Metagenome diversity illuminates the origins of pathogen effectors.

Recent metagenome-assembled genome (MAG) analyses have profoundly impacted Rickettsiology systematics. The discovery of basal lineages (novel families Mitibacteraceae and Athabascaceae) with predicted extracellular lifestyles exposed an evolutionary timepoint for the transition to host dependency, which seemingly occurred independent of mitochondrial evolution. Notably, these basal rickettsiae carry the Rickettsiales vir homolog (rvh) type IV secretion system and purportedly use rvh to kill congener microbes rather than parasitize host cells as described for later-evolving rickettsial pathogens. MAG analysis also substantially increased diversity for the genus Rickettsia and delineated a sister lineage (the novel genus Tisiphia) that stands to inform on the emergence of human pathogens from protist and invertebrate endosymbionts. Herein, we probed Rickettsiales MAG and genomic diversity for the distribution of Rickettsia rvh effectors to ascertain their origins. A sparse distribution of most Rickettsia rvh effectors outside of Rickettsiaceae lineages illuminates unique rvh evolution from basal extracellular species and other rickettsial families. Remarkably, nearly every effector was found in multiple divergent forms with variable architectures, indicating profound roles for gene duplication and recombination in shaping effector repertoires in Rickettsia pathogens. Lateral gene transfer plays a prominent role in shaping the rvh effector landscape, as evinced by the discovery of many effectors on plasmids and conjugative transposons, as well as pervasive effector gene exchange between Rickettsia and Legionella species. Our study exemplifies how MAGs can yield insight into pathogen effector origins, particularly how effector architectures might become tailored to the discrete host cell functions of different eukaryotic hosts.IMPORTANCEWhile rickettsioses are deadly vector-borne human diseases, factors distinguishing Rickettsia pathogens from the innumerable bevy of environmental rickettsial endosymbionts remain lacking. Recent metagenome-assembled genome (MAG) studies revealed evolutionary timepoints for rickettsial transitions to host dependency. The rvh type IV secretion system was likely repurposed from congener killing in basal extracellular species to parasitizing host cells in later-evolving pathogens. Our analysis of MAG diversity for over two dozen rvh effectors unearthed their presence in some non-pathogens. However, most effectors were found in multiple divergent forms with variable architectures, indicating gene duplication and recombination-fashioned effector repertoires of Rickettsia pathogens. Lateral gene transfer substantially shaped pathogen effector arsenals, evinced by the discovery of effectors on plasmids and conjugative transposons, as well as pervasive effector gene exchanges between Rickettsia and Legionella species. Our study exemplifies how MAGs yield insight into pathogen effector origins and evolutionary processes tailoring effectors to eukaryotic host cell biology.

Structural determination of Rickettsia lipid A without chemical extraction confirms shorter acyl chains in later-evolving spotted fever group pathogens.

