Disruption of the Rickettsia rickettsii Sca2 autotransporter inhibits actin-based motility

Abstract

Rickettsii rickettsii, the etiologic agent of Rocky Mountain spotted fever, replicates within the cytosol of infected cells and uses actin-based motility to spread inter- and intracellularly. Although the ultrastructure of the actin tail and host proteins associated with it are distinct from those of Listeria or Shigella, comparatively little is known regarding the rickettsial proteins involved in its organization. Here, we have used random transposon mutagenesis of R. rickettsii to generate a small-plaque mutant that is defective in actin-based motility and does not spread directly from cell to cell as is characteristic of spotted fever group rickettsiae. The transposon insertion site of this mutant strain was within Sca2, a member of a family of large autotransporter proteins. Sca2 exhibits several features suggestive of its apparent role in actin-based motility. It displays an N-terminal secretory signal peptide, a C-terminal predicted autotransporter domain, up to four predicted Wasp homology 2 (WH2) domains, and two proline-rich domains, one with similarity to eukaryotic formins. In a guinea pig model of infection, the Sca2 mutant did not elicit fever, suggesting that Sca2 and actin-based motility are virulence factors of spotted fever group rickettsiae.

FIG. 1.

Transposon mutagenesis of R. rickettsii resulted in the generation of 8 unique clones. (A) Southern blot demonstrating a single transposition event that occurred in the generation of each mutant. Purified genomic DNA from mutant clones 9-1 through 9-8 and the congenic parental R. rickettsii strain (R) was digested with HindIII and subjected to Southern blotting with a ³²P-labeled gfp-specific probe (GFP). Positive controls included pMW1650, as well as gfp-specific DNA used as the probe. Clone 9-2 did not appear to grow well and was not included in the Southern blot because of limited amounts of DNA recovered. (B) Diagram of the R. rickettsii chromosome depicting the relative locations of the insertion sites for each of the 8 mutants generated by transposon mutagenesis.

FIG. 2.

The Sca2 mutant displays a small-plaque phenotype but replicates at wild-type levels within Vero cells. (A) Plaques of the R. rickettsii parent strain R and mutants 9-3 and 9-7. Scale bar = 5 mm. (B) Growth curves of the R. rickettsii parent strain R and mutants 9-3 and 9-7 in Vero cells. PFU were enumerated in triplicate. The error bars represent standard errors of the mean.

FIG. 3.

The Sca2 mutant does not produce actin comet tails. Vero cells were infected with the parental R. rickettsii strain R, mutant strain 9-3, or Sca2 mutant 9-7. After 24 h, the cultures were fixed and stained for rickettsiae (green) and actin (red). Scale bar = 10 μm.

FIG. 4.

The Sca2 mutant displays reduced virulence in a guinea pig model. (A) Five guinea pigs were inoculated with either the congenic parental R. rickettsii strain (R), the mutant strain 9-3 (R 9-3), the Sca2 mutant 9-7 (R 9-7), or formalin-fixed R. rickettsii (R fixed) or were sham infected with K36 buffer alone (Buffer Only). Rectal temperatures were taken daily for 14 days beginning the day of inoculation. The error bars represent standard errors of the mean from 5 individual animals. (B) Sera collected at 30 days postinfection were used to determine anti-R. rickettsii titers by microimmunofluorescence. Bars indicate the means ± standard errors of the means.

FIG. 5.

Domain organization and sequence alignments of Sca2 WH2 and FH1-like domains. (A) Graphical illustration representing the domain organization of Sca2, a 1,821-amino-acid (aa) protein with a Sec-dependent secretion signal (SS) (aa 1 to 32), 4 putative WH2 domains (WH2-1, aa 454 to 479; WH2-2, aa 626 to 650; WH2-3, aa 702 to 730; and WH2-4, aa 1514 to 1538), an FH1 domain (aa 650 to 680), a proline-rich region (PRR) (aa 1057 to 1066), and an autotransporter domain (aa 1542 to 1821). The black arrowhead indicates the transposon insertion site in clone 9-7. (B) ClustalW amino acid sequence alignment of the four WH2 domains of Sca2 (R. rickettsii YP_001494228) and WH2-domain containing proteins from Homo sapiens, CAP1 (Q01518), CAP2 (P40123), WASP (P42768), N-WASP (O00401), WAVE1 (Q92558), WAVE2 (Q9Y6W5), and WAVE3 (Q9UPY6); from V. cholerae, VopF (AAZ32252); from V. parahaemolyticus, VopL (NP_800881); and from C. trachomatis, Tarp (YP_328278). (C) ClustalW amino acid sequence alignment of the Sca2 FH1-like domain and the portion of FH1 domain-containing proteins obtained through a BLASTp search. The accession numbers for proteins from humans are hDiap1, NP_001073280.1, and hFormin2, CAQ10135.1, and from mouse they are mDia1, AAH21396.1, and mFormin2, EDL13214.1. The consensus sequence was identified based upon a minimum of 75% amino acid identity at that position. Black shading indicates 100% similarity, and gray shading indicates >60% similarity.