Effects of environmental pH on membrane proteins in Borrelia burgdorferi

Abstract

Borrelia burgdorferi, the causative agent of Lyme disease, alternates between the microenvironments of the tick vector, Ixodes scapularis, and a mammalian host. The environmental conditions the spirochete encounters during its infectious cycle are suspected to differ greatly in many aspects, including available nutrients, temperature, and pH. Here we identify alterations in the membrane protein profile, as determined by immunoblotting and two-dimensional nonequilibrium pH gradient gel electrophoresis (2D-NEPHGE), that occur in virulent B. burgdorferi B31 as the pH of the medium is altered. Initial comparisons of cultures incubated at pHs 6.0, 7.0, and 8.0 yielded alterations in the expression of seven membrane proteins as determined by probing with hyperimmune rabbit serum. Six of these membrane proteins (54, 45, 44, 43, 35, and 24 kDa) were either present in increased amounts in or solely expressed by cultures incubated at pHs 6.0 and 7.0. The 24-kDa protein that decreased in expression at pH 8.0 was identified as outer surface protein C (OspC). In addition, a 42-kDa membrane protein increased in amount in cultures incubated at pH 8.0. Similar changes were observed with serum from a mouse infected by tick bite, with the recognition of two additional bands (48 and 46 kDa) unique to pHs 6. 0 and 7.0. When membrane fractions were analyzed by 2D-NEPHGE, at least 37 changes in the membrane protein profile between cells incubated at pHs 6.0, 7.0, and 8.0 were observed by immunoblotting and silver staining. Environmental cues such as pH may prove important in the regulation of virulence determinants and factors necessary for the adaptation of B. burgdorferi to the tick or mammalian microcosm.

FIG. 1

Silver stain (A) and comparable immunoblots probed with rabbit hyperimmune serum (B) or mouse serum infected by the bite of a tick (C) of total membrane proteins from cultures incubated at pHs 6.0, 7.0, and 8.0. The asterisks indicate similar-sized proteins recognized by both sera. The solid arrow marks the presence of an alkaline-induced protein detected by the hyperimmune serum. The open arrows mark immunoreactive proteins recognized by the tick bite immune serum. Molecular mass standards in kilodaltons are indicated to the left of each panel.

FIG. 2

Immunoblot of total membrane samples from cultures incubated at pH 6.0, pH 7.0, and pH 8.0 probed with polyclonal serum raised against OspC. Molecular mass standards in kilodaltons are indicated on the left.

FIG. 3

Immunoblots of total membrane samples from cultures incubated at pH 6.0, pH 7.0, and pH 8.0 separated by 2D-NEPHGE and probed with hyperimmune serum. The acidic ends are to the left. Alterations in the protein profiles are indicated by the prefix “I−” (for immunoblot) with a corresponding number designation for easy comparison between blots. The locations of OspA and OspC are indicated as reference marks. Molecular mass standards in kilodaltons are indicated on the left of each panel.

FIG. 4

Silver stains of total membrane samples from cultures incubated at pH 6.0, pH 7.0, and pH 8.0 separated by 2D-NEPHGE. The acidic ends are to the left. Alterations in the protein profiles are indicated by the prefix “S−” (for silver stain) with a corresponding number designation for easy comparison between panels. The locations of OspA and OspC are indicated as reference marks. Molecular mass standards in kilodaltons are indicated on the left of each panel.