RESUMEN
Rickettsia prowazekii is an intracellular, obligate, gram-negative coccobacillus responsible for epidemic typhus. Usually, the infected body louse or its excrement when rubbed into the skin abrasions transmits the disease. The infection with R. prowazekii causes the highest death rate (> 20% without antibiotic treatment and now 1-7%), followed by epidemic typhus, which often manifests in unsanitary conditions (up to 15-30%). Conventionally, vaccine design has required pathogen growth and both assays (in vivo and in vitro), which are costly and time-consuming. However, advancements in bioinformatics and computational biology have accelerated the development of effective vaccine designs, reducing the need for traditional, time-consuming laboratory experiments. Subtractive genomics and reverse vaccinology have become prominent computational methods for vaccine model construction. Therefore, the RefSeq sequence of Rickettsia prowazekii (strain Madrid E) (Proteome ID: UP000002480) was subjected to subtractive genomic analysis, including factors such as non-similarity to host proteome, essentiality, subcellular localization, antigenicity, non-allergenicity, and stability. Based on these parameters, the vaccine design process selected specific proteins such as outer membrane protein R (O05971_RICPR PETR; OmpR). Eventually, the OmpR was subjected to a reverse vaccinology approach that included molecular docking, immunological simulation, and the discovery of B-cell epitopes and MHC-I and MHC-II epitopes. Consequently, a chimeric or multi-epitope-based vaccine was proposed by selecting the V11 vaccine and its 3D structure modeling along with molecular docking against TLR and HLA protein, in silico simulation, and vector designing. The obtained results from this investigation resulted in a new perception of inhibitory ways against Rickettsia prowazekii by instigating novel immunogenic targets. To further assess the efficacy and protective ability of the newly designed V11 vaccine against Rickettsia prowazekii infections, additional evaluation such as in vitro or in vivo immunoassays is recommended.
Asunto(s)
Rickettsia prowazekii , Tifus Endémico Transmitido por Pulgas , Tifus Epidémico Transmitido por Piojos , Humanos , Proteómica , Rickettsia prowazekii/genética , Rickettsia prowazekii/metabolismo , Tifus Epidémico Transmitido por Piojos/microbiología , Simulación del Acoplamiento Molecular , Proteoma , Vacunología/métodos , Biología Computacional/métodos , Epítopos de Linfocito B , Epítopos de Linfocito T/genética , Vacunas de SubunidadRESUMEN
Murine typhus, which is caused by Rickettsia typhi, has a wide range of clinical manifestations. It has a low mortality rate but may result in meningoencephalitis and interstitial pneumonia in severe cases. Comparisons of complete genome sequences of R. typhi isolates from North Carolina, USA (Wilmington), Myanmar (B9991PP), and Thailand (TH1527) identified only 26 single nucleotide polymorphism (SNP) and 7 insertion-deletion (INDEL) sites in these highly syntenic genomes. Assays were developed to further define the distribution of these variant sites among 15 additional isolates of R. typhi with different histories from Asia, the USA, and Africa. Mismatch amplification mutation assays (MAMA) were validated for 22 SNP sites, while the 7 INDEL sites were analyzed directly on agarose gels. Six SNP types, 9 INDEL types, 11 total types were identified among these 18 isolates. Replicate DNA samples as well as comparisons of isolates with different passage and source histories gave consistent genetic typing profiles. Comparison of the SNP and INDEL markers to R. typhi's nearest neighbor Rickettsia prowazekii demonstrated that the majority of the SNPs represent intra-species variation that arose post divergence of these two species while several INDEL sites also exhibited intraspecies variability among the R. prowazekii genomes that have been completely sequenced. The assays for the presence of these SNP and INDEL sites, particularly the latter, comprise a low technology gel method for consistently distinguishing R. typhi and R. prowazekii as well as for differentiating genetic types of R. typhi.
