridization temperature was lowered to detect any possible cross-hybridization with Hartmannella, the other amoeba genus. The nonspecific hybridization (the bacterial srDNA gene does not have the RGPG target) could be avoided by using amoeba srDNA sequences, obtained by PCR amplification with eukaryotic-specific primers, rather than genomic DNA in the slot blots (not illustrated, but see below). RGP2/3 hybridized with genomic DNA from all 21 group-II and -111 isolates except A. lenticulata and with all 4 unidentified strains (Fig. lc; Table 1, A6). It did not hybridize with group-I strains (Al-3) or any controls except the probe-containing plasmid (F4). In the 23 cases where a positive signal was obtained, RGP2/3 exactly matched the target in 22 cases and only mismatched at 1 base in the other case (VOO6). Sequence analysis of 12 A. Zenticulutu strains (Diedrich and Byers, unpublished data) and of
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group-I species A. astronyxis and A. tubiashi revealed 7-nt mismatches between probe and target in each case. The basic sequences of A. lenticulata srDNAs are very different from those of other groupIII species and most strains of the species have unique srDNA introns (Ref. 16; and Diedrich, unpublished observations). The species probably should be reclassified into a different group. RGPA hybridized with DNA from 9 group II, 1 from group III and 2 unidentified species (Fig. Id; Table 1). Targets exactly matched the probe in all these strains except A. rhysodes where there was a single base mismatch. Based on comparisons of complete srDNA sequences, all these strains, including the group-III strain Diamond, can be placed in phylogenetically defined rcA, which appears to be a subset of group II. Thus, Diamond may belong in group II rather than III, a reassignment also supported by mitochondrial srDNA sequence comparisons (Awwad, Ledee and Byers, unpublished data). Where sequences are known, failure of hybridization by RGPA is due to mismatches at 2 or more positions (Table 1). RGPA, like RGPG, hybridized with P. aeruginosa genomic DNA (Fig. Id, E6). Again, the problem could be eliminated by hybridizing to PCR amplified srDNA rather than genomic DNA. It can take up to two weeks to culture amoebas from infections of the cornea for diagnosis. Thus, rapid and unequivocal direct detection of amoebas in tissue samples is very desirable. Tissue biopsies and epithelial scrapes used for microbiological evaluation of amoebic keratitis typically are very small samples and amoebas are greatly outnumbered by human cells. This suggests the need to develop an amoebas ecific PCR-based diagnostic technique. When Jr P-labelled RGP2/3 was hybridized to genomic DNA isolated from axenic amoeba cultures, lo’--lo3 amoebas could be detected (Fig. 2), but l-10 amoebas could be detected if PCR-amplified srDNA was the target. Detection sensitivity was reduced when RGP2/3 labelled at the 5’ end with digoxigenin was used for chemiluminescent detection (see the legend to Fig. 2). Detection sensitivity also was studied following PCR amplification of Acanthamoeba srDNA from mixtures containing l-lo4 amoebas plus lo5 HeLa cells plus or minus 1 pg of P. aeruginosa genomic DNA (not illustrated). The best results were obtained using RGPG and 892C
R.J. Gast, TJ. Byers/Molecular
1
2
and Biochemicaf Parasitology
3 IO4
103 102
10 1
0 g
is
g 2 Z
4
C Fig. 2. Minimum number of Ma strain amoebas detectable by hybridization of probe RGP2/3 to blots of Acanthamoeba DNA. Column 1, Genomic DNA from lo-fold dilutions of amoebas. Columns 2 and 3, PCR products obtained by amplifying a segment of amoeba srDNA using 892C and RGPG as primers and genomic DNA from IO-fold dilutions of amoebas as templates. Blots were probed as described in the legend to Fig. 1 with RGP2/3 5’-labelled with 32P in columns 1 and 2 or with digoxigenin (Genius System, Boehringer-Mannheim, Indianapolis, IN, uSA) in column-3. (C) Blot from control PCR reaction with no amoeba DNA added.
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2.59
oligonucleotide probes and genomic DNA, especially when axenic cultures are available. PCR amplified srDNAs, as used with Leishmania [9], would be the preferred probe targets if amoebas were mixed with other microorganisms or human cells. Lai et al. [17] developed a 125-bp nuclear large subunit ribosomal RNA gene probe that specifically hybridized to four Acanthamoeba isolates with a sensitivity similar to our probes. The advantage of our probes is that they have been designed to be more selective by basing their sequences on extensive knowledge of sequence variation and by making them smaller and more sensitive to target sequence differences.
Acknowledgements We thank many colleagues for cultures and helpful suggestions. This work was supported by NE1 Grant No. EY09073 to T.J.B. and P.A. Fuerst. R.J.G. submitted portions of this work to Ohio State University in partial fulfillment of requirements for the Ph.D.
