Outer membrane vesicle-associated lipase FtlA enhances cellular invasion and virulence in Francisella tularensis LVS

Abstract

Francisella tularensis is a highly infectious intracellular pathogen that infects a wide range of host species and causes fatal pneumonic tularemia in humans. ftlA was identified as a potential virulence determinant of the F. tularensis live vaccine strain (LVS) in our previous transposon screen, but its function remained undefined. Here, we show that an unmarked deletion mutant of ftlA was avirulent in a pneumonia mouse model with a severely impaired capacity to infect host cells. Consistent with its sequence homology with GDSL lipase/esterase family proteins, the FtlA protein displayed lipolytic activity in both E. coli and F. tularensis with a preference for relatively short carbon-chain substrates. FtlA thus represents the first F. tularensis lipase to promote bacterial infection of host cells and in vivo fitness. As a cytoplasmic protein, we found that FtlA was secreted into the extracellular environment as a component of outer membrane vesicles (OMVs). Further confocal microscopy analysis revealed that the FtlA-containing OMVs isolated from F. tularensis LVS attached to the host cell membrane. Finally, the OMV-associated FtlA protein complemented the genetic deficiency of the ΔftlA mutant in terms of host cell infection when OMVs purified from the parent strain were co-incubated with the mutant bacteria. These lines of evidence strongly suggest that the FtlA lipase promotes F. tularensis adhesion and internalization by modifying bacterial and/or host molecule(s) when it is secreted as a component of OMVs.

Bacterial lipoproteins and other factors released byFrancisella tularensismodulate human neutrophil lifespan: Effects of aTLR1SNP on apoptosis inhibition

“…Additionally or instead, BLPs may be released as components of OMVs. F. tularensis sheds very few OMVs as compared with other Gram‐negative bacteria, and their contribution to the pathogenesis of tularaemia is only beginning to be defined (Chen et al, ). We did not detect OMVs by scanning electron microscopy analysis of CM or neutrophils treated with CM, and no vesicles were apparent by transmission electron microscopy after ultracentrifugation of LVS‐CM at 400,000 × g (our unpublished data), results that are in keeping with the outcome of our [ 14 C]acetate labelling experiments.…”

Microbial esterases and ester prodrugs: An unlikely marriage for combating antibiotic resistance

“…Follow‐up experiments have confirmed these putative esterases as active esterases with unusual substrate specificity and proposed biological roles in xenobiotic or host ester degradation (Chen et al, ; Farberg, Hart, & Johnson, ). These F. tularensis esterases are also membrane bound and excreted in outer membrane vesicles making them accessible for ester prodrug activation (Chen et al, ; Filippova et al, ; Smith, Phillips, Rabin, & Johnson, ). Conversion of weakly active antibacterials into prodrug esters to target F. tularensis is already a confirmed strategy for increasing the cell penetration of polar antibacterials (McKenney et al, ).…”

“…Another antibacterial target with a growing connection between its esterases, its virulence, and its survival is Francisella tularensis , a highly toxic, endemic Gram‐negative bacterium (Llewellyn, Jones, Napier, Bina, & Weiss, ; Su et al, ; Weiss et al, ). Screens of virulence factors for F. tularensis identified multiple putative esterases with potential roles in virulence (Chen et al, ; Su et al, ; Weiss et al, ). Follow‐up experiments have confirmed these putative esterases as active esterases with unusual substrate specificity and proposed biological roles in xenobiotic or host ester degradation (Chen et al, ; Farberg, Hart, & Johnson, ).…”

“…Screens of virulence factors for F. tularensis identified multiple putative esterases with potential roles in virulence (Chen et al, ; Su et al, ; Weiss et al, ). Follow‐up experiments have confirmed these putative esterases as active esterases with unusual substrate specificity and proposed biological roles in xenobiotic or host ester degradation (Chen et al, ; Farberg, Hart, & Johnson, ). These F. tularensis esterases are also membrane bound and excreted in outer membrane vesicles making them accessible for ester prodrug activation (Chen et al, ; Filippova et al, ; Smith, Phillips, Rabin, & Johnson, ).…”

“…Screens of virulence factors for F. tularensis identified multiple putative esterases with potential roles in virulence (Chen et al, 2017;Su et al, 2007;Weiss et al, 2007). Follow-up experiments have confirmed these putative esterases as active esterases with unusual substrate specificity and proposed biological roles in xenobiotic or host ester degradation (Chen et al, 2017;Farberg, Hart, & Johnson, 2016).…”