Altered host immune responses, not lowered growth, are the mechanism of attenuation of Francisella tularensis clpB (P3126)

Abstract

Abstract Bacterial infections typically initiate both innate and adaptive immune responses. Bacterial attenuation is thought of as altered bacterial growth in the presence of constant immune pressure. In vivo screens have identified F. tularensis genes necessary for virulence. While many of these mutations render F. tularensis incapable of intracellular growth, some mutations have no impact on intracellular growth. We hypothesize that these F. tularensis mutants are attenuated because they induce an altered host immune response. We were particularly interested in the F. tularensis LVS mutant clpB (FTL_0094). Our LVS clpB strain is attenuated in pneumonic tularemia yet we did not observe an intracellular growth defect in bone marrow derived macrophages. We showed that LVS clpB induced an altered innate, but equivalent adaptive, immune response compared to LVS. In B6 mice, LVS clpB induced pro-inflammatory cytokine production in the lung early after infection in contrast to wild-type LVS infection. LVS clpB provoked a robust protective adaptive immune response similar in magnitude to LVS, but with increased IFN-γ and IL-17A production in the lung as measured by mean fluorescence intensity. Altogether, our results indicate that LVS clpB is attenuated due to altered host immunity and not an intrinsic growth defect. These results also indicate that disruption of non-essential gene(s) that cause attenuation, like F. tularensis clpB, can help elucidate bacterial immune evasion mechanisms.