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. 2002 Jan;70(1):323-34.
doi: 10.1128/iai.70.1.323-334.2002.

Novel 45-kilodalton Leptospiral Protein That Is Processed to a 31-kilodalton Growth-Phase-Regulated Peripheral Membrane Protein

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Novel 45-kilodalton Leptospiral Protein That Is Processed to a 31-kilodalton Growth-Phase-Regulated Peripheral Membrane Protein

James Matsunaga et al. Infect Immun. .
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Abstract

Leptospiral protein antigens are of interest as potential virulence factors and as candidate serodiagnostic and immunoprotective reagents. We identified leptospiral protein antigens by screening a genomic expression library with serum from a rabbit hyperimmunized with formalin-killed, virulent Leptospira kirschneri serovar grippotyphosa. Genes expressing known outer membrane lipoproteins LipL32 and LipL41, the heat shock protein GroEL, and the alpha, beta, and beta' subunits of RNA polymerase were isolated from the library. In addition, a new leptospiral gene that in Escherichia coli expressed a 45-kDa antigen with an amino-terminal signal peptide followed by the spirochetal lipobox Val(-4)-Phe(-3)-Asn(-2)-Ala(-1) (downward arrow)Cys(+1) was isolated. We designated this putative lipoprotein LipL45. Immunoblot analysis of a panel of Leptospira strains probed with LipL45 antiserum demonstrated that many low-passage strains expressed LipL45. In contrast, LipL45 was not detected in high-passage, culture-attenuated strains, suggesting that LipL45 is a virulence-associated protein. In addition, all leptospiral strains tested, irrespective of culture passage, expressed a 31-kDa antigen that was recognized by LipL45 antiserum. Southern blot and peptide mapping studies indicated that this 31-kDa antigen was derived from the carboxy terminus of LipL45; therefore, it was designated P31(LipL45). Membrane fractionation studies demonstrated that P31(LipL45) is a peripheral membrane protein. Finally, we found that P31(LipL45) levels increased as Leptospira entered the stationary phase, indicating that P31(LipL45) levels were regulated. Hamsters infected with L. kirschneri formed an antibody response to LipL45, indicating that LipL45 was expressed during infection. Furthermore, the immunohistochemistry of kidneys from infected hamsters indicated that LipL45 was expressed by L. kirschneri that colonized the renal tubule. These observations suggest that expression of LipL45 responds to environmental cues, including those encountered during infection of a mammalian host.

