Using PCR and an automated laser fluorescent DNA sequencer, we amplified and sequenced a 1,234-bp fragment of the citrate synthase-encoding gene ( g U ) of 28 bacteria belonging to the genus Rickettsia. Comparative sequence analysis showed that most of the spotted fever group (SFG) rickettsiae belonged to one of two subgroups. The first subgroup included Rickettsia massiliae, strain Bar 29, Rickettsia rhipicephali, "Rickettsia aeschlimanni," and Rickettsia montana, which have been isolated only from ticks. The second subgroup was larger and included the majority of the human pathogens and also rickettsiae isolated only from ticks; the members of this subgroup were strain S, Rickettsia apicae, "Rickettsia mongolotimonae," Rickettsia sibirica, Rickettsia parkeri, Rickettsia conorii, Rickettsia rickettsii, the Thai tick typhus rickettsia, the Israeli tick typhus rickettsia, the Astrakhan fever rickettsia, "Rickettsia slovaca," and Rickettsia japonica. The sequence analysis also showed that the tick-borne organisms Rickettsia helvetica and Rickettsia australis and the mite-borne organism Rickettsia akari were associated with the SFG cluster; that Rickettsia prowazekii and Rickettsia typhi, two representatives of the typhus group, clustered together; and that Rickettsia Canada, Rickettsia bellii, and the AB bacterium probably represent three new groups. We compared the phylogenetic trees inferred from citrate synthase gene sequences and from 16s ribosomal DNA (rDNA) sequences. For rickettsial phylogeny, the citrate synthase approach was more suitable, as demonstrated by significant bootstrap values for all of the nodes except those in the larger subgroup defined above. We also compared phylogenetic analysis results obtained in a comparison of the sequences of both genes for all of the representatives of the domain Bacteria for which the gZtA sequence was determined. We believe that comparison of glL4 sequences could be a complementary approach to 16s rDNA sequencing for inferring bacterial evolution, especially when unstable phylogenetic models are obtained from ribosomal sequences because of high levels of sequence similarity between the bacteria studied.Rickettsiae belong to the order Rickettsiales, whose members were described as obligately intracellular gram-negative microorganisms (55). Several species cause disease in humans or in other vertebrate and invertebrate hosts and have a worldwide distribution. Recently, on the basis of 16s rRNA sequence similarity, Rickettsia tsutsugamushi was removed from the genus Rickettsia and was renamed Orientia tsutsugarnushi (48). The genus Rickettsia is presently divided into two groups: the typhus group (TG), which includes three species (Rickettsia prowazekii, the agent of the epidemic typhus, Rickettsia typhi, which causes murine typhus, and Rickettsia Canada, which has been isolated only from ticks), and the spotted fever group (SFG). The number of recognized members of the SFG increased following the development of an improved cell culture isolation technique (the shell...

Senescent cells (SCs) have been considered a source of age-related chronic sterile systemic inflammation and a target for anti-aging therapies. To understand mechanisms controlling the amount of SCs, we analyzed the phenomenon of rapid clearance of human senescent fibroblasts implanted into SCID mice, which can be overcome when SCs were embedded into alginate beads preventing them from immunocyte attack. To identify putative SC killers, we analyzed the content of cell populations in lavage and capsules formed around the SC-containing beads. One of the major cell types attracted by secretory factors of SCs was a subpopulation of macrophages characterized by p16(Ink4a) gene expression and β-galactosidase activity at pH6.0 (β-galpH6), thus resembling SCs. Consistently, mice with p16(Ink4a) promoter-driven luciferase, developed bright luminescence of their peritoneal cavity within two weeks following implantation of SCs embedded in alginate beads. p16(Ink4a)/β-galpH6-expressing cells had surface biomarkers of macrophages F4/80 and were sensitive to liposomal clodronate used for the selective killing of cells capable of phagocytosis. At the same time, clodronate failed to kill bona fide SCs generated in vitro by genotoxic stress. Old mice with elevated proportion of p16(Ink4a)/β-galpH6-positive cells in their tissues demonstrated reduction of both following systemic clodronate treatment, indicating that a significant proportion of cells previously considered to be SCs are actually a subclass of macrophages. These observations point at a significant role of p16(Ink4a)/β-galpH6-positive macrophages in aging, which previously was attributed solely to SCs. They require re-interpretation of the mechanisms underlying rejuvenating effects following eradication of p16(Ink4a)/β-galpH6-positive cells and reconsideration of potential cellular target for anti-aging treatment.

Constitutive p16Ink4a expression, along with senescence-associated β-galactosidase (SAβG), are commonly accepted biomarkers of senescent cells (SCs). Recent reports attributed improvement of the healthspan of aged mice following p16Ink4a-positive cell killing to the eradication of accumulated SCs. However, detection of p16Ink4a/SAβG-positive macrophages in the adipose tissue of old mice and in the peritoneal cavity of young animals following injection of alginate-encapsulated SCs has raised concerns about the exclusivity of these markers for SCs. Here we report that expression of p16Ink4a and SAβG in macrophages is acquired as part of a physiological response to immune stimuli rather than through senescence, consistent with reports that p16Ink4a plays a role in macrophage polarization and response. Unlike SCs, p16Ink4a/SAβG-positive macrophages can be induced in p53-null mice. Macrophages, but not mesenchymal SCs, lose both markers in response to M1- [LPS, IFN-α, Poly(I:C)] and increase their expression in response to M2-inducing stimuli (IL-4, IL-13). Moreover, interferon-inducing agent Poly(I:C) dramatically reduced p16Ink4a expression in vivo in our alginate bead model and in the adipose tissue of aged mice. These observations suggest that the antiaging effects following eradication of p16Ink4a-positive cells may not be solely attributed to SCs but also to non-senescent p16Ink4a/SAβG-positive macrophages.

We describe the characterization of a novel Rickettsia species cultivated from Dermacentor ticks collected in Russia and France, for which we propose the name Rickettsia raoultii sp. nov. Using multigene sequencing, we demonstrated that five rickettsial isolates from Dermacentor silvarum, Dermacentor reticulatus, Dermacentor marginatus and Dermacentor nuttalli ticks were classified within this novel spotted fever rickettsia species. This rickettsia also exhibited a serotype distinct from previously described Rickettsia species. The type strain of Rickettsia raoultii sp. nov. is strain Khabarovsk T (5CSUR R3 T 5ATCC VR-1596 T ).