Dissolved oxygen levels alter gene expression and antigen profiles in Borrelia burgdorferi

Abstract

The Lyme disease spirochete, Borrelia burgdorferi, encounters many environmental signals as it cycles between the arthropod vector and mammalian hosts, including temperature, pH, and other host factors. To test the possibility that dissolved oxygen modulates gene expression in B. burgdorferi, spirochetes were exposed to differential levels of dissolved oxygen, and distinct alterations were observed at both the transcriptional and translational levels. Specifically NapA, a Dps/Dpr homologue involved in the oxidative stress response in other bacteria, was reduced when B. burgdorferi was grown under oxygen-limiting conditions. In contrast, several immunoreactive proteins were altered when tested with infection-derived sera from different hosts. Specifically, OspC, DbpA, and VlsE were synthesized at greater levels when cells were grown in limiting oxygen, whereas VraA was reduced. The levels of oxygen in the medium did not affect OspA production. Real-time reverse transcription-PCR analysis of RNA isolated from infectious isolates of strains B31 and cN40 indicated that the expression of ospC, dbpA, and vlsE increased while napA expression decreased under dissolved-oxygen-limiting conditions, whereas flaB was not affected. The reverse transcription-PCR results corroborated the immunoblot analyses and indicated that the increase in OspC, DbpA, and VlsE was due to regulation at the transcriptional level of the genes encoding these antigens. These results indicate that dissolved oxygen modulates gene expression in B. burgdorferi and imply that the redox environment may be an additional regulatory cue that spirochetes exploit to adapt to the disparate niches that they occupy in nature.

FIG. 1.

Level of oxidative stress response protein NapA (BB0690) in B. burgdorferi strains B31, 297, and cN40 grown in O₂-depleted medium. The B. burgdorferi isolates were grown under standard microaerophilic conditions in BSK-II medium (lanes −) or in BSK-II depleted of O₂ by treatment with Oxyrase (lanes +, panel A) or by displacement with N₂ gas (lanes +, panel B). Cultures were harvested after 96 h of treatment, separated on an SDS-PAGE gel stained with Coomassie blue (above), and analyzed by Western immunoblotting with NapA antiserum (shown below).

FIG. 2.

Immunoreactive proteins induced when B. burgdorferi strain B31 is grown under limiting oxygen. B. burgdorferi was grown under standard microaerophilic conditions in BSK-II medium (lanes −) or with added Oxyrase to reduce the level of dissolved oxygen (lanes +). Cells were grown as described in the text, and the samples were resolved by SDS-PAGE and stained with Coomassie blue (panel A) or immunoblotted and probed either with serum from a patient with chronic Lyme borreliosis (panel B) or with infection-derived mouse serum (panel C). Numbers on the left of each panel indicate the molecular mass of protein markers. The arrow denotes the location of the B. burgdorferi OspA lipoprotein.

FIG. 3.

Growth with reduced levels of dissolved oxygen modulates the synthesis of several B. burgdorferi antigens from B. burgdorferi sensu stricto strains B31 (MSK5), 297, and cN40. Equivalent numbers of B. burgdorferi cells, grown under standard microaerophilic conditions in BSK-II medium (lanes −, panels A and B) or treated with either Oxyrase (lanes +, panel A) or N₂ gas displacement (lanes +, panel B) were resolved by SDS-PAGE, immobilized onto membranes, and probed with antisera to the antigens indicated on the left. The strains tested are indicated above each immunoblot column.

FIG. 4.

Real-time reverse transcription-PCR indicates that redox regulation of B. burgdorferi genes occurs at the transcriptional level. RNA was isolated from B. burgdorferi strain B31 derivative MSK5 (panel A) and strain cN40 (panel B) following both microaerophilic and anaerobic growth and subjected to real-time reverse transcription-PCR as described in the text. All samples were normalized relative to the flaB transcript and are shown as the ratio of transcripts from cells grown anaerobically relative to cells grown under microaerophilic conditions (represented as fold difference on the y axis). The asterisks (*) indicate samples whose C_t values were statistically significant (i.e., P value less than 0.05) when transcripts from cells grown anaerobically were compared to the same gene transcripts from cells grown microaerophilically. The C_t values from both independent experimental sets were compared with a multiple paired t test. The white and black bars represent data from two independent experiments. The genes analyzed are listed below for each strain tested.