Joseph J. Gillespie

• Doctor of Philosophy
• University of Maryland, Baltimore

CinB nucleases are Wolbachia proteins that induce cytoplasmic incompatibility (CI) through tandem nuclease domains nuc 1 and nuc 2 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 t...

Pathogenic Bacillus and clostridial (i.e., Clostridium and Clostridioides) bacteria express a diverse repertoire of effector proteins to promote disease. This includes production of binary toxins, which enter host epithelial cells and seriously damage the intestinal tracts of insects, animals, and humans. In particular, binary toxins form an AB-typ...

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 mitochondri...

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, ri...

Pseudomonas aeruginosa is an opportunistic pathogen that causes severe infection in hospitalized and chronically ill individuals. During infection, P. aeruginosa undergoes adaptive changes to evade host defenses and therapeutic interventions, increasing mortality and morbidity. Lipid A structural alteration is one such change that P. aeruginosa iso...

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 composed of repetitive DNA that is derived from the highly proliferated Rickettsiales amplified genetic element (RAGE). RAG...

Unlabelled: 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 g...

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). RA...

Recent discoveries of basal extracellular Rickettsiales have illuminated divergent evolutionary paths to host dependency in later-evolving lineages. Family Rickettsiaceae, primarily comprised of numerous protist- and invertebrate-associated species, also includes human pathogens from two genera, Orientia and Rickettsia. Once considered sister taxa,...

Evolutionary algorithms (EAs) simulate Darwinian evolution and adeptly mimic natural evolution. Most EA applications in biology encode high levels of abstraction in top-down ecological population models. In contrast, our research merges protein alignment algorithms from bioinformatics into codon based EAs that simulate molecular protein string evol...

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...

Phylogeny estimation of newly discovered aquatic copiotrophic Rickettsiales indicates evolution of host dependency from extracellular species and the origin of mitochondria from an unknown proteobacterial lineage.

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-pat...

Pseudomonas aeruginosa (PA) is a Gram-negative nosocomial pathogen and one of the most prevalent organisms isolated from burn wounds worldwide. PA strain M2 (O5 serotype, type B flagella) is utilized for examining the murine model associated with burns. PA M2 is similar in lethality to common laboratory PA strains when infecting CD-1 mice. Converse...

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. ty...

Background 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-patho...

Peptidoglycan (PG) is a highly cross-linked peptide-glycan mesh that confers structural rigidity and shape to most bacterial cells. Polymerization of new PG is usually achieved by the concerted activity of two membrane-bound machineries, class-A penicillin binding proteins (aPBPs) and class-B penicillin binding proteins (bPBPs) in complex with shap...

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 t...

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, w CfeT and w CfeJ, found co-infecting...

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 f...

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, w...

Rickettsia species are Gram-negative obligate intracellular bacteria that infect a wide range of eukaryotes and vertebrates. In particular, human body louse-borne Rickettsia prowazekii and flea-borne Rickettsia typhi have historically plagued humankind and continue to reemerge globally. The unavailability of vaccines and limited effectiveness of an...

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, w CfeT and w CfeJ, found co-infecting...

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 s...

Rickettsia buchneri (formerly Rickettsia endosymbiont of Ixodes scapularis, or REIS) is an obligate intracellular endosymbiont 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...

Lipopolysaccharide (LPS) triggers an inflammatory response through the TLR4-MD2 receptor complex and inflammatory caspases, a process mediated by the lipid A moiety of LPS. Species of Rickettsia directly engage both extracellular and intracellular immunosurveillance, yet little is known about rickettsial lipid A. Here, we demonstrate that the alter...

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 plas...

Strains of Rickettsia rickettsii, the tick-borne agent of Rocky Mountain spotted fever, vary considerably in virulence. Genomic comparisons of R. rickettsii strains have identified a relatively small number of genes divergent in an avirulent strain. Among these is one annotated as Rickettsia ankyrin repeat protein 2 (RARP-2). Homologs of RARP-2 are...

Rickettsia species are obligate intracellular bacteria with both conserved and lineage-specific strategies for invading and surviving within eukaryotic cells. One variable component ofRickettsiabiology involves arthropod vectors: for instance, typhus group rickettsiae are principally vectored by insects (i.e., lice and fleas), whereas spotted fever...

