Acetyl-Phosphate Is Not a Global Regulatory Bridge between Virulence and Central Metabolism in Borrelia burgdorferi

In B. burgdorferi, the Rrp2-RpoN-RpoS signaling cascade is a distinctive system that coordinates the expression of virulence factors required for successful transition between its arthropod vector and mammalian hosts. Rrp2 (BB0763), an RpoN specific response regulator, is essential to activate this regulatory pathway. Previous investigations have attempted to identify the phosphate donor of Rrp2, including the cognate histidine kinase, Hk2 (BB0764), non-cognate histidine kinases such as Hk1, CheA1, and CheA2, and small molecular weight P-donors such as carbamoyl-phosphate and acetyl-phosphate (AcP). In a report by Xu et al., exogenous sodium acetate led to increased expression of RpoS and OspC and it was hypothesized this effect was due to increased levels of AcP via the enzyme AckA (BB0622). Genome analyses identified only one pathway that could generate AcP in B. burgdorferi: the acetate/mevalonate pathway that synthesizes the lipid, undecaprenyl phosphate (C55-P, lipid I), which is essential for cell wall biogenesis. To assess the role of AcP in Rrp2-dependent regulation of RpoS and OspC, we used a unique selection strategy to generate mutants that lacked ackA (bb0622: acetate to AcP) or pta (bb0589: AcP to acetyl-CoA). These mutants have an absolute requirement for mevalonate and demonstrate that ackA and pta are required for cell viability. When the ΔackA or Δpta mutant was exposed to conditions (i.e., increased temperature or cell density) that up-regulate the expression of RpoS and OspC, normal induction of those proteins was observed. In addition, adding 20mM acetate or 20mM benzoate to the growth media of B. burgdorferi strain B31 ΔackA induced the expression of RpoS and OspC. These data suggest that AcP (generated by AckA) is not directly involved in modulating the Rrp2-RpoN-RpoS regulatory pathway and that exogenous acetate or benzoate are triggering an acid stress response in B. burgdorferi

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

Borrelia burgdorferi OspC is required for the spirochete to establish infection in a mammal by tick transmission or needle inoculation. After a brief essential period, the protein no longer is required and the gene can be shut off. Using a system in which spirochetes contain only an unstable wild-type copy of the ospC gene, we can obtain mice persistently infected with bacteria lacking OspC. We implanted pieces of infected mouse skin subcutaneously in naïve mice, using donors carrying wild-type or ospC mutant spirochetes, and found that both could infect mice by this method, with similar numbers of wild-type or ospC mutant spirochetes disseminated throughout the tissues of recipient mice. Recipient mouse immune responses to tissue transfer-mediated infection with wild-type or ospC mutant spirochetes were similar. These experiments demonstrate that mammalian host-adapted spirochetes can infect and disseminate in mice in the absence of OspC, thereby circumventing this hallmark of tick-derived or in vitro-grown spirochetes. We propose a model in which OspC is one of a succession of functionally equivalent, essential proteins that are synthesized at different stages of mammalian infection. In this model, another protein uniquely present on host-adapted spirochetes performs the same essential function initially fulfilled by OspC. The strict temporal control of B. burgdorferi outer surface protein gene expression may reflect immunological constraints rather than distinct functions

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

<div><p><i>Borrelia burgdorferi</i>, the agent of Lyme disease, is a vector-borne pathogen that transits between <i>Ixodes</i> ticks and vertebrate hosts. During the natural infectious cycle, spirochetes must globally adjust their transcriptome to survive in these dissimilar environments. One way <i>B. burgdorferi</i> accomplishes this is through the use of alternative sigma factors to direct transcription of specific genes. RpoS, one of only three sigma factors in <i>B. burgdorferi</i>, 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 <i>rpoS</i> 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 <i>rpoS</i> transcript, indicating post-transcriptional regulation of RpoS by BBD18. Interestingly, BBD18-mediated repression of RpoS is independent of both the <i>rpoS</i> 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.</p></div

Synthesis of RpoS by wild type but not <i>bbd18</i>-expressing <i>B. burgdorferi</i>

<p>Immunoblot analysis of cell lysates from strains B31-S9 (wt), B31-S9Δ<i>rpoS</i> (Δ<i>rpoS</i>) and B31-S9/pBSV2*-<i>flaB</i>p-<i>bbd18</i> (wt/<i>flaB</i>p-<i>bbd18</i>) grown under <i>rpoS-</i>inducing conditions. Cell lysates were analyzed using RpoS antiserum (A) or a mouse monoclonal antibody to flagellin (B) to assess flagellin levels as a protein loading control. Cell lysates and immunoblot for flagellin were the same as those used in Fig. 1. Positions of molecular mass standards are shown on the left in kiloDaltons (kDa).</p

Analysis of BBD18 repression of an <i>rpoS</i> promoter <i>lacZ_Bb</i> transcriptional fusion.

