Antigenic and genetic characterization of lipoprotein LppQ from Mycoplasma mycoides subsp. mycoides SC

Abstract

Lipoprotein LppQ, a predominant 48-kDa antigen, and its corresponding gene, lppQ, were characterized in Mycoplasma mycoides subsp. mycoides SC, the etiological agent of contagious bovine pleuropneumonia. The lppQ gene is specific to M. mycoides subsp. mycoides SC and was found in the type strain and in field strains isolated in Europe, Africa, and Australia, as well as in vaccinal strains. LppQ is encoded as a precursor with a consensus sequence for prokaryotic signal peptidase II and a lipid attachment site. The leader sequence shows significant prominent transmembrane helix structure with a predicted outside-to-inside helix formation capacity. The N-terminal domain of the mature LppQ was shown to be surface exposed. It induced a strong, specific, early, and persistent immune response in naturally and experimentally infected animals. The C-terminal domain of LppQ possesses an integral membrane structure built up of repeated units, rich in hydrophobic and aromatic amino acids, which have a pore formation potential. A recombinant peptide representing the N-terminal domain of LppQ was obtained by site-directed mutagenesis of nine Mycoplasma-specific TGA (Trp) codons into universal TGG (Trp) codons and expression in Escherichia coli hosts. It was used for serodetection of cattle infected with M. mycoides subsp. mycoides SC, in which it was detected postinfection for significantly longer than conventional serological test reactions.

FIG. 1

Structure of LppQ. (Top) Genetic structure of the 1,764-bp segment of M. mycoides subsp. mycoides SC strain Afadé, cloned in plasmid pJFFmaO5. The box represents the ORF of lppQ. Dotted segment, precursor signal sequence; hatched segment, antigenic, surface-exposed N-terminal half; open segment, integral membrane C-terminal half. The circle with a stem represents the transcriptional stop signal. (Middle) Transmembrane helix prediction diagram; aa, amino acids. (Bottom) The solid line and left-hand scale represent the hydrophilicity diagram calculated according to the method of Hopp and Woods (20); the dotted line and right-hand scale show the predicted value of the coiled-coil tertiary structure calculated using a window size of 14 aa on the Lupas scale (22).

FIG. 2

Identification and characterization of the membrane lipoprotein LppQ. (A) Immunoblots containing total antigens of M. mycoides subsp. mycoides SC strain Afadé were reacted with serum from a cow experimentally infected with the homologous strain (1) (lane a) or with monospecific polyclonal rabbit anti-LppQ′ antibodies (lane b). (B) Autoradiography of [¹⁴C]palmitate-labeled M. mycoides subsp. mycoides SC strain Afadé. Lane 1, total antigens; lane 2, Triton X-114 detergent phase; lane 3, aqueous phase. (C) Filter containing the same samples as in panel B reacted with anti-LppQ′ antibodies. The scale to the left of panel A is in kilodaltons.

FIG. 3

Antigenic domains of LppQ. The immunoblots contain recombinant purified LppQ-C′ (lanes 1), LppQ-N′ (lanes 2), and whole LppQ′ (lanes 3). The filter in panel A was reacted with monospecific polyclonal rabbit anti-LppQ′. The filter in panel B was reacted with a field serum of a naturally infected cow suffering from CBPP. St, prestained molecular mass standard (in kilodaltons).

FIG. 4

Immunogenic specificity of LppQ. The immunoblots contain total-cell antigens. Lane 1, M. mycoides subsp. mycoides SC strain Afadé; lane 2, M. mycoides subsp. mycoides SC strain L2; lane 3, M. mycoides subsp. mycoides SC strain PG1; lane 4, M. mycoides subsp. mycoides LC strain Y-goat; lane 5, Mycoplasma sp. bovine group 7 strain PG50; lane 6, M. mycoides subsp. capri strain PG3; lane 7, M. capricolum subsp. capricolum strain California Kid; lane 8, M. capricolum subsp. capripneumoniae strain F38; lane 9, M. putrefaciens strain KS1. The blot in panel A was reacted with anti-LppQ-N′ antibodies. The blot in panel B was reacted with anti-LppQ-C′ antibodies. The blot in panel C (control) was reacted with a field serum from a cow which was infected experimentally with M. mycoides subsp. mycoides SC strain Afadé. St, prestained molecular mass standard (in kilodaltons).

FIG. 5

Immunogenicity of LppQ. The immunoblots contain purified recombinant LppQ-N′ reacted with cow sera taken sequentially before and after experimental infection with M. mycoides subsp. mycoides SC. (A) African strain Afadé; (B) European strain L2. The numbers indicate days before (−) or after infection.