Figure 1.
Venn diagram of the number of proteins predicted to be secreted by the indicated algorithms.
The entire R. typhi str. Wilmington proteome was submitted to SignalP, LipoP, Phobius and SecretomeP servers as outlined in the Materials & Methods section. Results from each program were compiled and the numbers of proteins predicted by each program individually or in all possible combinations were tabulated and are indicated in the above Venn diagram.
Figure 2.
Characteristics of rickettsial surface cell antigens (Scas).
A) Schema depicting the major features of R. typhi Sca1 (RT0015, YP_066986), Sca2 (RT0052, YP_067021), Sca3 (RT0438, YP_067397), Sca4 (RT0485, YP_067439), and Sca5 (RT0699, YP_067640). N-terminal circles depict predicted (SignalP) and/or experimentally-supported (PhoA fusion assay) sec dependent signal sequences. Divergent repeat regions [77] and other identified motifs are colored coded for Sca2, Sca3 and Sca4. B) Comparative analysis of Sca1-Sca5 across 16 rickettsia genomes. Dashed box encloses the R. typhi Scas. Phylogeny at left is based on whole genome analysis [82], with rickettsial groups as follows: red, ancestral group; aquamarine, typhus group; blue, transitional group, brown, spotted fever group. Principal host vectors (tick, flea, louse, mite) for each taxon are illustrated. Green boxes depict passenger domains (as illustrated in A) associated with an AT domain, with the length of each passenger given in amino acids. Skull and crossbones represent pseduogenes. Black boxes depict sca gene fragments encoded within the R. prowazekii str. Madrid E genome (all of which have a conserved downstream ORF encoding an AT domain). Accession numbers for all sequences are provided in Figure S1.
Figure 3.
Expression analysis by real-time RT-PCR of Sca genes during L929 cell infection and immunofluorescence assays of Sca protein expression in C. felis and rat spleens.
RNA was extracted from R. typhi infected L929 cells at different time points as described and analyzed by multiplex real-time qPCR for expression of Sca, rpsL and GAPDH genes. A) Transcript abundance of each Sca gene was normalized to rpsL transcript abundance and B) rickettsial burden (rpsL: GAPDH) confirmed increased infection over time. The results represent data from 3 experiments with each gene analyzed in duplicate. C) Cat fleas were housed in an artificial dog and fed uninfected or R. typhi infected whole sheep's blood for 48 h.; they were then fed uninfected blood for 8 days. Capsules were placed at −20°C to immobilize fleas before placing them in 3% PFA overnight. Fleas were embedded in OCT media, frozen and cryosectioned. Sections were labeled with anti-R. typhi rat immune serum (Alexa488-conjugated anti-rat secondary Ab – green), anti-serum to the sca protein indicated on the left (Alexa594-conjugated anti-rabbit secondary Ab – red) and mounted in VectaShield medium with DAPI (blue) to counterstain DNA. Arrowheads point to examples of positively stained rickettsiae. Bar = 5 µm. D) Spleens were harvested from 9 day infected female Sprague-Dawley rats and fixed and embedded as described. Sections (5 µm) were stained with AlexaFluor 532-labeled anti-R. typhi rat immune serum (red), AlexaFluor 350-labeled anti-serum to the sca protein indicated on the left (blue) and propidium iodide (green) to counterstain DNA (not shown). Sections were mounted in VectaShield medium. Arrowheads indicate examples of positively labeled rickettsiae.
Figure 4.
Transcriptional analysis of putative sca operons by RT-PCR.
RNA was extracted from 72 h-infected L929 fibroblasts and RT-PCR with primers designed to amplify the regions indicated in (A). A) Schematic of putative sca operons. Blue bars indicate regions successfully amplified, red bars indicate regions that could not be amplified. B) RT-PCR amplification of regions between ORFs in putative sca operons. Left panel – lanes 2 thru 4 – RT-PCR amplification of the 400 bp region within the encoded passenger domain of sca3 used in time course analyses ; lanes 5 thru 7 – amplification of Lon-sca3 co-transcript. Right panel: lanes 2 thru 4 – amplification of sca4 region used in time course analyses; lanes 5 thru 7 – amplification of tlcD-sca4 transcript. C) Analysis of sca5 operon; labels indicate genes spanned by primers. RT – reverse transcriptase reaction, NRT – no reverse transcriptase, D – DNA control.
Figure 5.
Immunogold electron microscopy of Sca expression on intact, negative stained R. typhi.
A suspension of rickettsiae was placed on grids and allowed to dry. Samples were labeled with antibodies against a Sca protein A) Sca1, B) Sca2, C) Sca3, D) Sca4 and E) Sca5 and negatively stained for contrast. Bar = 0.25 um.
Figure 6.
Evolution of rickettsial surface cell antigens (Scas).
All datasets were aligned with MUSCLE v3.6 [74], [75] and analyzed under maximum likelihood using RAxML [76] (see text for details). Branch support was assessed with 1000 bootstrap pseudoreplications, with values in intervals of 10 (>50% only) depicted with colored circles that are explained at the bottom left. Taxon color scheme according to major rickettsia group is explained in the Figure 2 legend. (A–C) Comparison of the phylogenies estimated for the passenger (left) and AT domains (right) for (A) Sca1, (B) Sca2 and (C) Sca5. For each tree, numbers above the line depict the total number of positions for each domain alignment, with numbers under the line representing the total number of informative sites within the alignment. The phylogenies of the AT domain better corroborate the rickettsia species tree (see Figure 2), thus the position of the taxa correspond with these trees. The taxa in the trees generated from the passenger domain are connected to their corresponding partner in the AT domain trees via dashed lines, with orange illustrating the major differences between the domain-generated trees. (D) Phylogeny estimation of an expanded set of rickettsial Sca4 proteins. The yellow box encloses the only known plasmid-encoded Sca4 proteins: pREIS1 and pREIS3 from REIS, rRPR from R. peacockii [83]. The gray arrow illustrates the replacement of the phylogram portion of the tree with the corresponding cladogram to account for the minimal divergence across the majority of SFG Rickettsia Sca4 proteins.
Table 1.
Primers designed for real-time quantitative PCR analysis of sca genes, rpsL and mouse GAPDH expression during R. typhi infection of mouse fibroblast cells and co-transcription of genes in putative sca operons.