An expanded genetic toolkit for inducible expression and targeted gene silencing in Rickettsia parkeri

Abstract

Pathogenic species within the Rickettsia genus are transmitted to humans through arthropod vectors and cause a spectrum of diseases ranging from mild to life-threatening. Despite rickettsiae posing an emerging global health risk, the genetic requirements of their infectious life cycles remain poorly understood. A major hurdle toward building this understanding has been the lack of efficient tools for genetic manipulation, owing to the technical difficulties associated with their obligate intracellular nature. To this end, we implemented the Tet-On system to enable conditional gene expression in Rickettsia parkeri. Using Tet-On, we show inducible expression of antibiotic resistance and a fluorescent reporter. We further used this inducible promoter to screen the ability of R. parkeri to express four variants of the catalytically dead Cas9 (dCas9). We demonstrate that all four dCas9 variants can be expressed in R. parkeri and used for CRISPR interference (CRISPRi)-mediated targeted gene knockdown. We show targeted knockdown of an antibiotic resistance gene as well as the endogenous virulence factor sca2. Altogether, we have developed systems for inducible gene expression and CRISPRi-mediated gene knockdown for the first time in rickettsiae, laying the groundwork for more scalable, targeted mechanistic investigations into their infectious life cycles.

Figure 1.. The Tet-On system enables conditional gene expression in R. parkeri.

(A) Anhydrotetracycline (aTc) toxicity curve in R. parkeri. Plaque assays were performed on Vero cell monolayers with varying concentrations of aTc indicated. The number of plaques formed at each aTc concentration was normalized to the no aTc control for each independent experiment (n = 3). *** represents p < 0.001 by ordinary one-way ANOVA with post hoc Tukey’s test. (B) Schematic of the Tet-On system cloned into pRAM18dSGA. The tet repressor, TetR, binds two tet operator sites (tetO) to block gene expression in the absence of aTc. The rparr-2 gene, which confers resistance to rifampicin, was placed under the control of Tet-On. Diagram not drawn to scale. (C) aTc induction of rifampicin resistance. Varying concentrations of aTc were added 30 mpi during plaque assays in Vero host cell monolayers. Each well shown had rifampicin added (200 ng/mL final concentration). All conditions shown were normalized to a no aTc and no rifampicin control well per independent experiment (n = 3). (D) Schematic of tagbfp cloned into the Tet-On system. The tagbfp gene was codon optimized for expression in R. conorii. Diagram not drawn to scale. (E & F) aTc induction of TagBFP during infection. A549 cell monolayers were infected with R. parkeri harboring a plasmid containing tagbfp under the control of Tet-On. aTc was added 24 hpi, then samples were fixed at 48 hpi and subsequently imaged. (D) All images were set to the same minimum and maximum grey values per channel for comparison of BFP intensity. Scale bar, 2 μm. (E) Blue fluorescence from the expression of tagbfp was quantified for each bacterium across three independent experiments. ** denotes p < 0.01 using an ordinary one-way ANOVA.

Figure 2.. pRAM18-Tet-On can be used to express dCas9 in R. parkeri.

(A) Schematic of pRAM18-based CRISPRi system. Expression of dCas9 is driven by the Tet-On promoter and the sgRNA is driven by the constitutive promoter P_rpsL. (B) Four dCas9 variants were cloned into pRAM18dSGA. Each dCas9 variant recognizes a distinct PAM, with each PAM found in varying instances in the R. parkeri genome. (C) Expression of dCas9 in R. parkeri. Each dCas9 variant was tagged with a C-terminal HA epitope and expression −/+ aTc was visualized by Western blot, as well as OmpA as a loading control.

Figure 3.. CRISPRi knockdown of a rifampicin resistance gene.

(A) Schematic of the engineered locus and screen to test knockdown of rifampicin resistance. The rpsL promoter driving expression of rparr-2 in the pMW1650 plasmid was modified to include additional PAMs to allow for testing of various dCas9 variants. Successful CRISPRi-mediated knockdown rparr-2 would sensitize strains to treatment with rifampicin, while strains with nonfunctional CRISPRi would remain resistant to rifampicin. Spectinomycin selection ensures that the strains maintain the plasmid encoding the CRISPRi components. (B-E) Quantification of CRISPRi-mediated knockdown of rifampicin resistance via plaque assay. A549 cell monolayers were infected with R. parkeri strains encoding the S. pyogenes dCas9 (B), S. thermophilus 01 dCas9 (C), S. thermophilus 03 dCas9 (D), S. pasteurianus dCas9 (E). For each dCas9 variant and sgRNA combination, the same volume of R. parkeri stock was added to each well, and then the number of plaques was normalized to the no aTc and no rifampicin condition for a total of n = 3 independent experiments. Statistical significance was determined by ordinary one-way ANOVA with post hoc Tukey’s test (* denotes p < 0.05, ** denotes p < 0.005, **** denotes p < 0.0001). Schematics below each bar graph depict the relative locations of each sgRNA tested for each dCas9.

Figure 4.. CRISPRi knockdown of the rickettsial virulence factor sca2.

(A) Schematic of sca2 knockdown experiment. Sca2 is a formin-like actin nucleator responsible for forming long actin tails during R. parkeri infection. CRISPRi-mediated knockdown of sca2 should result in decreased actin tail formation. (B) CRISPRi targeting leads to decreased expression of Sca2 protein. A549 host cell monolayers were infected with R. parkeri. aTc was added to infections 48 hpi and lysates were harvested at 72 hpi. Sca2 and OmpA (loading control) protein levels were visualized via Western blotting. (C & D) Measurement of actin tail formation by immunofluorescence. A549 cell monolayers were infected with R. parkeri for 28 h, with aTc being added to appropriate wells at the time of infection. These samples were then fixed, stained, and imaged to visualize (C) and quantify (D) actin tail formation. The white arrow indicates an actin tail, which is shown in greater detail in the inset. Scale bar, 10 μm and 5 μm in inset. For each condition, at least 300 bacteria were quantified in each of n = 3 independent experiments. *** denotes p < 0.001, determined by one-way ANOVA with post hoc Tukey’s test.