Supplementary Material for: <b><i>Francisella </i></b>Is Sensitive to Insect Antimicrobial Peptides

<i>Francisella tularensis</i> causes the zoonotic disease tularemia. Arthropod vectors are important transmission routes for the disease, although it is not known how <i>Francisella</i> survives the efficient arthropod immune response. Here, we used <i>Drosophila melanogaster</i> as a model host for <i>Francisella</i> infections and investigated whether the bacteria are resistant to insect humoral immune responses, in particular to the antimicrobial peptides (AMPs) secreted into the insect hemolymph. Moreover, we asked to what extent such resistance might depend on lipopolysaccharide (LPS) structure and surface characteristics of the bacteria. We analyzed <i>Francisella novicida</i> mutant strains in genes, directly or indirectly involved in specific steps of LPS biosynthesis, for virulence in wild-type and <i>Relish</i><sup><i>E20</i></sup> immune-deficient flies, and tested selected mutants for sensitivity to AMPs in vitro. We demonstrate that <i>Francisella</i> is sensitive to specific fly AMPs, i.e. Attacin, Cecropin, Drosocin and Drosomycin. Furthermore, six bacterial genes, <i>kpsF, manB, lpxF, slt, tolA </i>and<i> pal</i>, were found to be required for resistance to <i>Relish</i>-dependent immune responses, illustrating the importance of structural details of <i>Francisella</i> lipid A and Kdo core for interactions with AMPs. Interestingly, a more negative surface charge and lack of O-antigen did not render mutant bacteria more sensitive to cationic AMPs and did not attenuate virulence in flies.