Penicillin V acylases from gram-negative bacteria degrade N-acylhomoserine lactones and attenuate virulence in Pseudomonas aeruginosa

Abstract

Virulence pathways in gram-negative pathogenic bacteria are regulated by quorum sensing mechanisms, through the production and sensing of N-acylhomoserine lactone (AHL) signal molecules. Enzymatic degradation of AHLs leading to attenuation of virulence (quorum quenching) could pave the way for the development of new antibacterials. Penicillin V acylases (PVAs) belong to the Ntn hydrolase superfamily, together with AHL acylases. PVAs are exploited widely in the pharmaceutical industry, but their role in the natural physiology of their native microbes is not clearly understood. This report details the characterization of AHL degradation activity by homotetrameric PVAs from two gram-negative plant pathogenic bacteria, Pectobacterium atrosepticum (PaPVA) and Agrobacterium tumefaciens (AtPVA). Both the PVAs exhibited substrate specificity for degrading long-chain AHLs. Exogenous addition of these enzymes into Pseudomonas aeruginosa greatly diminished the production of elastase and pyocyanin and biofilm formation and increased the survival rate in an insect model of acute infection. Subtle structural differences in the PVA active site that regulate specificity for acyl chain length have been characterized, which could reflect the evolution of AHL-degrading acylases in relation to the environment of the bacteria that produce them and also provide strategies for enzyme engineering. The potential for using these enzymes as therapeutic agents in clinical applications and a few ideas about their possible significance in microbial physiology have also been discussed.

Plant growth-promoting activity and quorum quenching-mediated biocontrol of bacterial phytopathogens by Pseudomonas segetis strain P6

“…This gene was cloned into pGEX-4T-2 and expressed in E. coli DH5α demonstrating the AHL degradation activity of P6 strain. Although previous studies have reported that penicillin acylases are capable of degrading AHLs in other bacteria 57,58 , this activity had not previously been identified in the species Pseudomonas segetis. The actual physiological significance of AHL-degrading enzymes remains largely unclear 59 .…”

Deciphering Physiological Functions of AHL Quorum Quenching Acylases

“…The sequence similarity within the Ntn-hydrolase family is very low and the enzymes within this family exhibit quaternary structures ranging from heterodimers (PGAs), homotetramers (PVAs) to the complex four homoheptameric rings forming the 20S proteasome (Unno et al, 2002; Mukherji et al, 2014; Sunder et al, 2016). However, a conservation of their tertiary fold structure is strongly pointing toward their evolutionary relationship (Hewitt et al, 2000).…”

“…In addition, two tryptophan residues are thought to be important for the appropriate orientation of the substrate by providing a hydrophobic pocket for the acyl chain (PvdQ: Trpβ162 and Trpβ187; Pa PVA: Trp23 and Trp87). Ligand docking was performed in Discovery Studio 3.0 (Accelrys, United States) for PvdQ, and Glide’s extra precision for Pa PVA (Sunder et al, 2016). All structural figures were prepared in Pymol NRG studio.…”

“…Kc PGA from Kluyvera citrophila was found to be active on AHLs with 6–8 carbons with or without 3-oxo modification (Mukherji et al, 2014). Two PVAs from Gram-negative bacteria, namely At PVA (from Agrobacterium tumefaciens ) and Pa PVA (from Pectobacterium atrosepticum ) were found to be active on medium to long chain AHLs (Sunder et al, 2016). PVA and PGA differ in sequence, structural composition and N-terminal nucleophile residue.…”

“…Similar to PvdQ, bacterial PGAs are generally heterodimeric with a serine as the Ntn residue, while bacterial PVAs are homotetrameric with cysteine as the Ntn residue. In silico d ocking showed a suitable fit of long-chain AHLs in the crystal structure of At PVA and Pa PVA (Sunder et al, 2016). On the other side, AhlM, an extracellular AHL-acylase from Streptomyces sp.…”