Rickettsiae are Gram-negative obligate intracellular parasites of numerous eukaryotes. Human pathogens of the transitional group (TRG), typhus group (TG), and spotted fever group (SFG) rickettsiae infect blood-feeding arthropods, have dissimilar clinical manifestations, and possess unique genomic and morphological attributes. Lacking glycolysis, rickettsiae pilfer numerous metabolites from the host cytosol to synthesize peptidoglycan and lipopolysaccharide (LPS). For LPS, O-antigen immunogenicity varies between SFG and TG pathogens; however, lipid A proinflammatory potential is unknown. We previously demonstrated that Rickettsia akari (TRG), Rickettsia typhi (TG), and Rickettsia montanensis (SFG) produce lipid A with long 2' secondary acyl chains (C16 or C18) compared to short 2' secondary acyl chains (C12) in Rickettsia rickettsii (SFG) lipid A. To further probe this structural heterogeneity and estimate a time point when shorter 2' secondary acyl chains originated, we generated lipid A structures for two additional SFG rickettsiae (Rickettsia rhipicephali and Rickettsia parkeri) utilizing fast lipid analysis technique adopted for use with tandem mass spectrometry (FLATn). FLATn allowed analysis of lipid A structure directly from host cell-purified bacteria, providing a substantial improvement over lipid A chemical extraction. FLATn-derived structures indicate SFG rickettsiae diverging after R. rhipicephali evolved shorter 2' secondary acyl chains. While 2' secondary acyl chain lengths do not distinguish Rickettsia pathogens from non-pathogens, in silico analyses of Rickettsia LpxL late acyltransferases revealed discrete active sites and hydrocarbon rulers for long versus short 2' secondary acyl chain addition. Our collective data warrant determining Rickettsia lipid A inflammatory potential and how structural heterogeneity impacts lipid A-host receptor interactions.IMPORTANCEDeforestation, urbanization, and homelessness lead to spikes in Rickettsioses. Vector-borne human pathogens of transitional group (TRG), typhus group (TG), and spotted fever group (SFG) rickettsiae differ by clinical manifestations, immunopathology, genome composition, and morphology. We previously showed that lipid A (or endotoxin), the membrane anchor of Gram-negative bacterial lipopolysaccharide (LPS), structurally differs in Rickettsia rickettsii (later-evolving SFG) relative to Rickettsia montanensis (basal SFG), Rickettsia typhi (TG), and Rickettsia akari (TRG). As lipid A structure influences recognition potential in vertebrate LPS sensors, further assessment of Rickettsia lipid A structural heterogeneity is needed. Here, we sidestepped the difficulty of ex vivo lipid A chemical extraction by utilizing fast lipid analysis technique adopted for use with tandem mass spectrometry, a new procedure for generating lipid A structures directly from host cell-purified bacteria. These data confirm that later-evolving SFG pathogens synthesize structurally distinct lipid A. Our findings impact interpreting immune responses to different Rickettsia pathogens and utilizing lipid A adjuvant or anti-inflammatory properties in vaccinology.

Structural determination of Rickettsia lipid A without chemical extraction confirms shorter acyl chains in later-evolving Spotted Fever Group pathogens.

Rickettsiae are Gram-negative obligate intracellular parasites of numerous eukaryotes. Human pathogens of the Transitional Group (TRG), Typhus Group (TG), and Spotted Fever Group (SFG) rickettsiae infect blood-feeding arthropods, have dissimilar clinical manifestations, and possess unique genomic and morphological attributes. Lacking glycolysis, rickettsiae pilfer numerous metabolites from host cytosol to synthesize peptidoglycan and lipopolysaccharide (LPS). For LPS, O-antigen immunogenicity varies between SFG and TG pathogens; however, lipid A proinflammatory potential is unknown. We previously demonstrated that R. akari (TRG), R. typhi (TG), and R. montanensis (SFG) produce lipid A with long 2' secondary acyl chains (C16 or C18) compared to short 2' secondary acyl chains (C12) in R. rickettsii (SFG) lipid A. To further probe this structural heterogeneity and estimate a time point when shorter 2' secondary acyl chains originated, we generated lipid A structures for two additional SFG rickettsiae ( R. rhipicephali and R. parkeri ) utilizing Fast Lipid Analysis Technique adopted for use with tandem mass spectrometry (FLAT n ). FLAT n allowed analysis of lipid A structure directly from host cell-purified bacteria, providing substantial improvement over lipid A chemical extraction. FLAT n -derived structures indicate SFG rickettsiae diverging after R. rhipicephali evolved shorter 2' secondary acyl chains. Bioinformatics analysis of Rickettsia LpxL late acyltransferases revealed discrete active sites and hydrocarbon rulers for long versus short 2' secondary acyl chain addition. While the significance of different lipid A structures for diverse Rickettsia pathogens is unknown, our success using FLAT n will facilitate determining how structural heterogeneity impacts interactions with host lipid A receptors and overall inflammatory potential. IMPORTANCE: Deforestation, urbanization, and homelessness lead to spikes in Rickettsioses. Vector-borne human pathogens of Transitional Group (TRG), Typhus Group (TG), and Spotted Fever Group (SFG) rickettsiae differ by clinical manifestations, immunopathology, genome composition, and morphology. We previously showed that lipid A (or endotoxin), the membrane anchor of Gram-negative bacterial lipopolysaccharide (LPS), structurally differs in R. rickettsii (later-evolving SFG) relative to R. montanensis (basal SFG), R. typhi (TG), and R. akari (TRG). As lipid A structure influences recognition potential in vertebrate LPS sensors, further assessment of Rickettsia lipid A structural heterogeneity is needed. Here, we sidestepped the difficulty of ex vivo lipid A chemical extraction by utilizing FLAT n , a new procedure for generating lipid A structures directly from host cell-purified bacteria. These data confirm later-evolving SFG pathogens synthesize structurally distinct lipid A. Our findings impact interpreting immune responses to different Rickettsia pathogens and utilizing lipid A adjuvant or anti-inflammatory properties in vaccinology.