Asunto(s)
Rickettsia prowazekii , Rickettsia , Tifus Endémico Transmitido por Pulgas , Animales , Ratones , Rickettsia/genética , Rickettsia prowazekii/genética , Rickettsia typhi/genética , Análisis de Secuencia de ADN , TailandiaRESUMEN
Natural pathogen transmission of Rickettsia prowazekii, the etiologic agent of epidemic typhus, to humans is associated with arthropods, including human body lice, ticks, and ectoparasites of eastern flying squirrel. Recently, we have documented the presence of small RNAs in Rickettsia species and expression of R. prowazekii sRNAs during infection of cultured human microvascular endothelial cells (HMECs), which represent the primary target cells during human infections. Bacterial noncoding transcripts are now well established as critical post-transcriptional regulators of virulence and adaptation mechanisms in varying host environments. Despite their importance, little is known about the expression profile and regulatory activities of R. prowazekii sRNAs (Rp_sRs) in different host cells encountered as part of the natural life-cycle. To investigate the sRNA expression profile of R. prowazekii during infection of arthropod host cells, we employed an approach combining in vitro infection, bioinformatics, RNA sequencing, and PCR-based quantitation. Global analysis of R. prowazekii transcriptome by strand-specific RNA sequencing enabled us to identify 67 cis-acting (antisense) and 26 trans-acting (intergenic) Rp_sRs expressed during the infection of Amblyomma americanum (AAE2) cells. Comparative evaluation of expression during R. prowazekii infection of HMECs and AAE2 cells by quantitative RT-PCR demonstrated significantly higher expression of four selected Rp_sRs in tick AAE2 cells. Examination of the coding transcriptome revealed differential up-regulation of >150 rickettsial genes in either HMECs or AAE2 cells and yielded evidence for host cell-dependent utilization of alternative transcription start sites by 18 rickettsial genes. Our results thus suggest noticeable differences in the expression of both Rp_sRs as well as the coding transcriptome and the exploitation of multiple transcription initiation sites for select genes during the infection of human endothelium and tick vector cells as the host and yield new insights into rickettsial virulence and transmission mechanisms.
Asunto(s)
Vectores Arácnidos/microbiología , Regulación Bacteriana de la Expresión Génica , Ixodes/microbiología , ARN Bacteriano/genética , ARN Pequeño no Traducido/genética , Rickettsia prowazekii/genética , Transcriptoma , Animales , Perfilación de la Expresión Génica , Interacciones Huésped-Patógeno , Humanos , Rickettsia prowazekii/metabolismo , Análisis de Secuencia de ARN , Enfermedades por Picaduras de Garrapatas/transmisiónRESUMEN
Epidemic typhus caused by Rickettsia prowazekii is one of the oldest pestilential diseases of humankind. The disease is transmitted to human beings by the body louse Pediculus humanus corporis and is still considered a major threat by public health authorities, despite the efficacy of antibiotics, because poor sanitary conditions are conducive to louse proliferation. Epidemic typhus has accompanied disasters that impact humanity and has arguably determined the outcome of more wars than have soldiers and generals. The detection, identification, and characterization of microorganisms in ancient remains by paleomicrobiology has permitted the diagnosis of past epidemic typhus outbreaks through the detection of R. prowazekii. Various techniques, including microscopy and immunodetection, can be used in paleomicrobiology, but most of the data have been obtained by using PCR-based molecular techniques on dental pulp samples. Paleomicrobiology enabled the identification of the first outbreak of epidemic typhus in the 18th century in the context of a pan-European great war in the city of Douai, France, and supported the hypothesis that typhus was imported into Europe by Spanish soldiers returning from America. R. prowazekii was also detected in the remains of soldiers of Napoleon's Grand Army in Vilnius, Lithuania, which indicates that Napoleon's soldiers had epidemic typhus. The purpose of this article is to underscore the modern comprehension of clinical epidemic typhus, focus on the historical relationships of the disease, and examine the use of paleomicrobiology in the detection of past epidemic typhus outbreaks.
