Biology subjects

Gillespie, J. J.

Publications and source records attributed to Gillespie, J. J..

7 recordsLinked to original sources

bioRxiv2025

Origins of cin: Lateral Gene Transfer of Cytoplasmic Incompatibility Nuclease Operon to Orientia tsutsugamushi

CinB nucleases are Wolbachia proteins that induce cytoplasmic incompatibility (CI) through tandem nuclease domains nuc1 and nuc2 1. CI is a form of reproductive parasitism (RP) whereby males are conditionally sterilized 2. The system behaves as a toxin-antidote (TA) system 1-8 where operon gene cinA encodes an antidote and gene cinB encodes a toxin 1,5. Cin operons are purportedly the cause of gene-drive induced by wolbachiae infecting Drosophila simulans 1,9-12. An unanswered research question is whether lateral transfer of CI operons to bacteria outside wolbachiae would transfer RP phenotype and activate gene-drive. We demonstrate that a cin operon, capable of gene-drive, has jumped into the genome of Orientia tsutsugamushi, a human pathogen and causative agent of lethal scrub typhus. When expressed in transgenic Drosophila melanogaster, the wildtype cinBoTsu was capable of inducing CI independent of its partner antidote cinAoTsu. In addition, cinAoTsu rescued the phenotype in accordance with strict TA functionality. To understand the diverging roles of the tandem nuclease domains we mutated the domains and tested all permutations of active/inactive forms. Finally, we isolated IS5 transposon variants flanking the operon in O. tsutsugamushi and re-activated them to test their mobility. We demonstrate that these transposons can transfer genes and initiate lateral gene transfers into E. coli. These data demonstrate that active bacterial transposons can mobilize and transfer CI factors (cifs) to diverse bacteria. Overall, our data contribute mechanistic understanding in support of the TA model of CI and illuminate biochemical mechanisms that mobilize cifs across genomes from phylogenetically diverse taxa. Significance StatementCI operons are foundational genes that directly contribute to the success of Wolbachia-based bio-control strategies. Two applications of RP-inducing Wolbachia strains are insect population replacement and the incompatible insect technique. Both these techniques do not work if cifs do not function. Thus, a mechanistic understanding of cif function contributes to worldwide bio-control implementations. Furthermore, certain wolbachiae have long been studied as broad-spectrum inducers of diverse RP phenotypes, including CI, parthenogenesis, male-killing, and feminization. How these diverse phenotypes evolve and if they are all induced by cif genes is a long-standing field question. For example, Orientia tsutsugamushi, the causative agent of a deadly scrub typhus, induces parthenogenesis in Leptotrombidium mites, which putatively have reproductive advantages over uninfected mites. How Orientia induces RP is an important epidemiological question in vector biology. In our report, we show that an active cin operon is capable of CI and jumped into Orientia genomes via an IS5 transposon. We reconstructed this transposon and engineered it as a biotechnological tool. In toto, our study leads to the proposal of a new hypothesis whereby CI and parthenogenesis phenotypes might both be connected to the same cif expressed under divergent host genetic contexts.

molecular biology↗

bioRxiv2023

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 (FLATn). FLATn allowed analysis of lipid A structure directly from host cell-purified bacteria, providing substantial improvement over lipid A chemical extraction. FLATn-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 FLATn will facilitate determining how structural heterogeneity impacts interactions with host lipid A receptors and overall inflammatory potential. 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 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 FLATn, 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.

microbiology↗

bioRxiv2023

Orientia tsutsugamushi: analysis of the mobilome of a highly fragmented and repetitive genome reveals ongoing lateral gene transfer in an obligate intracellular bacterium.

The rickettsial human pathogen Orientia tsutsugamushi (Ot) is an obligate intracellular Gram-negative bacterium with one of the most highly fragmented and repetitive genomes of any organism. Around 50% of its [~]2.3 Mb genome is comprised of repetitive DNA that is derived from the highly proliferated Rickettsiales amplified genetic element (RAGE). RAGE is an integrative and conjugative element (ICE) that is present in a single Ot genome in up to 92 copies, most of which are partially or heavily degraded. In this report, we analysed RAGEs in eight fully sequenced Ot genomes and manually curated and reannotated all RAGE-associated genes, including those encoding DNA mobilisation proteins, P-type (vir) and F-type (tra) type IV secretion system (T4SS) components, Ankyrin repeat- and tetratricopeptide repeat-containing effectors, and other piggybacking cargo. Originally, the heavily degraded Ot RAGEs led to speculation that they are remnants of historical ICEs that are no longer active. Our analysis, however, identified two Ot genomes harbouring one or more intact RAGEs with complete F-T4SS genes essential for mediating ICE DNA transfer. As similar ICEs have been identified in unrelated rickettsial species, we assert that RAGEs play an ongoing role in lateral gene transfer within the Rickettsiales. Remarkably, we also identified in several Ot genomes remnants of prophages with no similarity to other rickettsial prophages. Together these findings indicate that, despite their obligate intracellular lifestyle and host range restricted to mites, rodents and humans, Ot genomes are highly dynamic and shaped through ongoing invasions by mobile genetic elements and viruses.

