Growth inhibition by bacterial Cas2Em proteins expressed in mammalian cells.

BACKGROUND: Clustered regularly interspaced short palindromic repeats (CRISPRs) and the CRISPR-associated (Cas) proteins are bacterial adaptive immune system for survival. In our previous study, we demonstrate that polyploid giant bacterial cells (PGBC) induced by Cas2 protein is a step required by new spacer acquisition reaction catalyzed by Cas1/Cas2 complex. We also demonstrated that a carboxyl terminal domain on Cas2Em (the protein Cas2 cloned from Elizabethkingia meningoseptica) is sufficient and enough for PGBC. Thus, the potential role of Cas2Em in microbial-host interaction was explored in this study. METHODS: The impacts of Cas2Em on growth of both CHO-K1 and Hela cells were investigated. The subcellular localization and potential molecular target of Ca2Em were studied. RESULTS: The growth of mammalian cells were inhibited by Cas2Em protein via G1 arresting and apoptosis. In addition, we also demonstrated that Cas2Em was tightly associated with nuclear outer membrane and could be immunoprecipitated with 14-3-3γ through a 30 amino acid domain (homology of CLK2). CONCLUSION: Cas2Em significantly suppressed the growth of mammalian cells indicating Cas2 proteins play an important role in mammalian cells.

Filamentation initiated by Cas2 and its association with the acquisition process in cells.

Cas1-and-Cas2-mediated new spacer acquisition is an essential process for bacterial adaptive immunity. The process is critical for the ecology of the oral microflora and oral health. Although molecular mechanisms for spacer acquisition are known, it has never been established if this process is associated with the morphological changes of bacteria. In this study, we demonstrated a novel Cas2-induced filamentation phenotype in E. coli that was regulated by co-expression of the Cas1 protein. A 30 amino acid motif at the carboxyl terminus of Cas2 is necessary for this function. By imaging analysis, we provided evidence to argue that Cas-induced filamentation is a step coupled with new spacer acquisition during which filaments are characterised by polyploidy with asymmetric cell division. This work may open new opportunities to investigate the adaptive immune response and microbial balance for oral health.

Identification and characterization of a novel glycoprotein core xylosidase from the bacterium Elizabethkingia meningoseptica.

Although core xylose on glycoproteins has been implicated in allergy, infection and other biological processes, research on core xylose modification is rare. The lack of a ß-d-xylosidase that can catalytically remove the core xylose directly from glycoproteins is a reason for this. Through functional genomic analysis, we identified a glycoprotein core xylosidase and named it gpcXase I. gpcXase I is located immediately downstream of glycoprotein core fucosidase cFase I in Elizabethkingia meningoseptica. These two genes form a functional operon for glycoprotein core modifications. Three acidic residues (Asp-200, Asp-304 and Glu-649) were identified as key catalytic sites for gpcXase I activity, suggesting a unique triacdic mechanize for its activity. Asp-200 was identified a novel and essential base catalysts in the catalytic process, Asp-304 and Glu-649 was function as catalytic nucleophiles and acid catalysts, respectively. In addition, IgE-specific reactions were detected in 55% of serum samples collected from 40 allergic patients, and the reactions were significantly attenuated by removal of the core xylose of the allergen by treatment with gpcXase I. gpcXase I is a novel tool for basic and clinical glycomics.

Identification and characterization of a core fucosidase from the bacterium Elizabethkingia meningoseptica.

All reported α-l-fucosidases catalyze the removal of nonreducing terminal l-fucoses from oligosaccharides or their conjugates, while having no capacity to hydrolyze core fucoses in glycoproteins directly. Here, we identified an α-fucosidase from the bacterium Elizabethkingia meningoseptica with catalytic activity against core α-1,3-fucosylated substrates, and we named it core fucosidase I (cFase I). Using site-specific mutational analysis, we found that three acidic residues (Asp-242, Glu-302, and Glu-315) in the predicted active pocket are critical for cFase I activity, with Asp-242 and Glu-315 acting as a pair of classic nucleophile and acid/base residues and Glu-302 acting in an as yet undefined role. These findings suggest a catalytic mechanism for cFase I that is different from known α-fucosidase catalytic models. In summary, cFase I exhibits glycosidase activity that removes core α-1,3-fucoses from substrates, suggesting cFase I as a new tool for glycobiology, especially for studies of proteins with core fucosylation.