Role of the ArcAB two-component system in the resistance of Escherichia coli to reactive oxygen stress

<p>Abstract</p> <p>Background</p> <p>The global regulatory system ArcAB controls the anaerobic growth of <it>E. coli</it>, however, its role in aerobic conditions is not well characterized. We have previously reported that ArcA was necessary for <it>Salmonella </it>to resist reactive oxygen species (ROS) in aerobic conditions.</p> <p>Results</p> <p>To investigate the mechanism of ROS resistance mediated by ArcAB, we generated deletion mutants of ArcA and ArcB in <it>E. coli</it>. Our results demonstrated that both ArcA and ArcB were necessary for resistance to hydrogen peroxide (H₂O₂), a type of ROS, and their function in this resistance was independent from H₂O₂scavenge. Mutagenesis analysis of ArcA indicated that ROS resistance was mediated through a distinct signaling pathway from that used in anaerobic conditions. An abundant protein flagellin was elevated at both the protein and mRNA levels in the <it>ΔarcA </it>mutant as compared to the wild type <it>E. coli</it>, and deletion of flagellin restored the resistance of the <it>ΔarcA </it>mutant to H₂O₂. The resistance of the <it>ΔarcA </it>mutant <it>E. coli </it>to H₂O₂can also be restored by amino acid supplementation, suggesting that a deficiency in amino acid and/or protein synthesis in the mutant contributed to its susceptibility to H₂O₂, which is consistent with the notion that protein synthesis is necessary for ROS resistance.</p> <p>Conclusion</p> <p>Our results suggest that in addition to its role as a global regulator for anaerobic growth of bacteria, ArcAB system is also important for bacterial resistance to ROS in aerobic conditions, possibly through its influence on bacterial metabolism, especially amino acid and/or protein assimilation and synthesis.</p

Inhibition of Murine Cytomegalovirus Infection in Animals by RNase P-Associated External Guide Sequences.

External guide sequence (EGS) RNAs are associated with ribonuclease P (RNase P), a tRNA processing enzyme, and represent promising agents for gene-targeting applications as they can direct RNase-P-mediated cleavage of a target mRNA. Using murine cytomegalovirus (MCMV) as a model system, we examined the antiviral effects of an EGS variant, which was engineered using in&nbsp;vitro selection procedures. EGSs were used to target the shared mRNA region of MCMV capsid scaffolding protein (mCSP) and assemblin. In&nbsp;vitro, the EGS variant was 60 times more active in directing RNase P cleavage of the target mRNA than the EGS originating from a natural tRNA. In MCMV-infected cells, the variant reduced mCSP expression by 92% and inhibited viral growth by 8,000-fold. In MCMV-infected mice hydrodynamically transfected with EGS-expressing constructs, the EGS variant was more effective in reducing mCSP expression, decreasing viral production, and enhancing animal survival than the EGS originating from a natural tRNA. These results provide direct evidence that engineered EGS variants with higher targeting activity in&nbsp;vitro are also more effective in reducing gene expression in animals. Furthermore, our findings imply the possibility of engineering potent EGS variants for therapy of viral infections

Mass spectrometry-based quantitative proteomic analysis of Salmonella enterica serovar Enteritidis protein expression upon exposure to hydrogen peroxide

<p>Abstract</p> <p>Background</p> <p><it>Salmonella </it><it>enterica</it>, a common food-borne bacterial pathogen, is believed to change its protein expression profile in the presence of different environmental stress such as that caused by the exposure to hydrogen peroxide (H₂O₂), which can be generated by phagocytes during infection and represents an important antibacterial mechanism of host cells. Among <it>Salmonella </it>proteins, the effectors of <it>Salmonella </it>pathogenicity island 1 and 2 (SPI-1 and SPI-2) are of particular interest since they are expressed during host infection <it>in vivo </it>and are important for invasion of epithelial cells and for replication in organs during systemic infection, respectively. However, the expression profiles of these proteins upon exposure to H₂O₂or to host cells <it>in vivo </it>during the established phase of systemic infection have not been extensively studied.</p> <p>Results</p> <p>Using stable isotope labeling coupled with mass spectrometry, we performed quantitative proteomic analysis of <it>Salmonella </it><it>enterica </it>serovar Enteritidis and identified 76 proteins whose expression is modulated upon exposure to H₂O₂. SPI-1 effector SipC was expressed about 3-fold higher and SopB was expressed approximately 2-fold lower in the presence of H₂O₂, while no significant change in the expression of another SPI-1 protein SipA was observed. The relative abundance of SipA, SipC, and SopB was confirmed by Western analyses, validating the accuracy and reproducibility of our approach for quantitative analysis of protein expression. Furthermore, immuno-detection showed substantial expression of SipA and SipC but not SopB in the late phase of infection in macrophages and in the spleen of infected mice.</p> <p>Conclusions</p> <p>We have identified <it>Salmonella </it>proteins whose expression is modulated in the presence of H₂O₂. Our results also provide the first direct evidence that SipC is highly expressed in the spleen at late stage of salmonellosis <it>in vivo</it>. These results suggest a possible role of SipC and other regulated proteins in supporting survival and replication of <it>Salmonella </it>under oxidative stress and during its systemic infection <it>in vivo</it>.</p

