Structure and function of REP34 implicates carboxypeptidase activity in Francisella tularensis host cell invasion.

Publication Type:

Journal Article


J Biol Chem, Volume 289, Issue 44, p.30668-30679 (2014)


Amino Acid Sequence, Bacterial Proteins, Carboxypeptidases, Catalytic Domain, Cell Line, Francisella tularensis, Host-Pathogen Interactions, Humans, Models, Molecular, Molecular Sequence Data, Monocytes, Protein Structure, Secondary, Structural Homology, Protein, X-Ray Diffraction


<p>Francisella tularensis is the etiological agent of tularemia, or rabbit fever. Although F. tularensis is a recognized biothreat agent with broad and expanding geographical range, its mechanism of infection and environmental persistence remain poorly understood. Previously, we identified seven F. tularensis proteins that induce a rapid encystment phenotype (REP) in the free-living amoeba, Acanthamoeba castellanii. Encystment is essential to the pathogen&#39;s long term intracellular survival in the amoeba. Here, we characterize the cellular and molecular function of REP34, a REP protein with a mass of 34 kDa. A REP34 knock-out strain of F. tularensis has a reduced ability to both induce encystment in A. castellanii and invade human macrophages. We determined the crystal structure of REP34 to 2.05-Å resolution and demonstrate robust carboxypeptidase B-like activity for the enzyme. REP34 is a zinc-containing monomeric protein with close structural homology to the metallocarboxypeptidase family of peptidases. REP34 possesses a novel topology and substrate binding pocket that deviates from the canonical funnelin structure of carboxypeptidases, putatively resulting in a catalytic role for a conserved tyrosine and distinct S1&#39; recognition site. Taken together, these results identify REP34 as an active carboxypeptidase, implicate the enzyme as a potential key F. tularensis effector protein, and may help elucidate a mechanistic understanding of F. tularensis infection of phagocytic cells.</p>