RESUMEN
Considering the influence of measurement error on target state estimation, there is an uncertain dispersion region for target position estimate, that is, the area of uncertainty (AOU, area of uncertainty). In underwater target tracking, the state estimation is point estimation without AOU estimation and its accuracy is poor in the early stage because of large measurement errors. Fast tracking with higher accuracy and AOU estimation are of great significance to time-sensitive target tracking. To improve the state estimation accuracy in the early stage, and estimate the AOU, a method of AOU estimation of underwater moving target is presented based on a stochastic maneuvering motion (SMM, stochastic maneuvering motion) model. The stochastic maneuvering motion model is established based on the Langevin equation to reflect the movement characteristics of an underwater moving target. Then, the target state is estimated with a noise adaptive Kalman filter by constructing the measurement equation and state equation according to measurement error characteristic and stochastic maneuvering model. Based on the physical significance of the error covariance matrix from the Kalman filter, the parameters of AOU are deduced. Simulation results of underwater target tracking and AOU estimation are presented to demonstrate the relative performance of the proposed algorithm compared with the adaptive Kalman filter. It is clearly shown from the results that SMM tracking algorithm achieves higher accuracy of state estimation in the initial stage of tracking, and the predicted AOU is consistent with the actual distribution of underwater moving targets while yielding more concentrated distribution, which reveals that estimated AOU can be precisely represented by the confidence ellipses. The presented approach and obtained results may be useful in time-sensitive target threat analysis and weapon strike applications.
RESUMEN
Arachnoid cysts are one of the benign spinal cystic lesions. Multiple nerve roots and spinal cord may be compressed by it, and operating is often recommended. Traditional surgical procedures often choose the posterior median approach, separating the paravertebral muscles, milling the lamina, fully exposing the cyst, partially or completely removing the cyst wall, looking for the leak, and then suturing and sealing. Here we present a case of giant spinal extradural arachnoid cyst in which a ultrafine flexible endoscope was used to visualize cystic spaces and identity the leaks. We repaired the leak after removing part of the cyst wall under the operating microscope, and the patient had an excellent recovery.
Asunto(s)
Quistes Aracnoideos , Enfermedades de la Médula Espinal , Quistes Aracnoideos/diagnóstico por imagen , Quistes Aracnoideos/cirugía , Endoscopios , Humanos , Imagen por Resonancia Magnética , Enfermedades de la Médula Espinal/cirugía , Columna VertebralRESUMEN
Burkholderia pseudomallei (Bp), causing a highly fatal disease called melioidosis, is a facultative intracellular pathogen that attaches and invades a variety of cell types. We previously identified BP1026B_I0091 as a surface attachment protein (Sap1) and an essential virulence factor, contributing to Bp pathogenesis in vitro and in vivo. The expression of sap1 is regulated at different stages of Bp intracellular lifecycle by unidentified regulator(s). Here, we identified SapR (BP1026B_II1046) as a transcriptional regulator that activates sap1, using a high-throughput transposon mutagenesis screen in combination with Tn-Seq. Consistent with phenotypes of the Δsap1 mutant, the ΔsapR activator mutant exhibited a significant reduction in Bp attachment to the host cell, leading to subsequent decreased intracellular replication. RNA-Seq analysis further revealed that SapR regulates sap1. The regulation of sap1 by SapR was confirmed quantitatively by qRT-PCR, which also validated the RNA-Seq data. SapR globally regulates genes associated with the bacterial membrane in response to diverse environments, and some of the genes regulated by SapR are virulence factors that are required for Bp intracellular infection (e.g., type III and type VI secretion systems). This study has identified the complex SapR regulatory network and its importance as an activator of an essential Sap1 attachment factor.
RESUMEN
Burkholderia pseudomallei (Bp) is the causative agent of melioidosis, a disease endemic to the tropics. Melioidosis manifests in various ways ranging from acute skin lesions to pneumonia and, in rare cases, infection of the central nervous system. Bp is a facultative intracellular pathogen and it can infect various cell types. The Bp intracellular lifecycle has been partially elucidated and is highly complex. Herein, we have identified a transcriptional regulator, BP1026B_II1198, that is differentially expressed as Bp transits through host cells. A deletion mutant of BP1026B_II1198 was attenuated in RAW264.7 cell and BALB/c mouse infection. To further characterize the function of this transcriptional regulator, we endeavored to determine the regulon of BP1026B_II1198. RNA-seq analysis showed the global picture of genes regulated while ChIP-seq analysis identified two specific BP1026B_II1198 binding regions on chromosome II. We investigated the transposon mutants of these genes controlled by BP1026B_II1198 and confirmed that these genes contribute to pathogenesis in RAW264.7 murine macrophage cells. Taken together, the data presented here shed light on the regulon of BP1026B_II1198 and its role during intracellular infection and highlights an integral portion of the highly complex regulation network of Bp during host infection.
