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.