Asunto(s)
Brotes de Enfermedades , Fósiles/microbiología , Paleopatología/métodos , Rickettsia prowazekii/aislamiento & purificación , Tifus Epidémico Transmitido por Piojos/epidemiología , Tifus Epidémico Transmitido por Piojos/historia , Animales , Técnicas Bacteriológicas/métodos , Salud Global , Historia del Siglo XVIII , Historia del Siglo XIX , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Pediculus/crecimiento & desarrollo , Pediculus/microbiología , Reacción en Cadena de la Polimerasa/métodos , Rickettsia prowazekii/genéticaRESUMEN
Rickettsia belong to a family of Gram-negative obligate intracellular infectious bacteria that are the causative agents of typhus and spotted fever. Outer membrane protein B (OmpB) occurs in all rickettsial species, serves as a protective envelope, mediates host cell adhesion and invasion, and is a major immunodominant antigen. OmpBs from virulent strains contain multiple trimethylated lysine residues, whereas the avirulent strain contains mainly monomethyllysine. Two protein-lysine methyltransferases (PKMTs) that catalyze methylation of recombinant OmpB at multiple sites with varying sequences have been identified and overexpressed. PKMT1 catalyzes predominantly monomethylation, whereas PKMT2 catalyzes mainly trimethylation. Rickettsial PKMT1 and PKMT2 are unusual in that their primary substrate appears to be limited to OmpB, and both are capable of methylating multiple lysyl residues with broad sequence specificity. Here we report the crystal structures of PKMT1 from Rickettsia prowazekii and PKMT2 from Rickettsia typhi, both the apo form and in complex with its cofactor S-adenosylmethionine or S-adenosylhomocysteine. The structure of PKMT1 in complex with S-adenosylhomocysteine is solved to a resolution of 1.9 Å. Both enzymes are dimeric with each monomer containing an S-adenosylmethionine binding domain with a core Rossmann fold, a dimerization domain, a middle domain, a C-terminal domain, and a centrally located open cavity. Based on the crystal structures, residues involved in catalysis, cofactor binding, and substrate interactions were examined using site-directed mutagenesis followed by steady state kinetic analysis to ascertain their catalytic functions in solution. Together, our data reveal new structural and mechanistic insights into how rickettsial methyltransferases catalyze OmpB methylation.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/química , N-Metiltransferasa de Histona-Lisina/química , Rickettsia prowazekii/química , Rickettsia typhi/química , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Catálisis , Cristalografía por Rayos X , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Cinética , Dominios Proteicos , Pliegue de Proteína , Rickettsia prowazekii/genética , Rickettsia prowazekii/metabolismo , Rickettsia typhi/genética , Rickettsia typhi/metabolismoRESUMEN
Louse borne typhus (also called epidemic typhus) was one of man's major scourges, and epidemics of the disease can be reignited when social, economic, or political systems are disrupted. The fear of a bioterrorist attack using the etiologic agent of typhus, Rickettsia prowazekii, was a reality. An attenuated typhus vaccine, R. prowazekii Madrid E strain, was observed to revert to virulence as demonstrated by isolation of the virulent revertant Evir strain from animals which were inoculated with Madrid E strain. The mechanism of the mutation in R. prowazekii that affects the virulence of the vaccine was not known. We sequenced the genome of the virulent revertant Evir strain and compared its genome sequence with the genome sequences of its parental strain, Madrid E. We found that only a single nucleotide in the entire genome was different between the vaccine strain Madrid E and its virulent revertant strain Evir. The mutation is a single nucleotide insertion in the methyltransferase gene (also known as PR028) in the vaccine strain that inactivated the gene. We also confirmed that the vaccine strain E did not cause fever in guinea pigs and the virulent revertant strain Evir caused fever in guinea pigs. We concluded that a single nucleotide insertion in the methyltransferase gene of R. prowazekii attenuated the R. prowazekii vaccine strain E. This suggested that an irreversible insertion or deletion mutation in the methyl transferase gene of R. prowazekii is required for Madrid E to be considered a safe vaccine.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas de Choque Térmico , Metiltransferasas/genética , Fragmentos de Péptidos , Rickettsia prowazekii/patogenicidad , Tifus Epidémico Transmitido por Piojos/veterinaria , Factores de Virulencia/genética , Animales , Análisis Mutacional de ADN/métodos , Genoma Bacteriano , Cobayas , Datos de Secuencia Molecular , Mutación , Rickettsia prowazekii/enzimología , Rickettsia prowazekii/genética , Tifus Epidémico Transmitido por Piojos/microbiología , Tifus Epidémico Transmitido por Piojos/prevención & control , Vacunas Atenuadas/genética , Vacunas Atenuadas/metabolismoRESUMEN
