Immunization with Ft-LPS provokes an antigen-specific, B-1a cell-derived antibody response that protects WT mice against an otherwise lethal challenge with Ft LVS. However, this same regimen offers limited protection to TLR2(-/-) mice, despite production of WT levels of anti-Ft-LPS antibodies. As Ft-LPS exhibits no TLR2 agonist activity, and macrophage-induced cytokine production in response to Ft LVS is overwhelmingly TLR2-dependent, we hypothesized that treatment of TLR2(-/-) mice with an alternative, MyD88-dependent TLR agonist would compensate for reduced recognition of Ft LVS in TLR2(-/-) mice and thereby, restore Ft-LPS-mediated protection. Administration of the nontoxic TLR4 agonist, synthetic Escherichia coli MPL, at the time of Ft-LPS immunization or Ft LVS challenge, fully protected TLR2(-/-) mice, whereas treatment of WT or TLR2(-/-) mice with MPL alone conferred partial protection. The TLR5 agonist, flagellin, also synergized with Ft-LPS to protect TLR2(-/-) mice from lethal Ft LVS challenge. In contrast to Ft LVS, Ft-LPS pretreatment failed to protect mice against i.n. challenge with Ft Schu S4, whereas MPL, administered in the absence or presence of Ft-LPS, conferred significant, albeit partial, protection. MPL treatment of macrophages increased the uptake of Ft LVS and decreased intracellular bacterial survival while shifting the macrophage-differentiation phenotype from "alternatively activated" to "classically activated". Collectively, our data suggest that optimal, Ft-LPS-mediated protection against Ft LVS infection requires two discrete events, i.e., production of Ft-LPS-specific antibody, as well as TLR-mediated macrophage activation, to fully control Francisella infection.