microbiology↗

bioRxiv2023

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.

microbiology↗

bioRxiv2022

Rickettsia typhi peptidoglycan mapping with data-dependent tandem mass spectrometry

Rickettsia species are diverse Gram-negative obligate intracellular bacteria often pervasive in numerous invertebrates, as well as fungal, nematode and microeukaryotic hosts. Certain species are etiological agents for well-known arthropod-borne illnesses; e.g., R. rickettsii (Rocky Mountain Spotted Fever), R. prowazekii (Epidemic Typhus), and R. typhi, (Endemic Typhus). Living freely in eukaryotic cytosol presumably exposes rickettsiae to host cell immune receptors, particularly those recognizing bacterial cell envelope glycoconjugates. However, the mechanics of host recognition of rickettsiae remain poorly defined. As rickettsiae synthesize a canonical Gram-negative cell envelope that includes peptidoglycan (PGN) and lipopolysaccharide (LPS), structural insight on these macromolecules is important for deciphering host responses to these pathogens. In this work, PGN from R. typhi was digested and the resultant subunits were analyzed by two different, albeit complementary, sample preparation methods. Both approaches were subsequently subjected to liquid chromatography/mass spectrometry analysis to infer PGN structure. R. typhi PGN was determined to be similar to most other Gram-negative bacteria, with mDAP-type muropeptide subunits. However, additional alanine residues were observed elongating the muropeptide stems, rather than the glycine residues usually observed in Gram-negative bacterial PGN. Despite this deviation, R. typhi contains a murein layer that is predicted to agonize host cellular PGN receptors and be susceptible to PGN-targeting antimicrobials. This same structure is likely synthesized by all Rickettsia species, as bioinformatics and comparative genomics analyses indicate the biosynthesis of PGN is highly conserved. Determining how host cells process this canonical glycoconjugate during infection is crucial for identifying factors behind rickettsial pathogenesis, including immunoavoidance or proinflammatory mechanisms possibly employed by rickettsiae with varying pathogenic potential.

microbiology↗

bioRxiv2020

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). 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 immediate-5 end genes are syntenic to large modular toxins encoded in CI-like operons of certain Wolbachia strains and Rickettsia species, signifying that CI toxins streamline by fission of larger 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 occasionally wCfeJ singly infects fleas in wild populations. Collectively, genomes of wCfeT, wCfeJ, and their cat flea host supply instances of lateral gene transfers that could drive transitions between parasitism and mutualism. ImportanceMany arthropod and certain nematode species are infected with wolbachiae which are intracellular bacteria well known for reproductive parasitism (RP). Like other RP strategies, Wolbachia-induced cytoplasmic incompatibility, CI, increases prevalence and frequency in host populations. Mutualism is another strategy employed by wolbachiae to maintain host infection, with some strains synthesizing and supplementing certain B vitamins (particularly biotin) to invertebrate hosts. Curiously, we discovered two novel Wolbachia strains that co-infect cat fleas (Ctenocephalides felis): wCfeT carries biotin synthesis genes, while wCfeJ carries a CI-inducing toxin-antidote operon. Our analyses of these genes highlight their mobility across the Wolbachia phylogeny and source to other intracellular bacteria. Remarkably, the C. felis genome also carries two CI-like antidote genes divergent from the wCfeJ antidote gene, indicating episodic RP in cat fleas. Collectively, wCfeT and wCfeJ inform on the rampant dissemination of diverse factors that mediate Wolbachia strategies for persisting in invertebrate host populations.

genomics↗

bioRxiv2020

Cat fleas in flux: Rampant gene duplication, genome size plasticity, and paradoxical Wolbachia infection

BackgroundFleas (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. ResultsBy combining Illumina and PacBio sequencing 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 4,000 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. ConclusionRampant 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.

genomics↗