Wholly Rickettsia! Reconstructed Metabolic Profile of the Quintessential Bacterial Parasite of Eukaryotic Cells

Reductive genome evolution has purged many metabolic pathways from obligate intracellular Rickettsia (Alphaproteobacteria; Rickettsiaceae). While some aspects of host-dependent rickettsial metabolism have been characterized, the array of host-acquired metabolites and their cognate transporters remains unknown. This dearth of information has thwarted efforts to obtain an axenic Rickettsia culture, a major impediment to conventional genetic approaches. Using phylogenomics and computational pathway analysis, we reconstructed the Rickettsia metabolic and transport network, identifying 51 host-acquired metabolites (only 21 previously characterized) needed to compensate for degraded biosynthesis pathways. In the absence of glycolysis and the pentose phosphate pathway, cell envelope glycocon- jugates are synthesized from three imported host sugars, with a range of additional host-acquired metabolites fueling the tricarboxylic acid cycle. Fatty acid and glycero- phospholipid pathways also initiate from host precursors, and import of both iso- prenes and terpenoids is required for the synthesis of ubiquinone and the lipid car- rier of lipid I and O-antigen. Unlike metabolite-provisioning bacterial symbionts of arthropods, rickettsiae cannot synthesize B vitamins or most other cofactors, accen- tuating their parasitic nature. Six biosynthesis pathways contain holes (missing en- zymes); similar patterns in taxonomically diverse bacteria suggest alternative en- zymes that await discovery. A paucity of characterized and predicted transporters emphasizes the knowledge gap concerning how rickettsiae import host metabolites, some of which are large and not known to be transported by bacteria. Collectively, our reconstructed metabolic network offers clues to how rickettsiae hijack host met- abolic pathways. This blueprint for growth determinants is an important step toward the design of axenic media to rescue rickettsiae from the eukaryotic cell

Isolation of a Rickettsial Pathogen from a Non-Hematophagous Arthropod

Rickettsial diversity is intriguing in that some species are transmissible to vertebrates, while others appear exclusive to invertebrate hosts. Of particular interest is Rickettsia felis, identifiable in both stored product insect pests and hematophagous disease vectors. To understand rickettsial survival tactics in, and probable movement between, both insect systems will explicate the determinants of rickettsial pathogenicity. Towards this objective, a population of Liposcelis bostrychophila, common booklice, was successfully used for rickettsial isolation in ISE6 (tick-derived cells). Rickettsiae were also observed in L. bostrychophila by electron microscopy and in paraffin sections of booklice by immunofluorescence assay using anti-R. felis polyclonal antibody. The isolate, designated R. felis strain LSU-Lb, resembles typical rickettsiae when examined by microscopy. Sequence analysis of portions of the Rickettsia specific 17-kDa antigen gene, citrate synthase (gltA) gene, rickettsial outer membrane protein A (ompA) gene, and the presence of the R. felis plasmid in the cell culture isolate confirmed the isolate as R. felis. Variable nucleotide sequences from the isolate were obtained for R. felis-specific pRF-associated putative tldD/pmbA. Expression of rickettsial outer membrane protein B (OmpB) was verified in R. felis (LSU-Lb) using a monoclonal antibody. Additionally, a quantitative real-time PCR assay was used to identify a significantly greater median rickettsial load in the booklice, compared to cat flea hosts. With the potential to manipulate arthropod host biology and infect vertebrate hosts, the dual nature of R. felis provides an excellent model for the study of rickettsial pathogenesis and transmission. In addition, this study is the first isolation of a rickettsial pathogen from a non-hematophagous arthropod

Detection of <i>Rickettsia</i> in booklice.

<p>(A) <i>Rickettsia</i> on formalin-fixed paraffin-embedded booklice sections were labeled with mouse polyclonal antibody against <i>R. felis</i> followed by FITC-conjugated goat anti-mouse IgG. Whole booklice tissues were counterstained with Evan's blue and nuclei were stained with DAPI as shown in red and blue, respectively. (B) Negative control staining using non-infected mouse serum. (C)–(F) Variation of mycetomes located in booklice abdomen. High magnification view of mycetomes shows densely packed rickettsiae. Bar = 100 µm.</p

Propagation of <i>Rickettsia</i> isolated from booklice in ISE6 cells.