Asunto(s)
Proteínas Bacterianas/genética , Burkholderia pseudomallei/patogenicidad , Regulación Bacteriana de la Expresión Génica , Melioidosis/microbiología , Proteínas Represoras/genética , Animales , Proteínas Bacterianas/metabolismo , Burkholderia pseudomallei/genética , Secuenciación de Inmunoprecipitación de Cromatina , Modelos Animales de Enfermedad , Humanos , Ratones , Mutación , Células RAW 264.7 , RNA-Seq , Regulón , Proteínas Represoras/metabolismo , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
Prokaryotic cell transcriptomics has been limited to mixed or sub-population dynamics and individual cells within heterogeneous populations, which has hampered further understanding of spatiotemporal and stage-specific processes of prokaryotic cells within complex environments. Here we develop a 'TRANSITomic' approach to profile transcriptomes of single Burkholderia pseudomallei cells as they transit through host cell infection at defined stages, yielding pathophysiological insights. We find that B. pseudomallei transits through host cells during infection in three observable stages: vacuole entry; cytoplasmic escape and replication; and membrane protrusion, promoting cell-to-cell spread. The B. pseudomallei 'TRANSITome' reveals dynamic gene-expression flux during transit in host cells and identifies genes that are required for pathogenesis. We find several hypothetical proteins and assign them to virulence mechanisms, including attachment, cytoskeletal modulation, and autophagy evasion. The B. pseudomallei 'TRANSITome' provides prokaryotic single-cell transcriptomics information enabling high-resolution understanding of host-pathogen interactions.
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Proteínas Bacterianas/genética , Burkholderia pseudomallei/genética , Biología Computacional/métodos , Perfilación de la Expresión Génica/métodos , Genes Bacterianos/genética , Factores de Virulencia/genética , Animales , Burkholderia pseudomallei/citología , Burkholderia pseudomallei/patogenicidad , Línea Celular Tumoral , Membrana Celular/microbiología , Citoplasma/microbiología , Células HEK293 , Interacciones Huésped-Patógeno , Humanos , Melioidosis/microbiología , Ratones , Ratones Endogámicos BALB C , Células RAW 264.7 , Análisis de la Célula Individual/métodos , Vacuolas/microbiología , Virulencia/genéticaRESUMEN
It is generally believed that the Pseudomonas aeruginosa biofilm matrix itself acts as a molecular sieve or sink that contributes to significant levels of drug resistance, but it is becoming more apparent that multidrug efflux pumps induced during biofilm growth significantly enhance resistance levels. We present here a novel transcriptional regulator, PA3898, which controls biofilm formation and multidrug efflux pumps in P. aeruginosa A mutant of this regulator significantly reduced the ability of P. aeruginosa to produce biofilm in vitro and affected its in vivo fitness and pathogenesis in Drosophila melanogaster and BALB/c mouse lung infection models. Transcriptome analysis revealed that PA3898 modulates essential virulence genes/pathways, including multidrug efflux pumps and phenazine biosynthesis. Chromatin immunoprecipitation sequencing (ChIP-seq) identified its DNA binding sequences and confirmed that PA3898 directly interacts with promoter regions of four genes/operons, two of which are mexAB-oprM and phz2 Coimmunoprecipitation revealed a regulatory partner of PA3898 as PA2100, and both are required for binding to DNA in electrophoretic mobility shift assays. PA3898 and PA2100 were given the names MdrR1 and MdrR2, respectively, as novel repressors of the mexAB-oprM multidrug efflux operon and activators for another multidrug efflux pump, EmrAB. The interaction between MdrR1 and MdrR2 at the promoter regions of their regulons was further characterized via localized surface plasmon resonance and DNA footprinting. These regulators directly repress the mexAB-oprM operon, independent of its well-established MexR regulator. Mutants of mdrR1 and mdrR2 caused increased resistance to multiple antibiotics in P. aeruginosa, validating the significance of these newly discovered regulators.
