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

Rickettsia 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 Rickettsia canadensis as a model organism. The screening data were utilized to train a Bayesian model to predict growth inhibition in this assay. This two-pronged methodology identified anti-rickettsial compounds, including duartin and JSF-3204 as highly specific, efficacious, and noncytotoxic compounds. Both molecules exhibited in vitro growth inhibition of R. prowazekii, the causative agent of epidemic typhus. These small molecules and the workflow, featuring a high-throughput phenotypic screen for growth inhibitors of intracellular Rickettsia spp. and machine learning models for the prediction of growth inhibition of an obligate intracellular Gram-negative bacterium, should prove useful in the search for new therapeutic strategies to treat infections from Rickettsia spp. and other obligate intracellular bacteria.

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., Rickettsia spp., Anaplasma spp., Ehrlichia spp. and Orientia spp. is, in part, due to their genetic intractability. Recently, genetic tools have been developed; however, optimizing the genomic manipulation of obligate intracellular bacteria remains challenging. In this Review, we describe the progress in, as well as the constraints that hinder, the systematic development of a genetic toolbox for obligate intracellular bacteria. We highlight how the use of genetically manipulated pathogens has facilitated a better understanding of microbial pathogenesis and immunity, and how the engineering of obligate intracellular bacteria could enable the discovery of novel signalling circuits in host-pathogen interactions.

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. prowazekii transformants. To reduce the time required and variability associated with propagation and harvesting of rickettsiae for each transformation experiment, characterized frozen stocks were used to generate electrocompetent rickettsiae. Transformation experiments employing these rickettsiae established that fluorescent rickettsial populations could be identified using a fluorescence activated cell sorter within one week following electroporation. Early detection was improved with increasing amounts of transforming DNA. In addition, we demonstrate that heterogeneous populations of rickettsiae-infected cells can be sorted into distinct sub-populations based on the number of rickettsiae per cell. Together our data suggest the combination of fluorescent reporters and cell sorting represent an important technical advance that will facilitate isolation of distinct R. prowazekii mutants and allow for closer examination of the effects of infection on host cells at various infectious burdens.

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. Significantly, the field is moving forward from a focus on the identification and development of genetic techniques to their application in addressing crucial questions related to mechanisms of bacterial pathogenicity and the requirements of obligate intracellular growth.

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. Theoretically, the C-terminal region of RalF could prevent nucleotide exchange activity by blocking the ability of Arf to interact with the Sec7 domain. For this reason, the C-terminal region of RalF has been termed a capping domain. Here, the role of the RalF capping domain was investigated by comparing biochemical and effector activities mediated by this domain in both the Legionella RalF protein (LpRalF) and in a RalF ortholog isolated from the unrelated intracellular pathogen Rickettsia prowazekii (RpRalF). These data indicate that both RalF proteins contain a functional Sec7 domain and that the capping domain regulates RalF GEF activity. The capping domain has intrinsic determinants that mediate localization of the RalF protein inside of host cells and confer distinct effector activities. Localization mediated by the capping domain of LpRalF enables the GEF to modulate membrane transport in the secretory pathway, whereas, the capping domain of RpRalF enables this bacterial GEF to modulate actin dynamics occurring near the plasma membrane. Thus, these data reveal that divergence in the function of the C-terminal capping domain alters the in vivo functions of the RalF proteins.

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. prowazekii, the existence of well-characterized plasmids in several less pathogenic rickettsial species provides an opportunity to expand the genetic systems available for the study of this human pathogen. Competent R. prowazekii were transformed with pRAM18dRGA, a 10.3 kb vector derived from pRAM18 of R. amblyommii. A plasmid-containing population of R. prowazekii was obtained following growth under antibiotic selection, and the rickettsial plasmid was maintained extrachromosomally throughout multiple passages. The transformant population exhibited a generation time comparable to that of the wild type strain with a copy number of approximately 1 plasmid per rickettsia. These results demonstrate for the first time that a plasmid can be maintained in R. prowazekii, providing an important genetic tool for the study of this obligate intracellular pathogen.

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. prowazekii indicated that insertions were random. Transposon mutagenesis provides a useful tool for rickettsial research.

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. With the use of a combination of commercially available algorithms and in-house software, quantitative MS-only data and comprehensive peptide coverage generated from MS-MS were integrated, resulting in the assignment of peptide identities with intensity values, allowing for the differential comparison of complex protein samples. With the use of these protocols, it was possible to directly compare protein abundance and analyze changes in the total proteome profile of R. prowazekii grown in different host backgrounds. Total protein extracted from rickettsiae grown in murine, tick, and insect cell lines or hen egg yolk sacs was analyzed. Here, we report the fold changes, including an upregulation of shock-related proteins, in rickettsiae cultivated in tissue culture compared to the level for rickettsiae harvested from hen yolk sacs. The ability to directly compare, in a complex sample, differential rickettsial protein expression provides a snapshot of host-specific proteomic profiles that will help to identify proteins important in intracellular growth and virulence.

