Genome Sequence of the Endosymbiont Rickettsia peacockii and Comparison with Virulent Rickettsia rickettsii: Identification of Virulence Factors

Rickettsia peacockii, also known as the East Side Agent, is a non-pathogenic obligate intracellular bacterium found as an endosymbiont in Dermacentor andersoni ticks in the western USA and Canada. Its presence in ticks is correlated with reduced prevalence of Rickettsia rickettsii, the agent of Rocky Mountain Spotted Fever. It has been proposed that a virulent SFG rickettsia underwent changes to become the East Side Agent. We determined the genome sequence of R. peacockii and provide a comparison to a closely related virulent R. rickettsii. The presence of 42 chromosomal copies of the ISRpe1 transposon in the genome of R. peacockii is associated with a lack of synteny with the genome of R. rickettsii and numerous deletions via recombination between transposon copies. The plasmid contains a number of genes from distantly related organisms, such as part of the glycosylation island of Pseudomonas aeruginosa. Genes deleted or mutated in R. peacockii which may relate to loss of virulence include those coding for an ankyrin repeat containing protein, DsbA, RickA, protease II, OmpA, ScaI, and a putative phosphoethanolamine transferase. The gene coding for the ankyrin repeat containing protein is especially implicated as it is mutated in R. rickettsii strain Iowa, which has attenuated virulence. Presence of numerous copies of the ISRpe1 transposon, likely acquired by lateral transfer from a Cardinium species, are associated with extensive genomic reorganization and deletions. The deletion and mutation of genes possibly involved in loss of virulence have been identified by this genomic comparison. It also illustrates that the introduction of a transposon into the genome can have varied effects; either correlating with an increase in pathogenicity as in Francisella tularensis or a loss of pathogenicity as in R. peacockii and the recombination enabled by multiple transposon copies can cause significant deletions in some genomes while not in others

Development of Shuttle Vectors for Transformation of Diverse Rickettsia Species

Plasmids have been identified in most species of Rickettsia examined, with some species maintaining multiple different plasmids. Three distinct plasmids were demonstrated in Rickettsia amblyommii AaR/SC by Southern analysis using plasmid specific probes. Copy numbers of pRAM18, pRAM23 and pRAM32 per chromosome in AaR/SC were estimated by real-time PCR to be 2.0, 1.9 and 1.3 respectively. Cloning and sequencing of R. amblyommii AaR/SC plasmids provided an opportunity to develop shuttle vectors for transformation of rickettsiae. A selection cassette encoding rifampin resistance and a fluorescent marker was inserted into pRAM18 yielding a 27.6 kbp recombinant plasmid, pRAM18/Rif/GFPuv. Electroporation of Rickettsia parkeri and Rickettsia bellii with pRAM18/Rif/GFPuv yielded GFPuv-expressing rickettsiae within 2 weeks. Smaller vectors, pRAM18dRG, pRAM18dRGA and pRAM32dRGA each bearing the same selection cassette, were made by moving the parA and dnaA-like genes from pRAM18 or pRAM32 into a vector backbone. R. bellii maintained the highest numbers of pRAM18dRGA (13.3 – 28.1 copies), and R. parkeri, Rickettsia monacensis and Rickettsia montanensis contained 9.9, 5.5 and 7.5 copies respectively. The same species transformed with pRAM32dRGA maintained 2.6, 2.5, 3.2 and 3.6 copies. pRM, the plasmid native to R. monacensis, was still present in shuttle vector transformed R. monacensis at a level similar to that found in wild type R. monacensis after 15 subcultures. Stable transformation of diverse rickettsiae was achieved with a shuttle vector system based on R. amblyommii plasmids pRAM18 and pRAM32, providing a new research tool that will greatly facilitate genetic and biological studies of rickettsiae

Long non-coding RNA LCAL62 / LINC00261 is associated with lung adenocarcinoma prognosis

Background: More than half of non-small cell lung cancer (NSCLC) patients present with metastatic disease at initial diagnosis with an estimated five-year survival rate of ~5%. Despite advances in understanding primary lung cancer oncogenesis metastatic disease remains poorly characterized. Recent studies demonstrate important roles of long non-coding RNAs (lncRNAs) in tumor physiology and as prognostic markers. Therefore, we present the first transcriptome analysis to identify lncRNAs altered in metastatic lung adenocarcinoma leading to the discovery and characterization of the lncRNA Patients and methods: RNA-Seq, microarray, nanoString expression, and clinical data from 1,116 LUAD patients across six independent cohorts and 83 LUAD cell lines were used to discover and evaluate the survival association of metastasis associated lncRNAs. Coexpression and gene set enrichment analyses were used to establish gene regulatory networks and implicate metastasis associated lncRNAs in specific biological processes. Results: Our integrative analysis discovered Conclusion: We discovered tha

Determining the Repertoire of Immunodominant Proteins via Whole-Genome Amplification of Intracellular Pathogens

