Genome-Wide Identification and Immune Response Analysis of Serine Protease Inhibitor Genes in the Silkworm, Bombyx mori

In most insect species, a variety of serine protease inhibitors (SPIs) have been found in multiple tissues, including integument, gonad, salivary gland, and hemolymph, and are required for preventing unwanted proteolysis. These SPIs belong to different families and have distinct inhibitory mechanisms. Herein, we predicted and characterized potential SPI genes based on the genome sequences of silkworm, Bombyx mori. As a result, a total of eighty SPI genes were identified in B. mori. These SPI genes contain 10 kinds of SPI domains, including serpin, Kunitz_BPTI, Kazal, TIL, amfpi, Bowman-Birk, Antistasin, WAP, Pacifastin, and alpha-macroglobulin. Sixty-three SPIs contain single SPI domain while the others have at least two inhibitor units. Some SPIs also contain non-inhibitor domains for protein-protein interactions, including EGF, ADAM_spacer, spondin_N, reeler, TSP_1 and other modules. Microarray analysis showed that fourteen SPI genes from lineage-specific TIL family and Group F of serpin family had enriched expression in the silk gland. The roles of SPIs in resisting pathogens were investigated in silkworms when they were infected by four pathogens. Microarray and qRT-PCR experiments revealed obvious up-regulation of 8, 4, 3 and 3 SPI genes after infection with Escherichia coli, Bacillus bombysepticus, Beauveria bassiana or B. mori nuclear polyhedrosis virus (BmNPV), respectively. On the contrary, 4, 11, 7 and 9 SPI genes were down-regulated after infection with E. coli, B. bombysepticus, B. bassiana or BmNPV, respectively. These results suggested that these SPI genes may be involved in resistance to pathogenic microorganisms. These findings may provide valuable information for further clarifying the roles of SPIs in the development, immune defence, and efficient synthesis of silk gland protein

Identification and Isolation of Brucella suis Virulence Genes Involved in Resistance to the Human Innate Immune System▿

Brucella strains are facultative intracellular pathogens that induce chronic diseases in humans and animals. This observation implies that Brucella subverts innate and specific immune responses of the host to develop its full virulence. Deciphering the genes involved in the subversion of the immune system is of primary importance for understanding the virulence of the bacteria, for understanding the pathogenic consequences of infection, and for designing an efficient vaccine. We have developed an in vitro system involving human macrophages infected by Brucella suis and activated syngeneic γ9δ2 T lymphocytes. Under these conditions, multiplication of B. suis inside macrophages is only slightly reduced. To identify the genes responsible for this reduced sensitivity, we screened a library of 2,000 clones of transposon-mutated B. suis. For rapid and quantitative analysis of the multiplication of the bacteria, we describe a simple method based on Alamar blue reduction, which is compatible with screening a large library. By comparing multiplication inside macrophages alone and multiplication inside macrophages with activated γ9δ2 T cells, we identified four genes of B. suis that were necessary to resist to the action of the γ9δ2 T cells. The putative functions of these genes are discussed in order to propose possible explanations for understanding their exact role in the subversion of innate immunity

Comparative gut transcriptome analysis reveals differences between virulent and avirulent Russian wheat aphids, Diuraphis noxia

Citation: Anathakrishnan, R., Sinha, D. K., Murugan, M., Zhu, K. Y., Chen, M. S., Zhu, Y. C., & Smith, C. M. (2014). Comparative gut transcriptome analysis reveals differences between virulent and avirulent Russian wheat aphids, Diuraphis noxia. Retrieved from http://krex.ksu.eduThe Russian wheat aphid, Diuraphis noxia, is a destructive pest of cereal crops that exhibits virulence to D. noxia resistance genes in wheat. Therefore, it is important to identify D. noxia virulence factors. The insect gut, the primary site of defense to ingested toxins, is also a likely site of differential gene expression in virulent insects. Comparative analyses of gut transcriptomes from virulent and avirulent D. noxia can improve an understanding of aphid gut physiology and may reveal factors critical to compatible D. noxia-wheat interactions. A total of 4, 600 clones were sequenced from gut cDNA libraries prepared from avirulent (biotype 1) and virulent (biotype 2) D. noxia feeding on biotype 1-resistant wheat. A majority of the sequences (66% in biotype 1, 64% in biotype 2) matched those from the NR database. BLASTX analysis of sequences with the highest E-values revealed that 59% of the biotype 1 sequences matched those of the pea aphid, Acyrthosiphon pisum. However, only 17% of the biotype 2 sequences were similar to those of A. pisum. RT-qPCR expression analyses confirmed that the biotype 2 gut transcriptome differs significantly from that of biotype 1. A transcript coding the tRNA-Leu gene was significantly up-regulated in the biotype 2 transcriptome, strongly suggesting that leucine metabolism is a critical factor in biotype 2 survival. Many more transcripts encoding protease inhibitors occurred in the avirulent biotype 1 gut than in the gut of virulent biotype 2. However, more protease transcripts occurred in the biotype 2 gut than in the biotype 1 gut, suggesting that the avirulent biotype produces protease inhibitors in response to plant proteases. The virulent biotype 2 produces trypsin-like and chymotrypsin-like serine protease counter-defenses to overcome biotype 1-resistant plants