Cloning and molecular characterization of an immunogenic LigA protein of Leptospira interrogans

Abstract

A clone expressing a novel immunoreactive leptospiral immunoglobulin-like protein A of 130 kDa (LigA) from Leptospira interrogans serovar pomona type kennewicki was isolated by screening a genomic DNA library with serum from a mare that had recently aborted due to leptospiral infection. LigA is encoded by an open reading frame of 3,675 bp, and the deduced amino acid sequence consists of a series of 90-amino-acid tandem repeats. A search of the NCBI database found that homology of the LigA repeat region was limited to an immunoglobulin-like domain of the bacterial intimin binding protein of Escherichia coli, the cell adhesion domain of Clostridium acetobutylicum, and the invasin of Yersinia pestis. Secondary structure prediction analysis indicates that LigA consists mostly of beta sheets with a few alpha-helical regions. No LigA was detectable by immunoblot analysis of lysates of the leptospires grown in vitro at 30 degrees C or when cultures were shifted to 37 degrees C. Strikingly, immunohistochemistry on kidney from leptospira-infected hamsters demonstrated LigA expression. These findings suggest that LigA is specifically induced only in vivo. Sera from horses, which aborted as a result of natural Leptospira infection, strongly recognize LigA. LigA is the first leptospiral protein described to have 12 tandem repeats and is also the first to be expressed only during infection. Thus, LigA may have value in serodiagnosis or as a protective immunogen in novel vaccines.

FIG. 1.

(A) Nucleotide sequence of ligA and its deduced amino acid sequence. Italics regions are the three possible translation start codons. Bold and underlined nucleotides indicate primer annealing sites for Fig. 2 and 6, respectively. Arrows show the potential transcription termination sequence. (B) Alignment of the predicted amino acid sequences for the 12 tandem repeats and the immunoglobulin-like domain of E. coli intimin-binding (receptor) protein (Ig11, CD:pfam02368; Ig12, CD:smart00635). Twelve repeat sequences of a 90-amino-acid sequence include residues from 136 to 218, 224 to 310, 311 to 400, 401 to 489, 490 to 580, 581 to 670, 671 to 760, 761 to 851, 852 to 942, 943 to 1033, 1034 to 1125, and 1126 to 1216, respectively.

FIG. 1.

(A) Nucleotide sequence of ligA and its deduced amino acid sequence. Italics regions are the three possible translation start codons. Bold and underlined nucleotides indicate primer annealing sites for Fig. 2 and 6, respectively. Arrows show the potential transcription termination sequence. (B) Alignment of the predicted amino acid sequences for the 12 tandem repeats and the immunoglobulin-like domain of E. coli intimin-binding (receptor) protein (Ig11, CD:pfam02368; Ig12, CD:smart00635). Twelve repeat sequences of a 90-amino-acid sequence include residues from 136 to 218, 224 to 310, 311 to 400, 401 to 489, 490 to 580, 581 to 670, 671 to 760, 761 to 851, 852 to 942, 943 to 1033, 1034 to 1125, and 1126 to 1216, respectively.

FIG. 1.

(A) Nucleotide sequence of ligA and its deduced amino acid sequence. Italics regions are the three possible translation start codons. Bold and underlined nucleotides indicate primer annealing sites for Fig. 2 and 6, respectively. Arrows show the potential transcription termination sequence. (B) Alignment of the predicted amino acid sequences for the 12 tandem repeats and the immunoglobulin-like domain of E. coli intimin-binding (receptor) protein (Ig11, CD:pfam02368; Ig12, CD:smart00635). Twelve repeat sequences of a 90-amino-acid sequence include residues from 136 to 218, 224 to 310, 311 to 400, 401 to 489, 490 to 580, 581 to 670, 671 to 760, 761 to 851, 852 to 942, 943 to 1033, 1034 to 1125, and 1126 to 1216, respectively.

FIG. 2.

Expression of LigA in E. coli. Whole-cell lysates of E. coli were subjected to SDS-PAGE, transferred to nitrocellulose, and blotted with a 1:100 dilution of rabbit antiserum to the 90-kDa truncated LigA. Lanes 1 and 2, E. coli with vector, pET22b only; lanes 3 and 4, E. coli harboring pET22b plus ligA construct; lanes 2 and 4, E. coli was induced with 0.4 mM IPTG; lane 5, prestained molecular size markers (Bio-Rad).

FIG. 3.

LipL32 and LipL36 but not LigA expression are temperature regulated. Lane 1, whole-cell lysate of leptospires grown at 30°C; lanes 2, 3, 4, 5, and 6, cultures 2, 3, 4, 5, and 6 days old, respectively, of leptospires grown at 37°C. Each lane was loaded with ∼5.0 μg of protein.

FIG. 4.

LigA expression in hamsters infected with L. interrogans serovar pomona. Sections of kidney were treated with rabbit antiserum specific for a 90-kDa truncated LigA (A), L. interrogans serovar pomona (B), LipL32 (C), LipL36 (D), and with preimmune serum (E). Kidney sections from noninfected hamsters were unreactive. Bar, 67 μm.

FIG. 5.

Recombinant LigA protein purified by using metal affinity chromatography and subjected to SDS-PAGE separation was probed with normal horse sera (lanes 1 to 4), equine lyme disease-positive sera (lanes 5 to 9), HGE-positive sera (lanes 10 to 11), aborted mare sera (lanes 12 to 19), and rabbit serum specific for a 90-kDa truncated LigA (lane 20). Each lane was loaded with ∼0.5 μg of protein.

FIG. 6.

Agarose gel showing PCR products and restriction analysis of ligA from different pathogenic serovars of Leptospira. (A) PCR products of ligA. (B) HindIII-digested PCR product of ligA. Lanes: 1, L. interrogans serovar pomona type kennewicki; 2, L. interrogans serovar pomona; 3, L. interrogans serovar hardjo; 4, L. interrogans serovar icterohemorrhagiae; 5, L. kirchneri serovar grippotyphosa; 6, L. interrogans serovar wolfii.