Production and Characterization of Photorin, a Novel Proteinaceous Protease Inhibitor from the Entomopathogenic Bacteria Photorhabdus laumondii

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Igor M. Berdyshev^1#, Anastasia O. Svetlova^1#, Ksenia N. Chukhontseva¹, Maria A. Karaseva¹, Anna M. Varizhuk², Vasily V. Filatov³, Sergey Y. Kleymenov^4,5, Sergey V. Kostrov¹, and Ilya V. Demidyuk^1,a*

¹National Research Centre “Kurchatov Institute”, 123182 Moscow, Russia

²Moscow Institute of Physics and Technology (National Research University), 141701 Dolgoprudny, Moscow Region, Russia

³Semenov Federal Research Center for Chemical Physics, Chernogolovka Branch, Russian Academy of Sciences, 142432 Chernogolovka, Moscow Region, Russia

⁴Bach Institute of Biochemistry, Research Center of Biotechnology, Russian Academy of Sciences, 119071 Moscow, Russia

⁵Koltzov Institute of Developmental Biology, Russian Academy of Sciences, 119334 Moscow, Russia

^* To whom correspondence should be addressed.

^# These authors contributed equally to this study.

Received April 13, 2023; Revised May 27, 2023; Accepted June 17, 2023

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Entomopathogenic bacteria of the genus Photorhabdus secrete protease S (PrtS), which is considered a virulence factor. We found that in the 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. The gene of emfourin-like inhibitor 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 activity of protealysin and thermolysin, which, similar to PrtS, belong to the M4 peptidase family. Inhibition constants were 1.0 ± 0.3 and 10 ± 2 µM, respectively. It was also demonstrated that Phin is able to suppress proteolytic activity of P. laumondii culture fluid (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 emfourin-like inhibitors, which probably regulate activity of the enzyme during infection. Strict regulation of the activity of proteolytic enzymes is essential for 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 promising models 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 protease activity regulation by proteinaceous inhibitors in the context of bacterial interactions with different types of hosts.

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KEY WORDS: proteinaceous protease inhibitor, Photorhabdus, entomopathogenic bacteria, protealysin, emfourin, photorin

DOI: 10.1134/S0006297923090158

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