The intracellular bacterial pathogen Legionella pneumophila (Lp) evades destruction in macrophages by camouflaging in a specialized organelle, the Legionella-containing vacuole (LCV), where it replicates. The LCV maturates by incorporating ER vesicles, which are diverted by effectors that Lp injects to take control of host cell membrane transport processes. One of these effectors, RalF, recruits the trafficking small GTPase Arf1 to the LCV. LpRalF has a Sec7 domain related to host ArfGEFs, followed by a capping domain that intimately associates with the Sec7 domain to inhibit GEF activity. How RalF is activated to function as a LCV-specific ArfGEF is unknown. We combined the reconstitution of Arf activation on artificial membranes with cellular expression and Lp infection assays, to analyze how auto-inhibition is relieved for LpRalF to function in vivo. We find that membranes activate LpRalF by about 1000 fold, and identify the membrane-binding region as the region that inhibits the Sec7 active site. It is enriched in aromatic and positively charged residues, which establish a membrane sensor to control the GEF activity in accordance with specific lipid environments. A similar mechanism of activation is found in RalF from Rickettsia prowazekii (Rp), with a different aromatic/charged residues ratio that results in divergent membrane preferences. The membrane sensor is the primary determinant of the localization of LpRalF on the LCV, and drives the timing of Arf activation during infection. Finally, we identify a conserved motif in the capping domain, remote from the membrane sensor, which is critical for RalF activity presumably by organizing its active conformation. These data demonstrate that RalF proteins are regulated by a membrane sensor that functions as a binary switch to derepress ArfGEF activity when RalF encounters a favorable lipid environment, thus establishing a regulatory paradigm to ensure that Arf GTPases are efficiently activated at specific membrane locations.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Factores de Intercambio de Guanina Nucleótido/química , Factores de Intercambio de Guanina Nucleótido/metabolismo , Legionella pneumophila/química , Legionella pneumophila/metabolismo , Enfermedad de los Legionarios/metabolismo , Proteínas Bacterianas/genética , Sitios de Unión , Factores de Intercambio de Guanina Nucleótido/genética , Células HEK293 , Humanos , Legionella pneumophila/genética , Enfermedad de los Legionarios/genética , Estructura Terciaria de Proteína , Rickettsia prowazekii/genética , Rickettsia prowazekii/metabolismo , Vacuolas/genética , Vacuolas/metabolismo , Vacuolas/microbiologíaRESUMEN
Rickettsia prowazekii has been tested for biological warfare due to the high mortality that it produces after aerosol transmission of very low numbers of rickettsiae. Epidemic typhus, the infection caused by these obligately intracellular bacteria, continues to be a threat because it is difficult to diagnose due to initial non-specific symptoms and the lack of commercial diagnostic tests that are sensitive and specific during the initial clinical presentation. A vaccine to prevent epidemic typhus would constitute an effective deterrent to the weaponization of R. prowazekii; however, an effective and safe vaccine is not currently available. Due to the cytoplasmic niche of Rickettsia, CD8(+) T-cells are critical effectors of immunity; however, the identification of antigens recognized by these cells has not been systematically addressed. To help close this gap, we designed an antigen discovery strategy that uses cell-based vaccination with antigen presenting cells expressing microbe's proteins targeted to the MHC class I presentation pathway. We report the use of this method to discover a protective T-cell rickettsial antigen, RP884, among a test subset of rickettsial proteins.
Asunto(s)
Antígenos Bacterianos/inmunología , Linfocitos T CD8-positivos/inmunología , Rickettsia prowazekii/inmunología , Tifus Epidémico Transmitido por Piojos/inmunología , Tifus Epidémico Transmitido por Piojos/prevención & control , Animales , Células Presentadoras de Antígenos/inmunología , Proteínas Bacterianas/metabolismo , Linfocitos T CD8-positivos/microbiología , Biología Computacional , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos/genética , Vectores Genéticos/metabolismo , Ratones , Reproducibilidad de los Resultados , Rickettsia prowazekii/genética , Vacunas contra Rickettsia/inmunología , Tifus Epidémico Transmitido por Piojos/microbiologíaRESUMEN
Rickettsia prowazekii is an obligate intracytosolic pathogen and the causative agent of epidemic typhus fever in humans. As an evolutionary model of intracellular pathogenesis, rickettsiae are notorious for their use of transport systems that parasitize eukaryotic host cell biochemical pathways. Rickettsial transport systems for substrates found only in eukaryotic cell cytoplasm are uncommon among free-living microorganisms and often possess distinctive mechanisms. We previously reported that R. prowazekii acquires triose phosphates for phospholipid biosynthesis via the coordinated activities of a novel dihydroxyacetone phosphate transport system and an sn-glycerol-3-phosphate dehydrogenase (K. M. Frohlich et al., J. Bacteriol. 192:4281-4288, 2010). In the present study, we have determined that R. prowazekii utilizes a second, independent triose phosphate acquisition pathway whereby sn-glycerol-3-phosphate is directly transported and incorporated into phospholipids. Herein we describe the sn-glycerol-3-phosphate and dihydroxyacetone phosphate transport systems in isolated R. prowazekii with respect to kinetics, energy coupling, transport mechanisms, and substrate specificity. These data suggest the existence of multiple rickettsial triose phosphate transport systems. Furthermore, the R. prowazekii dihydroxyacetone phosphate transport systems displayed unexpected mechanistic properties compared to well-characterized triose phosphate transport systems from plant plastids. Questions regarding possible roles for dual-substrate acquisition pathways as metabolic virulence factors in the context of a pathogen undergoing reductive evolution are discussed.
