Aglow: A Fluorescence Assay and Machine Learning Model to Identify Inhibitors of Intracellular Infection.

is a genus of Gram-negative bacteria that has for centuries caused large-scale morbidity and mortality. In recent years, the resurgence of rickettsial diseases as a major cause of pyrexias of unknown origin, bioterrorism concerns, vector movement, and concerns over drug resistance is driving a need to identify novel treatments for these obligate intracellular bacteria. Utilizing an uvGFP plasmid reporter, we developed a screen for identifying anti-rickettsial small molecule inhibitors using as a model organism.

Engineering of obligate intracellular bacteria: progress, challenges and paradigms.

It is estimated that approximately one billion people are at risk of infection with obligate intracellular bacteria, but little is known about the underlying mechanisms that govern their life cycles. The difficulty in studying Chlamydia spp., Coxiella spp.

Fluorescence Activated Cell Sorting of Rickettsia prowazekii-Infected Host Cells Based on Bacterial Burden and Early Detection of Fluorescent Rickettsial Transformants.

Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligate intracellular bacterium that replicates only within the cytosol of a eukaryotic host cell. Despite the barriers to genetic manipulation that such a life style creates, rickettsial mutants have been generated by transposon insertion as well as by homologous recombination mechanisms. However, progress is hampered by the length of time required to identify and isolate R.

Genetic systems for studying obligate intracellular pathogens: an update.

Rapid advancements in the genetic manipulation of obligate intracellular bacterial pathogens have been made over the past two years. In this paper we attempt to summarize the work published since 2011 that documents these exciting accomplishments. Although each genus comprising this diverse group of pathogens poses unique problems, requiring modifications of established techniques and the introduction of new tools, all appear amenable to genetic analysis.

The capping domain in RalF regulates effector functions.

The Legionella pneumophila effector protein RalF functions as a guanine nucleotide exchange factor (GEF) that activates the host small GTPase protein ADP-ribosylation factor (Arf), and recruits this host protein to the vacuoles in which this pathogen resides. GEF activity is conferred by the Sec7 domain located in the N-terminal region of RalF. Structural studies indicate that the C-terminal region of RalF makes contacts with residues in the Sec7 domain important for Arf interactions.

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.

Transformation frequency of a mariner-based transposon in Rickettsia rickettsii.

Transformation frequencies of a mariner-based transposon system in Rickettsia rickettsii were determined using a plaque assay system for enumeration and isolation of mutants. Sequence analysis of insertion sites in both R. rickettsii and R.

Differential proteomic analysis of Rickettsia prowazekii propagated in diverse host backgrounds.

The obligate intracellular growth of Rickettsia prowazekii places severe restrictions on the analysis of rickettsial gene expression. With a small genome, predicted to code for 835 proteins, identifying which proteins are differentially expressed in rickettsiae that are isolated from different hosts or that vary in virulence is critical to an understanding of rickettsial pathogenicity. We employed a liquid chromatography (LC)-linear trap quadrupole (LTQ)-Orbitrap mass spectrometer for simultaneous acquisition of quantitative mass spectrometry (MS)-only data and tandem mass spectrometry (MS-MS) sequence data.

Analysis of convergent gene transcripts in the obligate intracellular bacterium Rickettsia prowazekii.

Termination of transcription is an important component of bacterial gene expression. However, little is known concerning this process in the obligate intracellular pathogen and model for reductive evolution, Rickettsia prowazekii. To assess transcriptional termination in this bacterium, transcripts of convergent gene pairs, some containing predicted intrinsic terminators, were analyzed.

Directed mutagenesis of the Rickettsia prowazekii pld gene encoding phospholipase D.

Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligately intracytoplasmic bacterium, a lifestyle that imposes significant barriers to genetic manipulation. The key to understanding how this unique bacterium evades host immunity is the mutagenesis of selected genes hypothesized to be involved in virulence. The R.

Mariner-based transposon mutagenesis of Rickettsia prowazekii.

Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligate intracellular bacterium that grows directly within the cytoplasm of its host cell, unbounded by a vacuolar membrane. The obligate intracytoplasmic nature of rickettsial growth places severe restrictions on the genetic analysis of this distinctive human pathogen. In order to expand the repertoire of genetic tools available for the study of this pathogen, we have employed the versatile mariner-based, Himar1 transposon system to generate insertional mutants of R.

