Production and characterisation of photorin, a new protein protease inhibitor from the entomopathogenic bacteria <i>photorhabdus laumondii</i>

Abstract

Entomopathogenic bacteria of the genus Photorhabdus secrete protease S (PrtS), which is considered as a virulence factor. We found that in Photorhabdus genomes, immediately after the prtS genes, there are genes that encode small hypothetical proteins homologous to emfourin, a recently discovered protein inhibitor of metalloproteases. Emfourin-like inhibitor gene from Photorhabdus laumondii subsp. laumondii TT01 was cloned and expressed in Escherichia coli cells. The recombinant protein, named photorin (Phin), was purified by metal chelate affinity and gel permeation chromatography and characterized. It has been established that Phin is a monomer and inhibits the activity of protealysin and thermolysin, which, like PrtS, belong to the M4 peptidase family. The inhibition constants were 1.0 ± 0.3 and 10 ± 2 µM, respectively. It was also demonstrated that Phin is able to suppress the proteolytic activity of P. laumondii culture broth (half-maximal inhibition concentration 3.9 ± 0.3 nM). Polyclonal antibodies to Phin were obtained, and it was shown by immunoblotting that P. laumondii cells produce Phin. Thus, the prtS genes in entomopathogenic bacteria of the genus Photorhabdus are colocalized with the genes of emphorin-like inhibitors, which probably regulate the activity of the enzyme during infection. Strict regulation of the activity of proteolytic enzymes is essential for the functioning of all living systems. At the same time, the principles of regulation of protease activity by protein inhibitors remain poorly understood. Bacterial protease-inhibitor pairs, such as the PrtS and Phin pair, are a promising model for in vivo studies of these principles. Bacteria of the genus Photorhabdus have a complex life cycle with multiple hosts, being both nematode symbionts and powerful insect pathogens. This provides a unique opportunity to use the PrtS and Phin pair as a model for studying the principles of regulation of protease activity by proteinaceous inhibitors in the context of bacterial interactions with different types of hosts.