A Salmonella Small Non-Coding RNA Facilitates Bacterial Invasion and Intracellular Replication by Modulating the Expression of Virulence Factors

Small non-coding RNAs (sRNAs) that act as regulators of gene expression have been identified in all kingdoms of life, including microRNA (miRNA) and small interfering RNA (siRNA) in eukaryotic cells. Numerous sRNAs identified in Salmonella are encoded by genes located at Salmonella pathogenicity islands (SPIs) that are commonly found in pathogenic strains. Whether these sRNAs are important for Salmonella pathogenesis and virulence in animals has not been reported. In this study, we provide the first direct evidence that a pathogenicity island-encoded sRNA, IsrM, is important for Salmonella invasion of epithelial cells, intracellular replication inside macrophages, and virulence and colonization in mice. IsrM RNA is expressed in vitro under conditions resembling those during infection in the gastrointestinal tract. Furthermore, IsrM is found to be differentially expressed in vivo, with higher expression in the ileum than in the spleen. IsrM targets the mRNAs coding for SopA, a SPI-1 effector, and HilE, a global regulator of the expression of SPI-1 proteins, which are major virulence factors essential for bacterial invasion. Mutations in IsrM result in disregulation of expression of HilE and SopA, as well as other SPI-1 genes whose expression is regulated by HilE. Salmonella with deletion of isrM is defective in bacteria invasion of epithelial cells and intracellular replication/survival in macrophages. Moreover, Salmonella with mutations in isrM is attenuated in killing animals and defective in growth in the ileum and spleen in mice. Our study has shown that IsrM sRNA functions as a pathogenicity island-encoded sRNA directly involved in Salmonella pathogenesis in animals. Our results also suggest that sRNAs may represent a distinct class of virulence factors that are important for bacterial infection in vivo

Molecular Characterization of Highly Pathogenic H5N1 Avian Influenza A Viruses Isolated from Raccoon Dogs in China

The highly pathogenic avian influenza H5N1 virus can infect a variety of animals and continually poses a threat to animal and human health. While many genotypes of H5N1 virus can be found in chicken, few are associated with the infection of mammals. Characterization of the genotypes of viral strains in animal populations is important to understand the distribution of different viral strains in various hosts. This also facilitates the surveillance and detection of possible emergence of highly pathogenic strains of specific genotypes from unknown hosts or hosts that have not been previously reported to carry these genotypes.Two H5N1 isolates were obtained from lung samples of two raccoon dogs that had died from respiratory disease in China. Pathogenicity experiments showed that the isolates were highly pathogenic to chicken. To characterize the genotypes of these viruses, their genomic sequences were determined and analyzed. The genetic contents of these isolates are virtually identical and they may come from the same progenitor virus. Phylogenetic analysis indicated that the isolates were genetically closely related to genotype V H5N1 virus, which was first isolated in China in 2003, and were distinct from the dominant virus genotypes (e.g. genotype Z) of recent years. The isolates also contain a multibasic amino acid motif at their HA cleavage sites and have an E residue at position 627 of the PB2 protein similar to the previously-identified avian viruses.This is the first report that genotype V H5N1 virus is found to be associated with a mammalian host. Our results strongly suggest that genotype V H5N1 virus has the ability to cross species barriers to infect mammalian animals. These findings further highlight the risk that avian influenza H5N1 virus poses to mammals and humans, which may be infected by specific genotypes that are not known to infect these hosts

A Novel Performance Adaptation and Diagnostic Method for Aero-Engines Based on the Aerothermodynamic Inverse Model

