Comparison of type 5d autotransporter phospholipases demonstrates a correlation between high activity and intracellular pathogenic lifestyle

Abstract

Autotransporters, or type 5 secretion systems, are widespread surface proteins of Gram-negative bacteria often associated with virulence functions. Autotransporters consist of an outer membrane β-barrel domain and an exported passenger. In the poorly studied type 5d subclass, the passenger is a patatin-like lipase. The prototype of this secretion pathway is PlpD of Pseudomonas aeruginosa, an opportunistic human pathogen. The PlpD passenger is a homodimer with phospholipase A1 (PLA1) activity. Based on sequencing data, PlpD-like proteins are present in many bacterial species. We characterized the enzymatic activity, specific lipid binding and oligomeric status of PlpD homologs from Aeromonas hydrophila (a fish pathogen), Burkholderia pseudomallei (a human pathogen) and Ralstonia solanacearum (a plant pathogen) and compared these with PlpD. We demonstrate that recombinant type 5d-secreted patatin domains have lipase activity and form dimers or higher-order oligomers. However, dimerization is not necessary for lipase activity; in fact, by making monomeric variants of PlpD, we show that enzymatic activity slightly increases while protein stability decreases. The lipases from the intracellular pathogens A. hydrophila and B. pseudomallei display PLA2 activity in addition to PLA1 activity. Although the type 5d-secreted lipases from the animal pathogens bound to intracellular lipid targets, phosphatidylserine and phosphatidylinositol phosphates, hydrolysis of these lipids could only be observed for FplA of Fusobacterium nucleatum. Yet, we noted a correlation between high lipase activity in type 5d autotransporters and intracellular lifestyle. We hypothesize that type 5d phospholipases are intracellularly active and function in modulation of host cell signaling events.

Rv3091, An Extracellular Patatin-Like Phospholipase in Mycobacterium tuberculosis, Prolongs Intracellular Survival of Recombinant Mycolicibacterium smegmatis by Mediating Phagosomal Escape

“…PLA 1 of VipD is dependent on Rab5 and Rab22 (Ku et al, 2012). In this study, activity characterization revealed that unlike many other PLPs, Rv3091 exhibited PLA 1 activity in addition to PLA 2 activity and did not need the participation of eukaryotic cofactors, which is consistent with the PLP AhPIA and BpPIA from the intracellular pathogens Aeromonas hydrophila and Burkholderia pseudomallei that display both PLA 1 and PLA 2 activity (Trunk et al, 2019). We failed to detect robust PLA 2 activity in Rv3091 as compared with PLA 1 activity.…”

“…The PLA 1 and PLA 2 activity of purified recombinant protein (Rv3091NS, S214A, D407A, and S214AD407A) was detected using the fluorogenic phospholipid substrate [PLA 1 and PLA 2 substrates PED-A1 (Invitrogen, USA) and BODIPY PC-A2 (Invitrogen, USA)] according to the manufacturer's protocol (EnzChek Phospholipase A 1 Assay Kit and EnzChek Phospholipase A 2 Assay Kit, Invitrogen, USA) as previously described with minor modifications (Trunk et al, 2019). Briefly, the cleavage of the sn-1/sn-2 bond of PED-A1/BODIPY PC-A 2 using the PLA 1 /PLA 2 enzyme induces an increase in fluorescence emission.…”

The patatin-like protein PlpD forms novel structurally dynamic homodimers in thePseudomonas aeruginosaouter membrane

“…In contrast, PlpD and its homologs appear to lack a lateral gate that might widen the dimer to allow PL-domain translocation. Curiously, a small portion of the first strand of the PlpD b-barrel binds to a lid sequence (Ile-Ser-Phe) that is similar to the last three residues of the "b-signal", a motif that is Although the exact physiological function of PlpD and its homologs is unknown, the PLdomains of PlpD and FplA (expressed without the b-barrel and POTRA domains) have been shown to bind various lipids and to have a phospholipase A1 activity that, like that of other phosphatases, is dependent on a Ser-Asp catalytic dyad [43]. Neither PlpD nor FplA, however, appears to function as a secreted virulence factor.…”

“…Neither PlpD nor FplA, however, appears to function as a secreted virulence factor. A study on the effects of purified PlpD and FlpA injected into G. mellonella larvae reported no direct toxic effects, even at very high concentrations [43].…”

“…The PL-domain is similar to the potatoderived patatin protein, a phospholipase characterized by an a/β hydrolase fold and a Ser-Asp catalytic dyad, as well as four conserved sequence motifs [41,42]. Recombinant forms of the PL-domain and its homologs have been shown to have phospholipase activity in vitro [42,43].…”