Asunto(s)
Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Fosfatos/metabolismo , Rickettsia prowazekii/metabolismo , Triosas/metabolismo , Proteínas Bacterianas/genética , Transporte Biológico Activo/fisiología , Fosfatos/química , Rickettsia prowazekii/genética , Especificidad por Sustrato , Triosas/químicaRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Membrana Celular/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Legionella pneumophila/metabolismo , Animales , Proteínas Bacterianas/genética , Células CHO , Membrana Celular/genética , Cricetinae , Cricetulus , Factores de Intercambio de Guanina Nucleótido/genética , Células HeLa , Humanos , Legionella pneumophila/genética , Unión Proteica , Estructura Terciaria de Proteína , Rickettsia prowazekii/genética , Rickettsia prowazekii/metabolismoRESUMEN
Rickettsia prowazekii, the etiologic agent of epidemic typhus, is a potential biological threat agent. Its outer membrane protein B (OmpB) is an immunodominant antigen and plays roles as protective envelope and as adhesins. The observation of the correlation between methylation of lysine residues in rickettsial OmpB and bacterial virulence has suggested the importance of an enzymatic system for the methylation of OmpB. However, no rickettsial lysine methyltransferase has been characterized. Bioinformatic analysis of genomic DNA sequences of Rickettsia identified putative lysine methyltransferases. The genes of the potential methyltransferases were synthesized, cloned, and expressed in Escherichia coli, and expressed proteins were purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. The methyltransferase activities of the purified proteins were analyzed by methyl incorporation of radioactively labeled S-adenosylmethionine into recombinant fragments of OmpB. Two putative recombinant methyltransferases (rRP789 and rRP027-028) methylated recombinant OmpB fragments. The specific activity of rRP789 is 10- to 30-fold higher than that of rRP027-028. Western blot analysis using specific antibodies against trimethyl lysine showed that both rRP789 and rRP027-028 catalyzed trimethylation of recombinant OmpB fragments. Liquid chromatography-tandem mass spectrometry (LC/MS-MS) analysis showed that rRP789 catalyzed mono-, di-, and trimethylation of lysine, while rRP027-028 catalyzed exclusively trimethylation. To our knowledge, rRP789 and rRP027-028 are the first biochemically characterized lysine methyltransferases of outer membrane proteins from Gram-negative bacteria. The production and characterization of rickettsial lysine methyltransferases provide new tools to investigate the mechanism of methylation of OmpB, effects of methylation on the structure and function of OmpB, and development of methylated OmpB-based diagnostic assays and vaccine candidates.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Rickettsia prowazekii/enzimología , Rickettsia prowazekii/metabolismo , Western Blotting , Cromatografía de Afinidad , Cromatografía Liquida , Clonación Molecular , Biología Computacional , Escherichia coli/genética , Expresión Génica , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/aislamiento & purificación , Marcaje Isotópico , Cinética , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Rickettsia prowazekii/genética , S-Adenosilmetionina/metabolismo , Espectrometría de Masas en TándemRESUMEN
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.