<p>(A) A schematic diagram of the transcriptional fusion of the <i>rpoS</i> promoter and 5’ untranslated region (UTR) fused directly to the <i>lacZ</i>_Bb open reading frame (ORF). The position of the <i>rpoS</i> transcriptional start site <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093141#pone.0093141-Lybecker1" target="_blank">[58]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093141#pone.0093141-Burtnick1" target="_blank">[63]</a> is indicated by a filled arrowhead. The Shine-Dalgarno sequence (RBS), and the translational start site of β-galactosidase (<i>lacZ_Bb</i>), indicated by the ATG, are also shown. Regions corresponding to the <i>rpoS</i> promoter and 5’ UTR (141bp) or the <i>lacZ</i>_Bb ORF are indicated with brackets. The "G" in the RBS marked with an asterisk is identified because the reporter construct harbored an A->G mutation relative to the published sequence at that position. Cell lysates from strains B31-S9 (wt), B31-S9/pBSV2G-<i>rpoS</i>p<i>₁₄₁</i>-<i>lacZ_Bb</i> (wt/<i>rpoS</i>p-<i>lacZ_Bb</i>), and B31-S9/pBSV2G- <i>rpoS</i>p<i>₁₄₁</i>-<i>lacZ_Bb</i>/pBSV28-<i>flaB</i>p-<i>bbd18</i> (wt/<i>rpoS</i>p-<i>lacZ_Bb</i>/<i>flaB</i>p-<i>bbd18</i>) were grown under <i>rpoS</i>-inducing conditions and analyzed with OspC antisera (B) or stained with Coomassie blue (C) to demonstrate equivalent protein loads in each lane. The positions of molecular mass standards are shown on the left in kiloDaltons (kDa). (D) β-galactosidase activity in cell lysates from strains B31-S9 (wt), B31-S9/pBSV2G-<i>rpoS</i>p<i>₁₄₁</i>-<i>lacZ_Bb</i> (wt/<i>rpoS</i>p-<i>lacZ_Bb</i>), and B31-S9/pBSV2G- <i>rpoS</i>p<i>₁₄₁</i>-<i>lacZ_Bb</i>/pBSV28-<i>flaB</i>p-<i>bbd18</i> (wt/<i>rpoS</i>p-<i>lacZ_Bb</i>/<i>flaB</i>p-<i>bbd18</i>) grown under <i>rpoS</i>-inducing conditions.</p

Analysis of the transcript level of alternative sigma factors <i>rpoN</i> and <i>rpoS</i>.

<p>qRT-PCR data displaying the transcript levels of <i>rpoS</i> and <i>rpoN</i> in strains B31-S9 (wt), B31-S9Δ<i>rpoS</i> (Δ<i>rpoS</i>) and B31-S9/pBSV2*-<i>flaB</i>p-<i>bbd18</i> (wt/<i>flaB</i>p-<i>bbd18</i>) grown under <i>rpoS</i>-inducing conditions. Levels of <i>rpoS</i> and <i>rpoN</i> transcripts are displayed in relative units per 1000 copies of <i>flaB</i> transcript. Transcript levels were analyzed using Student’s unpaired t-test and no statistically significant difference was detected (p>.05).</p

Weak Organic Acids Decrease Borrelia burgdorferi Cytoplasmic pH, Eliciting an Acid Stress Response and Impacting RpoN- and RpoS-Dependent Gene Expression

The spirochete Borrelia burgdorferi survives in its tick vector, Ixodes scapularis, or within various hosts. To transition between and survive in these distinct niches, B. burgdorferi changes its gene expression in response to environmental cues, both biochemical and physiological. Exposure of B. burgdorferi to weak monocarboxylic organic acids, including those detected in the blood meal of fed ticks, decreased the cytoplasmic pH of B. burgdorferi in vitro. A decrease in the cytoplasmic pH induced the expression of genes encoding enzymes that have been shown to restore pH homeostasis in other bacteria. These include putative coupled proton/cation exchangers, a putative Na+/H+ antiporter, a neutralizing buffer transporter, an amino acid deaminase and a proton exporting vacuolar-type VoV1 ATPase. Data presented in this report suggested that the acid stress response triggered the expression of RpoN- and RpoS-dependent genes including important virulence factors such as outer surface protein C (OspC), BBA66, and some BosR (Borreliaoxidative stress regulator)-dependent genes. Because the expression of virulence factors, like OspC, are so tightly connected by RpoS to general cellular stress responses and cell physiology, it is difficult to separate transmission-promoting conditions in what is clearly a multifactorial and complex regulatory web

Effect of BBD18 on proteins in the RpoS regulon.

<p><i>B. burgdorferi</i> strains B31-S9 (wt), B31-S9Δ<i>rpoS</i> (Δ<i>rpoS</i>) and B31-S9/pBSV2*-<i>flaB</i>p-<i>bbd18</i> (wt/<i>flaB</i>p-<i>bbd18</i>) were grown under <i>rpoS</i>-inducing conditions (BSKII medium, pH 6.8). Cell lysates were subjected to SDS-PAGE, Coomassie blue staining (A), and immunoblot analysis (B-G). Membranes were probed for the presence of OspC (B), BBD18 (C), BBA66 (D), or DbpA (E), using protein-specific antibodies or antisera. Pooled sera from mice infected with <i>B. burgdorferi</i> by tick bite was used to detect changes in the antigenic protein profile (F). A mouse monoclonal α-flagellin antibody (H9724) was used to detect flagellin as a protein loading control (G). Positions of molecular mass standards are shown on the left in kiloDaltons (kDa).</p

Analysis of RpoS-dependent and RpoS-independent gene transcription.

<p>Quantitative reverse transcriptase-PCR (qRT-PCR) analysis of gene expression in strains B31-S9 (wt), B31-S9Δ<i>rpoS</i> (Δ<i>rpoS</i>) and B31-S9/pBSV2*-<i>flaB</i>p-<i>bbd18</i> (wt/<i>flaB</i>p-<i>bbd18</i>), grown under <i>rpoS-</i>inducing conditions (BSKII medium, pH6.8). The transcript level of RpoS-dependent genes <i>ospC</i>, <i>bba66</i>, <i>bba72</i>, <i>bbg01</i>, <i>bbj24</i>, and <i>bba34</i> (A) and RpoS-independent genes <i>bbj41</i>, <i>bba62</i> (<i>lp6</i>.6) and <i>bba15</i> (<i>ospA</i>), (B) are shown as relative units, and normalized to the constitutively expressed <i>flaB</i> transcript. Data were analyzed using Student’s unpaired t-test and brackets marked with asterisks represent a statistically significant difference (p<.05).</p