Conjugative Transposons and Their Cargo Genes Vary across Natural Populations of Rickettsia buchneri Infecting the Tick Ixodes scapularis

Rickettsia buchneri (formerly Rickettsia endosymbiont of Ixodes scapularis, or REIS) is an obligate intracellular endoparasite of the black-legged tick, the primary vector of Lyme disease in North America. It is noteworthy among the rickettsiae for its relatively large genome (1.8 Mb) and extraordinary proliferation of mobile genetic elements (MGEs), which comprise nearly 35% of its genome. Previous analysis of the R. buchneri genome identified several integrative conjugative elements named Rickettsiales amplified genomic elements (RAGEs); the composition of these RAGEs suggests that continued genomic invasions by MGEs facilitated the proliferation of rickettsial genes related to an intracellular lifestyle. In this study, we compare the genomic diversity at RAGE loci among sequenced rickettsiae that infect three related Ixodes spp., including two strains of R. buchneri and Rickettsia endosymbiont of Ixodes pacificus strain Humboldt, as well as a closely related species R. tamurae infecting Amblyomma testudinarium ticks. We further develop a novel multiplex droplet digital PCR assay and use it to quantify copy number ratios of chromosomal R. buchneri RAGE-A and RAGE-B to the single-copy gene gltA within natural populations of I. scapularis. Our results reveal substantial diversity among R. buchneri at these loci, both within individual ticks as well as in the I. scapularis population at large, demonstrating that genomic rearrangement of MGEs is an active process in these intracellular bacteria

A Tangled Web: Origins of Reproductive Parasitism

While typically a flea parasite and opportunistic human pathogen, the presence of Rickettsia felis (strain LSU-Lb) in the non-blood- feeding, parthenogenetically reproducing booklouse, Liposcelis bostrychophila, provides a system to ascertain factors governing not only host transitions but also obligate reproductive parasitism (RP). Analysis of plasmid pLbAR, unique to R. felis str. LSU-Lb, revealed a toxin–antitoxin module with similar features to prophage-encoded toxin–antitoxin modules utilized by parasitic Wolbachia strains to induce another form of RP, cytoplasmic incompatibility, in their arthropod hosts. Curiously, multiple deubiquitinase and nuclease domains of the large (3,841 aa) pLbAR toxin, as well the entire antitoxin, facilitated the detection of an assortment of related proteins from diverse intracellular bacteria, including other reproductive parasites. Our description of these remarkable components of the intracellular mobilome, including their presence in certain arthropod genomes, lends insight on the evolution of RP, while invigo- rating research on parasite-mediated biocontrol of arthropod-borne viral and bacterial pathogens

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

Differential Rickettsial Transcription in Bloodfeeding and Non-Bloodfeeding Arthropod Hosts.

Crucial factors influencing the epidemiology of Rickettsia felis rickettsiosis include pathogenesis and transmission. Detection of R. felis DNA in a number of arthropod species has been reported, with characterized isolates, R. felis strain LSU and strain LSU-Lb, generated from the cat flea, Ctenocephalides felis, and the non-hematophagous booklouse, Liposcelis bostrychophila, respectively. While it is realized that strain influence on host biology varies, the rickettsial response to these distinct host environments remained undefined. To identify a panel of potential rickettsial transmission determinants in the cat flea, the transcriptional profile for these two strains of R. felis were compared in their arthropod hosts using RNAseq. Rickettsial genes with increased transcription in the flea as compared to the booklouse were identified. Genes previously associated with bacterial virulence including LPS biosynthesis, Type IV secretion system, ABC transporters, and a toxin-antitoxin system were selected for further study. Transcription of putative virulence-associated genes was determined in a flea infection bioassay for both strains of R. felis. A host-dependent transcriptional profile during bloodfeeding, specifically, an increased expression of selected transcripts in newly infected cat fleas and flea feces was detected when compared to arthropod cell culture and incubation in vertebrate blood. Together, these studies have identified novel, host-dependent rickettsial factors that likely contribute to successful horizontal transmission by bloodfeeding arthropods

Primers for qPCR validation of RNAseq genes of interest (GOI).

<p>Gene sequences from URRWXCal2 <i>R</i>. <i>felis</i> reference genome (accession CP000053.1, CP000054.1) were used for primer design.</p

qPCR validation of a subset of upregulated genes determined by RNAseq in <i>R</i>. <i>felis</i> str. LSU infecting cat fleas as compared to <i>R</i>. <i>felis</i> str. LSU-Lb constitutively infecting booklice.

<p>Copy number for each transcript is divided by the copy number of the <i>Rf</i>17kDa gene. Data shown are mean ratio of transcript per rickettsia (GOI/<i>Rf</i>17kDa) from two independent experiments. The asterisk denotes a significant difference between the two strains of <i>R</i>. <i>felis</i> for each target gene as determined by t-test with p ≤0.05 considered significant. Error bars represent the standard error of the mean.</p

Expression of selected genes of interest by both <i>R</i>. <i>felis</i> str. LSU and <i>R</i>. <i>felis</i> str. LSU-Lb during the flea infection bioassay.

<p>Cultured <i>R</i>. <i>felis</i> (in ISE6) was added to bovine blood and fleas were exposed for 36 hours. Cultured bacteria, infectious bloodmeal, fleas, and flea feces were then collected for qPCR. Transcript expression of each gene is divided by the number of rickettsiae in each sample (GOI/<i>Rf</i>17kDa) and compared to the ratio of GOI/<i>Rf</i>17kDa in ISE6 cells. The fold-change between treatment(s) and culture for the respective <i>R</i>. <i>felis</i> strains are calculated using two independent experiments. The asterisk denotes significant expression as determined by one way ANOVA followed by a Tukey’s post-hoc test with a p ≤0.05 considered significant. Error bars represent the standard error of the mean.</p

Selected <i>R</i>. <i>felis</i> transcripts with increased expression in cat fleas as compared to booklice chosen for validation by qPCR and transcriptional analysis in the flea infection bioassay.

<p>Fold change is reported as the greatest fold-change identified by RNAseq across three biological replicates.</p

Clusters of Orthologous Groups (COG) containing <i>R</i>. <i>felis</i> genes upregulated in the cat flea as compared to booklice.

<p>COG assignments shown are determined from the URRXWCal2 genome annotation and are represented as the percentage of total differentially upregulated transcripts in fleas.</p

The complete mitochondrial genome of the cat flea,

The cat flea, , is widely recognized as a global veterinary pest and a vector of pathogenic bacteria. We recently reported on the . nuclear genome, which is characterized by over 38% protein coding gene duplication, extensive tRNA gene family expansion, and remarkable gene copy number variation (CNV) between individual fleas. Herein, we describe the assembly of the . mitochondrial genome, a novel resource for comparative genomics of fleas and other insects. The order and content of mitochondrial genes is highly consistent with four previously sequenced flea mitochondrial genomes, limiting CNV to siphonapteran nuclear genomes