Identification and characterization of the mammalian association and actin-nucleating domains in the Rickettsia conorii autotransporter protein, Sca2

Abstract

Establishment of infection by spotted fever group rickettsial species is dependent on the ability of these bacteria to adhere to and invade the host endothelium. Recent studies have attributed these processes to a handful of rickettsial surface proteins from the surface cell antigen (sca) family of autotransporters. A rickettsial autotransporter from Rickettsia conorii, Sca2, has been shown to be sufficient to mediate both adherence and invasion of human endothelial cells and to participate in intracellular actin-based motility. Here we identify a region of Sca2 capable of interacting with the mammalian cell surface and show that this function of Sca2 is independent and separable from its actin nucleation activity. Furthermore, pre-incubation of mammalian cells with the Sca2 mammalian association region prior to R. conorii infection can competitively inhibit rickettsial invasion, suggesting that Sca2 plays an important role in the initial interaction with mammalian cells. Together, our results demonstrate that the Sca2 autotransporter protein in R. conorii contains distinct functional domains that likely are involved in mediating cellular interactions at the plasma membrane and the host cytosol.

Figure 1. Sca2_34–556 is sufficient to mediate adherence to the surface of mammalian cells

A. Immunofluorescence microscopy of purified GST-fused Sca2 peptides adhered to the surface of a HeLa (left panels) or EA.hy 926 (right panels) cell monolayers. Scale bar represents 20 μm. B. Flow cytometric analysis of titration of GST-fused Sca2 peptides bound to the surface of HeLa cells. C. Graph of Mean FITC signal of data presented in Figure B as a function of protein concentration. D and E. Flow cytometric analysis of GST-fused Sca2 peptides (at 1.25 μM) bound to the surface of EA.hy cells (D) and HeLa cells (E). Twenty thousand events were recorded for all flow cytometry samples. All subscripted text refers to amino acid numbers.

Figure 2

Amino acid alignment of the N-terminal third of the passenger domain of Sca2 from R. conorii, R. parkeri, and R. peacockii. Numbers above the alignment indicate the amino acid numbers of Sca2 from R. conorii. Identical amino acids are shaded dark gray and boxed. Deletions are indicated by a dash.

Figure 3. The adherence function of Sca2 is separable from its actin nucleation function

A. Schematic of Sca2 peptides produced as N-terminal GST fusions, their names as used in this manuscript and the vector they are expressed from. GST is represented in green and portions of Sca2 in blue. Numbers located at the ends of the blue bars indicate amino acid numbers. B. Flow cytometric analysis of adherence of GST-fused Sca2 fragments diagramed in A to the surface of HeLa and EA.hy 926 cells. Twenty thousand events were recorded for each sample. C. Spontaneous actin assembly assays measuring actin polymerization over time in the presence of indicated concentrations of Sca2 fragments.

Figure 4. Inhibition of R. conorii invasion by recombinant Sca2 protein in vitro is governed by the mammalian association region (MAR)

HeLa cell monolayers were incubated with 10 μM recombinant Sca2 protein prior to infection with R. conorii for 45 min. Total R. conorii associated per cell (A) and percent bacteria invaded out of total associated (B) was determined by differential immunofluorescent microscopy. ^*, P<0.05. The data presented is representative of three independent experiments. Error bars represent the standard deviation from the average.