Aero-engines are faced with severe challenges of availability and reliability in the increasing operation, and traditional gas path filtering diagnostic methods have limitations restricted by various factors such as strong nonlinearity of the system and lack of critical sensor information. A method based on the aerothermodynamic inverse model (AIM) is proposed to improve the adaptation accuracy and fault diagnostic dynamic estimation response speed in this paper. Thermodynamic mechanisms are utilized to develop AIM, and scaling factors are designed to be calculated iteratively in the presence of measurement correction. In addition, the proposed method is implemented in combination with compensation of the nonlinear filter for real-time estimation of health parameters under the hypothesis of estimated dimensionality reduction. Simulations involved experimental datasets revealed that the maximum average simulated error decreased from 13.73% to 0.46% through adaptation. It was also shown that the dynamic estimated convergence time of the improved diagnostic method reached 2.183 s decrease averagely without divergence compared to the traditional diagnostic method. This paper demonstrates the proposed method has the capacity to generalize aero-engine adaptation approaches and to achieve unbiased estimation with fast convergence in performance diagnostic techniques

A Novel Performance Adaptation and Diagnostic Method for Aero-Engines Based on the Aerothermodynamic Inverse Model

Aero-engines are faced with severe challenges of availability and reliability in the increasing operation, and traditional gas path filtering diagnostic methods have limitations restricted by various factors such as strong nonlinearity of the system and lack of critical sensor information. A method based on the aerothermodynamic inverse model (AIM) is proposed to improve the adaptation accuracy and fault diagnostic dynamic estimation response speed in this paper. Thermodynamic mechanisms are utilized to develop AIM, and scaling factors are designed to be calculated iteratively in the presence of measurement correction. In addition, the proposed method is implemented in combination with compensation of the nonlinear filter for real-time estimation of health parameters under the hypothesis of estimated dimensionality reduction. Simulations involved experimental datasets revealed that the maximum average simulated error decreased from 13.73% to 0.46% through adaptation. It was also shown that the dynamic estimated convergence time of the improved diagnostic method reached 2.183 s decrease averagely without divergence compared to the traditional diagnostic method. This paper demonstrates the proposed method has the capacity to generalize aero-engine adaptation approaches and to achieve unbiased estimation with fast convergence in performance diagnostic techniques

Association of Salmonella enterica Serovar Enteritidis YafD with Resistance to Chicken Egg Albumen

Salmonella enterica serovar Enteritidis is a major cause of food-borne diseases in industrialized countries. The incidence of S. enterica serovar Enteritidis infections has increased substantially in recent decades, and S. enterica serovar Enteritidis is now one of the leading serovars of Salmonella in the United States. A unique epidemiological characteristic of S. enterica serovar Enteritidis is its association with chicken shell eggs, since approximately 80% of all human gastrointestinal diseases can be traced to contaminated egg products. Eggs are contaminated when bacteria from reproductive tissues of infected hens are packaged into the eggs and persist inside the hostile egg albumen environment. Therefore, resistance to egg albumen is an important aspect in the transmission of S. enterica serovar Enteritidis. We identified a gene, yafD from S. enterica serovar Enteritidis, whose overexpression conferred upon S. enterica serovar Typhimurium enhanced resistance to egg albumen, while disruption of this gene in S. enterica serovar Enteritidis rendered the organism more susceptible to egg albumen. YafD is homologous to members of an exonuclease-endonuclease-phosphatase family, including some enzymes involved in DNA repair. Furthermore, we discovered that egg albumen has nuclease activities and uses both circular and linear DNA as substrates. We propose that YafD provides a survival advantage to S. enterica serovar Enteritidis in eggs by repairing DNA damage caused by egg albumen and that it may be one of the biologic determinants that contribute to the epidemiological association of S. enterica serovar Enteritidis with egg products

A Novel Adaptive Generation Method for Initial Guess Values of Component-Level Aero-Engine Start-Up Models

To solve the difficult problem of selecting initial guess values for component-level aero-engine start-up models, a novel method based on the flow-based back-calculation algorithm (FBBCA) is investigated. By exploiting the monotonic feature of low-speed aero-engine component characteristics and the principle of flow balance abided by components in the start-up process, this method traverses all the flows in each component characteristic at a given engine rotor speed. This method also limits the pressure ratios and flow rates of each component, along with the surplus power of the high-pressure rotor. Finally, a set of “fake initial values” for iterative calculation of the aero-engine start-up model can be generated and approximate true initial guess values that meet the accuracy requirement according to the Newton–Raphson iteration method. Extensive simulation verifies the low computational cost and high computational accuracy of this method as a solver for the initial guess values of the aero-engine start-up model