(~‘-GTcAGAGGTGAAA’ITC~GG)
as primers to amplify a fragment of approx. 350 bp. These eukaryotic specific primers prevented amplification of bacterial srDNA and the amoeba-specific RGPG prevented amplification of human srDNA. The amount of amoeba PCR product was reduced, however, by the presence of relatively large amounts of either human or bacterial DNA possibly due to nonspecific competition for the primers. A minimum of l-10 amoebas could be detected when pure amoeba cultures were used, but the minimum was lo-100 when amoebas were mixed with human cells and 1000 when amoebas were mixed with human cells plus relatively large amounts of bacterial DNA. RGPG, RGP2/3 and RGPA recognize Acanthamoeba targets with high specificity and are most useful for identifying all members of the genus or of subgenus clusters of strains. Individual strains can be identified readily by srDNA sequencing since the sequences have been unique for each of the more than 50 strains that we have examined. We have shown that detection specificity and sensitivity can be achieved for Acanthamoeba using
References [l] John, D.T. (1993) Opportunistically pathogenic free-living amebae. In: Parasitic Protozoa (Kreier, J.P. and Baker, J.R.. eds), 2nd edn., pp. 143-246. Academic Press, San Diego, CA. [2] Palmer, M.L. and Hyndiuk, R.A. (1993) Contact lens-related infectious keratitis. Int. OphthaImoI. Clin. 33, 23-49. [3] Gordon, Y.J. (1993) Rapid diagnostic tests for infectious ocular disease. Int. Ophthalmol. Clin. 33, 153-161. [4] Bacon, AS., Dart, J.K., Ficker, L.A., Matheson, M.M. and Wright, P. (1993) Acanthamoeba keratitis. The value of early diagnosis. Opthalmology 100, 1238-1243. [5] MacPherson, J.M. and Gajadhar, A.A. (1993) Ribosomal RNA sequences for the specific detection of Toxoplasma gondii by hybridization assay. Mol. Cell. Probes 7, 97-103. [6] Holmdahl, O.J.M., Mattsson, J.G., Uggla, A. and Johansson, K.-E. (1993) Oligonucleotide probes complementary to variable regions of 18s rRNA from Sarcocystis species. Mol. Cell. Probes 7, 481-486. [7] Benavides, G.R., Sullivan, J.J., Steurer, F., McGraw, R.A. and Tarleton, R.L. (1993) Differentiation of trypanosomatid species by hybridization to selected rRNA probes. Mol. Cell. Probes 7, 89-96.
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[81 Uliana, S.R.B., Nelson, K., Beverley, S.M., Camargo, E.P. and Floerer-Winter, L.M. (1994) Discrimination amongst Ieishmania by polymerase chain reaction and hybridization with small subunit ribosomal DNA derived oligonucleotides. J. Euk. Microbial. 41, 324-330. [9] Visvesvara, G.S. (1991) Classification of Acanthamoeba. Rev. Infect. Dis. 13, S369-S372. [lo] O’Day, D.M. (1983) Fungal keratitis. In: The Cornea: Scientific foundation and clinical practice (O’Day, D.M., ed.), pp. 420-428. Little&Brown, Boston, MA. [ll] Weekers, P.H.H., Gast, R.J., Fuerst, P.A. and Byers, T.J. (19941 Sequence variations in small-subunit ribosomal RNAs of Hartmannella uermiformis and their phylogenetic implications. Mol. Biol. Evol. 11, 684-690. [121 Byers, T.J., Maynard, B.J., L&en, R.A., Martin, S.M. and Akins, R.A. (1980) Rapid growth of Acanthamoeba in defined media; induction of encystment by glucose-acetate starvation. J. Protozool. 27, 216-219.
Parasitology
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[13] Hugo, E.R., Gast, R.J., Byers, T.J. and Stewart, V.J. (19921 Purification of amoeba mtDNA using the UNSET procedure. In: Protocols in Protozoology (Lee, J.J. and Soldo, A.T., eds.), pp. D7.1-7.2. Allen Press, Lawrence. 1141 Walsh, P.S., Metzger, D.A. and Higuchi, R. (1991) Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material. BioTechniques 10, 506-513. [15] Giovannoni, S.J., DeLong, E.R., Olsen, G.J. and Pace, N.R. (1988) Phylogenetic group specific oligonucleotide probes for identification of single microbial cells. J. Bacterial. 170, 720-26. [16] Gast, R.J., Fuerst, P.A. and Byers, T.J. (1994) Discovery of group I introns in the nuclear small subunit ribosomal RNA genes of Acanthamoeba. Nucleic Acids Res. 22, 592-596. [17] Lai, S., Asgari, M. and Henney Jr., H.R. (1994) Non-radioactive DNA probe and polymerase chain reaction procedures for the specific detection of Acanthamoeba. Mol Cell. Probes 8, 81-89.