Identification, characterization, and synthesis of peptide epitopes and a recombinant six-epitope protein for Trichomonas vaginalis serodiagnosis

Figures & data

Table 1 Epitopesa,b of Trichomonas vaginalis fructose-1,6-bisphosphate aldolase are reactive with MAbs and human sera

SPOTs^i no	Amino acid sequence	Epitope sequence	Positive control women sera	Positive control men sera	MAb reactions
13–14	40–47	AIITASVK	A-W1c
19–21	58–65	AGARKYAN		A-M1c,d
21	61–71	RKYANQTMLRY	A-W2c,d
31	91–101	PIVLHLDHGDS	A-W3c,d,e
47–49	142–149	RPDYVTVE			ALD12c
55–57	166–173	KHTYTRPE			ALD64c,f
57	169–179	YTRPEEVQDFV	A-W4c
64–65	193–203	TSHGAYKF	A-W5c,f,g,h
78	232–242	SIPQEYVEMVN	A-W6c
93	277–287	RMVMTGTIRRL		A-M2c
99–100	298–305	RQYLGEAR	A-W7c,d,h	A-M3c,d,h
101–103	304–311	ARTKLTEM	A-W8

Notes:

a This refers to epitopes that have ≥50% amino acid sequence identity with the indicated human and microbial pathogen homologues;

b there is minimal amino acid sequence identity of these epitopes with the human homologue;

c Treponema pallidum;

d Neisseria gonorrhoeae;

e Candida albicans, Saccharomyces cerevisiae, Streptococcus pyogenes, S. pneumoniae, Staphylococcus aureus, and Escherichia coli;

f C. albicans and S. cerevisiae;

g C. albicans, S. aureus, and E. coli;

h S. aureus; ⁱnumbers of amino acids for the immobilized peptides (Materials and methods).

Abbreviations: A, aldolase; M, positive control men sera; MAb, monoclonal antibody; W, positive control women sera; ALD12 and ALD64, designate MAbs to aldolase.

Table 3 Epitopesa of Trichomonas vaginalis glyceraldehyde-3-phosphate dehydrogenase are reactive with MAbs and human sera

SPOTsl no	Amino acid sequence	Epitope sequence	Positive control women seral	Positive control men seral	MAb reactions
3–5	13–17	LYPKD		G-M1
12	34–44	YLLKYDTAHRA	G-W1e,h,i,k		ALD32Ce,h,i,k
20	58–68	FTVGEGADKWV		G-M2
23–24	70–77	KSIGGRLG	G-W2		ALD30A
25–27	79–83	SQLPW		G-M3b,d–g
31–33	94–101	STGIFRTK	G-W3c–e,j
35–37	106–113	AEGKIKKD	G-W4
39–41	118–125	HLVSGAKK	G-W5c–k	G-M4c–k
51–53	157–161	SNASC		G-M5c–k
57–59	175–182	NAFGIRNG	G-W6b–e,i–k
67–69	205–212	RRARAAGM	G-W7h,i		B43i,j
72–74	217–224	TSTGAAIA	G-W8b–k
75–77	229–233	CHGLP		G-M6
84	250–260	SLVDLTVNVNA	G-W9c–e,j,k
97–98	292–299	VSSDIIGC		G-M7b–e,h,i,k
99–100	298–305	GCQYSSIV		G-M8b–f
101	301–311	YSSIVDALSTK	G-W10e,f,h,i
109	325–335	VLSWYDNEWMY	G-W11b–k	G-M9b–k
111–113	337–341	CRCAD		G-M10

Notes:

a This refers to epitopes that have ≥50% amino acid sequence identity with the indicated human and microbial pathogen homologues;

b human homologue;

c Candida Albicans;

d Saccharomyces Cerevisiae;

e Chlamydia Trachomatis;

F Treponema Pallidum;

g Neisseria Gonorrhoeae;

h Streptococcus Pneumoniae;

i Streptococcus Pyogenes;

j Staphylococcus Aureus;

k Escherichia Coli;

l numbers of amino acids for the immobilized peptides (Materials and methods).

