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.

Overexpression of CsrA (BB0184) Alters the Morphology and Antigen Profiles ofBorrelia burgdorferi

“…RNA was extracted as previously described (73). Briefly, B. burgdorferi cultures were grown to a density of 2 ϫ 10 7 to 3 ϫ 10 7 spirochetes per ml and RNA was extracted by resuspending the bacterial pellets in RNA-Bee (Tel-Test, Inc., Friendswood, TX) at a ratio of 0.2 ml to every 10 6 cells.…”

“…The induction of each gene in ES25 relative to that in MSK5 or ML23/pBSV2 was normalized to the levels of recA as previously described (55,80). The threshold cycle (C T ) values of each of the genes from three independent experiments were averaged following normalization, and the levels of induction were determined with the ⌬⌬C T method, where the quantity of each transcript was determined by the equation 2 Ϫ⌬⌬CT , where C T is the cycle number of the detection threshold, as described previously (42,73). To determine whether our real-time reverse transcription-PCR (RT-PCR) data were statistically significant, the differences between the normalized C T values obtained for ES25 and those for the ML23/ pBSV2 or MSK5 strain were subjected to a two-way analysis of variance followed by the Bonferroni post hoc test implemented in PRISM.…”

Role of the BBA64 Locus of Borrelia burgdorferi in Early Stages of Infectivity in a Murine Model of Lyme Disease

“…B. burgdorferi whole-cell lysates were prepared and separated by SDS-12.5% polyacrylamide gel electrophoresis as described previously (59). The separated proteins were either visualized by Coomassie brilliant blue staining or transferred onto a polyvinylidene difluoride membrane (Amersham Hybond-P; GE Healthcare, Buckinghamshire, United Kingdom) and subjected to immunoblot analysis.…”

Alternate Sigma Factor RpoS Is Required for the In Vivo-Specific Repression ofBorrelia burgdorferiPlasmid lp54-BorneospAandlp6.6Genes

“…Not unexpectedly, the entry of a bloodmeal into a B. burgdorferi-infected tick is accompanied by extensive changes in the borrelial transcriptome and proteome, a process referred to as mammalian host adaptation (1,4,43). While a number of studies have demonstrated that the expression of many borrelial loci can be modulated by the manipulation of in vitro growth conditions (1,2,5,8,10,11,17,27,32,41,48,51,58,60,(63)(64)(65)68), we and others have shown that as-yet-undefined mammalian hostspecific cues are essential for triggering the genetic program(s) underlying host adaptation (1,2,8,18,27,32,46,49,51,52,63,64,66).…”