Expression of an Epitope-Tagged Virulence Protein in Rickettsia parkeri Using Transposon Insertion

Despite recent advances in our ability to genetically manipulate Rickettsia, little has been done to employ genetic tools to study the expression and localization of Rickettsia virulence proteins. Using a mariner-based Himar1 transposition system, we expressed an epitope-tagged variant of the actin polymerizing protein RickA under the control of its native promoter in Rickettsia parkeri, allowing the detection of RickA using commercially-available antibodies. Native RickA and epitope-tagged RickA exhibited similar levels of expression and were specifically localized to bacteria. To further facilitate protein expression in Rickettsia, we also developed a plasmid for Rickettsia insertion and expression (pRIE), containing a variant Himar1 transposon with enhanced flexibility for gene insertion, and used it to generate R. parkeri strains expressing diverse fluorescent proteins. Expression of epitope-tagged proteins in Rickettsia will expand our ability to assess the regulation and function of important virulence factors

Detection of FLAG-RickA in bacteria.

<p><i>R. parkeri</i> strains that were not transformed (<i>Rp</i>) or transformed with pMW1650-FLAG-RickA (<i>Rp</i> FLAG-RickA) were used to infect Vero cells and then (A) labeled by immunofluorescence with anti-RickA antibody and stained for DNA with DAPI, or (B) labeled by immunofluorescence with anti-FLAG antibody and stained for DNA with DAPI. In the merged images, RickA or FLAG are labeled in green, and DNA in red. Scale bar 10 µm. Higher magnification images of individual bacteria (highlighted in boxes in the lower magnification images) are shown on the right.</p

Expression of fluorescent proteins in <i>R. parkeri</i>.

<p><i>R. parkeri</i> strains expressing (A) GFP_UV, (B) EGFP, (C) mCherry, and (D) 3XmCherry are shown in infected Cos7 cells alone (left) and together with actin filaments (right). Colors are indicated in the image. Scale bar 10 µm.</p

Expression of FLAG-RickA does not affect actin-based motility.

<p><i>R. parkeri</i> strains (green) transformed with (A) pMW1650 to express GFP_UV, or (B) pMW1650-FLAG-RickA to express GFP_UV and FLAG-RickA, were imaged in live Cos7 cells expressing the actin marker mCherry-Lifeact (red). Scale bar 10 µm. (C) Scatter plots of the rates of actin-based motility for untransformed bacteria (control) or strains expressing GFP_UV or GFP_UV and FLAG-RickA.</p

Expression of FLAG-RickA in <i>R. parkeri</i>.

<p>Immunoblots of <i>R. parkeri</i> strains that are not transformed (untransformed), transformed with pMW1650 to express GFP_UV (GFP-1, 2), and transformed with pMW1650-FLAG-RickA to express GFP_UV and FLAG-RickA (FLAG-RickA-1, 2, 3), probed with anti-RickA (top blot; higher molecular mass band is FLAG-RickA, lower is endogenous RickA), anti-FLAG (middle blot) or anti-GFP antibodies (bottom blot). Equal volumes were loaded into each lane, and the levels of endogenous RickA serve as a built in loading control.</p

Transposon insertion sites for <i>R. parkeri</i> strains.

<p>Transposon insertion sites for <i>R. parkeri</i> strains.</p

Physical maps for pMW1650 and pRIE.

<p>Both pMW1650 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037310#pone.0037310-Liu1" target="_blank">[9]</a> and pRIE contain a <i>Himar1</i> transposase gene under the control of the <i>Borrelia burgdorferi flgB</i> promoter, and a kanamycin resistance gene (KanR). Within the transposable element, bounded by inverted repeats (IR), are an <i>E. coli</i> ColE1 origin of replication, a GFP_UV gene under the control of the <i>R. rickettsii ompA</i> promoter, and a rifampicin resistance gene (RifR) under the control of the <i>rpsL</i> promoter. In addition, pRIE contains two multiple cloning sites: MCS1 for insertion of fluorescent protein genes under the control of the <i>R. rickettsii ompA</i> promoter, and MCS2 for insertion of other genes to be expressed under the control of their native promoter.</p