Abbreviations: G, glyceraldehyde-3-phosphate dehydrogenase; M, positive control men sera; MAb, monoclonal antibody; W, positive control women sera; ALD32C and ALD30A, designate MAbs to glyceraldehyde-3-phosphate dehydrogenase.

Table 4 List of synthetic 15-mer peptides of representative epitopes of ALD, ENO, GAP, and ACT that are reactive with positive control women and men sera

No	Women/men epitope designationa	Peptide name	Amino acid numbers	Amino acid sequenceb
1	A-W1	ALD1	36–50	EQLQAIITASVKTES
2	A-ALD12	ALD2	138–152	EAHSRPDYVTVEGEL
3	A-ALD64	ALD3	159–173	EDDVKAEKHTYTRPE
4	A-W4	ALD4	167–181	HTYTRPEEVODFVSK
5	A-W6	ALD5	230–244	SSSIPOEYVEMVNKY
6	A-M2	ALD6	275–289	DGRMVMTGTIRRLFV
7	A-W8	ALD7	301–315	LGEARTKLTEMYMRK
8	E-W1	ENO1	57–71	VKYLGRVTLAARSSA
9	E-M1	ENO2	70–84	VTLAARSSAPSGAST
10	E-W4, E-M4	ENO3	182–196	TDGTVLKKNIGGNAC
11	E-M6	ENO4	224–238	DKVPKKFKLPSPFFN
12	E-W5	ENO5	236–250	KLGGLLVKKYGLSAK
13	E-M8	ENO6	295–309	SSEFYDEEKKLYEVE
14	E-W7	ENO7	344–358	DYENWTKLNARLGQR
15	E-W8, E-M10	ENO8	366–380	LYTTNPITIKKGLEG
16	G-M1	GAP1	8–22	RACRKLYPKDIQVVA
17	G-M2	GAP2	56–70	QEFTVGEGADKWVVK
18	G-W2	GAP3	67–81	WVVKSIGGRLGPSQL
19	G-W4	GAP4	104–118	KDAEGKIKKDDGYDGH
20	G-M6	GAP5	224–238	ALPKVCHGLPPKSLD
21	G-M10	GAP6	332–346	EWMYSCRCADIFHRL
22	ACT-W9, M1	ACT1	463–467	SVNRHHSQLITYIKH
23	ACT-W10, M2	ACT2	492–506	AQPLYDEAIAFKEEV
24	ACT-W13, M5	ACT3	803–817	FKDTFKYFDKDKSNS

Notes:

a Designations are those as indicated in Tables 1 through 3;

b underlined amino acids represent epitope sequences, as determined by the SPOTs assay (Materials and methods).

Abbreviations: A and ALD1 through ALD7, aldolase peptides; ACT and ACT1 through ACT3, alpha-actinin peptides; E and ENO1 through ENO8, alpha-enolase peptides; G and GAP1 through GAP6, glyceraldehyde-3-phosphate dehydrogenase peptides; M, positive control men sera; W, positive control women sera.

Figure 1 Sequence analyses of Trichomonas vaginalis fructose-1,6-bisphosphate aldolase.

Notes: The amino acid sequence identity of T. vaginalis enzyme was compared with the ALD proteins of Treponema pallidum, Neisseria gonorrhoeae, Streptococcus pyogenes, Streptococcus pneumoniae, Staphylococcus aureus, Escherichia coli, Candida albicans, Saccharomyces cerevisiae, and Homo sapiens. The percent identity was found to be 68%, 39%, 30%, 30%, 38%, 32%, 23%, 21%, and 10%, respectively. Boxed amino acids indicate epitopes of T. vaginalis detected by women and men sera and MAbs ALD12 and ALD64 (Table 1). Blue-colored amino acid residues represent high sequence identity ranging from 68% to 100%. Red residues are low sequence identity ranging from 0 to 33%, and dashed lines are gaps within the sequences of the corresponding ALD proteins of other organisms. Black lettering signifies approximately 34% to 67% identity. A-W1 through A-W8 are epitopes detected by women sera; A-M1 through A-M3 are epitopes detected by men sera.
Abbreviations: ALD, aldolase; Ca, Candida albicans; Ec, Escherichia coli; Hs, Homo sapiens; M, men; MAbs, monoclonal antibodies; Ng, Neisseria gonorrhoeae; Sa, Staphylococcus aureus; Sc, Saccharomyces cerevisiae; Spn, S. pneumoniae; Spy, Streptococcus pyogenes; Tp, Treponema pallidum; W, women; ALD12 and ALD64, designate MAbs to aldolase as per Table 1.