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. These analyses revealed that, rather than terminating at a specific site within the intervening region between the convergent genes, most of the transcripts demonstrated either a lack of termination within this region, which generated antisense RNA, or a putative non-site-specific termination that occurred throughout the intervening sequence. Transcripts terminating at predicted intrinsic terminators, as well as at a putative Rho-dependant terminator, were also examined and found to vary based on the rickettsial host environment. These results suggest that transcriptional termination, or lack thereof, plays a role in rickettsial gene regulation.

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. prowazekii pld gene, encoding a protein with phospholipase D activity, has been associated with phagosomal escape. To demonstrate the feasibility of site-directed knockout mutagenesis of rickettsial genes and to generate a nonrevertible vaccine strain, we utilized homologous recombination to generate a pld mutant of the virulent R. prowazekii strain Madrid Evir. Using linear DNA for transformation, a double-crossover event resulted in the replacement of the rickettsial wild-type gene with a partially deleted pld gene. Linear DNA was used to prevent potentially revertible single-crossover events resulting in plasmid insertion. Southern blot and PCR analyses were used to confirm the presence of the desired mutation and to demonstrate clonality. While no phenotypic differences were observed between the mutant and wild-type strains when grown in tissue culture, the pld mutant exhibited attenuated virulence in the guinea pig model. In addition, animals immunized with the mutant strain were protected against subsequent challenge with the virulent Breinl strain, suggesting that this transformant could serve as a nonrevertible, attenuated vaccine strain. This study demonstrates the feasibility of generating site-directed rickettsial gene mutants, providing a new tool for understanding rickettsial biology and furthering advances in the prevention of epidemic typhus.

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. prowazekii. A transposon containing the R. prowazekii arr-2 rifampin resistance gene and a gene coding for a green fluorescent protein (GFP(UV)) was constructed and placed on a plasmid expressing the Himar1 transposase. Electroporation of this plasmid into R. prowazekii resulted in numerous transpositions into the rickettsial genome. Transposon insertion sites were identified by rescue cloning, followed by DNA sequencing. Random transpositions integrating at TA sites in both gene coding and intergenic regions were identified. Individual rickettsial clones were isolated by the limiting-dilution technique. Using both fixed and live-cell techniques, R. prowazekii transformants expressing GFP(UV) were easily visible by fluorescence microscopy. Thus, a mariner-based system provides an additional mechanism for generating rickettsial mutants that can be screened using GFP(UV) fluorescence.

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. An internal position of the chromophore was made possible by attaching it to the extra hydroxyl arm in the branched monomer 4'-C-hydroxymethylthymidine. Upon excitation of the donor fluorophore, it was demonstrated that electrons could be injected into the side of an M-DNA helix thereby extending the range of nanoelectronic structures that can be prepared from DNA.

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. From the mass action analysis, it was determined that approximately 1.4 protons are released per base pair, with k(eq) on the order of 10(-8), indicative of a strong interaction. As resonance energy transfer is shown to be unlikely over the distances involved in this work, the observed quenching in M-DNA is discussed in terms of an electron hopping mechanism for electron transfer, with k(hop)=2.5 x 10(11)s(-1).

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. prowazekii, which contain recognizable inactivating mutations. However, several strains of the typhus group rickettsiae possess metK genes lacking obvious mutations. In order to determine if these genes code for a product that retains MAT function, an Escherichia coli metK deletion mutant was constructed in which individual rickettsial metK genes were tested for the ability to complement the methionine adenosyltransferase deficiency. Both the R. prowazekii Breinl and R. typhi Wilmington metK genes complemented at a level comparable to that of an E. coli metK control, demonstrating that the typhus group rickettsiae have the capability of synthesizing as well as transporting SAM. However, the appearance of mutations that affect the function of the metK gene products (a stop codon in the Madrid E strain and a 6-bp deletion in the Breinl strain) provides experimental support for the hypothesis that these typhus group genes, like the more degenerate spotted fever group orthologs, are in the process of gene degradation.

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. In addition, the examination of rickettsial gene expression through characterization of the rickettsial proteome will be described. This will include a description of a high-throughput, accurate mass approach that has identified 596 rickettsial proteins in a complex rickettsial protein sample.

