TolC-Dependent Modulation of Host Cell Death by the Francisella tularensis Live Vaccine Strain

Abstract

Francisella tularensis is a facultative intracellular, Gram-negative pathogen and the causative agent of tularemia. We previously identified TolC as a virulence factor of the F. tularensis live vaccine strain (LVS) and demonstrated that a ⌬tolC mutant exhibits increased cytotoxicity toward host cells and elicits increased proinflammatory responses compared to those of the wild-type (WT) strain. TolC is the outer membrane channel component used by the type I secretion pathway to export toxins and other bacterial virulence factors. Here, we show that the LVS delays activation of the intrinsic apoptotic pathway in a TolC-dependent manner, both during infection of primary macrophages and during organ colonization in mice. The TolC-dependent delay in host cell death is required for F. tularensis to preserve its intracellular replicative niche. We demonstrate that TolC-mediated inhibition of apoptosis is an active process and not due to defects in the structural integrity of the ⌬tolC mutant. These findings support a model wherein the immunomodulatory capacity of F. tularensis relies, at least in part, on TolC-secreted effectors. Finally, mice vaccinated with the ⌬tolC LVS are protected from lethal challenge and clear challenge doses faster than WT-vaccinated mice, demonstrating that the altered host responses to primary infection with the ⌬tolC mutant led to altered adaptive immune responses. Taken together, our data demonstrate that TolC is required for temporal modulation of host cell death during infection by F. tularensis and highlight how shifts in the magnitude and timing of host innate immune responses may lead to dramatic changes in the outcome of infection.

Macrophage cell death upon intracellular bacterial infection

“…F. holarctica live vaccine strain (LVS) and its Δ tol C mutant induces J774 apoptosis by different kinetics but similar mechanism via an intrinsic apoptotic pathway with mitochondrial damage, PS expression, casp-3 activation, and DNA fragmentation [28–33] . The Δ tol C mutant is hypercytotoxic with a kinetic as early as 7 h PI and causes apoptosis with faster cleavage of casp-3, casp-9, and PARP but independent of casp-1 and casp-8 in murine macrophages [29, 31–34] whereas most hypercytotoxic Francisella mutants induce pyroptosis [35] .…”

“…In vivo , casp-3 activation and apoptotic cell death have been observed in C57BL/6 mice challenged with type A F. tularensis by the intranasal route [31] and in female C3H/HeN mice infected with the hypercytotoxic Δ tol C mutant [33] . At 4 days PI, extensive cell death is within tissues of type A F. tularensis -infected WT and casp-1 −/− but not casp-3 −/− mice, and dying cells express activated casp-3 but very little activated casp-1, confirming that apoptosis in vivo was not mediated by activated casp-1 [31] .…”

“…At 4 days PI, extensive cell death is within tissues of type A F. tularensis -infected WT and casp-1 −/− but not casp-3 −/− mice, and dying cells express activated casp-3 but very little activated casp-1, confirming that apoptosis in vivo was not mediated by activated casp-1 [31] . In infected female C3H/HeN mice, 80% splenocytes are casp-3-positive at day 3 PI with the Δ tol C mutant as compared to the 8% for the WT LVS [33] .…”

Strategies Used by Bacteria to Grow in Macrophages

“…Similarly, caspase-1-deficient macrophages infected with F. tularensis undergo AIM2/ASC-dependent caspase-8-mediated apoptosis (295). As already mentioned, F. tularensis delays inflammasome activation during the early stages of infection (279), but also uses TolC-secreted effectors to delay the activation of the intrinsic apoptotic pathway in order to preserve the host cell (296). This suggests that cytosolic bacteria have to interact with several host pathways in order to inhibit, or at least delay, cell death.…”

Identification of novel vaccine candidates againstAcinetobacter baumanniiusing reverse vaccinology

“…42 ABAYE3514 which has sequences homology to TolC protein of Samonella paratyphi and belongs to the type I secretion outer membrane channel component could be the vaccine target. 43 ABAYE3820 comprises of a rotamase domain that may plays an important role in pathological processes 24 and is similar to Haemophilus parasuis FKBP-type peptidyL-prolyl cis/trans isomerase proposed as a vaccine candidate for Gl€ asser's disease in swine. 44 ABAYE2564 is similar to a putative alkyl hydroperoxide reductase subunit C that has been identified as a potential target for multivalent anthrax vaccines.…”