Metagenome diversity illuminates origins of pathogen effectors.

Recent metagenome assembled genome (MAG) analyses have profoundly impacted Rickettsiology systematics. Discovery of basal lineages (Mitibacteraceae and Athabascaceae) with predicted extracellular lifestyles reveals an evolutionary timepoint for the transition to host dependency, which occurred independent of mitochondrial evolution. Notably, these basal rickettsiae carry the Rickettsiales vir homolog (rvh) type IV secretion system (T4SS) and purportedly use rvh to kill congener microbes rather than parasitize host cells as described for derived rickettsial pathogens. MAG analysis also substantially increased diversity for genus Rickettsia and delineated a basal lineage (Tisiphia) that stands to inform on the rise of human pathogens from protist and invertebrate endosymbionts. Herein, we probed Rickettsiales MAG and genomic diversity for the distribution of Rickettsia rvh effectors to ascertain their origins. A sparse distribution of most Rickettsia rvh effectors outside of Rickettsiaceae lineages indicates unique rvh evolution from basal extracellular species and other rickettsial families. Remarkably, nearly every effector was found in multiple divergent forms with variable architectures, illuminating profound roles for gene duplication and recombination in shaping effector repertoires in Rickettsia pathogens. Lateral gene transfer plays a prominent role shaping the rvh effector landscape, as evinced by the discover of many effectors on plasmids and conjugative transposons, as well as pervasive effector gene exchange between Rickettsia and Legionella species. Our study exemplifies how MAGs can provide incredible insight on the origins of pathogen effectors and how their architectural modifications become tailored to eukaryotic host cell biology.

Transposon mutagenesis of Rickettsia felis sca1 confers a distinct phenotype during flea infection.

Since its recognition in 1994 as the causative agent of human flea-borne spotted fever, Rickettsia felis, has been detected worldwide in over 40 different arthropod species. The cat flea, Ctenocephalides felis, is a well-described biological vector of R. felis. Unique to insect-borne rickettsiae, R. felis can employ multiple routes of infection including inoculation via salivary secretions and potentially infectious flea feces into the skin of vertebrate hosts. Yet, little is known of the molecular interactions governing flea infection and subsequent transmission of R. felis. While the obligate intracellular nature of rickettsiae has hampered the function of large-scale mutagenesis strategies, studies have shown the efficiency of mariner-based transposon systems in Rickettsiales. Thus, this study aimed to assess R. felis genetic mutants in a flea transmission model to elucidate genes involved in vector infection. A Himar1 transposase was used to generate R. felis transformants, in which subsequent genome sequencing revealed a transposon insertion near the 3' end of sca1. Alterations in sca1 expression resulted in unique infection phenotypes. While the R. felis sca1::tn mutant portrayed enhanced growth kinetics compared to R. felis wild-type during in vitro culture, rickettsial loads were significantly reduced during flea infection. As a consequence of decreased rickettsial loads within infected donor fleas, R. felis sca1::tn exhibited limited transmission potential. Thus, the use of a biologically relevant model provides evidence of a defective phenotype associated with R. felis sca1::tn during flea infection.

Cryptic Genes for Interbacterial Antagonism Distinguish Rickettsia Species Infecting Blacklegged Ticks From Other Rickettsia Pathogens.

