ABSTRACT
Shewanella putrefaciens CN-32 contains a single Type I-Fv CRISPR-Cas system which confers adaptive immunity against bacteriophage infection. Three Cas proteins (Cas6f, Cas7fv, Cas5fv) and mature CRISPR RNAs were shown to be required for the assembly of an interference complex termed Cascade. The Cas protein-CRISPR RNA interaction sites within this complex were identified via mass spectrometry. Additional Cas proteins, commonly described as large and small subunits, that are present in all other investigated Cascade structures, were not detected. We introduced this minimal Type I system in Escherichia coli and show that it provides heterologous protection against lambda phage. The absence of a large subunit suggests that the length of the crRNA might not be fixed and recombinant Cascade complexes with drastically shortened and elongated crRNAs were engineered. Size-exclusion chromatography and small-angle X-ray scattering analyses revealed that the number of Cas7fv backbone subunits is adjusted in these shortened and extended Cascade variants. Larger Cascade complexes can still confer immunity against lambda phage infection in E. coli Minimized Type I CRISPR-Cas systems expand our understanding of the evolution of Cascade assembly and diversity. Their adjustable crRNA length opens the possibility for customizing target DNA specificity.
Subject(s)
Bacterial Proteins/chemistry , CRISPR-Associated Proteins/chemistry , CRISPR-Cas Systems , Clustered Regularly Interspaced Short Palindromic Repeats , Escherichia coli/genetics , Shewanella putrefaciens/genetics , Amino Acid Sequence , Bacterial Proteins/genetics , Bacterial Proteins/immunology , Bacteriophage lambda/physiology , CRISPR-Associated Proteins/genetics , CRISPR-Associated Proteins/immunology , Escherichia coli/immunology , Escherichia coli/metabolism , Escherichia coli/virology , Gene Expression , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/immunology , Protein Subunits/chemistry , Protein Subunits/genetics , Protein Subunits/immunology , RNA, Bacterial/chemistry , RNA, Bacterial/genetics , RNA, Bacterial/immunology , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Sequence Alignment , Shewanella putrefaciens/immunology , Shewanella putrefaciens/metabolism , Shewanella putrefaciens/virology , Transformation, BacterialABSTRACT
Complement factor I (Cfi) is a soluble serine protease which plays a crucial role in the modulation of complement cascades. In the presence of substrate modulating cofactors (such as complement factor H, C4bp, CR1, etc), Cfi cleaves and inactivates C3b and C4b, thereby controlling the complement-mediated processes. In this study, we sequenced and characterized Cfi gene from Cynoglossus Semilaevis (designated as CsCfi) for the first time. The full-length cDNA of CsCfi was 2230 bp in length, including a 98 bp 5'-untranslated region (UTR), a 164 bp 3'-UTR and a 1968 bp open reading frame (ORF). It encoded a polypeptide of 656 amino acids, with a molecular mass of 72.28 kDa and an isoelectric point of 7.71. A signal peptide was defined at N-terminus, resulting in a 626-residue mature protein. Multiple sequence alignment revealed that Cfi proteins were well conserved with the typical modular architecture and identical active sites throughout the vertebrates, which suggested the conserved function of Cfi. Phylogenetic analysis indicated that CsCfi and the homologous Cfi sequences from teleosts clustered into a clade, separating from another clade from the cartilaginous fish and other vertebrates. Tissue expression profile analysis by quantitative real-time PCR (qRT-PCR) showed that CsCfi mRNA constitutively expressed in all tested tissues, with the predominant expression in liver and the lowest in stomach. Temporal expression levels of CsCfi after challenging with Vibrio anguillarum showed different expression patterns in intestine, spleen, skin, blood, head kidney and liver. The recombinant CsCfi (rCsCfi) protein showed broad-spectrum antimicrobial activities against the Gram-positive bacteria Staphylococcus aureus and the Gram-negative bacteria Escherichia coli, Pseudomonas aeruginosa and Shewanella putrefaciens. The research revealed that CsCfi plays an important role in C. Semilaevis immunity.
Subject(s)
Complement Factor I/genetics , Complement Factor I/immunology , Fish Diseases/immunology , Flatfishes/immunology , Animals , Base Sequence , Conserved Sequence/genetics , Escherichia coli/immunology , Fish Diseases/microbiology , Flatfishes/genetics , Gene Expression Profiling , Molecular Sequence Data , Phylogeny , Pseudomonas aeruginosa/immunology , Sequence Alignment , Sequence Analysis, DNA , Shewanella putrefaciens/immunology , Staphylococcus aureus/immunology , Vibrio/immunologyABSTRACT
BACKGROUND: In our previous studies the specific egg yolk antibody (IgY) against Shewanella putrefaciens (one of the specific spoilage organisms for marine products during aerobic chilling storage) demonstrated significant activity to prolong the shelf life of refrigerated fish. The exploitation of the antigen-binding fragment plus the hinge region (IgY-Fab') is now considered a promising method for improving the efficiency of such natural antimicrobial agents. RESULTS: The antimicrobial activity of IgY-Fab' against S. putrefaciens was investigated using refrigerated turbot as samples. By microbial, chemical and sensory tests, it was shown to be able to effectively inhibit bacterial growth and prolong the shelf life of samples, with an efficiency evaluated significantly higher than that of whole IgY with the same molarity. The interaction between IgY agents and S. putrefaciens cells was also investigated, and the IgY-Fab' showed a much greater ability to damage cell membranes than the whole IgY. CONCLUSION: Compared to whole IgY with the same molarity, IgY-Fab' demonstrated higher and more durable antimicrobial efficiency. Such a result was assumed to be closely related to its structural properties (such as the much lower molecular weight), which may enhance its ability to influence physiological activities of antigen bacteria, especially the property or/and structure of cell membranes.
