Leptospiral proteins recognized during the humoral immune response to leptospirosis in humans

Abstract

Leptospirosis is an emerging zoonosis caused by pathogenic spirochetes belonging to the genus Leptospira. An understanding of leptospiral protein expression regulation is needed to develop new immunoprotective and serodiagnostic strategies. We used the humoral immune response during human leptospirosis as a reporter of protein antigens expressed during infection. Qualitative and quantitative immunoblot analysis was performed using sera from 105 patients from Brazil and Barbados. Sera from patients with other diseases and healthy individuals were evaluated as controls. Seven proteins, p76, p62, p48, p45, p41, p37, and p32, were identified as targets of the humoral response during natural infection. In both acute and convalescent phases of illness, antibodies to lipopolysaccharide were predominantly immunoglobulin M (IgM) while antibodies to proteins were exclusively IgG. Anti-p32 reactivity had the greatest sensitivity and specificity: positive reactions were observed in 37 and 84% of acute- and convalescent-phase sera, respectively, while only 5% of community control individuals demonstrated positive reactions. Six immunodominant antigens were expressed by all pathogenic leptospiral strains tested; only p37 was inconsistently expressed. Two-dimensional immunoblots identified four of the seven infection-associated antigens as being previously characterized proteins: LipL32 (the major outer membrane lipoprotein), LipL41 (a surface-exposed outer membrane lipoprotein), and heat shock proteins GroEL and DnaK. Fractionation studies demonstrated LipL32 and LipL41 reactivity in the outer membrane fraction and GroEL and DnaK in the cytoplasmic fraction, while p37 appeared to be a soluble periplasmic protein. Most of the other immunodominant proteins, including p48 and p45, were localized to the inner membrane. These findings indicate that leptospiral proteins recognized during natural infection are potentially useful for serodiagnosis and may serve as targets for vaccine design.

FIG. 1

Representative immunoblots of sera from patients with leptospirosis during urban epidemics in the city of Salvador, Brazil. SDS-PAGE (10% polyacrylamide) was used to separate antigen extracts of a clinical isolate of L. interrogans serovar copenhageni. Immunoblots were incubated with serum samples of individual patients (patients 235 [lane 1], 66 [lanes 2 and 3], 7 [lanes 4 and 5], 134 [lanes 6 and 7], 205 [lane 8], and 66 [lane 9]) and probed with either anti-IgM (IgM) or anti-IgG (IgG) secondary antibody conjugate. Lane 10 was incubated with serum from a healthy control individual. The locations of molecular mass markers (in kilodaltons) and selected leptospiral proteins are shown on the right and left, respectively. In addition, the positions of leptospiral proteins are marked (○) within the immunoblot and correspond to the following, in descending order: p76/82, p70, p62, p48, p45, the p41/42 complex, p37, p32, and p31 in lane 3; p44 and the p41/42 complex in lane 5; p58 and the p41/42 complex in lane 8; and the p41/42 complex and p25 in lane 9.

FIG. 2

Receiver-operator characteristic curve showing the sensitivity and false-positive rate for immunoblot seroreactivity to combinations of leptospiral proteins. Sensitivity and 1-specificity were defined as the proportion of sera from confirmed cases and community control individuals, respectively, which recognized at least one leptospiral antigen in a combination. The best order of antigen combinations for acute-phase sera was as follows: p32 alone (a); p32 or p62 (b); p32, p62, or p76 (c); p32, p62, p76, or p41 (d); p32, p62, p76, p41, or p45 (e). For convalescent-phase sera the best order was as follows: p32 (A); p32 or p76 (B); p32, p76 or p41 (C); p32, p76, p41, or p45 (D); p32, p76, p41, p45, or p62 (E).

FIG. 3

Immunoblot panel of Leptospira species probed with pooled convalescent-phase sera from leptospirosis patients in Salvador, Brazil. The locations of molecular mass markers (in kilodaltons) and selected leptospiral proteins are indicated to the right and left, respectively. Most proteins were conserved among pathogenic Leptospira species L. interrogans, L. kirschneri, L. borgpetersenii, L. noguchii, L. santarosai, L. weilii, and L. inadai. Two proteins, p62 and p76, which were identified as heat shock proteins GroEL and DnaK, respectively, were exceptions to this pattern. Their expression is conserved in all Leptospira species including the nonpathogens L. biflexa, L. meyeri, and L. wolbachii and the related organism Leptonema illini.

FIG. 4

A two-dimensional immunoblot of L. interrogans serovar copenhageni proteins probed with pooled convalescent-phase sera from leptospirosis patients in Salvador, Brazil. The locations of DnaK, GroEL, LipL41, OmpL1, LipL32, and three as yet unidentified antigens (p45, p25, and p22) are indicated. The locations of molecular mass markers (in kilodaltons) and reference isoelectric points (pI) are indicated on the right and top of the figure, respectively.

FIG. 5

Localization of leptospiral protein antigens by fractionation of cellular components. Extracts of whole organisms (W) were used as starting material. The total membrane fraction (MP) contains both the cytoplasmic membrane and outer membrane, while the soluble fraction (MS) contains cytoplasmic and periplasmic material. The detergent Triton X-114 selectively releases the outer membrane, leaving the cytoplasm and cytoplasmic membrane in the Triton X-114 insoluble pellet (TP). Phase partitioning of the Triton X-114 extract separates periplasmic material in the aqueous phase (TA) from outer membrane components in the detergent phase (TD). The locations of molecular mass markers (in kilodaltons) and leptospiral protein antigens are shown on the left and right, respectively. In addition, the positions of leptospiral proteins are marked by arrows (←) within the immunoblot and correspond to, in descending order, p58, p48, p45, and p42 in the p41/42 complex in lane TP and LipL41, part of the p41/42 complex, and LipL45/31 in lane TD.