The Rickettsial Ankyrin Repeat Protein 2 Is a Type IV Secreted Effector That Associates with the Endoplasmic Reticulum.

Strains of Rickettsia rickettsii, the tick-borne agent of Rocky Mountain spotted fever, vary considerably in virulence. Genomic comparisons of R. rickettsii strains have identified a relatively small number of genes divergent in an avirulent strain. Among these is one annotated as Rickettsia ankyrin repeat protein 2 (RARP-2). Homologs of RARP-2 are present in all strains of R. rickettsii, but the protein in the avirulent strain Iowa contains a large internal deletion relative to the virulent Sheila Smith strain. RARP-2 is secreted in a type IV secretion system-dependent manner and exposed to the host cell cytosol. RARP-2 of Sheila Smith colocalizes with multilamellar membranous structures bearing markers of the endoplasmic reticulum (ER), whereas the Iowa protein shows no colocalization with host cell organelles and evidence of proteolytic degradation is detected. Overexpression of Sheila Smith RARP-2 in R. rickettsii Iowa converts this avirulent strain's typically nonlytic or opaque plaque type to a lytic plaque phenotype similar to that of the virulent Sheila Smith strain. Mutation of a predicted proteolytic active site of Sheila Smith RARP-2 abolished the lytic plaque phenotype but did not eliminate association with host membrane. RARP-2 is thus a type IV secreted effector and released from the rickettsiae into the host cytosol to modulate host processes during infection. Overexpression of Sheila Smith RARP-2 did not, however, restore the virulence of the Iowa strain in a guinea pig model, likely due to the multifactorial nature of rickettsial virulence.IMPORTANCE Members of the genus Rickettsia are obligate intracellular bacteria that exhibit a range of virulence from harmless endosymbionts of arthropods to the etiologic agents of severe disease. Despite the growing number of available genomes, little is known regarding virulence determinants of rickettsiae. Here, we have characterized an ankyrin repeat-containing protein, RARP-2, which differs between a highly virulent and an avirulent strain of R. rickettsii, the agent of Rocky Mountain spotted fever. RARP-2 is secreted by a type IV secretion system into the cytosol of the host cell, where it interacts with and manipulates the structure of the endoplasmic reticulum. RARP-2 from the avirulent strain is truncated by the loss of seven of 10 ankyrin repeat units but, although secreted, fails to alter ER structure. Recognition of those rickettsial factors associated with virulence will facilitate understanding of regional and strain-specific variation in severity of disease.

Comparative genomic analysis of Rickettsia rickettsii for identification of drug and vaccine targets: tolC as a proposed candidate for case study.

BACKGROUND: Antibiotic resistance is increasing rapidly in pathogenic organisms, creating more complications for treatment of diseases. Rocky Mountain spotted fever (RMSF) is a neglected tropical disease in humans caused by Rickettsia rickettsii for which no effective therapeutic is available. Subtractive genomics methods facilitate the characterization of non-homologous essential proteins that could be targeted for the discovery of potential therapeutic compounds against R. rickettsii to combat RMSF. Present study followed an in-silico based methodology, involving scanning and filtering the complete proteome of Rickettsia rickettsii by using several prioritization parameters in the search of potential candidates for drug development. Further the putative targets were subjected to series of molecular dockings with ligands obtained from PDB ligand database to identify suitable potential inhibitors. The comparative genomic analysis revealed 606 non-homologous proteins and 233 essential non-homologous proteins of R. rickettsii. The metabolic pathway analysis predicted 120 proteins as putative drug targets, out of which 56 proteins were found to be associated with metabolic pathways unique to the bacteria and further subcellular localization analysis revealed that 9 proteins as potential drug targets which are secretion proteins, involved in peptidoglycan biosynthesis, folate biosynthesis and bacterial secretion system. As secretion proteins are more feasible as vaccine candidates, we have selected a most potential target i.e. tolC, an outer membrane efflux protein that belongs to type I secretion system and has major role in pathogen survival as well as MDR persistence. So for case study, we have modelled the three dimensional structure of tolC (tunnel protein). The model was further subjected to virtual screening and in-silico docking. The study identified three potential inhibitors having PDB Id 19V, 6Q8 and 39H. Further we have suggested that the above study would be most important while considering the selection of candidate targets and drug or vaccine designing against R. rickettsii.

Immunogenicity of OmpA and OmpB antigens from Rickettsia rickettsii on mononuclear cells from Rickettsia positive Mexican patients.

