Transformation frequency of a mariner-based transposon in Rickettsia rickettsii.

Transformation frequencies of a mariner-based transposon system in Rickettsia rickettsii were determined using a plaque assay system for enumeration and isolation of mutants. Sequence analysis of insertion sites in both R. rickettsii and R. prowazekii indicated that insertions were random. Transposon mutagenesis provides a useful tool for rickettsial research.

Complementation of Rickettsia rickettsii RelA/SpoT restores a nonlytic plaque phenotype.

Spotted fever group rickettsiae are known to produce distinct plaque phenotypes. Strains that cause lytic infections in cell culture form clear plaques, while nonlytic strains form opaque plaques in which the cells remain intact. Clear plaques have historically been associated with more-virulent species or strains of spotted fever group rickettsiae. We have selected spontaneous mutant pairs from two independent strains of Rickettsia rickettsii, the virulent R strain and the avirulent Iowa strain. A nonlytic variant of R. rickettsii R, which typically produces clear plaques, was isolated and stably maintained. A lytic variant of the Iowa strain, which characteristically produces opaque plaques, was also selected and maintained. Genomic resequencing of the variants identified only a single gene disrupted in each strain. In both cases, the mutation was in a gene annotated as relA/spoT-like. In the Iowa strain, a single mutation introduced a premature stop codon upstream from region encoding the predicted active site of RelA/SpoT and caused the transition to a lytic plaque phenotype. In R. rickettsii R, the nonlytic plaque phenotype resulted from a single-nucleotide substitution that shifted a tyrosine residue to histidine near the active site of the enzyme. The intact relA/spoT gene thus occurred in variants with the nonlytic plaque phenotype. Complementation of the truncated relA/spoT gene in the Iowa lytic plaque variant restored the nonlytic phenotype. The relA/spoT mutations did not affect the virulence of either strain in a Guinea pig model of infection; R strain lytic and nonlytic variants both induced fever equally, and the mutation in Iowa to a lytic phenotype did not cause them to become virulent.

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

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.

Chlamydial effector proteins localized to the host cell cytoplasmic compartment.

Disease-causing microbes utilize various strategies to modify their environment in order to create a favorable location for growth and survival. Gram-negative bacterial pathogens often use specialized secretion systems to translocate effector proteins directly into the cytosol of the eukaryotic cells they infect. These bacterial proteins are responsible for modulating eukaryotic cell functions. Identification of the bacterial effectors has been a critical step toward understanding the molecular basis for the pathogenesis of the bacteria that use them. Chlamydiae are obligate intracellular bacterial pathogens that have a type III secretion system believed to translocate virulence effector proteins into the cytosol of their host cells. Selective permeabilization of the eukaryotic cell membrane was used in conjunction with metabolic labeling of bacterial proteins to identify chlamydial proteins that localize within the cytosol of infected cells. More than 20 Chlamydia trachomatis and C. pneumoniae proteins were detected within the cytoplasmic compartment of infected cells. While a number of cytosolic proteins were shared, others were unique to each species, suggesting that variation among cytosolic chlamydial proteins contributes to the differences in the pathogenesis of the chlamydial species. The spectrum of chlamydial proteins exported differed concomitant with the progress of the developmental cycle. These data confirm that a dynamic relationship exists between Chlamydia and its host and that translocation of bacterial proteins into the cytosol is developmentally dependent.

Bacteria-associated fibronectin does not enhance Chlamydia trachomatis infectivity in vitro.

The molecules responsible for mediating attachment of pathogenic bacteria to their host cells are essential determinants of the pathogen's success. Staphylococci, streptococci, and gonococci are known to utilize fibronectin to mediate adhesion and/or entry into eukaryotic cells. It has been shown that Chlamydia trachomatis binds fibronectin via a heparan sulfate-lyase sensitive molecule on the surface of the bacteria. Because heparan sulfate-lyase treated Chlamydia are markedly reduced in infectivity, we hypothesized that fibronectin might be acting as an intermediate to form a molecular bridge between heparan sulfate-like molecules on the surface of Chlamydia and integrins on the host cell. To test this question, fibronectin-deficient C. trachomatis were used in a series of infectivity assays whereby exogenous fibronectin was added to the inoculum. At concentrations up to 100 ug/ml fibronectin did not modulate C. trachomatis infectivity. However, at high fibronectin concentrations (1 mg/ml) bacterial infectivity was reduced. These data suggest that, unlike several other microbial pathogens, C. trachomatis does not utilize fibronectin to mediate infectivity in vitro.

Recruitment of mammalian cell fibronectin to the surface of Chlamydia trachomatis.

Pathogenic bacteria exploit the presence of various host cell molecules in order to colonize new tissues. Fibronectin is involved in a wide range of cell functions in vivo, and staphylococci, streptococci, and gonococci have evolved mechanisms to utilize this glycoprotein to mediate host cell binding. We show that elementary bodies (EB) from two biovars of Chlamydia trachomatis recruit fibronectin to their surfaces upon lysis of the host cell. We also demonstrate that a heparan sulfate lyase-sensitive molecule on chlamydial EB is responsible for binding at least a portion of this fibronectin.

Phage-display antibody detection of Chlamydia trachomatis-associated antigens.

A phage-displayed human single-chain Fv antibody library (6.7x10(9) members) was used to select probes specific to components associated with the surface of Chlamydia trachomatis elementary bodies (EBs). Each of 15 antibodies was characterized by ELISA, dot-blot, immunoblot and immunocytochemistry, resulting in the identification of several new chlamydial components associated with the surface of EBs. In addition, six antibodies were specific for host-cell components associated with the surface of EBs. While phage display has been used effectively to produce specific antibodies for purified components, these data show that this technology is suitable for selection of specific probes from complex antigens such as the surface of a microbial pathogen.