<p>(A) ISE6 cells infected with <i>Rickettsia</i> isolated from booklice were Cytospin centrifuged and rickettsiae were visualized by Diff-Quik staining. (B) The infected cells on coverslips were fixed and permeabilized prior to stain with mouse polyclonal antibody against <i>R. felis</i> and FITC-conjugated goat anti-mouse IgG. Host cell actin and nuclei were stained with with Rhodamine-Phalloidin and DAPI as shown in red and blue, respectively. Bar = 5 µm.</p

Electron micrographs of <i>Rickettsia</i> in booklice.

<p>(A) Rickettsiae free in the cytosol of gut epithelial cells. MV represents microvilli. (B) Higher magnification of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016396#pone-0016396-g002" target="_blank">Fig. 2A</a> shows typical rickettsial ultrastructure: rickettsial cell wall including trilaminar cell wall membrane associated with the external surface microcapsule layer and an internal trilaminar cytoplasmic membrane (arrowheads), surrounded by an outermost “halo” zone (h). Solid arrow indicates a small vacuole inside rickettsial cytoplasm. (C) Rickettsiae (R) in ovary. Inset: higher magnification view of the boxed rickettsiae. (D) Ultrathin section of a mycetome-like, several cells of which contain large vacuoles tightly packed with rickettsiae of irregular shape with dense cytoplasm. Bar = 500 nm.</p

Electron micrographs of <i>Rickettsia</i> isolated from booklice in ISE6 cells.

<p>(A) Typical infected cells containing rickettsiae in cytosol. (B) Typical ultrastructure of <i>Rickettsia</i>. Inset: higher magnification view of rickettsial cell wall including trilaminar cell wall associated with the external surface microcapsule layer (solid arrows) and an internal trilaminar cytoplasmic membrane (arrowheads). m represents mitochondria. (C) Rickettsiae surrounded by a halo zone (h). (D) Rickettsiae being destroyed in a phagolysosome. Long arrows indicate phagolysosomal membrane. Bar = 500 nm.</p

Identity of sequenced <i>R. felis</i> LSU-Lb isolate genes compared to <i>R. felis</i> URRWXcal2 Accession numbers CP000053 (genome) and CP000054 (plasmid pRF).

<p>Identity of sequenced <i>R. felis</i> LSU-Lb isolate genes compared to <i>R. felis</i> URRWXcal2 Accession numbers CP000053 (genome) and CP000054 (plasmid pRF).</p

Primers used for PCR amplification and sequencing of <i>R. felis</i> (LSU-Lb) and primer/probe combination used for quantitative real-time PCR for <i>Rickettsia</i>.

<p>*IUB codes for mixed bases at a given position: N = A+T+C+G; H = A+T+C; Y = C+T; K = G+T; R = A+G.</p

Novel Identification of <i>Dermacentor variabilis</i> Arp2/3 Complex and Its Role in Rickettsial Infection of the Arthropod Vector

<div><p>Tick-borne spotted fever group (SFG) <i>Rickettsia</i> species must be able to infect both vertebrate and arthropod host cells. The host actin-related protein 2/3 (Arp2/3) complex is important in the invasion process and actin-based motility for several intracellular bacteria, including SFG <i>Rickettsia</i> in <i>Drosophila</i> and mammalian cells. To investigate the role of the tick Arp2/3 complex in tick-<i>Rickettsia</i> interactions, open reading frames of all subunits of the protein including Arp2, Arp3, ARPC1, ARPC2, ARPC3, ARPC4, and ARPC5 were identified from <i>Dermacentor variabilis</i>. Amino acid sequence analysis showed variation (ranging from 25–88%) in percent identity compared to the corresponding subunits of the complex from <i>Drosophila melanogaster</i>, <i>Mus musculus</i>, <i>Homo sapiens</i>, and <i>Saccharomyces cerevisiae</i>. Potential ATP binding sites were identified in <i>D. variabilis</i> (<i>Dv</i>) Arp2 and Arp3 subunits as well as five putative WD (Trp-Asp) motifs which were observed in <i>Dv</i>ARPC1. Transcriptional profiles of all subunits of the <i>Dv</i>Arp2/3 complex revealed greater mRNA expression in both <i>Rickettsia</i>-infected and -uninfected ovary compared to midgut and salivary glands. In response to <i>R. montanensis</i> infection of the tick ovary, the mRNA level of only <i>Dv</i>ARPC4 was significantly upregulated compared to uninfected tissues. Arp2/3 complex inhibition bioassays resulted in a decrease in the ability of <i>R. montanensis</i> to invade tick tissues with a significant difference in the tick ovary, indicating a role for the Arp2/3 complex in rickettsial invasion of tick cells. Characterization of tick-derived molecules associated with rickettsial infection is imperative in order to better comprehend the ecology of tick-borne rickettsial diseases.</p></div