Background: The genus Rickettsia (Alphaproteobacteria: Rickettsiales) encompasses numerous obligate intracellular species with predominantly ciliate and arthropod hosts. Notable species are pathogens transmitted to mammals by blood-feeding arthropods. Mammalian pathogenicity evolved from basal, non-pathogenic host-associations; however, some non-pathogens are closely related to pathogens. One such species, Rickettsia buchneri, is prevalent in the blacklegged tick, Ixodes scapularis. While I. scapularis transmits several pathogens to humans, it does not transmit Rickettsia pathogens. We hypothesize that R. buchneri established a mutualism with I. scapularis, blocking tick superinfection with Rickettsia pathogens. Methods: To improve estimates for assessing R. buchneri infection frequency in blacklegged tick populations, we used comparative genomics to identify an R. buchneri gene (REIS_1424) not present in other Rickettsia species present throughout the I. scapularis geographic range. Bioinformatic and phylogenomics approaches were employed to propose a function for the hypothetical protein (263 aa) encoded by REIS_1424. Results: REIS_1424 has few analogs in other Rickettsiales genomes and greatest similarity to non-Proteobacteria proteins. This cohort of proteins varies greatly in size and domain composition, possessing characteristics of Recombination hotspot (Rhs) and contact dependent growth inhibition (CDI) toxins, with similarity limited to proximal C-termini (~145 aa). This domain was named CDI-like/Rhs-like C-terminal toxin (CRCT). As such proteins are often found as toxin-antidote (TA) modules, we interrogated REIS_1423 (151 aa) as a putative antidote. Indeed, REIS_1423 is similar to proteins encoded upstream of CRCT domain-containing proteins. Accordingly, we named these proteins CDI-like/Rhs-like C-terminal toxin antidotes (CRCA). R. buchneri expressed both REIS_1423 and REIS_1424 in tick cell culture, and PCR assays showed specificity for R. buchneri over other rickettsiae and utility for positive detection in three tick populations. Finally, phylogenomics analyses uncovered divergent CRCT/CRCA modules in varying states of conservation; however, only R. buchneri and related Tamurae/Ixodes Group rickettsiae carry complete TA modules. Conclusion: We hypothesize that Rickettsia CRCT/CRCA modules circulate in the Rickettsia mobile gene pool, arming rickettsiae for battle over arthropod colonization. While its functional significance remains to be tested, R. buchneri CRCT/CRCA serves as a marker to positively identify infection and begin deciphering the role this endosymbiont plays in the biology of the blacklegged tick.

Genome sequencing of Pseudomonas aeruginosa strain M2 illuminates traits of an opportunistic pathogen of burn wounds.

Pseudomonas aeruginosa is a Gram-negative nosocomial pathogen and one of the most prevalent organisms isolated from burn wounds worldwide. Pseudomonas aeruginosa strain M2 (O5 serotype, type B flagella) is utilized for examining the murine model associated with burns. Pseudomonas aeruginosa M2 is similar in lethality to common laboratory P. aeruginosa strains when infecting CD-1 mice. Conversely, we recently showed that, relative to these strains, P. aeruginosa M2-infected mice are more susceptible to sepsis and demonstrate a 6-log reduction in LD50 from subcutaneous infection at the infection site directly after 10% total body surface area burn. To better understand this striking phenotypic difference from other P. aeruginosa strains employed in burn models, we sequenced the P. aeruginosa M2 genome. A total of 4,136,641 read pairs were obtained, providing an average genome coverage of 97.5X; subsequent assembly yielded a draft genome with 187 contigs comprising 6,360,304 bp with a G + C content of 66.45%. Genome-based phylogeny estimation of 92 P. aeruginosa strains placed P. aeruginosa M2 with P. aeruginosa-12-4-4(59), a nonairway clinical strain isolated from the blood culture of a burn patient. Phylogenomic analyses identified genes shared between P. aeruginosa M2 and P. aeruginosa 14, another strain exhibiting increased lethality in thermal tissues, as well as P. aeruginosa M2 unique genes with diverse functions like degradation of toxic aromatic compounds, iron scavenging, swarming motility and biofilm formation, defense against invasive DNA, and host assault. Predicted lateral gene transfers illuminate proteins heretofore uncharacterized for roles in P. aeruginosa biology. Our work yields a rich resource for assessing P. aeruginosa genes required for increased lethality in burn tissue seroma.

Lipid A Structural Divergence in Rickettsia Pathogens.