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 thwarte...

Rickettsiae are obligate intracellular pathogens that induce their uptake into non-phagocytic cells; however, the events instigating this process are incompletely understood. Importantly, diverse Rickettsia species are predicted to utilize divergent mechanisms to colonize host cells, as nearly all adhesins and effectors involved in host cell entry...

Many prokaryotes utilize type IV secretion systems (T4SSs) to translocate substrates (e.g., nucleoprotein, DNA, protein) across the cell envelope, and/or to elaborate surface structures (i.e., pili or adhesins). Among eight distinct T4SS classes, P-T4SSs are typified by the Agrobacterium tumefaciens vir T4SS, which is comprised of 12 scaffold compo...

Ticks transmit more pathogens to humans and animals than any other arthropod. We describe the 2.1 Gbp nuclear genome of the tick, Ixodes scapularis (Say), which vectors pathogens that cause Lyme disease, human granulocytic anaplasmosis, babesiosis and other diseases. The large genome reflects accumulation of repetitive DNA, new lineages of retro-tr...

Ticks transmit more pathogens to humans and animals than any other arthropod. We describe the 2.1 Gbp nuclear genome of the tick, Ixodes scapularis (Say), which vectors pathogens that cause Lyme disease, human granulocytic anaplasmosis, babesiosis and other diseases. The large genome reflects accumulation of repetitive DNA, new lineages of retro-tr...

Supplementary Figures 1-25, Supplementary Tables 1-38, Supplementary Note 1, Supplementary Methods and Supplementary References

Prokaryotes use type IV secretion systems (T4SSs) to translocate substrates (e.g., nucleoprotein, DNA, and protein) and/or elaborate surface structures (i.e., pili or adhesins). Bacterial genomes may encode multiple T4SSs, e.g., there are three functionally divergent T4SSs in some Bartonella species ( vir , vbh , and trw ). In a unique case, most r...

Prokaryotes use type IV secretion systems (T4SSs) to translocate substrates (e.g., nucleoprotein, DNA, and protein) and/or elaborate surface structures (i.e., pili or adhesins). Bacterial genomes may encode multiple T4SSs, e.g., there are three functionally divergent T4SSs in some Bartonella species (vir, vbh, and trw). In a unique case, most ricke...

Bacterial Sec7-domain-containing proteins (RalF) are known only from species of Legionella and Rickettsia, which have facultative and obligate intracellular lifestyles, respectively. L. pneumophila RalF, a type IV secretion system (T4SS) effector, is a guanine nucleotide exchange factor (GEF) of ADP-ribosylation factors (Arfs), activating and recru...

Bacteria of the order Rickettsiales (Alphaproteobacteria) are gram-negative, small, rod-shaped, and coccoid, with all described species existing as obligate intracellular parasites of a wide range of eukaryotic organisms (Gillespie et al. 2012b). Before the DNA revolution, bacteria were assigned to Rickettsiales based primarily on chemical composit...

Amblyomma americanum (Lone star tick) is an important disease vector in the United States. It transmits several human pathogens, including the agents of human monocytic ehrlichiosis, tularemia and Southern Tick-Associated Rash Illness. Blood-feeding insects (Class Insecta) depend on bacterial endosymbionts to provide vitamins and cofactors that are...

Rickettsia felis (Alphaproteobacteria: Rickettsiales) is the causative agent of an emerging flea-borne rickettsiosis with worldwide occurrence. Originally described from the cat flea, Ctenocephalides felis, recent reports have identified R. felis from other flea species, as well as other insects and ticks. This diverse host range for R. felis may i...

The genus Rickettsia (Alphaproteobacteria; Rickettsiales; Rickettsiaceae) is comprised of obligate intracellular parasites, with virulent species of interest both as causes of emerging infectious diseases and for their potential deployment as bioterrorism agents. Currently there are no effective commercially available vaccines, with treatment limit...

Background Bacteria of the order Rickettsiales (Alphaproteobacteria) are obligate intracellular parasites that infect species from virtually every major eukaryotic lineage. Several rickettsial genera harbor species that are significant emerging and re-emerging pathogens of humans. As species of Rickettsiales are associated with an extremely diverse...

