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

The Role of the Aryl Hydrocarbon Receptor (AhR) in the Immune Response against Microbial Infections

Aryl hydrocarbon receptor (AhR), an important nuclear receptor, regulates the cellular response to environmental stressors. It is well known for its critical functions in toxicology, but is currently considered an essential regulator of diseases, with specific modulatory effects on immune, antimicrobial and inflammatory responses. The present chapter discusses AhR’s function and mechanism in the immune response against microbial infections

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

Effects of N-G-monomethyl-L-arginine (NMMA) and catalase on intracellular survival of Vibrio alginolyticus in macrophages of large yellow croaker Pseudosciaena crocea

The intracellular survival of Vibrio alginolyticus and Vibrio parahaemolyticus in large yellow croaker macrophages of Pseudosciaena crocea was investigated. In addition, the effects of N-G-monomethyl-L-arginine (NMMA) and catalase on the interaction of V. alginolyticus with macrophages from head kidney and on macrophages reactive nitrogen intermediate (RNI) and reactive oxygen intermediate (ROI) in vitro were determined. V. alginolyticus ND-01 was able to survive in macrophages from head kidney of large yellow croakers for at least 3 h, while V. paraheamolyticus 1.1614 could not survive in the macrophages for 1 h. Intracellular bacterial survival was affected by the addition of specific inhibitors of macrophage oxidative function. Exposure of macrophages to NMMA and catalase decreased the number of viable cells of V. alginolyticus inside large yellow croaker macrophages. Furthermore, a close correlation was observed between the number of intracellular survival bacteria with the amount of NO and H2O2 produced by macrophages.National Department Public Benefit Research Foundation of China [200903029]; Natural Science Foundation of Fujian Province [2011J06014

Non-specific immune response of bullfrog Rana catesbeiana to intraperitoneal injection of bacterium Aeromonas hydrophila

Non-specific immune response of bullfrog Rana catesbeiana to pathogenic Aeromonas hydrophila was studied to 60 individuals in two groups. Each bullfrog in bacterium-injected group was injected intraperitoneally (i.p.) with 0.2 ml bacterial suspension at a density of 5.2 x 10(6) CFU/ml, hile each one in control group injected i.p. with 0.2 ml sterile saline solution (0.85%, w/v). Three bullfrogs in both groups were sampled at 0, 1, 3, 7, 11, 15 and 20 days post-injection (dpi) for the evaluation of non-specific immune parameters. It was observed that intraperitoneal injection of A. hydrophila significantly increased the number of leucocytes and that of NBT-positive cells in peripheral blood. Significant increases in serum bactericidal activity and serum acid phosphatase activity were also observed in the bacterium-injected frogs when compared with those in the control group. However, a significant reduction was detected in vitro in phagocytosis activity of peripheral blood phagocytes. No significant difference in changes in the number of peripheral erythrocytes, serum superoxide dismutase (SOD) activity, and lysozyme activity was detected between the two groups. It is suggested that bullfrogs may produce a series of non-specific immune reactions in response to the A. hydrophila infection.National High-Tech Research and Development Program of China [2001AA5070, 2002AA639600]; Natural Science Foundation of Fujian Province of China [B0410022, 2006F5066

Community Change and Pathogenicity of <em>Vibrio</em>

Vibrio is a rod-shaped Gram-negative bacteria, which is widely distributed in marine and estuarine environments worldwide. It is an important component of the aquatic ecosystem and plays an important role in biogeochemical cycle. Its population dynamics are usually affected by climate and seasonal factors. Most of the Vibrios in the environment are not pathogenic, but some of them are pathogenic bacteria for human and animal, such as Vibrio cholerae, Vibrio vulnificus, Vibrio parahaemolyticus, and Vibrio anguillarum, etc., which are generally reported to be related to aquatic animal diseases and human food-borne diseases. Over the last couple of years, due to the influence of the rising seawater temperature and climate change, the incidence of diseases caused by Vibrio infection has increased significantly, which poses a great threat to human health and aquaculture. The research on pathogenic Vibrio has attracted more and more attention. The abundance and community changes of Vibrio in the environment are usually controlled by many biological and abiotic factors. The Vibrio pathogenicity is related to the virulence factors encoded by virulence genes. The process of Vibrio infecting the host and causing host disease is determined by multiple virulence factors acting together, instead of being determined by a single virulence factor. In this chapter, community changes of Vibrio, as well as the virulence factors of Vibrio and the related virulence genes of Vibiro are summarized, and their important roles in Vibrio infection are also discussed

A simplified system without purification for selection of aptamers against Vibrio alginolyticus

We have explored a simplified system without purification for systematic evolution of ligands by exponential enrichment (SELEX) against Vibrio alginolyticus to select single-stranded DNA ligands (aptamers) from a random 82-nt library. The DNA content was quantified by agarose gel electrophoresis and follow-up image analyses with the BandScan 5.0 software. Anti-digoxigenin/HRP system was used to determine the binding activity of aptamers for V. alginolyticus. The results showed that the affinity of aptamers increased gradually with the increase of screening rounds, indicating that the lack of purification did not affect screening results but rather made SELEX screening much more convenient and significantly increased the efficiency