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Proteínas Bacterianas/genética , Pseudomonas aeruginosa/fisiología , Animales , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/metabolismo , Sitios de Unión , Biopelículas , Drosophila melanogaster , Regulación Bacteriana de la Expresión Génica , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Mutación , Fenazinas/metabolismo , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/patogenicidad , Proteínas Represoras/genética , Proteínas Represoras/metabolismoRESUMEN
Natural competency requires uptake of exogenous DNA from the environment and the integration of that DNA into recipient bacteria can be used for DNA-repair or genetic diversification. The Burkholderia genus is unique in that only some of the species and strains are naturally competent. We identified and characterized two genes, comE and crp, from naturally competent B. pseudomallei 1026b that play a role in DNA uptake and catabolism. Single-copies of rhamnose-inducible comE and crp genes were integrated into a Tn7 attachment-site in non-naturally competent Burkholderia including pathogens B. pseudomallei K96243, B. cenocepacia K56-2, and B. mallei ATCC23344. Strains expressing comE or crp were assayed for their ability to uptake and catabolize DNA. ComE and Crp allowed non-naturally competent Burkholderia species to catabolize DNA, uptake exogenous gfp DNA and express GFP. Furthermore, we used synthetic comE and crp to expand the utility of the λ-red recombineering system for genetic manipulation of non-competent Burkholderia species. A newly constructed vector, pKaKa4, was used to mutate the aspartate semialdehyde dehydrogenase (asd) gene in four B. mallei strains, leading to the complete attenuation of these tier-1 select-agents. These strains have been excluded from select-agent regulations and will be of great interest to the field.
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Burkholderia pseudomallei/genética , Genes Bacterianos/genética , Animales , Aspartato-Semialdehído Deshidrogenasa/genética , Línea Celular , Reparación del ADN/genética , ADN Bacteriano/genética , Técnicas Genéticas , Vectores Genéticos/genética , Ratones , Ratones Endogámicos BALB C , Células RAW 264.7RESUMEN
Gene regulation network in Pseudomonas aeruginosa is complex. With a relatively large genome (6.2 Mb), there is a significant portion of genes that are proven or predicted to be transcriptional regulators. Many of these regulators have been shown to play important roles in biofilm formation and maintenance. In this study, we present a novel transcriptional regulator, PA1226, which modulates biofilm formation and virulence in P. aeruginosa. Mutation in the gene encoding this regulator abolished the ability of P. aeruginosa to produce biofilms in vitro, without any effect on the planktonic growth. This regulator is also essential for the in vivo fitness and pathogenesis in both Drosophila melanogaster and BALB/c mouse lung infection models. Transcriptome analysis revealed that PA1226 regulates many essential virulence genes/pathways, including those involved in alginate, pili, and LPS biosynthesis. Genes/operons directly regulated by PA1226 and potential binding sequences were identified via ChIP-seq. Attempts to confirm the binding sequences by electrophoretic mobility shift assay led to the discovery of a co-regulator, PA1413, via co-immunoprecipitation assay. PA1226 and PA1413 were shown to bind collaboratively to the promoter regions of their regulons. A model is proposed, summarizing our finding on this novel dual-regulation system.
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Biopelículas/crecimiento & desarrollo , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/fisiología , Pseudomonas aeruginosa/patogenicidad , Factores de Transcripción/metabolismo , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Modelos Animales de Enfermedad , Drosophila melanogaster , Perfilación de la Expresión Génica , Humanos , Enfermedades Pulmonares/microbiología , Masculino , Ratones , Ratones Endogámicos BALB C , Pseudomonas aeruginosa/genética , Factores de Transcripción/genética , VirulenciaRESUMEN
Bacterial cooperative associations and dynamics in biofilm microenvironments are of special interest in recent years. Knowledge of localized gene-expression and corresponding bacterial behaviors within the biofilm architecture at a global scale has been limited, due to a lack of robust technology to study limited number of cells in stratified layers of biofilms. With our recent pioneering developments in single bacterial cell transcriptomic analysis technology, we generated herein an unprecedented spatial transcriptome map of the mature in vitro Pseudomonas aeruginosa biofilm model, revealing contemporaneous yet altered bacterial behaviors at different layers within the biofilm architecture (i.e., surface, middle and interior of the biofilm). Many genes encoding unknown functions were highly expressed at the biofilm-solid interphase, exposing a critical gap in the knowledge of their activities that may be unique to this interior niche. Several genes of unknown functions are critical for biofilm formation. The in vivo importance of these unknown proteins was validated in invertebrate (fruit fly) and vertebrate (mouse) models. We envisage the future value of this report to the community, in aiding the further pathophysiological understanding of P. aeruginosa biofilms. Our approach will open doors to the study of bacterial functional genomics of different species in numerous settings.