Figure 2 Hydrophobicity and antigenicity profiles of Trichomonas vaginalis α-enolase, aldolase, and glyceraldehyde-3-phosphate dehydrogenase proteins.

Notes: The individual epitopes detected by women and men sera and the MAbs are labeled above the numbered linear protein. Hydrophilicity is presented as values <0, and the corresponding antigenicity is >0.
Abbreviations: A and ALD, fructose-1,6-bisphosphate aldolase; E and ENO, α-enolase; G and GAP, glyceraldehyde-3-phosphate dehydrogenase; M, men; MAb, monoclonal antibody; W, women; ALD13, ALD55, ALD11, ALD25, and B44, designate MAbs to alpha-enolase, ALD12/64 designates a MAb to aldolase, and ALD32, ALD30, and B43 designate MAbs to glyceraldehyde-3-phosphate dehydrogenase.

Figure 3 Representative experiment showing dot blots of individual 15-mer peptide epitopes.

Notes: The peptides were derived from the proteins fructose-1,6-bisphosphate aldolase (A), α-enolase (B), glyceraldehyde-3-phosphate dehydrogenase (C), and α-actinin (D). Individual 15-mer peptides were spotted onto nitrocellulose prior to reacting with the sera of women and men (indicated below the spots), as detailed in Materials and methods. The individual epitopes are those indicated in Table 4.
Abbreviations: ACT, α-actinin; ALD, fructose-1,6-bisphosphate aldolase; ENO, α-enolase; GAP, glyceraldehyde-3-phosphate dehydrogenase.

Figure 4 Representative experiments in duplicate dot blots of combinations of 15-mer peptide epitopes (A) and densitometric scans of reactive dot blots (B).

Notes: (A) shows duplicate dot blots of combinations of peptides (A1 though A10) incubated with men and women sera that were highly seroreactive (labeled 4+/5+) with α-actinin by ELISA (34) as well as with the negative control nonreactive sera (labeled -0-). Only one dot blot each is shown for the negative control reaction. (B) presents the densitometric scans performed on the dot blots from experiment 1 for the combinations of peptides (bars numbered 1 through 7) as well as for the positive control ACT2 plus ACT3 (bar numbered 8). Bars 9 and 10 show the absence of any reactivity, using the negative control women and/or men sera. The individual epitopes are those indicated in Table 4.
Abbreviations: ACT, α-actinin; ALD, fructose-1,6-bisphosphate aldolase; con, control; ELISA, enzyme-linked immunosorbent assay; ENO, α-enolase; GAP, glyceraldehyde-3-phosphate dehydrogenase glyceraldehyde; neg, negative.

Figure 5 The 111 amino acid sequence and expression and purification of the rSOE encoding epitopes of GAP, ENO, and ALD.

Notes: (A) The sequence of two 15-mer epitopes (underlined) for each protein, as shown in Table 4. The six H residues at the carboxy terminus represent the hexahistidine added for both purification using Ni²⁺-NTA affinity chromatography and detection using MAb. (B) The SDS-PAGE and Coomassie-brilliant blue stained gels of recombinant Escherichia coli expressing the rSOE (lane 1) and the lysate prepared for chromatography (lane 2), as described in Materials and methods. Flow through is the lysate obtained after chromatography (lane 3), followed by two representative washes of the column (lanes 4 and 5). Finally, lanes 6–11 show the rSOE eluted from the column, and the single band indicates specific purification of the rSOE::His₆.
Abbreviations: ALD, fructose-1,6-bisphosphate aldolase; ENO, α-enolase; GAP, glyceraldehyde-3-phosphate dehydrogenase; His₆, hexahistidine; MAb, monoclonal antibody; mw, molecular weight; Ni2⁺, nickel; NTA, nitrilotriacetic acid; rSOE, recombinant protein of sequences of epitopes; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; SOE, sequences of epitopes.

