Reciprocal Expression of ospA and ospC in Single Cells of Borrelia burgdorferi

Abstract

Outer surface proteins (Osp) A and C of the Lyme disease spirochete (Borrelia burgdorferi) are selectively produced and of functional significance in the tick vector and mammalian host, respectively. Some studies indicate a simple, reciprocal relationship where the signals and pathways that turn on ospC also turn off ospA. Other studies indicate a more complex regulation where many spirochetes produce both proteins and others produce one of the proteins or neither protein. Here, we have used flow cytometry to characterize ospA and ospC transcript and protein levels in individual bacterial cells grown in culture. The results support a simple, reciprocal model where, at the level of single cells, the transcription of ospC is linked to the repression of ospA. We also demonstrate that under conditions conducive for OspC production, spirochetes display an "all or none" response, with some cells displaying high levels of ospC transcription and others demonstrating little or no transcription. Despite the reciprocal regulation of ospA and ospC at the single-cell level, we propose that spirochetes display an array of phenotypes due to stochasticity in the pathways that regulate osp expression and the slow turnover of outer surface proteins.Borrelia burgdorferi, the spirochete responsible for Lyme disease, is transmitted by Ixodes ticks (3). B. burgdorferi persistently colonizes the gut of ticks. When infected ticks feed, the spirochetes multiply within the gut, migrate to the tick's salivary glands, and infect the vertebrate host (1). Within the feeding tick, the spirochetes alter the expression of many genes in preparation for transmission and infection of the new host (1, 26). B. burgdorferi outer surface proteins (Osp) A and C have served as a paradigm for understanding the regulation of bacterial gene expression within feeding ticks. In ticks, ospA is predominantly expressed before the blood meal, whereas ospC is induced during the blood meal (9,17,22,23). The functions of these two proteins are consistent with their pattern of expression, where OspA is required for colonizing the vector and OspC is required for infecting the host (12,19,29).Temperature, pH, and cell density act as signals for regulating the expression of ospA and -C in culture, and these signals are likely to play a role in the feeding tick as well (26). Proteomic and microarray studies with cultured spirochetes grown in "tick-like" (low temperature, high pH) or "host-like" (high temperature, low pH) conditions have led to the identification of large subsets of Borrelia proteins and genes with "OspAlike" or "OspC-like" patterns of expression (18,20). The bacterial signaling pathway regulating the expression of ospC and ospC-like genes has been characterized in some detail (6,7,14,24,27,28). The pathway is activated by a two-component system consisting of a sensor with a histidine kinase domain (HK2) and a cytoplasmic response regulator protein (Rrp2) (14, 27). Activated Rrp2, together with the alternative sigma factor RpoN, induces the expression of...

lacZ Reporter System for Use in Borrelia burgdorferi

“…This observation has not been reported for B312 previously, although earlier studies did report variation in ospC expression on an individual cell basis in other B. burgdorferi backgrounds (12,35). In order to use B312, we first confirmed OspC production in B312 transformants with immunoblotting before proceeding with further experiments on those clones (data not shown).…”

OspC-Independent Infection and Dissemination by Host-Adapted Borrelia burgdorferi

“…While it is possible that OspC production facilitates infection by tissue transfer, we have observed no reproducible difference in the efficiency of infection by tissue transfer of wild-type and ospC mutant spirochetes. Since ospC gene regulation appears to have a stochastic component (23,37,49), the immunological response may just be the consequence of sporadic OspC production by infecting bacteria in the mammalian host, which can persist when B. burgdorferi infects mice that are unable to mount an acquired immune response (31). We attempted to use an ELISA with OspC as the antigen to analyze the responses of needle-inoculated and tissue-transferred animals, but the reactivity of infected mouse sera from these experiments was not sufficiently higher than that of uninfected sera, so we could not draw quantitative conclusions about possible differences in response.…”

Surface Localization Determinants of Borrelia OspC/Vsp Family Lipoproteins

“…Outer surface protein A (OspA), e.g., is expressed by the Lyme disease spirochete Borrelia burgdorferi during the vector phase, where its immunoprotective and adhesive properties appear to ensure continuity of the infectious cycle (6,7,44,45). Upon tick feeding and transmission to a new mammalian host, complex regulatory mechanisms lead to the replacement of OspA by OspC (45,46,60,61,65,66). OspC is required for the establishment of mammalian infection (24), which appears to be further enhanced by binding to Salp15, an immune-modulating tick salivary gland protein (2, 53).…”