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Biopelículas/crecimiento & desarrollo , Regulación Bacteriana de la Expresión Génica , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidad , Animales , Proteínas Bacterianas/genética , Drosophila/genética , Ratones , Mutación , Transcriptoma , Factores de Virulencia/genéticaRESUMEN
Pseudomonas aeruginosa can grow to very high-cell-density (HCD) during infection of the cystic fibrosis (CF) lung. Phosphatidylcholine (PC), the major component of lung surfactant, has been hypothesized to support HCD growth of P. aeruginosa in vivo. The phosphorylcholine headgroup, a glycerol molecule, and two long-chain fatty acids (FAs) are released by enzymatic cleavage of PC by bacterial phospholipase C and lipases. Three different bacterial pathways, the choline, glycerol, and fatty acid degradation pathways, are then involved in the degradation of these PC components. Here, we identified five potential FA degradation (Fad) related fadBA-operons (fadBA1-5, each encoding 3-hydroxyacyl-CoA dehydrogenase and acyl-CoA thiolase). Through mutagenesis and growth analyses, we showed that three (fadBA145) of the five fadBA-operons are dominant in medium-chain and long-chain Fad. The triple fadBA145 mutant also showed reduced ability to degrade PC in vitro. We have previously shown that by partially blocking Fad, via mutagenesis of fadBA5 and fadDs, we could significantly reduce the ability of P. aeruginosa to replicate on FA and PC in vitro, as well as in the mouse lung. However, no studies have assessed the ability of mutants, defective in choline and/or glycerol degradation in conjunction with Fad, to grow on PC or in vivo. Hence, we constructed additional mutants (ΔfadBA145ΔglpD, ΔfadBA145ΔbetAB, and ΔfadBA145ΔbetABΔglpD) significantly defective in the ability to degrade FA, choline, and glycerol and, therefore, PC. The analysis of these mutants in the BALB/c mouse lung infection model showed significant inability to utilize PC in vitro, resulted in decreased replication fitness and competitiveness in vivo compared to the complement strain, although there was little to no variation in typical virulence factor production (e.g., hemolysin, lipase, and protease levels). This further supports the hypothesis that lung surfactant PC serves as an important nutrient for P. aeruginosa during CF lung infection.
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Colina/metabolismo , Ácidos Grasos/metabolismo , Glicerol/metabolismo , Fosfatidilcolinas/metabolismo , Pseudomonas aeruginosa/metabolismo , 3-Hidroxiacil-CoA Deshidrogenasa/genética , 3-Hidroxiacil-CoA Deshidrogenasa/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Fibrosis Quística/metabolismo , Fibrosis Quística/microbiología , Interacciones Huésped-Patógeno , Pulmón/metabolismo , Pulmón/microbiología , Pulmón/patología , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Mutagénesis , Mutación , Operón , Infecciones por Pseudomonas/metabolismo , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/fisiología , Homología de Secuencia de Aminoácido , Transducción de Señal/genéticaRESUMEN
The fatty acid (FA) degradation pathway of Pseudomonas aeruginosa, an opportunistic pathogen, was recently shown to be involved in nutrient acquisition during BALB/c mouse lung infection model. The source of FA in the lung is believed to be phosphatidylcholine, the major component of lung surfactant. Previous research indicated that P. aeruginosa has more than two fatty acyl-CoA synthetase genes (fadD; PA3299 and PA3300), which are responsible for activation of FAs using ATP and coenzyme A. Through a bioinformatics approach, 11 candidate genes were identified by their homology to the Escherichia coli FadD in the present study. Four new homologues of fadD (PA1617, PA2893, PA3860, and PA3924) were functionally confirmed by their ability to complement the E. coli fadD mutant on FA-containing media. Growth phenotypes of 17 combinatorial fadD mutants on different FAs, as sole carbon sources, indicated that the four new fadD homologues are involved in FA degradation, bringing the total number of P. aeruginosa fadD genes to six. Of the four new homologues, fadD4 (PA1617) contributed the most to the degradation of different chain length FAs. Growth patterns of various fadD mutants on plant-based perfumery substances, citronellic and geranic acids, as sole carbon and energy sources indicated that fadD4 is also involved in the degradation of these plant-derived compounds. A decrease in fitness of the sextuple fadD mutant, relative to the ΔfadD1D2 mutant, was only observed during BALB/c mouse lung infection at 24 h.
