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

The enigmatic biology of rickettsiae: recent advances, open questions and outlook

ABSTRACT Rickettsiae are obligate intracellular bacteria that can cause life-threatening illnesses and are among the oldest known vector-borne pathogens. Members of this genus are extraordinarily diverse and exhibit a broad host range. To establish intracellular infection, Rickettsia species undergo complex, multistep life cycles that are encoded by heavily streamlined genomes. As a result of reductive genome evolution, rickettsiae are exquisitely tailored to their host cell environment but cannot survive extracellularly. This host-cell dependence makes for a compelling system to uncover novel host–pathogen biology, but it has also hindered experimental progress. Consequently, the molecular details of rickettsial biology and pathogenesis remain poorly understood. With recent advances in molecular biology and genetics, the field is poised to start unraveling the molecular mechanisms of these host–pathogen interactions. Here, we review recent discoveries that have shed light on key aspects of rickettsial biology. These studies have revealed that rickettsiae subvert host cells using mechanisms that are distinct from other better-studied pathogens, underscoring the great potential of the Rickettsia genus for revealing novel biology. We also highlight several open questions as promising areas for future study and discuss the path toward solving the fundamental mysteries of this neglected and emerging human pathogen.</jats:p

Actin-based motility and cell-to-cell spread of bacterial pathogens

Subversion of the host actin cytoskeleton is a critical virulence mechanism used by a variety of intracellular bacterial pathogens during their infectious life cycles. These pathogens manipulate host actin to promote actin-based motility and coordinate motility with cell-to-cell spread. Growing evidence suggests that the tactics employed by pathogens are surprisingly diverse. Here, we review recent advances suggesting that bacterial surface proteins exhibit divergent biochemical mechanisms of actin polymerization and recruit distinct host protein networks to drive motility, and that bacteria deploy secreted effector proteins that alter host cell mechanotransduction pathways to enable spread. Further investigation into the divergent strategies used by bacterial pathogens to mobilize actin will reveal new insights into pathogenesis and cytoskeleton regulation

A streamlined method for transposon mutagenesis of Rickettsia parkeri yields numerous mutations that impact infection.

The rickettsiae are obligate intracellular alphaproteobacteria that exhibit a complex infectious life cycle in both arthropod and mammalian hosts. As obligate intracellular bacteria, rickettsiae are highly adapted to living inside a variety of host cells, including vascular endothelial cells during mammalian infection. Although it is assumed that the rickettsiae produce numerous virulence factors that usurp or disrupt various host cell pathways, they have been challenging to genetically manipulate to identify the key bacterial factors that contribute to infection. Motivated to overcome this challenge, we sought to expand the repertoire of available rickettsial loss-of-function mutants, using an improved mariner-based transposon mutagenesis scheme. Here, we present the isolation of over 100 transposon mutants in the spotted fever group species Rickettsia parkeri. Transposon insertions disrupted genes whose products are implicated in a variety of pathways, including bacterial replication and metabolism, the type IV secretion system, factors with previously established roles in host cell interactions and pathogenesis, or are of unknown function. Given the need to identify critical virulence factors, forward genetic screens such as this will provide an excellent platform to more directly investigate rickettsial biology and pathogenesis

Mapping the transposon insertion sites.

<p>(A) Diagram showing the insertion of the transposon cassette into a chromosomal region (in grey). Primers specific to the transposon ends were paired with universal primers to amplify the chromosome- transposon junctions (red triangles), using semi-random nested PCR. Two nested PCR reactions were done to improve amplification of the chromosome-transposon junction directly from boiled bacteria. (B) <i>R</i>. <i>parkeri</i> chromosomal map showing all transposon insertion sites (see red lines) identified in this screen.</p

Transposon mutagenesis of <i>R</i>. <i>parkeri</i>.

<p>(A) Map of the pMW1650 plasmid used in this study for transposon mutagenesis (IR, inverted repeats). (B) Experimental scheme for transposon mutagenesis and isolation of individual mutants.</p

Transposon insertion sites in <i>R</i>. <i>parkeri</i>.

<p>Transposon insertion sites in <i>R</i>. <i>parkeri</i>.</p

Rickettsia Actin-Based Motility Occurs in Distinct Phases Mediated by Different Actin Nucleators

Many intracellular bacterial pathogens undergo actin-based motility to promote cell-cell spread during infection [1]. For each pathogen, motility was assumed to be driven by a single actin polymerization pathway. Curiously, spotted fever group Rickettsia differ from other pathogens in possessing two actin-polymerizing proteins. RickA, an activator of the host Arp2/3 complex, was initially proposed to drive motility [2, 3]. Sca2, a mimic of host formins [4, 5], was later shown to be required for motility [6]. Whether and how their activities are coordinated has remained unclear. Here, we show that each protein directs an independent mode of Rickettsia parkeri motility at different times during infection. Early after invasion, motility is slow and meandering, generating short, curved actin tails that are enriched with Arp2/3 complex and cofilin. Early motility requires RickA and Arp2/3 complex and is correlated with transient RickA localization to the bacterial pole. Later in infection, motility is faster and directionally persistent, resulting in long, straight actin tails. Late motility is independent of Arp2/3 complex and RickA and requires Sca2, which accumulates at the bacterial pole. Both motility pathways facilitate cell-to-cell spread. The ability to exploit two actin assembly pathways may allow Rickettsia to establish an intracellular niche and spread between diverse cells throughout a prolonged infection