Temperature-regulated protein synthesis by Leptospira interrogans

Abstract

Leptospira interrogans is an important mammalian pathogen. Transmission from an environmental source requires adaptations to a range of new environmental conditions in the organs and tissues of the infected host. Since many pathogenic bacteria utilize temperature to discern their environment and regulate the synthesis of appropriate proteins, we investigated the effects of temperature on protein synthesis in L. interrogans. Bacteria were grown for several days after culture temperatures were shifted from 30 to 37 degrees C. Triton X-114 cellular fractionation identified several proteins of the cytoplasm, periplasm, and outer membrane for which synthesis was dependent on the culture temperature. Synthesis of a cytoplasmic protein of 20 kDa was switched off at 37 degrees C, whereas synthesis of a 66-kDa periplasmic protein was increased at the higher temperature. Increased synthesis of a 25-kDa outer membrane protein was observed when the organisms were shifted from 30 to 37 degrees C. A 36-kDa protein synthesized at 30 but not at 37 degrees C was identified as LipL36, an outer membrane lipoprotein. In contrast, expression of another lipoprotein, LipL41, was the same at either temperature. Immunoblotting with convalescent equine sera revealed that some proteins exhibiting thermoregulation of synthesis elicited antibody responses during infection. Our results show that sera from horses which aborted as a result of naturally acquired infection with L. interrogans serovar pomona type kennewicki recognize periplasmic and outer membrane proteins which are differentially synthesized in response to temperature and which therefore may be important in the host-pathogen interaction during infection.

FIG. 1

(A) Antigens in lysates of L. interrogans serovar pomona type kennewicki JEN4 grown at 30°C and shifted to 24, 30, or 37°C. The lysates were separated by SDS-PAGE and immunoblotted with serum from a convalescent mare. A 57-kDa antigen apparently upregulated at 37°C is indicated with an arrow. The asterisks indicate antigens present in greater amounts at 24 and 30°C than at 37°C. (B) To establish that equivalent amounts of protein were loaded in the gel shown in panel A, lysates were probed with antiserum to endoflagella of T. pallidum, a non-temperature-regulated protein. Molecular mass markers are indicated in kilodaltons.

FIG. 2

Immunoblot analysis of leptospiral heat shock proteins DnaK and GroEL in lysates of L. interrogans JEN4 expressed at 30 and 37°C following SDS-PAGE. Molecular mass markers are indicated in kilodaltons.

FIG. 3

(A) Silver stain of Triton X-114 extracts of L. interrogans JEN4 at 30 or 37°C separated by SDS-PAGE and showing proteins in fractions consisting of protoplasmic cylinder (P), aqueous phase (A), and detergent phase (D). (B) Fractions probed with antiserum to T. pallidum endoflagella to demonstrate that the protoplasmic cylinder was intact. Molecular mass markers are indicated in kilodaltons. Upregulated proteins are indicated with arrows; downregulated proteins are indicated with asterisks.

FIG. 4

Triton X-114 extract of L. interrogans JEN4 at 30 and 37°C immunoblotted with serum from a convalescent mare following SDS-PAGE. Fractions consisting of protoplasmic cylinder (P), aqueous phase (A), and detergent phase (D) are reactive. Molecular mass markers are indicated in kilodaltons. Upregulated antigens are indicated with arrows; downregulated antigens are indicated with asterisks.

FIG. 5

Lysates of JEN4 grown at 30°C, grown at 30°C and shifted to 37°C, grown at 37°C and maintained at 37°C, or grown at 37°C and shifted to 30°C. Following SDS-PAGE, the lysates were immunoblotted with LipL36- and LipL41-specific antisera.