Culturing many obligate intracellular bacteria is difficult or impossible. However, these organisms have numerous adaptations allowing for infection persistence and immune system evasion, making them some of the most interesting to study. Recent advancements in genome sequencing, pyrosequencing and Phi29 amplification, have allowed for examination of whole-genome sequences of intracellular bacteria without culture. We have applied both techniques to the model obligate intracellular pathogen Anaplasma marginale and the human pathogen Anaplasma phagocytophilum, in order to examine the ability of phi29 amplification to determine the sequence of genes allowing for immune system evasion and long-term persistence in the host. When compared to traditional pyrosequencing, phi29-mediated genome amplification had similar genome coverage, with no additional gaps in coverage. Additionally, all msp2 functional pseudogenes from two strains of A. marginale were detected and extracted from the phi29-amplified genomes, highlighting its utility in determining the full complement of genes involved in immune evasion

Epigenetic Silencing of Host Cell Defense Genes Enhances Intracellular Survival of the Rickettsial Pathogen Anaplasma phagocytophilum

Intracellular bacteria have evolved mechanisms that promote survival within hostile host environments, often resulting in functional dysregulation and disease. Using the Anaplasma phagocytophilum–infected granulocyte model, we establish a link between host chromatin modifications, defense gene transcription and intracellular bacterial infection. Infection of THP-1 cells with A. phagocytophilum led to silencing of host defense gene expression. Histone deacetylase 1 (HDAC1) expression, activity and binding to the defense gene promoters significantly increased during infection, which resulted in decreased histone H3 acetylation in infected cells. HDAC1 overexpression enhanced infection, whereas pharmacologic and siRNA HDAC1 inhibition significantly decreased bacterial load. HDAC2 does not seem to be involved, since HDAC2 silencing by siRNA had no effect on A. phagocytophilum intracellular propagation. These data indicate that HDAC up-regulation and epigenetic silencing of host cell defense genes is required for A. phagocytophilum infection. Bacterial epigenetic regulation of host cell gene transcription could be a general mechanism that enhances intracellular pathogen survival while altering cell function and promoting disease

Establishment of a Replicating Plasmid in Rickettsia prowazekii

Rickettsia prowazekii, the causative agent of epidemic typhus, grows only within the cytosol of eukaryotic host cells. This obligate intracellular lifestyle has restricted the genetic analysis of this pathogen and critical tools, such as replicating plasmid vectors, have not been developed for this species. Although replicating plasmids have not been reported in R. prowazekii, the existence of well-characterized plasmids in several less pathogenic rickettsial species provides an opportunity to expand the genetic systems available for the study of this human pathogen. Competent R. prowazekii were transformed with pRAM18dRGA, a 10.3 kb vector derived from pRAM18 of R. amblyommii. A plasmid-containing population of R. prowazekii was obtained following growth under antibiotic selection, and the rickettsial plasmid was maintained extrachromosomally throughout multiple passages. The transformant population exhibited a generation time comparable to that of the wild type strain with a copy number of approximately 1 plasmid per rickettsia. These results demonstrate for the first time that a plasmid can be maintained in R. prowazekii, providing an important genetic tool for the study of this obligate intracellular pathogen

Evolutionary Genomics of a Temperate Bacteriophage in an Obligate Intracellular Bacteria (Wolbachia)

Genome evolution of bacteria is usually influenced by ecology, such that bacteria with a free-living stage have large genomes and high rates of horizontal gene transfer, while obligate intracellular bacteria have small genomes with typically low amounts of gene exchange. However, recent studies indicate that obligate intracellular species that host-switch frequently harbor agents of horizontal transfer such as mobile elements. For example, the temperate double-stranded DNA bacteriophage WO in Wolbachia persistently transfers between bacterial coinfections in the same host. Here we show that despite the phage's rampant mobility between coinfections, the prophage's genome displays features of constraint related to its intracellular niche. First, there is always at least one intact prophage WO and usually several degenerate, independently-acquired WO prophages in each Wolbachia genome. Second, while the prophage genomes are modular in composition with genes of similar function grouping together, the modules are generally not interchangeable with other unrelated phages and thus do not evolve by the Modular Theory. Third, there is an unusual core genome that strictly consists of head and baseplate genes; other gene modules are frequently deleted. Fourth, the prophage recombinases are diverse and there is no conserved integration sequence. Finally, the molecular evolutionary forces acting on prophage WO are point mutation, intragenic recombination, deletion, and purifying selection. Taken together, these analyses indicate that while lateral transfer of phage WO is pervasive between Wolbachia with occasional new gene uptake, constraints of the intracellular niche obstruct extensive mixture between WO and the global phage population. Although the Modular Theory has long been considered the paradigm of temperate bacteriophage evolution in free-living bacteria, it appears irrelevant in phages of obligate intracellular bacteria