Global Repression of Host-Associated Genes of the Lyme Disease Spirochete through Post-Transcriptional Modulation of the Alternative Sigma Factor RpoS

Abstract

Borrelia burgdorferi, the agent of Lyme disease, is a vector-borne pathogen that transits between Ixodes ticks and vertebrate hosts. During the natural infectious cycle, spirochetes must globally adjust their transcriptome to survive in these dissimilar environments. One way B. burgdorferi accomplishes this is through the use of alternative sigma factors to direct transcription of specific genes. RpoS, one of only three sigma factors in B. burgdorferi, controls expression of genes required during tick-transmission and infection of the mammalian host. How spirochetes switch between different sigma factors during the infectious cycle has remained elusive. Here we establish a role for a novel protein, BBD18, in the regulation of the virulence-associated sigma factor RpoS. Constitutive expression of BBD18 repressed transcription of RpoS-dependent genes to levels equivalent to those observed in an rpoS mutant. Consistent with the global loss of RpoS-dependent transcripts, we were unable to detect RpoS protein. However, constitutive expression of BBD18 did not diminish the amount of rpoS transcript, indicating post-transcriptional regulation of RpoS by BBD18. Interestingly, BBD18-mediated repression of RpoS is independent of both the rpoS promoter and the 5’ untranslated region, suggesting a mechanism of protein destabilization rather than translational control. We propose that BBD18 is a novel regulator of RpoS and its activity likely represents a first step in the transition from an RpoS-ON to an RpoS-OFF state, when spirochetes transition from the host to the tick vector.

Phosphoenolpyruvate Phosphotransferase System Components Modulate Gene Transcription and Virulence of Borrelia burgdorferi

“…Although several studies have demonstrated that the alternative sigma factors RpoN and RpoS and other factors play key roles in the control of gene expression in Borrelia (13,40,41), additional, as-yet-unidentified regulatory factors are likely to be involved in this complex regulatory network. In many Gram-positive and Gram-negative organisms, the PEP-PTS acts as an important signal transduction system that controls gene expression depending on carbohydrate availability.…”

Use of an Endogenous Plasmid Locus for Stable in trans Complementation in Borrelia burgdorferi

“…One microgram of total RNA was subjected to DNase treatment as outlined in the manufacturer's protocol (Life Technologies) and used to synthesize cDNA using the High Capacity DNA Reverse Transcriptase (RT) kit (Life Technologies). A control reaction mixture lacking reverse transcriptase (no RT) was included for each cDNA synthesis reaction, and each sample was tested with three technical replicates by quantitative PCR (qPCR) using a 1:10 dilution of cDNA, the TaqMan Universal PCR Mastermix (Life Technologies), and ABI Viia7 software, as previously described (47). The constitutively expressed flaB gene was used as the PCR endogenous control to which ospC and pncA values were normalized.…”

Long-Term Survival of Borrelia burgdorferi Lacking the Hibernation Promotion Factor Homolog in the Unfed Tick Vector

“…Total RNA was extracted from B. burgdorferi cultures using TRIzol reagent, according to the manufacturer's protocol (Life Technologies, Grand Island, NY, USA), and cDNA was produced as previously described (54). Briefly, RNA was extracted by lysing spirochetes in TRIzol reagent, extracting RNA in chloroform, precipitating it in isopropanol, and resuspending it in RNase-free water.…”

“…Contaminating DNA was removed by treatment with the Turbo DNA-free kit according to the manufacturer's instructions (Life Technologies), and cDNA was produced using the high-capacity cDNA reverse transcriptase kit (Life Technologies) according to the manufacturer's instructions. Quantitative reverse transcriptase PCRs (qRT-PCRs) were performed as described previously (54) with TaqMan Universal PCR master mix (Life Technologies) and gene-specific primers and probe sets (see Table S1 in the supplemental material) (Integrated DNA Technologies, Coralville, IA, USA, or SigmaAldrich, St. Louis, MO, USA). Experiments were performed in biological and technical triplicates, and experimental results were analyzed on a Viia7 real-time PCR system with the Viia7 software package (Life Technologies).…”