Phylogenic position and low genomic diversity of "Candidatus Rickettsia kotlanii" inferred by complete genome sequences of two Japanese isolates.

Many Rickettsia species of the spotted fever group (SFG) cause tick-borne diseases known as "spotted fever." One of the candidate SFG Rickettsia species is "Candidatus Rickettsia kotlanii," which was first detected in Haemaphysalis concinna in Hungary in 2006. However, its precise phylogenetic position in the SFG is not clear because only single-gene sequence-based phylogenetic analyses were performed using very limited genes. Here, we present the complete genome sequences of two Japanese "Ca. R. kotlanii" isolates, which differed only by a 135 bp insertion/deletion (InDel). Using these genomes and publicly available whole genome sequences of other Rickettsia species, the precise phylogenetic position of "Ca. R. kotlanii" in Rickettsia was determined to be in a clade of the SFG. The phylogenetic relationships and average nucleotide identity of "Ca. R. kotlanii" relative to the other species indicated that "Ca. R. kotlanii" is an independent taxon in the SFG. Notably, although the genomes of the two isolates were almost identical, the isolates were obtained from different tick species in different regions and years, suggesting extremely low genomic diversity in "Ca. R. kotlanii." While the genome of "Ca. R. kotlanii" is the smallest in the transitional group and SFG Rickettsia sequenced to date, we identified genes uniquely present or absent in "Ca. R. kotlanii," but most were apparently degraded. Therefore, analyses of differences at the sequence (single nucleotide polymorphisms and small InDels) or gene expression level will be required to understand the functional or physiological features unique to "Ca. R. kotlanii."

Activation of Mechanistic Target of Rapamycin (mTOR) in Human Endothelial Cells Infected with Pathogenic Spotted Fever Group Rickettsiae.

Attributed to the tropism for host microvascular endothelium lining the blood vessels, vascular inflammation and dysfunction represent salient features of rickettsial pathogenesis, yet the details of fundamentally important pathogen interactions with host endothelial cells (ECs) as the primary targets of infection remain poorly appreciated. Mechanistic target of rapamycin (mTOR), a serine/threonine protein kinase of the phosphatidylinositol kinase-related kinase family, assembles into two functionally distinct complexes, namely mTORC1 (Raptor) and mTORC2 (Rictor), implicated in the determination of innate immune responses to intracellular pathogens via transcriptional regulation. In the present study, we investigated activation status of mTOR and its potential contributions to host EC responses during Rickettsia rickettsii and R. conorii infection. Protein lysates from infected ECs were analyzed for threonine 421/serine 424 phosphorylation of p70 S6 kinase (p70 S6K) and that of serine 2448 on mTOR itself as established markers of mTORC1 activation. For mTORC2, we assessed phosphorylation of protein kinase B (PKB or Akt) and protein kinase C (PKC), respectively, on serine 473 and serine 657. The results suggest increased phosphorylation of p70 S6K and mTOR during Rickettsia infection of ECs as early as 3 h and persisting for up to 24 h post-infection. The steady-state levels of phospho-Akt and phospho-PKC were also increased. Infection with pathogenic rickettsiae also resulted in the formation of microtubule-associated protein 1A/1B-light chain 3 (LC3-II) puncta and increased lipidation of LC3-II, a response significantly inhibited by introduction of siRNA targeting mTORC1 into ECs. These findings thus yield first evidence for the activation of both mTORC1 and mTORC2 during EC infection in vitro with Rickettsia species and suggest that early induction of autophagy in response to intracellular infection might be regulated by this important pathway known to function as a central integrator of cellular immunity and inflammation.

Identification of a Ribosomal Protein RpsB as a Surface-Exposed Protein and Adhesin of Rickettsia heilongjiangensis.

Rickettsia heilongjiangensis is an obligate intracellular bacterium that is responsible for far-eastern spotted fever. Surface-exposed proteins (SEPs) play important roles in its pathogenesis. Previous work identified a ribosomal protein RpsB as an SEP by biotin-avidin affinity, a seroreactive antigen, and a diagnostic candidate protein, indicating that it might play an important role in the pathogenesis of rickettsiae. However, in the absence of other evidence, its subcellular location of being surface-exposed was puzzling because ribosomal proteins are located in the cytoplasm. In the present study, the subcellular location of RpsB was analyzed with bioinformatics tools coupled with immunoelectron microscopy. The adhesion ability of RpsB was evaluated by protein microarray and cellular ELISA. Consequently, different bioinformatics tools gave different location predication results. Thus, RpsB was found in the cytoplasma and inner and outer membranes of R. heilongjiangensis by transmission electron microscopy. Protein microarray and cellular ELISA showed that RpsB binds to the host cell surface and its adhesion ability was even stronger than the known adhesin Adr1. In conclusion, RpsB was visually and directly shown for the time to be an SEP of rickettsiae and might be an important ligand and adhesin of rickettsiae. Its roles in pathogenesis warrant further study.

Diversity of spotted fever group rickettsiae and their association with host ticks in Japan.

Spotted fever group (SFG) rickettsiae are obligate intracellular Gram-negative bacteria mainly associated with ticks. In Japan, several hundred cases of Japanese spotted fever, caused by Rickettsia japonica, are reported annually. Other Rickettsia species are also known to exist in ixodid ticks; however, their phylogenetic position and pathogenic potential are poorly understood. We conducted a nationwide cross-sectional survey on questing ticks to understand the overall diversity of SFG rickettsiae in Japan. Out of 2,189 individuals (19 tick species in 4 genera), 373 (17.0%) samples were positive for Rickettsia spp. as ascertained by real-time PCR amplification of the citrate synthase gene (gltA). Conventional PCR and sequencing analyses of gltA indicated the presence of 15 different genotypes of SFG rickettsiae. Based on the analysis of five additional genes, we characterised five Rickettsia species; R. asiatica, R. helvetica, R. monacensis (formerly reported as Rickettsia sp. In56 in Japan), R. tamurae, and Candidatus R. tarasevichiae and several unclassified SFG rickettsiae. We also found a strong association between rickettsial genotypes and their host tick species, while there was little association between rickettsial genotypes and their geographical origins. These observations suggested that most of the SFG rickettsiae have a limited host range and are maintained in certain tick species in the natural environment.