Figures

FIG. 1.
FIG. 1.
Expression of leptospiral antigens in E. coli. Several clones obtained from the expression library were transformed as plasmid DNA into E. coli XL1-Blue. Overnight cultures of the transformants were resuspended in final sample buffer and analyzed in an immunoblot with serum (1:5,000) from a rabbit hyperimmunized with formalin-killed, virulent L. kirschneri. The clone number is indicated above each lane (lanes 1 and 4, LipL32; lanes 5, 9, and 29, LipL45; lane 14, unknown; lane 26, GroEL; lane 27, unknown; lane 34, LipL41; lane C, negative control that does not express an antigen). The positions of molecular mass standards (in kilodaltons) are indicated on the left.
FIG. 2.
FIG. 2.
Sequence of the leptospiral gene encoding LipL45. The nucleotide sequence of clone 5 (accession number AF379683) from position 760 through position 2109 is shown. Probable −35 and −10 promoter sequences are indicated by large letters. The Shine-Dalgarno sequence is underlined. The spirochetal lipobox is indicated by boldface type, and the proposed signal peptidase II processing site is indicated by an arrow. The sequences of the PCR primers used to generate the probe for the Southern blot are indicated by multiple arrowheads. The putative ρ-independent transcriptional terminator is indicated by dashed arrows. The amino acid sequence used to generate serum to the C terminus of LipL45 (5ORF2C) is shaded.
FIG. 3.
FIG. 3.
LipL45 levels in low- and high-passage L. kirschneri isolates. L. kirschneri was cultured from kidney (K) or blood (B) samples, and isolates are designated by the hamster (H1 or H2) from which they were isolated and the number of passages in culture (P2 to P5). High-passage L. kirschneri (HP) was obtained by multiple passages of the organism in vitro. The immunoblot was probed with LipL45 antiserum (1:5,000).
FIG. 4.
FIG. 4.
LipL45 expression in low-passage isolates of various serovars of Leptospira. Leptospira isolates were cultured from the kidneys of infected hamsters and passaged serially in Bovuminar PLM-5 medium up to five times (P2 to P5). Leptospires were resuspended in final sample buffer prior to immunoblot analysis with LipL45 antiserum (1:4,000). Lane 1, L. kirschneri serovar grippotyphosa strain RM52; lane 2, L. interrogans serovar pomona strain P10637-46; lane 3, L. interrogans serovar canicola/portlandvere strain Mex 1; lane 4, L. interrogans serovar canicola/portlandvere strain CDC Nic 1808; lane 5, L. kirschneri serovar grippotyphosa strain ISU 82; lane 6, L. kirschneri serovar grippotyphosa strain RM52, high passage.
FIG. 5.
FIG. 5.
LipL45 expression in high-passage pathogenic and saprophytic isolates of various serovars of Leptospira. A panel of leptospiral serovar isolates was examined by immunoblot analysis with LipL45 (1:5,000) and ImpL63 (1:4,000) antisera. Lane 1, L. kirschneri serovar grippotyphosa strain Moskva V; lane 2, L. interrogans serovar pomona strain PO-01; lane 3, L. interrogans serovar bratislava strain AS-05; lane 4, Leptospira noguchii serovar proechymis strain LT 796; lane 5, L. noguchii serovar fort bragg strain Fort Bragg; lane 6, L. kirschneri serovar mozdok strain 5621; lane 7, L. kirschneri serovar grippotyphosa strain RM52; lane 8, Leptospira borgpetersenii serovar hardjo strain HB-15B/93U; lane 9, Leptospira santarosai serovar canalzonae strain CZ 188; lane 10, L. santarosai serovar bakeri strain 79; lane 11, Leptospira wolbachii serovar biflexa strain codice; lane 12, Leptonema illini 3055; lane 13, Leptospira inadai serovar lyme strain 10; lane 14, L. borgpetersenii serovar javanica strain Veldrat Batavia 46; lane 15, L. borgpetersenii serovar tarassovi strain Perepelicin; lane 16, L. interrogans serovar pomona strain RZ11; lane 17, Leptospira weilii serovar celledoni strain Celledoni; lane 18, Leptospira biflexa serovar patoc strain Patoc I; lane 19, Leptospira meyeri serovar semaranga strain Veldrat Semarang 173. The lanes containing the saprophytic species L. wolbachii, L. illini, L. inadai, L. weilii, L. biflexa, and L. meyeri are indicated by asterisks (lanes 11 to 13 and 17 to 19).
FIG. 6.
FIG. 6.
Peptide mapping of recombinant LipL45 and P31LipL45. Recombinant His6-LipL45 (rLipL45) and native P31LipL45 (P31) were gel purified, digested with V8 protease, and subjected to immunoblot analysis. The membrane was probed with serum generated against the C-terminal 88 amino acid residues of LipL45 (1:4,000). Lane 1, purified recombinant His6-LipL45; lane 2, high-passage L. kirschneri RM52; lane 3, gel-purified P31 treated with V8 protease; lane 4, gel-purified His6-LipL45 treated with V8 protease. The positions of molecular mass standards (in kilodaltons) are indicated on the right. The His6-LipL45 protein migrated as a 46-kDa band (lane 1). Note that LipL45 was detected when a large number of high-passage leptospires were loaded onto the gel (lane 2).
FIG. 7.
FIG. 7.
Low-stringency Southern blot analysis of the gene encoding LipL45. Genomic DNA isolated from low-passage (LP) (lanes 1 and 3) or high-passage (HP) (lanes 2 and 4) L. kirschneri isolates were digested with EcoRI (lanes 1 and 2) or ClaI (lanes 3 and 4) and subjected to a low-stringency Southern blot analysis with a 5orf2 probe (Fig. 2). The positions of molecular weight standards (in kilobases) are indicated on the left.
FIG. 8.
FIG. 8.
Localization of LipL45 and P31LipL45. (A) High-passage L. kirschneri was fractionated with Triton X-114, and each fraction was subjected to immunoblot analysis with LipL45 (1:5,000) and LipL36 (1:4,000) antisera. Lane W, whole cells; lane P, insoluble pellet; lane A, aqueous fraction; lane D, detergent fraction. (B) Membrane fractions of virulent L. kirschneri were washed with buffer (lanes 2 and 3), NaCl (lanes 4 and 5), urea (lanes 6 and 7), Na2CO3 (lanes 8 and 9), or Triton X-100 (lanes 10 and 11) for 15 min. After this, the membrane was pelleted by centrifugation, and the pellets (P) and supernatant fluids (S) were subjected to immunoblot analysis with LipL45 and LipL41 antisera. Lane 1 contained unfractionated L. kirschneri (W).
FIG. 9.
FIG. 9.
Variation of P31LipL45 levels with growth phase. (A) L. interrogans serovar pomona and L. kirschneri serovar grippotyphosa were inoculated into Bovuminar PLM-5 medium at a density of 5 × 105 cells/ml, and the cultures were incubated at 30°C. The number of cells in each culture was determined by dark-field microscopy at different times. (B and C) Samples (1 × 108 cells) obtained from the cultures at different times were examined by immunoblot analysis with LipL45 (1:4,000) and LipL41 (1:8,000) antisera. The strains used were L. interrogans serovar pomona strain P10637-46 (B) and L. kirschneri serovar grippotyphosa strain RM52 (C).
FIG. 10.
FIG. 10.
Immunohistochemistry of the kidney of an infected hamster. A kidney from an infected Golden Syrian hamster obtained 28 days after infection with virulent L. kirschneri was examined by performing an immunohistochemistry analysis with LipL45 antibody. The arrows indicate the reactive lumina of three kidney tubules. The lumen of one tubule (left) and one venule (top, containing a red blood cell) did not react with the LipL45 antibody.

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