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 7.5, while Mg(2+) and Ca(2+) caused small changes at both pHs. The concentrations that gave 50% of the maximum responses were 0.06 and 0.50 mM for Zn(2+) and Ni(2+), respectively. At pH 8.5, Cd(2+) denatured over 40% of the dsDNA, while other metals denatured less than 5% of the DNA. Smaller pH-dependent signals were induced by Zn(2+), Ni(2+) or Cd(2+) with 50% GC single-stranded DNA (ssDNA), and with a homopolymer of d(T)30. Homopolymers d(A)30 and d(C)30 showed small signals that were largely independent of pH in the presence of Zn(2+) or Ni(2+).

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. The arr-2 gene codes for an enzyme that ADP-ribosylates rifampin, thereby destroying its antibacterial activity. Based on the published sequence, this gene was synthesized by PCR with overlapping primers that contained rickettsial codon usage base changes. This R. prowazekii-adapted arr-2 gene (Rparr-2) was placed downstream of the strong rickettsial rpsL promoter (rpsL(P)), and the entire construct was inserted into the Epicentre EZ::TN transposome system. A purified transposon containing rpsL(P)-Rparr-2 was combined with transposase, and the resulting DNA-protein complex (transposome) was electroporated into competent rickettsiae. Following selection with rifampin, rickettsiae with transposon insertions in the genome were identified by PCR and Southern blotting and the insertion sites were determined by rescue cloning and inverse PCR. Multiple insertions into widely spaced areas of the R. prowazekii genome were identified. Three insertions were identified within gene coding sequences. Transposomes provide a mechanism for generating random insertional mutations in R. prowazekii, thereby identifying nonessential rickettsial genes.

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. prowazekii Madrid E genome sequence revealed the presence of a mutation in the rickettsial metK gene, the gene encoding the enzyme responsible for the synthesis of S-adenosylmethionine (AdoMet). Since AdoMet is required for rickettsial processes, the apparent inability of this strain to synthesize AdoMet suggested the presence of a rickettsial AdoMet transporter. We have confirmed the presence of an AdoMet transporter in the rickettsiae which, to our knowledge, is the first bacterial AdoMet transporter identified. The influx of AdoMet into rickettsiae was a saturable process with a K(T) of 2.3 micro M. Transport was inhibited by S-adenosylethionine and S-adenosylhomocysteine but not by sinfungin or methionine. Transport was also inhibited by 2,4-dinitrophenol, suggesting an energy-linked transport mechanism, and by N-ethylmaleimide. AdoMet transporters with similar properties were also identified in the Breinl strain of R. prowazekii and in Rickettsia typhi. By screening Escherichia coli clone banks for AdoMet transport, the R. prowazekii gene coding for a transporter, RP076 (sam), was identified. AdoMet transport in E. coli containing the R. prowazekii sam gene exhibited kinetics similar to that seen in rickettsiae. The existence of a rickettsial transporter for AdoMet raises intriguing questions concerning the evolutionary relationship between the synthesis and transport of this essential metabolite.

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 8.5 exhibited an endothermic cooperative binding process at Zn(2+) concentrations of approximately 0.1 mM, indicating an entropy driven process. This binding process is consistent with a site-specific binding interaction, similar in nature to Z-DNA formation; however, the interaction occurs at much lower metal ion concentrations. The enthalpy of M-DNA formation for calf thymus DNA was determined to be 10.5+/-0.7 and 9+/-2 kJ/mbp at DNA concentrations of 100 and 50 microg ml(-1), respectively. An enthalpy of 13+/-3 kJ/mbp was obtained for M-DNA formation for 50 microg ml(-1) E. coli DNA. No evidence of M-DNA formation was observed in either DNA at pH 7.5 with Zn(2+) or at either pH 7.5 or 8.5 with Mg(2+).

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. The dismutation of calf thymus DNA exhibits hysteresis with the formation of M-DNA occurring at a higher pH than the reconversion of M-DNA back to B-DNA. Hysteresis is most prominent with the Ni form of M-DNA where complete reconversion to B-DNA takes several hours even in the presence of EDTA. Increasing the DNA concentration leads to an increase in the metal ion concentration required for M-DNA formation. Both poly(dG)*poly(dC) and poly(dA)*poly(dT) formed M-DNA more readily than the corresponding mixed sequence DNAs. For poly(dG)*(poly(dC) M-DNA formation was observed at pH 7.4 with 0.5 mM ZnCl2. Modified bases were incorporated into a 500 bp fragment of phage lambda DNA by polymerase chain reaction. DNAs in which guanine was replaced with hypoxanthine or thymine with 5-fluorouracil formed M-DNA at pHs below 8 whereas substitutions such as 2-aminoadenine and 5-methylcytosine had little effect. Poly[d(A5FU)] also formed a very stable M-DNA duplex as judged from T(m) measurements. It is evident that the lower the pK(a) of the imino proton of the base, the lower the pH at which M-DNA will form; a finding that is consistent with the replacement of the imino proton with the metal ion.