Making and Breaking Leupeptin Protease Inhibitors in Pathogenic Gammaproteobacteria

Abstract

Leupeptin is a bacterial small molecule that is used worldwide as a protease inhibitor. However, its biosynthesis and genetic distribution remain unknown. We identified a family of leupeptins in gammaproteobacterial pathogens, including Photorhabdus, Xenorhabdus, and Klebsiella species, amongst others. Through genetic, metabolomic, and heterologous expression analyses, we established their construction by discretely expressed ligases and accessory enzymes. In Photorhabdus species, a hypothetical protein required for colonizing nematode hosts was established as a new class of proteases. This enzyme cleaved the tripeptide aldehyde protease inhibitors, leading to the formation of “pro‐pyrazinones” featuring a hetero‐tricyclic architecture. In Klebsiella oxytoca, the pathway was enriched in clinical isolates associated with respiratory tract infections. Thus, the bacterial production and proteolytic degradation of leupeptins can be associated with animal colonization phenotypes.

Improved diagnosis of antibiotic-associated haemorrhagic colitis (AAHC) in faecal specimens by a new qualitative real-time PCR assay detecting relevant toxin genes of Klebsiella oxytoca sensu lato

“…Cytotoxic K. oxytoca s.l. isolates are mainly affiliated with the species K. oxytoca and K. grimontii and despite the presence of npsA/npsB homologues in K. michiganensis, toxin production by K. michiganensis has rarely been observed [6,13,31]. In line with this, lower BLAST scores for npsA/npsB and negative in silico PCRs were observed for K. michiganensis and K. pasteurii.…”

Convergent evolution of Streptomyces protease inhibitors involving a tRNA-mediated condensation-minus NRPS

“…On the other hand, the occurrence of convergent evolution in natural products biosynthesis is a recurring theme (Fischbach 2009), although its significance and rate remains to be determined (Chevrette et al 2020). It is nevertheless tempting to speculate that the degree of convergent evolution correlates with chemical scaffolds that contribute towards the fitness of the host organisms, as previously suggested for protease inhibitors (Chater et al 2010;Guo et al 2017;Li et al 2020)…”

“…Livipetin was found to be chemically related to bacithrocins and thiolstatin, reported previously to be produced by Brevibacillus laterosporus (Kamiyama et al 1994); and to leupeptin, whose BGC in Streptomyces is also identified herein. Interestingly, leupeptin and livipeptin were shown to be synthesized by unrelated pathways, demonstrating convergent evolution within Streptomyces and with other taxonomically unrelated leupeptin-producing proteobacteria (Li et al 2020). Genetic and semisynthetic chemical experiments in the presence of RNAse, as done previously for other tRUEs (Ortega and van der Donk 2016), confirmed the involvement of an L/F transferase, typically involved in protein tagging during proteolysis (N-end rule) within an essential process carried out by many organisms that regulate the half-life of proteins and morphological differentiation (Leibowitz and Soffer 1969; Varshavsky 2011), during livipeptin biosynthesis.…”

Global analysis of biosynthetic gene clusters reveals conserved and unique natural products in entomopathogenic nematode-symbiotic bacteria

“…To identify products derived from this GCF, we focused on rdb1 that contains five out of eight BGCs in this GCF, and attempted to activate the rdb1 in Xenorhabdus budapestensis DSM 16342 by inserting a P BAD promoter in front of rdb1A. The X. budapestensis P BAD rdb1A mutant yielded four N-myristoyl-D-asparagine congeners (19)(20)(21)(22), as well as a non-XAD-resin-bound hydrophilic compound with a low production level (23; Supplementary Fig. 18).…”

“…Our previous metabolic analysis on 30 XP strains preliminarily revealed their biosynthetic capacity of natural products, via linking the metabolic profile of wild-type strains to known natural product biosynthetic gene clusters (BGCs) 16 . Intense research efforts have also been devoted to the characterization of unknown BGCs for natural product discovery, as well as functional assignments of natural products in the context of bacteria-nematode-insect interactions 8,[17][18][19][20][21][22] . However, these studies either mostly revolved around the identification and characterization of individual BGCs on a single-genome basis, or lacked a comprehensive comparison of intra/interspecies BGCs.…”