Asunto(s)
Replicación del ADN , Plásmidos , Rickettsia prowazekii/genética , Animales , Línea Celular , Embrión de Pollo , Dosificación de Gen , Ratones , Rickettsia prowazekii/crecimiento & desarrolloRESUMEN
BACKGROUND: Detecting the borders between coding and non-coding regions is an essential step in the genome annotation. And information entropy measures are useful for describing the signals in genome sequence. However, the accuracies of previous methods of finding borders based on entropy segmentation method still need to be improved. METHODS: In this study, we first applied a new recursive entropic segmentation method on DNA sequences to get preliminary significant cuts. A 22-symbol alphabet is used to capture the differential composition of nucleotide doublets and stop codon patterns along three phases in both DNA strands. This process requires no prior training datasets. RESULTS: Comparing with the previous segmentation methods, the experimental results on three bacteria genomes, Rickettsia prowazekii, Borrelia burgdorferi and E.coli, show that our approach improves the accuracy for finding the borders between coding and non-coding regions in DNA sequences. CONCLUSIONS: This paper presents a new segmentation method in prokaryotes based on Jensen-Rényi divergence with a 22-symbol alphabet. For three bacteria genomes, comparing to A12_JR method, our method raised the accuracy of finding the borders between protein coding and non-coding regions in DNA sequences.
Asunto(s)
Algoritmos , Borrelia burgdorferi/genética , ADN Bacteriano/metabolismo , Escherichia coli/genética , Rickettsia prowazekii/genética , Codón de Terminación , ADN Bacteriano/química , ADN Bacteriano/genética , Nucleótidos/química , Sistemas de Lectura AbiertaRESUMEN
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.
Asunto(s)
Elementos Transponibles de ADN/genética , Rickettsia rickettsii/genética , Transformación Genética , ADN Bacteriano/genética , Mutagénesis Insercional , Rickettsia prowazekii/genética , Ensayo de Placa ViralRESUMEN
Here we have characterized the Rickettsia prowazekii RP534 protein, a homologue of the Pseudomonas aeruginosa ExoU phospholipase A (PLA) secreted cytotoxin. Our studies showed that purified recombinant RP534 PLA possessed the predicted PLA(2) and lyso-PLA(2) activities based on what has been published for P. aeruginosa ExoU. RP534 also displayed PLA(1) activity under the conditions tested, whereas ExoU did not. In addition, recombinant RP534 displayed a basal PLA activity that could hydrolyze phosphatidylcholine in the absence of any eukaryotic cofactors. Interestingly, the addition of bovine liver superoxide dismutase 1 (SOD1), a known activator of P. aeruginosa ExoU, resulted in an increased rate of RP534-catalyzed phospholipid hydrolysis, indicating that mechanisms of activation of the ExoU family of PLAs may be evolutionarily conserved. The mechanism of SOD1-dependent stimulation of RP534 was further examined using active site mutants and a fluorogenic phospholipid substrate whose hydrolysis by RP534 over a short time course is measureable only in the presence of SOD1. These studies suggest a mechanism by which SOD1 stimulates RP534 activity once it has bound to the substrate. We also show that antibody raised against RP534 was useful for immunoprecipitating active RP534 from R. prowazekii lysed cell extracts, thus verifying that this protein is expressed and active in rickettsiae isolated from embryonated hen egg yolk sacs.
Asunto(s)
Lisofosfolipasa/metabolismo , Fosfolipasas A1/metabolismo , Fosfolipasas A2/metabolismo , Rickettsia prowazekii/enzimología , Hidrólisis , Lisofosfolipasa/genética , Lisofosfolipasa/aislamiento & purificación , Fosfatidilcolinas/metabolismo , Fosfolipasas A1/genética , Fosfolipasas A1/aislamiento & purificación , Fosfolipasas A2/genética , Fosfolipasas A2/aislamiento & purificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Rickettsia prowazekii/genética , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1RESUMEN
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.
Asunto(s)
Proteínas Bacterianas/análisis , Proteoma/análisis , Proteómica/métodos , Rickettsia prowazekii/química , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Línea Celular , Embrión de Pollo , Cromatografía Liquida/métodos , Expresión Génica , Genoma Bacteriano , Espectrometría de Masas/métodos , Ratones , Biosíntesis de Proteínas , Proteoma/genética , Proteoma/metabolismo , Rickettsia prowazekii/genética , Rickettsia prowazekii/metabolismo , Spodoptera , Espectrometría de Masas en Tándem , Garrapatas/microbiologíaRESUMEN
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.
