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.Emerging Microbes & Infections (2017) 6, e66; doi:10.1038/emi.2017.53; published online 26 July 2017
Highlights
Francisella tularensis is a Gram-negative bacterium that causes zoonotic tularemia
Because the transposon used in our previous study terminates the transcription of the sequence downstream of its insertion site gene,[22] we first attempted to clarify the possibility of the polar effect associated with these ΔftlA mutants by constructing an unmarked deletion mutant in F. tularensis live vaccine strain (LVS), in which a 771-bp coding sequence of FtlA was removed (Supplementary Figure S1B)
This study has revealed that the FtlA lipase plays an important role in F. tularensis infection of host cells and in vivo fitness based on the following results: (i) genetic deletion of the F. tularensis lipase A (ftlA) gene in the genome of F. tularensis LVS leads to significant impairment in the ability to infect host cells and complete loss of virulence in the pneumonia mouse model; (ii) FtlA possesses a lipolytic activity in both E. coli and F. tularensis; (iii) FtlA can be translocated to the extracellular environment as a component of F. tularensis outer membrane vesicles (OMVs); and (iv) the OMV-associated FtlA promotes bacterial adhesion to, and entry into, host cells
Summary
Francisella tularensis is a Gram-negative bacterium that causes zoonotic tularemia. It infects a wide range of hosts, including amoebae, insects, fish, amphibians, birds, small mammals, lagomorphs and primates.[1,2]Humans are accidental hosts that are infected by multiple routes, such as the bites of arthropod vectors, contact with infected animals, and inhalation of aerosolized bacteria.[3]. Francisella tularensis is a Gram-negative bacterium that causes zoonotic tularemia. It infects a wide range of hosts, including amoebae, insects, fish, amphibians, birds, small mammals, lagomorphs and primates.[1,2]. Humans are accidental hosts that are infected by multiple routes, such as the bites of arthropod vectors, contact with infected animals, and inhalation of aerosolized bacteria.[3] Francisella is divided into four subspecies: tularensis (type A), holarctica (type B), mediasiatica and novicida.[4,5] The strains of types A and B cause the vast majority of human infections due to their relatively higher virulence.[6] F. tularensis subsp. When humans inhale F. tularensis, the bacteria invade primarily alveolar macrophages and epithelial cells.[4]
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