Species of Rickettsia (Alphaproteobacteria: Rickettsiales) are obligate intracellular parasites of a wide range of eukaryotes, with recognized arthropod-borne human pathogens belonging to the transitional group (TRG), typhus group (TG), and spotted fever group (SFG) rickettsiae. Growing in the host cytosol, rickettsiae pilfer numerous metabolites to make a typical Gram-negative bacterial cell envelope. The O-antigen of rickettsial lipopolysaccharide (LPS) is immunogenic and has been shown to tether the S-layer to the rickettsial surface; however, little is known about the structure and immunogenicity of the Rickettsia lipid A moiety. The structure of lipid A, the membrane anchor of LPS, affects the ability of this molecule to interact with components of the host innate immune system, specifically the MD-2/TLR4 receptor complex. To dissect the host responses that can occur during Rickettsia in vitro and in vivo infection, structural analysis of Rickettsia lipid A is needed. Lipid A was extracted from four Rickettsia species and structurally analyzed. R. akari (TRG), R. typhi (TG), and R. montanensis (SFG) produced a similar structure, whereas R. rickettsii (SFG) altered the length of a secondary acyl group. While all structures have longer acyl chains than known highly inflammatory hexa-acylated lipid A structures, the R. rickettsii modification should differentially alter interactions with the hydrophobic internal pocket in MD2. The significance of these characteristics toward inflammatory potential as well as membrane dynamics between arthropod and vertebrate cellular environments warrants further investigation. Our work adds lipid A to the secretome and O-antigen as variable factors possibly correlating with phenotypically diverse rickettsioses.IMPORTANCE Spikes in rickettsioses occur as deforestation, urbanization, and homelessness increase human exposure to blood-feeding arthropods. Still, effective Rickettsia vaccines remain elusive. Recent studies have determined that Rickettsia lipopolysaccharide anchors the protective S-layer to the bacterial surface and elicits bactericidal antibodies. Furthermore, growing immunological evidence suggests vertebrate sensors (MD-2/TLR4 and noncanonical inflammasome) typically triggered by the lipid A portion of lipopolysaccharide are activated during Rickettsia infection. However, the immunopotency of Rickettsia lipid A is unknown due to poor appreciation for its structure. We determined lipid A structures for four distinct rickettsiae, revealing longer acyl chains relative to highly inflammatory bacterial lipid A. Surprisingly, lipid A of the Rocky Mountain spotted fever agent deviates in structure from other rickettsiae. Thus, lipid A divergence may contribute to variable disease phenotypes, sounding an alarm for determining its immunopotency and possible utility (i.e., as an adjuvant or anti-inflammatory) for development of more prudent rickettsiacidal therapies.

Evolution of Wolbachia mutualism and reproductive parasitism: insight from two novel strains that co-infect cat fleas.

Wolbachiae are obligate intracellular bacteria that infect arthropods and certain nematodes. Usually maternally inherited, they may provision nutrients to (mutualism) or alter sexual biology of (reproductive parasitism) their invertebrate hosts. We report the assembly of closed genomes for two novel wolbachiae, wCfeT and wCfeJ, found co-infecting cat fleas (Ctenocephalides felis) of the Elward Laboratory colony (Soquel, CA, USA). wCfeT is basal to nearly all described Wolbachia supergroups, while wCfeJ is related to supergroups C, D and F. Both genomes contain laterally transferred genes that inform on the evolution of Wolbachia host associations. wCfeT carries the Biotin synthesis Operon of Obligate intracellular Microbes (BOOM); our analyses reveal five independent acquisitions of BOOM across the Wolbachia tree, indicating parallel evolution towards mutualism. Alternately, wCfeJ harbors a toxin-antidote operon analogous to the wPip cinAB operon recently characterized as an inducer of cytoplasmic incompatibility (CI) in flies. wCfeJ cinB and three adjacent genes are collectively similar to large modular toxins encoded in CI-like operons of certain Wolbachia strains and Rickettsia species, signifying that CI toxins streamline by fission of large modular toxins. Remarkably, the C. felis genome itself contains two CI-like antidote genes, divergent from wCfeJ cinA, revealing episodic reproductive parasitism in cat fleas and evidencing mobility of CI loci independent of WO-phage. Additional screening revealed predominant co-infection (wCfeT/wCfeJ) amongst C. felis colonies, though fleas in wild populations mostly harbor wCfeT alone. Collectively, genomes of wCfeT, wCfeJ, and their cat flea host supply instances of lateral gene transfers that could drive transitions between parasitism and mutualism.

A chromosome-level assembly of the cat flea genome uncovers rampant gene duplication and genome size plasticity.