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Ácidos Grasos/metabolismo , Redes y Vías Metabólicas , Pseudomonas aeruginosa/metabolismo , Animales , Coenzima A Ligasas/genética , Coenzima A Ligasas/metabolismo , Ratones , Mutación , Fenotipo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crecimiento & desarrollo , Terpenos/metabolismo , Factores de Tiempo , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
Pseudomonas aeruginosa is a well-known clinical pathogen for its recalcitrant infection caused by biofilm formation which are initiated by flagella-mediated attachment. Sodium houttuyfonate (SH) is a natural phytoanticipin derivative of houttuynin and has anti-pathogenic effect on P. aeruginosa biofilm formation. In this paper, when using 1/2 × MIC SH, the diameter of P. aeruginosa swimming motility was sharply shortened to 36 % in 24 h incubation, and the fold changes of fliC required for swimming motility was 0.36 in 24 h cultivation, the adherence inhibition accounted for about 46 %, and the pyocyanin production decreased to 47 % after 1-day treatment and 56 % after 3-day treatment with obvious visual changes from dark green to light green, compared with the negative control. With the help of mass spectra and scanning electronic microscope, 1/2 × MIC SH was further testified to be enough to eradicate flagella and inhibit pyocyanin secretion of P. aeruginosa. The results do not only re-affirm the close interplay of attachment and virulence (i.e. swimming motility and pyocyanin), but also unravel the potential mechanism of SH on anti-biofilm of P. aeruginosa.
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Alcanos/farmacología , Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Flagelos/efectos de los fármacos , Pseudomonas aeruginosa/efectos de los fármacos , Piocianina/metabolismo , Sulfitos/farmacología , Proteínas Bacterianas/genética , Biopelículas/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Flagelos/fisiología , Pruebas de Sensibilidad Microbiana , Microscopía Electrónica de Rastreo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidad , Pseudomonas aeruginosa/fisiología , VirulenciaRESUMEN
OBJECTIVE: To study antimicrobial effect of Sodium houttuyfonate (SH) on Staphylococcus epidermidis (SE) and Candida albicans (CA). METHODS: The prepared strain broths (OD,o=0.05) containing SE and CA were firstly used to test the minimal inhibitory concentrations (MICs) of SH, azithromycin (AZM) and fluconazole (FLU) by micro-dilution method. Then the biofilms of SE and CA were matured in 96-well plates, and co-cultured with SH, AZM and FLU for 1, 2 and 3 days to assess the antibiofilm efficacies of the agents with different concentrations by crystal violet staining method. At last, the treated biofilms of SE and CA by 2x MIC agents were observed by scanning electronic microscope. RESULTS: The MICs of SE and CA were 256 and 1024 microg/ml, respectively. After the 1st, 2nd and 3rd day of medications, the suppressions of biofilm were about 60% (P < 0.01), 76% (P = 0.000) and 75% (P = 0.000) by 2 x MIC SH, the suppressions of biofilm were about 90% (P = 0.000), 88% (P = 0.000) and 90% (P = 0.000) by 2 x MIC SH, which could be testified by scanning electron microscope results. However, the inhibitions of biofilm attachment had no significant difference for SE by SH and azithromycin and CA by SH and fluconazole. CONCLUSION: SH had widely anti-pathogenic effect on pathogenic biofilm formation of either bacteria or fungus, had more influence on enclosed cells of SE and CA than the traditional antibiotics, revealing its target might be the extracellular polymeric substances, and was more active to inhibit the growth of CA than SE.