Asunto(s)
Genes Bacterianos , ARN Bacteriano/genética , Rickettsia prowazekii/genética , Transcripción Genética , Regulación Bacteriana de la Expresión Génica , Interacciones Huésped-PatógenoRESUMEN
BACKGROUND: Rickettsia prowazekii is the etiological agent of epidemic typhus and is an obligate intracellular bacterium that grows as a parasite freely within the cytoplasm of a eukaryotic host cell. Previous studies have shown that rOmpA and rOmpB which belong to the family of rickettsial cell surface antigens are involved in vitro in the adhesion of Rickettsiae to epithelial cells. Recently, two putative rickettsial adhesins have been identified using high resolution 2D-PAGE coupled with mass spectrometry. In this study, we further characterize and describe the adhesin Adr2 from R. prowazekii. METHODOLOGY/PRINCIPAL FINDINGS: Using an overlay assay coupled with mass spectrometry two adhesins, Adr1 (RP827) and Adr2 (RP828), were identified from the R. prowazekii proteome Recombinant R. prowazekii Adr2 was expressed through fusion with Dsbc in Escherichia coli, purified and concentrated, thus allowing production of specific monoclonal antibodies, as confirmed by western blot assays. Finally, inhibition of rickettsiae-induced cytotoxicity with monoclonal anti-Adr2 antibody has showed a greatest impact on bacterial cell entry at 8 h post-infection (ca50%) and then decreased progressively to attempt 18% of inhibition at day 7. These, correlated to the inhibition of rickettsiae-induced cytotoxicity with monoclonal anti-rOmpB antibody. Thus, Adr2 is sufficient to mediate R. prowazekii entry into the cell at early stage of mammalian cell infection. CONCLUSIONS: Our results suggest that R. prowazekii Adr2 could be the main actor promoting the entry of rickettsiae into the host cells. The present study opens the framework for future investigations for better understanding of the Adr2 -mediated mechanisms involved in adhesion/invasion or intracellular survival of R. prowazekii.
Asunto(s)
Adhesinas Bacterianas/metabolismo , Adhesión Bacteriana , Fibroblastos/microbiología , Rickettsia prowazekii/genética , Rickettsia prowazekii/patogenicidad , Adhesinas Bacterianas/aislamiento & purificación , Animales , Anticuerpos Antibacterianos/inmunología , Células Cultivadas , Clonación Molecular , Electroforesis en Gel Bidimensional , Escherichia coli/genética , Expresión Génica , Espectrometría de Masas , Ratones , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
While new sequencing technologies have ushered in an era where microbial genomes can be easily sequenced, the goal of routinely producing high-quality draft and finished genomes in a cost-effective fashion has still remained elusive. Due to shorter read lengths and limitations in library construction protocols, shotgun sequencing and assembly based on these technologies often results in fragmented assemblies. Correspondingly, while draft assemblies can be obtained in days, finishing can take many months and hence the time and effort can only be justified for high-priority genomes and in large sequencing centers. In this work, we revisit this issue in light of our own experience in producing finished and nearly-finished genomes for a range of microbial species in a small-lab setting. These genomes were finished with surprisingly little investments in terms of time, computational effort and lab work, suggesting that the increased access to sequencing might also eventually lead to a greater proportion of finished genomes from small labs and genomics cores.
Asunto(s)
Gammaproteobacteria/genética , Genoma Bacteriano , Rickettsia prowazekii/genética , Análisis de Secuencia de ADN/métodos , Yersinia/genética , Humanos , Análisis de Secuencia de ADN/economíaRESUMEN
Rickettsia prowazekii, the agent of epidemic typhus, is an obligate intracellular bacterium that is transmitted to human beings by the body louse. Several strains that differ considerably in virulence are recognized, but the genetic basis for these variations has remained unknown since the initial description of the avirulent vaccine strain nearly 70 yr ago. We use a recently developed murine model of epidemic typhus and transcriptomic, proteomic, and genetic techniques to identify the factors associated with virulence. We identified four phenotypes of R. prowazekii that differed in virulence, associated with the up-regulation of antiapoptotic genes or the interferon I pathway in the host cells. Transcriptional and proteomic analyses of R. prowazekii surface protein expression and protein methylation varied with virulence. By sequencing a virulent strain and using comparative genomics, we found hotspots of mutations in homopolymeric tracts of poly(A) and poly(T) in eight genes in an avirulent strain that split and inactivated these genes. These included recO, putative methyltransferase, and exported protein. Passage of the avirulent Madrid E strain in cells or in experimental animals was associated with a cascade of gene reactivations, beginning with recO, that restored the virulent phenotype. An area of genomic plasticity appears to determine virulence in R. prowazekii and represents an example of adaptive mutation for this pathogen.