BACKGROUND: Fleas (Insecta: Siphonaptera) are small flightless parasites of birds and mammals; their blood-feeding can transmit many serious pathogens (i.e., the etiological agents of bubonic plague, endemic and murine typhus). The lack of flea genome assemblies has hindered research, especially comparisons to other disease vectors. Accordingly, we sequenced the genome of the cat flea, Ctenocephalides felis, an insect with substantial human health and veterinary importance across the globe. RESULTS: By combining Illumina and PacBio sequencing of DNA derived from multiple inbred female fleas with Hi-C scaffolding techniques, we generated a chromosome-level genome assembly for C. felis. Unexpectedly, our assembly revealed extensive gene duplication across the entire genome, exemplified by ~ 38% of protein-coding genes with two or more copies and over 4000 tRNA genes. A broad range of genome size determinations (433-551 Mb) for individual fleas sampled across different populations supports the widespread presence of fluctuating copy number variation (CNV) in C. felis. Similarly, broad genome sizes were also calculated for individuals of Xenopsylla cheopis (Oriental rat flea), indicating that this remarkable "genome-in-flux" phenomenon could be a siphonapteran-wide trait. Finally, from the C. felis sequence reads, we also generated closed genomes for two novel strains of Wolbachia, one parasitic and one symbiotic, found to co-infect individual fleas. CONCLUSION: Rampant CNV in C. felis has dire implications for gene-targeting pest control measures and stands to complicate standard normalization procedures utilized in comparative transcriptomics analysis. Coupled with co-infection by novel Wolbachia endosymbionts-potential tools for blocking pathogen transmission-these oddities highlight a unique and underappreciated disease vector.

The complete mitochondrial genome of the cat flea, Ctenocephalides felis.

The cat flea, Ctenocephalides fells, is widely recognized as a global veterinary pest and a vector of pathogenic bacteria. We recently reported on the C. felis 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 C. felis 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.

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.

Molecular Confirmation of Ascaris suum: Further Investigation into the Zoonotic Origin of Infection in an 8-Year-Old Boy with Loeffler Syndrome.

An 8-year-old male from south Louisiana was diagnosed with Loeffler syndrome of suspected Ascaris origin. Further investigation of the farm recovered larvated, infective Ascaris eggs from the soil in drains surrounding pens on the family's small hog farm. Molecular analysis of the recovered eggs, in conjunction with Ascaris-specific IgE, inadequate farm management and sanitation, and behavioral risk factors indicate the patient had an Ascaris suum soil-transmitted infection.

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 invigorating research on parasite-mediated biocontrol of arthropod-borne viral and bacterial pathogens.

Role of Sca2 and RickA in the Dissemination of Rickettsia parkeri in Amblyomma maculatum.

The Gram-negative obligate intracellular bacterium Rickettsia parkeri is an emerging tick-borne human pathogen. Recently, R. parkeri Sca2 and RickA have been implicated in adherence and actin-based motility in vertebrate host cell infection models; however, the rickettsia-derived factors essential to tick infection are unknown. Using R. parkeri mutants lacking functional Sca2 or RickA to compare actin polymerization, replication, and cell-to-cell spread in vitro, similar phenotypes in tick and mammalian cells were observed. Specifically, actin polymerization in cultured tick cells is controlled by the two separate proteins in a time-dependent manner. To assess the role of Sca2 and RickA in dissemination in the tick host, Rickettsia-free Amblyomma maculatum, the natural vector of R. parkeri, was exposed to wild-type, R. parkeri rickA::tn, or R. parkeri sca2::tn bacteria, and individual tick tissues, including salivary glands, midguts, ovaries, and hemolymph, were analyzed at 12 h and after continued bloodmeal acquisition for 3 or 7 days postexposure. Initially, ticks exposed to wild-type R. parkeri had the highest rickettsial load across all organs; however, rickettsial loads decreased and wild-type rickettsiae were cleared from the ovaries at 7 days postexposure. In contrast, ticks exposed to R. parkeririckA::tn or R. parkerisca2::tn had comparatively lower rickettsial loads, but bacteria persisted in all organs for 7 days. These data suggest that while RickA and Sca2 function in actin polymerization in tick cells, the absence of these proteins did not change dissemination patterns within the tick vector.

