AtolCMutant ofFrancisella tularensisIs Hypercytotoxic Compared to the Wild Type and Elicits Increased Proinflammatory Responses from Host Cells

Abstract

The highly infectious bacterium Francisella tularensis is a facultative intracellular pathogen and the causative agent of tularemia. TolC, which is an outer membrane protein involved in drug efflux and type I protein secretion, is required for the virulence of the F. tularensis live vaccine strain (LVS) in mice. Here, we show that an LVS ⌬tolC mutant colonizes livers, spleens, and lungs of mice infected intradermally or intranasally, but it is present at lower numbers in these organs than in those infected with the parental LVS. For both routes of infection, colonization by the ⌬tolC mutant is most severely affected in the lungs, suggesting that TolC function is particularly important in this organ. The ⌬tolC mutant is hypercytotoxic to murine and human macrophages compared to the wild-type LVS, and it elicits the increased secretion of proinflammatory chemokines from human macrophages and endothelial cells. Taken together, these data suggest that TolC function is required for F. tularensis to inhibit host cell death and dampen host immune responses. We propose that, in the absence of TolC, F. tularensis induces excessive host cell death, causing the bacterium to lose its intracellular replicative niche. This results in lower bacterial numbers, which then are cleared by the increased innate immune response of the host.

Subversion of Host Recognition and Defense Systems by Francisella spp

“…Interestingly, TolC mutants stimulate an increased proinflammatory response compared to that of wild-type Francisella (178). This may be due to increased sensitivity to antimicrobials in vivo leading to increased PAMP release or to a general membrane instability defect eventually leading to PAMP release.…”

“…This multidrug efflux pump is important for Francisella resistance to ␤-lactams, tetracyclines, aminoglycosides, quinolones, detergents (notably bile salts), and antimicrobial dyes (30,91). Highlighting the importance of this system in pathogenesis, mutants with mutations in pump components are severely attenuated in vivo (30,91,178). While the AcrAB/TolC system is known to provide resistance against host antimicrobial peptides in bacteria (95) and it is assumed that Francisella mutants would be more sensitive to host-derived antimicrobials during infection, to the best of our knowledge this has not been directly tested.…”

“…The AcrAB inner membrane efflux platform (30), coupled with the TolC outer membrane transporter (91,178), forms a pump that facilitates the active efflux of toxic compounds and detergents from the bacteria, preventing their antimicrobial action. This multidrug efflux pump is important for Francisella resistance to ␤-lactams, tetracyclines, aminoglycosides, quinolones, detergents (notably bile salts), and antimicrobial dyes (30,91).…”

Francisella tularensisSchu S4 O-Antigen and Capsule Biosynthesis Gene Mutants Induce Early Cell Death in Human Macrophages

“…Weiss and coworkers identified two mutants of F. novicida (with mutations in homologs of FTT0584 and FTT0748) that exhibited increased caspase-1-and ASC-mediated cytotoxicity in primed murine macrophages as well as decreased virulence in mice (70). In addition, an LVS tolC mutant is defective for growth within murine macrophages (25) and exhibits increased cytotoxicity in vitro (54) and diminished virulence in the mouse model (25,54). The intracellular localization of the tolC mutant and the precise mechanism for this increased cell death are not yet understood but are thought to involve caspase-3 and not caspase-1 (54).…”

Identification of a Novel Francisella tularensis Factor Required for Intramacrophage Survival and Subversion of Innate Immune Response

“…A recent study has shown that an unknown Francisella factor suppresses proinflammatory cytokine production from infected as well as uninfected bystander cells (6). Another report has speculated that this factor may be secreted in a TolCdependent fashion to cause immune suppression (15). To date, a limited number of F. tularensis factors including intracellular growth locus C, RipA, and antioxidant enzyme catalase (KatG) of F. tularensis LVS have been shown to cause innate immune subversion through inhibition of MAPK and NF-B signaling (16 -19).…”