The Pathosystems Resource Integration Center (PATRIC) is the all-bacterial Bioinformatics Resource Center (BRC) (http://www.patricbrc.org). A joint effort by two of the original National Institute of Allergy and Infectious Diseases-funded BRCs, PATRIC provides researchers with an online resource that stores and integrates a variety of data types [e...

Protein regulation by ubiquitin has been extensively described in model organisms. However, characterization of the ubiquitin machinery in disease vectors remains mostly unknown. This fundamental gap in knowledge presents a concern because new therapeutics are needed to control vector-borne diseases, and targeting the ubiquitin machinery as a means...

Phylogeny estimation of Rickettsia Pat1 and Pat1-like patatin phospholipases (cd07199). The conserved domain cd07199 includes PNPLA8, PNPLA9, and Pat17 patatin-like phospholipases. See text for alignment and tree-building methods. Tree is final optimization likelihood: (−61591.367104) using WAG substitution model with GAMMA and proportion of invari...

NCBI and PATRIC accession numbers for Rickettsia Pat1 and Pat2 sequences. (PDF)

Purified recombinant proteins for phospholipase A2 assay. Imperial Protein Stained (Pierce) 4 to 12% Tris-glycine precast gel (Invitrogen) using 1×Tris-glycine-SDS running buffer (BioRad). Purified recombinant proteins (including C-terminal myc epitope and 6×His tag) expressed in E. coli TOP10 cells shown: Lane 1, Pat2 (70.2 kD from pTrc-522HS); La...

The long-standing proposal that phospholipase A2 (PLA2) enzymes are involved in rickettsial infection of host cells has been given support by the recent characterization of a patatin phospholipase (Pat2) with PLA2 activity from the pathogens Rickettsia prowazekii and R. typhi. However, pat2 is not encoded in all Rickettsia genomes; yet another unch...

Eukaryotic genome sequencing projects often yield bacterial DNA sequences, data typically considered as microbial contamination. However, these sequences may also indicate either symbiont genes or lateral gene transfer (LGT) to host genomes. These bacterial sequences can provide clues about eukaryote-microbe interactions. Here, we used the genome o...

Background Transmission of arthropod-borne apicomplexan parasites that cause disease and result in death or persistent infection represents a major challenge to global human and animal health. First described in 1901 as Piroplasma equi, this re-emergent apicomplexan parasite was renamed Babesia equi and subsequently Theileria equi, reflecting an un...

Figure. Depiction of the T. equi apicoplast genome gene arrangement, showing unidirectional coding of genes. Known enzymes shown in red, ribosomal proteins in green, rRNA sequences in yellow, and groupings of tRNA molecules in blue. Location of conserved hypothetical (gray), and hypothetical (black) protein-encoding genes are shown by arrows or bar...

Table. 30 largest protein families.

Table. Proteins used in phylogenetic analysis.

Table. GPI anchored proteins predicted by GPI-SOM.

Table. Nuclear encoded genes potentially targeted to the apicoplast.

Figure. Alignment of EMA family sequences. Residues highlighted in yellow are conserved among all family members, and those in blue conserved among the majority of family members. Dashes represent gaps introduced to accommodate non-conserved stretches of sequence.

Table. Transporter comparison B.bovis, T. equi, and T.parva.

Figure. Phylogenetic trees. A: Most frequently recovered trees from maximum parsimony analysis of 150 polypeptides conserved among eight taxa, showing the number of times tree recovered out of 210 total topologies. Value above the line represents the number times that branch was recovered out of total times whole topology was recovered , and value...

Rickettsia typhi, the causative agent of murine (endemic) typhus, is an obligate intracellular pathogen with a life cycle involving both vertebrate and invertebrate hosts. In this study, we characterized a gene (RT0218) encoding a C-terminal ankyrin repeat domain-containing protein, named Rickettsia ankyrin repeat protein 1 (RARP-1), and identified...

Surface proteins of the obligate intracellular bacterium Rickettsia typhi, the agent of murine or endemic typhus fever, comprise an important interface for host-pathogen interactions including adherence, invasion and survival in the host cytoplasm. In this report, we present analyses of the surface exposed proteins of R. typhi based on a suite of p...

