Changes in bacterial growth rate govern expression of the Borrelia burgdorferi OspC and Erp infection-associated surface proteins

Abstract

The Lyme disease spirochete controls production of its OspC and Erp outer surface proteins, repressing protein synthesis during colonization of vector ticks but increasing expression when those ticks feed on vertebrate hosts. Early studies found that the synthesis of OspC and Erps can be stimulated in culture by shifting the temperature from 23°C to 34°C, leading to a hypothesis that Borrelia burgdorferi senses environmental temperature to determine its location in the tick-mammal infectious cycle. However, borreliae cultured at 34°C divide several times faster than do those cultured at 23°C. We developed methods that disassociate bacterial growth rate and temperature, allowing a separate evaluation of each factor's impacts on B. burgdorferi gene and protein expression. Altogether, the data support a new paradigm that B. burgdorferi actually responds to changes in its own replication rate, not temperature per se, as the impetus to increase the expression of the OspC and Erp infection-associated proteins.

Fig 1

Effects of culture temperature and medium composition on B. burgdorferi growth. Culture densities were determined by enumerating cultured bacteria using a Petroff-Hausser counting chamber. Cultures were counted in quadruplicate at each time point. Culture conditions were as follows (unless otherwise noted, BSK-II medium contains 6% rabbit serum): 34°C to 23°C, grown to late exponential phase in BSK-II medium at 34°C, diluted into BSK-II medium, and then grown at 23°C; 23°C to 34°C, grown to late exponential phase in BSK-II medium at 23°C, diluted into BSK-II medium, and then grown at 34°C; −80°C to 23°C, stored at −80°C for >1 month, diluted into BSK-II medium, and then grown at 23°C; 1.2% R.S., grown to late exponential phase in BSK-II medium at 34°C, diluted into BSK-II medium plus 1.2% rabbit serum, and then grown at 34°C; 1.2% R.S. to 6% R.S., grown to late exponential phase in BSK-II medium plus 1.2% rabbit serum at 34°C, diluted into BSK-II medium, and then grown at 34°C; 25% BSK-II, grown to late exponential phase in BSK-II medium plus rabbit serum at 34°C, diluted into 25%-strength BSK-II medium, and then grown at 34°C; 25% BSK-II to 100% BSK-II, grown to late exponential phase in 25%-strength BSK-II medium at 34°C, diluted into full-strength BSK-II medium, and then grown at 34°C.

Fig 2

Increases in B. burgdorferi growth rate correlate with increased production of OspC and Erp proteins. Effects of changing culture conditions on B. burgdorferi protein expression profiles were examined by immunoblotting. For each column, two conditions were kept constant, while a third was varied. For all studies, bacteria were first cultured to late exponential phase under a condition that impaired growth (complete BSK-II medium at 23°C, BSK-II medium containing only 1.2% rabbit serum at 34°C, or 25%-strength BSK-II medium with 6% serum at 34°C) and then diluted 1:100 into fresh, complete BSK-II medium and cultured at 34°C. All cultures were harvested at late exponential phase. The constitutively expressed flagellar component FlaB served as a control, and fold changes of other proteins were calculated relative to the FlaB band intensities (44). Illustrated data for each condition are from analyses of the same paired bacterial lysates.

Fig 3

Further indications that changes in borrelial growth rates lead to alterations in gene expression. Shown are data from quantitative reverse transcription-PCR (Q-RT-PCR) analyses of bacterial ospC, erpG, and rpoS mRNAs. Results are presented relative to mRNA levels of the constitutively expressed flaB gene (45). All analyses were performed in triplicate. Error bars (±1 standard deviation) are below the resolution of the figure. Differences within paired conditions were all statistically significant (P < 0.001).

Fig 4

Absolute temperature does not control production of OspC or Erp proteins. B. burgdorferi bacteria were cultured in complete BSK-II medium under various temperature regimens, and protein contents were examined by immunoblotting. Conditions examined were cultures inoculated with bacteria that had been frozen for >1 month at −80°C and then cultured at 23°C (left lanes), cultures inoculated with bacteria that had been grown to late exponential phase at 34°C and then cultured at 23°C (center lanes), and cultures inoculated with bacteria that had been grown to late exponential phase at 23°C and then cultured at 34°C (right lanes). All cultures were harvested at late exponential phase. The constitutively expressed flagellar component FlaB served as a control, and fold changes of other proteins were calculated relative to the FlaB band intensities (44). Illustrated data for each condition are from analyses of the same paired bacterial lysates.