Dual RNA-Seq Unveils Pseudomonas plecoglossicida htpG Gene Functions During Host-Pathogen Interactions With Epinephelus coioides

Pseudomonas plecoglossicida is a temperature-dependent opportunistic pathogen which is associated with a variety of diseases in fish. During the development of “white nodules” disease, the expression of htpG in P. plecoglossicida was found to be significantly up-regulated at its virulent temperature of 18°C. The infection of htpG-RNAi strain resulted in the onset time delay, reduction in mortality and infection symptoms in spleen of Epinephelus coioides, and affected the bacterial tissue colonization. In order to reveal the effect of htpG silencing of P. plecoglossicida on the virulence regulation in P. plecoglossicida and immune response in E. coioides, dual RNA-seq was performed and a pathogen-host integration network was constructed. Our results showed that infection induced the expression of host genes related to immune response, but attenuated the expression of bacterial virulence genes. Novel integration was found between host immune genes and bacterial virulence genes, while IL6, IL1R2, IL1B, and TLR5 played key roles in the network. Further analysis with GeneMANIA indicated that flgD and rplF might play key roles during the htpG-dependent virulence regulation, which was in accordance with the reduced biofilm production, motility and virulence in htpG-RNAi strain. Meanwhile, IL6 and IL1B were found to play key roles during the defense against P. plecoglossicida, while CELA2, TRY, CPA1, CPA2, and CPB1 were important targets for P. plecoglossicida attacking to the host

Integration of Transcriptomic and Proteomic Approaches Reveals the Temperature-Dependent Virulence of Pseudomonas plecoglossicida

Pseudomonas plecoglossicida is a facultative pathogen that is associated with diseases of multiple fish, mainly at 15–20°C. Although fish disease caused by P. plecoglossicida has led to significant economic losses, the mechanisms of the temperature-dependent virulence are unclear. Here, we identify potential pathogenicity mechanisms and demonstrate the direct regulation of several virulence factors by temperature with transcriptomic and proteomic analyses, quantitative real-time PCR (qRT-PCR), RNAi, pyoverdine (PVD) quantification, the chrome azurol S (CAS) assay, growth curve measurements, a biofilm assay, and artificial infection. The principal component analysis, the heat map generation and hierarchical clustering, together with the functional annotations of the differentially expressed genes (DEGs) demonstrated that, under different growth temperatures, the animation and focus of P. plecoglossicida are quite different, which may be the key to pathogenicity. Genes involved in PVD synthesis and in the type VI secretion system (T6SS) are specifically upregulated at the virulent temperature of 18°C. Silencing of the PVD-synthesis-related genes reduces the iron acquisition, growth, biofilm formation, distribution in host organs and virulence of the bacteria. Silencing of the T6SS genes also leads to the reduction of biofilm formation, distribution in host organs and virulence. These findings reveal that temperature regulates multiple virulence mechanisms in P. plecoglossicida, especially through iron acquisition and T6SS secretion. Meanwhile, integration of transcriptomic and proteomic data provide us with a new perspective into the pathogenesis of P. plecoglossicida, which would not have been easy to catch at either the protein or mRNA differential analyses alone, thus illustrating the power of multi-omics analyses in microbiology

A High Performance Torque Sensor for Milling Based on a Piezoresistive MEMS Strain Gauge

In high speed and high precision machining applications, it is important to monitor the machining process in order to ensure high product quality. For this purpose, it is essential to develop a dynamometer with high sensitivity and high natural frequency which is suited to these conditions. This paper describes the design, calibration and performance of a milling torque sensor based on piezoresistive MEMS strain. A detailed design study is carried out to optimize the two mutually-contradictory indicators sensitivity and natural frequency. The developed torque sensor principally consists of a thin-walled cylinder, and a piezoresistive MEMS strain gauge bonded on the surface of the sensing element where the shear strain is maximum. The strain gauge includes eight piezoresistances and four are connected in a full Wheatstone circuit bridge, which is used to measure the applied torque force during machining procedures. Experimental static calibration results show that the sensitivity of torque sensor has been improved to 0.13 mv/Nm. A modal impact test indicates that the natural frequency of torque sensor reaches 1216 Hz, which is suitable for high speed machining processes. The dynamic test results indicate that the developed torque sensor is stable and practical for monitoring the milling process

Disruption of chemotaxis-related genes affects multiple cellular processes and the virulence of pathogenic Vibrio harveyi

National Natural Science Foundation of China [31272699, 41176115]; National Department Public Benefit Research Foundation of China [200903029]; Natural Science Foundation of Fujian Province [2011J06014]; National Hi-Tech Research and Development Program of China (863 Program) [2007AA09Z115]Chemotactic motility is involved in the virulence of many pathogenic bacteria. In order to understand the role of chemotactic motility of Vibrio harveyi in cellular processes and virulence, mini-Tn10/Kan transposon-induced mutants with deficient chemotactic motility were constructed, screened, and identified. Sequence analysis revealed that the 465-bp fragment (Gen Bank accession number HM630274) flanking the transposon insertion site in mutant TS-CM1 had the highest identity (96.9%) with a hypothetical protein gene of V. harveyi ATCC BAA-1116 and the second-highest identity (91.8%) with the pgk gene of V. parahaemolyticus RIMD 2210633. In another mutant, TS-CM2, 356 bp of transposon-flanking sequence (Gen Bank accession number HM630275) also showed the highest identity (94.6%) with a hypothetical protein gene of V. harveyi ATCC BAA-1116 and the second-highest identity (92.4%) with the flaB gene of V. alginolyticus HY9901. Studies on virulence-related biological characteristics such as growth, motility, adhesion, and infectivity of themutants showed that disruption of either the flagellin gene or energy metabolism gene led to subsequent loss of chemotactic motility and changes in growth, motility, adhesion, and virulence of the pathogenic V. harveyi. Hence, the flagellin gene and crucial energy metabolism gene played an important role in the chemotactic motility of V. harveyi