Immunofluorescence assays of Sca expression in uninfected C. felis. Cat fleas were housed in an artificial feeding unit and fed uninfected whole sheep's blood for 14 days. Capsules were placed at −20°C to immobilize fleas before placing them in 3% PFA overnight. Fleas were embedded in OCT medium, frozen and cryosectioned. Sections were labeled with...

Immunofluorescence assays of Sca expression in uninfected rats. Spleens were harvested from 9 day uninfected female Sprague-Dawley rats and fixed and embedded as described. Sections were labeled with anti-R. typhi rat immune serum (Alexa532-conjugated - red), anti-serum to the Sca protein indicated on the left (Alexa350-conjugated - blue) and stain...

Proteins predicted to be secreted by only the SecretomeP method. Predictions for proteins secreted by non-classical methods were made using the SecretomeP 2.0 server. (XLS)

Surface-exposed proteins of R. typhi str. Wilmington identified by LC/MS/MS. Proteins were run on SDS-PAGE gels and analyzed as described in the methods and section and Text S1. The presence of signal peptides and predicted subcellular localizations are included. (XLS)

Prediction of co-transcription of sca genes. The OperonDB algorithm (http://operondb.cbcb.umd.edu/cgi-bin/operondb/operons.cgi) was utilized to determine the probability of each sca gene being transcribed with genes immediately upstream or downstream from them. (DOC)

Supporting information – methods and references. Details on the processing of Coomassie-stained protein bands for LC-MS/MS analysis and the software used for protein identifications are outlined in file Text S1. References for supporting figures are also included in this Supporting Information file. (DOC)

Immunoblotting of Sca proteins in uninfected and infected L929 cell fractions. L929 cells were infected with R. typhi for 4 days as described or left uninfected. Cells were washed and harvested for partial purification of rickettsiae and the resulting fractions separated by SDS-PAGE and processed for immunoblotting. Antiserum to each Sca (indicated...

Immunogold post-embedding electron microscopy of Sca expression in uninfected L929 cells. Bar = 0.25 um. Uninfected L929 cells were left uninfected and processed for immunogold electron microscopy as described in the methods. Sections were labeled with antibodies against the Sca protein indicated in the black box within each image. (EPS)

Multiple sequence alignment of rickettsial surface cell antigens (Scas). For each analysis, sequences were aligned using MUSCLE v3.6 [1], [2] (default parameters). Alignments were not manually adjusted. Sca1-5 alignments are noted above each alignment. All R. typhi sequences are bolded. Each alignment contains two rows above the sequences. Top row,...

We present the draft genome for the Rickettsia endosymbiont of Ixodes scapularis (REIS), a symbiont of the deer tick vector of Lyme disease in North America. Among Rickettsia species (Alphaproteobacteria: Rickettsiales), REIS has the largest genome sequenced to date (>2 Mb) and contains 2,309 genes across the chromosome and four plasmids (pREIS1 to...

Funded by the National Institute of Allergy and Infectious Diseases, the Pathosystems Resource Integration Center (PATRIC) is a genomics-centric relational database and bioinformatics resource designed to assist scientists in infectious-disease research. Specifically, PATRIC provides scientists with (i) a comprehensive bacterial genomics database,...

Research on specialized biological systems is often hampered by a lack of consistent terminology, especially across species. In bacterial Type IV secretion systems genes within one set of orthologs may have over a dozen different names. Classifying research publications based on biological processes, cellular components, molecular functions, and mi...

With an obligate intracellular lifestyle, Alphaproteobacteria of the order Rickettsiales have inextricably coevolved with their various eukaryotic hosts, resulting in small, reductive genomes and strict dependency on host resources. Unsurprisingly, large portions of Rickettsiales genomes encode proteins involved in transport and secretion. One part...

Anaplasma and related Ehrlichia spp. are important tick-borne, Gram-negative bacterial pathogens of livestock and humans that cause acute infection and disease and can persist. Immunization of cattle with an Anaplasma marginale fraction enriched in outer membranes (OM) can provide complete protection against disease and persistent infection. Serolo...