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 glycoconjugates are synthesized from three imported host sugars, with a range of additional host-acquired metabolites fueling the tricarboxylic acid cycle. Fatty acid and glycerophospholipid pathways also initiate from host precursors, and import of both isoprenes and terpenoids is required for the synthesis of ubiquinone and the lipid carrier of lipid I and O-antigen. Unlike metabolite-provisioning bacterial symbionts of arthropods, rickettsiae cannot synthesize B vitamins or most other cofactors, accentuating their parasitic nature. Six biosynthesis pathways contain holes (missing enzymes); similar patterns in taxonomically diverse bacteria suggest alternative enzymes 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 metabolic pathways. This blueprint for growth determinants is an important step toward the design of axenic media to rescue rickettsiae from the eukaryotic cell.IMPORTANCE A hallmark of obligate intracellular bacteria is the tradeoff of metabolic genes for the ability to acquire host metabolites. For species of Rickettsia, arthropod-borne parasites with the potential to cause serious human disease, the range of pilfered host metabolites is unknown. This information is critical for dissociating rickettsiae from eukaryotic cells to facilitate rickettsial genetic manipulation. In this study, we reconstructed the Rickettsia metabolic network and identified 51 host metabolites required to compensate patchwork Rickettsia biosynthesis pathways. Remarkably, some metabolites are not known to be transported by any bacteria, and overall, few cognate transporters were identified. Several pathways contain missing enzymes, yet similar pathways in unrelated bacteria indicate convergence and possible novel enzymes awaiting characterization. Our work illuminates the parasitic nature by which rickettsiae hijack host metabolism to counterbalance numerous disintegrated biosynthesis pathways that have arisen through evolution within the eukaryotic cell. This metabolic blueprint reveals what a Rickettsia axenic medium might entail.

Comparative vertical transmission of Rickettsia by Dermacentor variabilis and Amblyomma maculatum.

The geographical overlap of multiple Rickettsia and tick species coincides with the molecular detection of a variety of rickettsial agents in what may be novel tick hosts. However, little is known concerning transmissibility of rickettsial species by various tick hosts. To examine the vertical transmission potential between select tick and rickettsial species, two sympatric species of ticks, Dermacentor variabilis and Amblyomma maculatum, were exposed to five different rickettsial species, including Rickettsia rickettsii, Rickettsia parkeri, Rickettsia montanensis, Rickettsia amblyommatis, or flea-borne Rickettsia felis. Fitness-related metrics including engorgement weight, egg production index, nutrient index, and egg hatch percentage were then assessed. Subsamples of egg clutches and unfed larvae, nymphs, and adults for each cohort were assessed for transovarial and transstadial transmission of rickettsiae by qPCR. Rickettsial exposure had a minimal fitness effect in D. variabilis and transovarial transmission was observed for all groups except R. rickettsii. In contrast, rickettsial exposure negatively influenced A. maculatum fitness and transovarial transmission of rickettsiae was demonstrated only for R. amblyommatis- and R. parkeri-exposed ticks. Sustained maintenance of rickettsiae via transstadial transmission was diminished from F1 larvae to F1 adults in both tick species. The findings of this study suggest transovarial transmission specificity may not be tick species dependent, and sustained vertical transmission is not common.

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.

Identification of host proteins involved in rickettsial invasion of tick cells.

Tick-borne spotted fever group (SFG) Rickettsia species are obligate intracellular bacteria capable of infecting both vertebrate and invertebrate host cells, an essential process for subsequent bacterial survival in distinct hosts. The host cell signaling molecules involved in the uptake of Rickettsia into mammalian and Drosophila cells have been identified; however, invasion into tick cells is understudied. Considering the movement of SFG Rickettsia between vertebrate and invertebrate hosts, the hypothesis is that conserved mechanisms are utilized for host cell invasion. The current study employed biochemical inhibition assays to determine the tick proteins involved in Rickettsia montanensis infection of tick-derived cells from a natural host, Dermacentor variabilis. The results revealed several tick proteins important for rickettsial invasion, including actin filaments, actin-related protein 2/3 complex, phosphatidylinositol-3'-kinase, protein tyrosine kinases (PTKs), Src family PTK, focal adhesion kinase, Rho GTPase Rac1, and neural Wiskott-Aldrich syndrome protein. Delineating the molecular mechanisms of rickettsial infection is critical to a thorough understanding of rickettsial transmission in tick populations and the ecology of tick-borne rickettsial diseases.