Background & objectives: The nature of the rickettsial antigens and the immune response generated by them, have been the subject of exhaustive research so that a suitable vaccine can be developed. Till date evaluations of Rickettsia rickettsii antigens that induce both humoral and cellular responses in animal models have only shown partial protection and short-term immunological memory. This study was aimed to evaluate the immune response induced by DNA plasmids generated from the OmpA and OmpB genes of R. rickettsii in peripheral blood mononuclear cells of rickettsial (sensitized) patients compared to healthy subjects. Methods: Plasmids OmpA-49, OmpB-15 and OmpB-24 were generated in the pVAX vector. Macrophages derived from the THP-1 cell line were transfected in vitro with the plasmids and were co-cultured with T-lymphocytes from sensitized subjects and healthy subjects to evaluate cell proliferation and cytokine production. Results: The OmpB-24 plasmid induced proliferative response in human lymphocytes, with production of IL-2, IFN-γ, IL-12p70, IL-6 and TNF-α, likely due to the presence of conserved epitopes among R. rickettsii, R. typhi and R. felis (differing from 1 to 3 amino acids) during the construction of the plasmids. Interpretation & conclusion: DNA sequences of rickettsial epitopes can be cloned into the pVAX vector. Constructed plasmids can generate a proliferative response and produce cytokines in vitro, in co-culture of transfected macrophages with sensitized human lymphocytes. Plasmid OmpB-24 proved to be the most immunogenic with respect to plasmids OmpA-49 and OmpB-15.

Crystal structure of the N-terminal domains of the surface cell antigen 4 of Rickettsia.

The obligate intracellular, gram-negative bacterium Rickettsia is the causative agent of spotted fevers and typhus in humans. Surface cell antigen (sca) proteins surround these bacteria. We recently reported the co-localization of one of these proteins, sca4, with vinculin in cells at sites of focal adhesions and demonstrated that two vinculin binding sites directed the sca4/vinculin interaction. Here we report the 2.2 Å crystal structure of the conserved N-terminal 38 kDa domain of sca4 from Rickettsia rickettsii. The structure reveals two subdomains. The first is an all-helical domain that is folded in a fashion similar to the dimeric assembly chaperone for rubisco, namely RbcX. The following and highly conserved ß-strand domain lacks significant structural similarity with other known structures and to the best of our knowledge represents a new protein fold.

Genomic comparison of virulent Rickettsia rickettsii Sheila Smith and avirulent Rickettsia rickettsii Iowa.

Rickettsia rickettsii is an obligate intracellular pathogen that is the causative agent of Rocky Mountain spotted fever. To identify genes involved in the virulence of R. rickettsii, the genome of an avirulent strain, R. rickettsii Iowa, was sequenced and compared to the genome of the virulent strain R. rickettsii Sheila Smith. R. rickettsii Iowa is avirulent in a guinea pig model of infection and displays altered plaque morphology with decreased lysis of infected host cells. Comparison of the two genomes revealed that R. rickettsii Iowa and R. rickettsii Sheila Smith share a high degree of sequence identity. A whole-genome alignment comparing R. rickettsii Iowa to R. rickettsii Sheila Smith revealed a total of 143 deletions for the two strains. A subsequent single-nucleotide polymorphism (SNP) analysis comparing Iowa to Sheila Smith revealed 492 SNPs for the two genomes. One of the deletions in R. rickettsii Iowa truncates rompA, encoding a major surface antigen (rickettsial outer membrane protein A [rOmpA]) and member of the autotransporter family, 660 bp from the start of translation. Immunoblotting and immunofluorescence confirmed the absence of rOmpA from R. rickettsii Iowa. In addition, R. rickettsii Iowa is defective in the processing of rOmpB, an autotransporter and also a major surface antigen of spotted fever group rickettsiae. Disruption of rompA and the defect in rOmpB processing are most likely factors that contribute to the avirulence of R. rickettsii Iowa. Genomic differences between the two strains do not significantly alter gene expression as analysis of microarrays revealed only four differences in gene expression between R. rickettsii Iowa and R. rickettsii strain R. Although R. rickettsii Iowa does not cause apparent disease, infection of guinea pigs with this strain confers protection against subsequent challenge with the virulent strain R. rickettsii Sheila Smith.

rOmpA is a critical protein for the adhesion of Rickettsia rickettsii to host cells.

rOmpA and rOmpB are immunodominant, surface-exposed proteins of Rickettsia rickettsii. Prior evidence suggests that adhesion of R. rickettsii to the host cell is mediated by a rickettsial protein. Five monoclonal antibodies to rOmpA, five to rOmpB, and one to the rickettsial lipopolysaccharide (LPS) were tested for inhibition of rickettsial attachment. All the monoclonal antibodies to rOmpA inhibited adhesion of rickettsiae to the L-929 cells with some inhibition rates as high as 90%. In contrast, monoclonal antibodies to rOmpB and LPS did not block attachment. When Fab fragments of monoclonal antibodies against rOmpA and rOmpB were used, similar results were observed as for the intact monoclonals, non-adhesion and adhesion, respectively. Purified rOmpA showed a competitive inhibitive effect on the attachment of R. rickettsii to host cells. Trypsin completely digested rOmpA but not rOmpB from the surface of intact R. rickettsii, resulting in loss of the ability of the rickettsiae to attach to the host cell. rOmpA appears to play an important role in the initial adhesion of R. rickettsii to the host cell.