Functional characterization and localization of a Bacillus subtilis sortase and its substrate and use of this sortase system to covalently anchor a heterologous protein to the B. subtilis cell wall for surface display.

Sortases catalyze the covalent anchoring of proteins to the cell surface on Gram-positive bacteria. Bioinformatic analysis suggests the presence of structural genes encoding sortases and their substrates in the Bacillus subtilis genome. In this study, a β-lactamase reporter was fused to the cell wall anchoring domain from a putative sortase substrate, YhcR.

Development of a LytE-based high-density surface display system in Bacillus subtilis.

The three N-terminal, tandemly arranged LysM motifs from a Bacillus subtilis cell wall hydrolase, LytE, formed a cell wall-binding module. This module, designated CWBM(LytE), was demonstrated to have tight cell wall-binding capability and could recognize two classes of cell wall binding sites with fivefold difference in affinity. The lower-affinity sites were approximately three times more abundant.

Centrosome replication in hydroxyurea-arrested CHO cells expressing GFP-tagged centrin2.

Centrosome duplication is tightly coupled with the cell cycle and neither too many nor too few centrosomes are induced in a normal cell. To study how centrosome assembly is regulated, we analyzed the abnormal process of multiple centrosome replications in Chinese hamster ovary (CHO) cells induced by hydroxyurea (HU), which is known to uncouple the centrosome cycle from the cell cycle. Green fluorescent protein (GFP)-tagged centrin2 expressed in CHO cells labels both centrioles and the pericentriolar material (PCM).

NK cells use perforin rather than granulysin for anticryptococcal activity.

Cytotoxic lymphocytes have the capacity to kill microbes directly; however, the mechanisms involved are poorly understood. Using Cryptococcus neoformans, which causes a potentially fatal fungal infection in HIV-infected patients, our previous studies showed that granulysin is necessary, while perforin is dispensable, for CD8 T lymphocyte fungal killing. By contrast, the mechanisms by which NK cells exert their antimicrobial activity are not clear, and in particular, the contribution of granulysin and perforin to NK-mediated antifungal activity is unknown.