Identification of a novel family of cysteine proteases and their functions in bacterial pathogenesis.

Abstract

Pathogenic <italic>Yersinia</italic> utilizes a type III secretion system to inject six effectors into the eukaryotic host cell. These proteins function to paralyze the host immune response, thereby ensuring the survival and proliferation of the bacteria. One of the effectors, YopT, is a cytotoxin capable of disrupting the host cell cytoskeleton. My thesis research was initially aimed at revealing the biochemical function of YopT. Using bioinformatics approaches I have identified a group of more than 20 proteins from a variety of bacterial pathogens, each showing distant sequence identity to YopT. This novel family of proteins was termed the YopT family. Sequence alignment shows a characteristic Cys/His/Asp triad conserved in all the YopT family members. Mutation of the Cys/His/Asp residues completely abolishes the cytotoxicity of YopT. A yeast suppressor screen and subsequent protein-protein interaction studies suggest that Rho GTPases could be the target of YopT. Biochemical and functional studies demonstrate that YopT functions as a cysteine protease, cleaving N-terminal to the prenylated cysteine at the C-termini of Rho GTPases. The proteolytic activity of YopT relies on the invariant Cys/His/Asp residues. Proteolytic cleavage of Rho GTPases by YopT leads to the loss of their membrane attachment, which results in the collapse of the actin cytoskeleton of the host cells. This is the first example of proteolytic inactivation of Rho GTPases by a bacterial pathogen, suggesting a novel mechanism in bacterial pathogenesis. Further biochemical studies show that the C-terminal polybasic sequence and prenylated cysteine methylester of the GTPases are important for YopT recognition and cleavage. The proteolytic triad (Cys/His/Asp) is conserved in the entire YopT family, suggesting that YopT defines a novel family of cysteine proteases. AvrPphB is another member in the YopT family from the plant bacterial pathogen <italic>P. syringae</italic>. AvrPphB undergoes auto-proteolytic cleavage to expose an N-myristoylation motif. This cleavage requires the invariant Cys/His/Asp triad. I further demonstrate that AvrPphB proteolytically cleaves its resistance kinase PBS1 in <italic>Arabidopsis</italic> both <italic>in vitro</italic> and <italic>in vivo</italic>. Inactivation of PBS1 kinase leads to the disease resistance triggered by AvrPphB. This finding provides the beginning of a detailed molecular understanding of plant disease resistance.