Differential expression of Borrelia burgdorferi proteins during growth in vitro - PubMed (original) (raw)

Differential expression of Borrelia burgdorferi proteins during growth in vitro

R Ramamoorthy et al. Infect Immun. 1998 Nov.

Abstract

In an earlier paper we described the transcriptionally regulated differential levels of expression of two lipoproteins of Borrelia burgdorferi, P35 and P7.5, during growth of the spirochetes in culture from logarithmic phase to stationary phase (K. J. Indest, R. Ramamoorthy, M. Solé, R. D. Gilmore, B. J. B. Johnson, and M. T. Philipp, Infect. Immun. 65:1165-1171, 1997). Here we further assess this phenomenon by investigating whether the expression of other antigens of B. burgdorferi, including some well-characterized ones, are also regulated in a growth-phase-dependent manner in vitro. These studies revealed 13 additional antigens, including OspC, BmpD, and GroEL, that were upregulated 2- to 66-fold and a 28-kDa protein that was downregulated 2- to 10-fold, during the interval between the logarithmic- and stationary-growth phases. Unlike with these in vitro-regulated proteins, the levels of expression of OspA, OspB, P72, flagellin, and BmpA remained unchanged throughout growth of the spirochetes in culture. Furthermore, ospAB, bmpAB, groEL, and fla all exhibited similar mRNA profiles, which is consistent with the constitutive expression of these genes. By contrast, the mRNA and protein profiles of ospC and bmpD indicated regulated expression of these genes. While bmpD exhibited a spike in mRNA expression in early stationary phase, ospC maintained a relatively higher level of mRNA throughout culture. These findings demonstrate that there are additional genes besides P7.5 and P35 whose regulated expression can be investigated in vitro and which may thus serve as models to facilitate the study of regulatory mechanisms in an organism that cycles between an arthropod and a vertebrate host.

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Figures

FIG. 1

FIG. 1

(A) Growth curve of B. burgdorferi JD1 (passage 6) in vitro. The arrows indicate the cell densities at which spirochetes were harvested and processed for Western and Northern blotting analyses. Numbers above the arrows designate each sample. The open arrow depicts the passage 8 logrev sampling point. (B) Silver-stained gel showing the profiles of proteins expressed at different times during culture. The filled arrowhead indicates OspC, and the open arrowhead indicates P28. The lane numbers correspond to the numbers in panel A and designate the time point at which each sample was collected. (C) Western blot developed with serum from a monkey with an acute infection of B. burgdorferi JD1 (22). Arrowheads indicate antigens whose levels of expression steadily increased with the increasing age of the culture. Lane numbers are as described for panel B.

FIG. 2

FIG. 2

Western blots showing the patterns of expression of specific B. burgdorferi proteins during growth of the spirochetes in vitro. The blots were developed with monoclonal or monospecific polyclonal antibodies specific for the following proteins: OspA, OspB, OspC, GroEL, P72, flagellin, BmpA, BmpD, and P35. Lanes 1 to 5 contain samples of the initial culture taken on days 3, 4, 6, and 8 and of the second subculture taken on day 5, respectively.

FIG. 3

FIG. 3

Northern blots showing the mRNA profiles for ospAB, ospC, groEL, fla, bmpAB, bmpD, and P35. It should be noted that lanes 2 to 5 contain samples of the initial culture taken on days 4, 6, and 8 of the second subculture taken on day 5, respectively.

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