Figure 6 Immunodetection of the rSOE by ELISA (A), dot blot (B), and immunoblotting after SDS-PAGE (C).

Notes: (A) shows a representative ELISA for detection of rSOE immobilized onto individual wells of 96-well microtiter plates (Materials and methods) by sera of women and men highly reactive to the trichomonad protein α-actinin (34) (labeled 4+) compared with the negative control, unreactive sera (labeled 0). HA423 is a MAb to α-actinin, which was unreactive to the rSOE. In contrast, the MAb to hexahistidine of the fusion protein readily bound to the rSOE, as evidenced by the very high absorbance values (A_{405 nm}). (C) Representative immunoblot after SDS-PAGE and blotting onto nitrocellulose, of rSOE detected by pooled sera of women and men reactive with α-actinin, as above in (A) (lane 1). Lane 2 is a duplicate of the nitrocellulose blot stained with Ponceau S to show efficient transfer onto nitrocellulose, and lane 3 is the Coomassie-brilliant blue-stained gel of a duplicate sample of the rSOE used for blotting.
Abbreviations: ELISA, enzyme-linked immunosorbent assay; His₆, hexahistidine; MAb, monoclonal antibody; mw, molecular weight; rSOE, recombinant protein of sequences of epitopes; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Table 2 Epitopesa of Trichomonas vaginalis α-enolase are reactive with MAbs and human sera

SPOTsm no	Amino acid sequence	Epitope sequence	Positive control women sera	Positive control men sera	MAb reactions
2–3	7–14	AIVKECIA			ALD13
21–23	64–71	YLGRVTLA	E-W1		ALD55/97
23–25	70–77	LAARSSAP		E-M1d
31–33	94–101	DKARYGGK	E-W2b,f–l	E-M2b,f–l
46–47	139–146	TVLKKNIG	E-W3h	E-M3h
62	184–194	VPKKFKLPSPF	E-W4d	E-M4d
67	199–209	NGGKHAGGNLK		E-M5b,d–f,h,l
76	226–236	QLRMVAEVYQK		E-M6
79–81	238–245	GGLLVKKY	E-W5d
81	241–251	LVKKYGLSAKN		E-M7d,e
99–100	298–305	FYDEEKKL		E-M8d
110	328–338	KKHPAIVSIED	E-W6c,f
114–116	343–350	LDYENWTK	E-W7
117–118	352–359	NARLGQRV		E-M9f,i–k
121–123	364–371	DDLYTTNPb			ALD11c,f
123–124	370–377	NPITIKKG	E-W8d	E-M10d
149–151	448–455	ERIQKYTR	E-W9b,i–k	E-M11b,h–j	B44b,i–k
154–155	463–470	LKEHDMLA			ALD25

Notes:

a This refers to epitopes that have ≥50% amino acid sequence identity with the indicated human and microbial pathogen homologues;

b human homologue;

c microbial pathogens listed in Table 1;

d Candida Albicans;

e Saccharomyces cerevisiae;

F Chlamydia Trachomatis;

g Treponema Pallidum;

h Neisseria gonorrhoeae;

i Streptococcus Pneumoniae;

j as per ^“a” above except for Streptococcus pyogenes;

k as per ^“a” above except for Staphylococcus aureus;

l as per ^“a” above except for Escherichia coli;

m numbers of amino acids for the immobilized peptides (Materials and methods).

Abbreviations: E, α-enolase; M, positive control men sera; MAb, monoclonal antibody; W, positive control women sera; ALD13, ALD55/97, ALD11, and ALD25, designate MAbs to enolase.