Dissecting the Rickettsia prowazekii genome: genetic and proteomic approaches.

The obligate nature of Rickettsia prowazekii intracellular growth places severe restrictions on the analysis of rickettsial gene function and gene expression. Fortunately, this situation is improving as methods for the genetic manipulation and proteomic analysis of this fascinating human pathogen become available. In this paper, we review the current status of rickettsial genetics and the isolation of rickettsial mutants using a genetic approach.

Electron injection from the side of an M-DNA duplex.

M-DNA is a complex formed between duplex DNA and divalent metal ions (Zn2+, Cu2+ or Ni2+) at pHs above 8. Previous results showed that the fluorescence of an electron donor fluorophore was quenched when an acceptor flourophore was placed in the opposite end of an M-DNA duplex suggesting electron transfer through the duplex and indicating M-DNA may operate as a better conductor than B-DNA. To further investigate the properties of M-DNA, oligodeoxynucleotides were prepared with fluorescein (Fl) as an electron donor placed at different positions along the helix.

M-DNA: A novel metal ion complex of DNA studied by fluorescence techniques.

M-DNA, a complex formed in solution between divalent metal ions (M) and duplex DNA, has been studied extensively using fluorescence quenching. This review examines the methods used to examine the formation of M-DNA, and its ability to serve as a pathway for electron transfer between donor and acceptor chromaphores. A mass action model for M-DNA formation is presented based upon the results of fluorescence quenching studies using fluorescein/QSY-7 labeled duplexes.

Rickettsial metK-encoded methionine adenosyltransferase expression in an Escherichia coli metK deletion strain.

The obligate intracellular bacterium Rickettsia prowazekii has recently been shown to transport the essential metabolite S-adenosylmethionine (SAM). The existence of such a transporter would suggest that the metK gene, coding for the enzyme that synthesizes SAM, is unnecessary for rickettsial growth. Genome sequencing has revealed that this is the case for the metK genes of the spotted fever group and the Madrid E strain of R.

Investigation of pH-dependent DNA-metal ion interactions by surface plasmon resonance.

Ni(II) and Zn(II) M-DNA formation and denaturation of double-stranded DNA (dsDNA) by Cd(2+) were monitored by surface plasmon resonance (SPR). When exposed to immobilized 30 bp 50% GC dsDNA, Zn(2+) and Ni(2+) were found to give signals indicative of a conformational change at pH 8.5 but not 7.

Transposon mutagenesis of the obligate intracellular pathogen Rickettsia prowazekii.

Genetic analysis of Rickettsia prowazekii has been hindered by the lack of selectable markers and efficient mechanisms for generating rickettsial gene knockouts. We have addressed these problems by adapting a gene that codes for rifampin resistance for expression in R. prowazekii and by incorporating this selection into a transposon mutagenesis system suitable for generating rickettsial gene knockouts.

S-adenosylmethionine transport in Rickettsia prowazekii.

Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligate, intracellular, parasitic bacterium that grows within the cytoplasm of eucaryotic host cells. Rickettsiae exploit this intracellular environment by using transport systems for the compounds available in the host cell's cytoplasm. Analysis of the R.

Thermodynamic investigation of M-DNA: a novel metal ion-DNA complex.

The thermodynamics of formation of a novel divalent metal ion-DNA complex known as M-DNA have been investigated using an ethidium bromide (EB) fluorescence assay, and with isothermal titration calorimetry. The process of M-DNA formation was observed from the EB assay to be strongly temperature-dependent. The binding of Zn(2+) to calf thymus (42% GC content) and Escherichia coli (50% GC content) DNA at pH 8.

M-DNA is stabilised in G*C tracts or by incorporation of 5-fluorouracil.

M-DNA is a complex between the divalent metal ions Zn2+, Ni2+ and Co2+ and duplex DNA which forms at a pH of approximately 8.5. The stability and formation of M-DNA was monitored with an ethidium fluorescence assay in order to assess the relationship between pH, metal ion concentration, DNA concentration and the base composition.