Abstract
The foodborne pathogen Listeria monocytogenes employs a number of virulence determinants including metalloproteases to infect hosts. Here for the first time, we identified an M29 family aminopeptidase T (encoded by lmo1603) from L. monocytogenes that possesses a typical feature to catalyze the cleavage of amino acids from peptide substrates, with a preference for arginine. The purified recombinant Lmo1603 was activated by Fe3+, Zn2+ and Mn2+, but strongly stimulated by Co2+, indicating that Lmo1603 is a cobalt-dependent aminopeptidase. Single mutation at any of the Glu216, Glu281, His308, Tyr315, His327, and Asp329 completely abolished the enzymatic activity of Lmo1603. More importantly, we showed that Lmo1603 was mainly involved in Listeria infection, but not required for growth in rich laboratory medium and minimal defined medium. Disruption of Lmo1603 resulted in almost complete attenuation of Listeria virulence in a mouse infection model. In addition, we demonstrated that Lmo1603 was mainly localized in the bacterial cytosol and required for invasion and survival inside human epithelial cells and murine macrophages. We conclude that Lmo1603 encodes a functional aminopeptidase T of M29 family, which acts as a novel intracellular virulence factor essential in the successful establishment of L. monocytogenes infections in a mouse model.
Highlights
Listeria monocytogenes (L. monocytogenes) is a foodborne bacterial pathogen capable of invasion and replication in phagocytic and non-phagocytic cells
Based on sequence alignment using the MEROPS database, Lmo1603 is a member of the M29 family aminopeptidases
Lmo1603 shows relatively low amino acid sequence homologies (19.7%–22.1%) to other members of the M29 family, including aminopeptidase T (M29.001), aminopeptidase II (M29.002), PepS aminopeptidase (M29.004) and aminopeptidase S (M29.005)[11,12], the residues of the predicted catalytic site and ligand binding site are 100% conserved between Lmo1603 and the other members of the M29 family (Fig. 1A)
Summary
Listeria monocytogenes (L. monocytogenes) is a foodborne bacterial pathogen capable of invasion and replication in phagocytic and non-phagocytic cells. The ability of L. monocytogenes to cause disease in a mammalian host depends upon expression of a number of virulence determinants that enable this pathogen to successfully gain entry into host cells, escape from host cell vacuoles, replicate within the cytosol, and spread to adjacent cells[4,5,6]. These virulence factors mainly include internalins, listeriolysin O, phospholipases, actin polymerization protein, and, metalloproteases families[5,7]. We conclude that Lmo1603 is a novel virulence factor essential for Listeria pathogenesis
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