Francisella is sensitive to insect antimicrobial peptides

Abstract

Francisella tularensis causes the zoonotic disease tularemia. Arthropod vectors are important transmission routes for the disease, although it is not known how Francisella survives the efficient arthropod immune response. Here, we used Drosophila melanogaster as a model host for Francisella 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 Francisella novicida mutant strains in genes, directly or indirectly involved in specific steps of LPS biosynthesis, for virulence in wild-type and Relish(E20) immune-deficient flies, and tested selected mutants for sensitivity to AMPs in vitro. We demonstrate that Francisella is sensitive to specific fly AMPs, i.e. Attacin, Cecropin, Drosocin and Drosomycin. Furthermore, six bacterial genes, kpsF, manB, lpxF, slt, tolA and pal, were found to be required for resistance to Relish-dependent immune responses, illustrating the importance of structural details of Francisella 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.

Fig. 1

Protective effect of antimicrobial peptides on survival of flies infected with F. tularensis LVS. Antimicrobial peptides were ectopically expressed using the UAS/GAL4 system with the ubiquitously active daughterless (da) promoter indicated by ‘da>'. UAS-constructs were Attacin A (AttA), Cecropin A (CecA), Cecropin B (CecB), Drosocin (Drc), Drosomycin (Drs). a Survival of LVS-injected imd; spz immune-deficient flies which overexpress single antimicrobial peptides. Control flies were w¹¹¹⁸; b pr imd; da-GAL4 spz^rm7 with an MLL of 1.7 ± 0.1 days. The difference in MLL (ΔMLL) was 0.5 ± 0.2 days for da>AttA, 0.3 ± 0.1 days for da>CecA, 0.1 ± 0.0 days for da>Drc and 0.6 ± 0.1 days for da>Drs. b Survival of LVS-injected wild-type flies overexpressing Cecropin A or B with an MLL of 9.9 ± 0.5 and 10.3 ± 0.4 days, respectively, in comparison to 7.9 ± 0.4 days for LVS-infected genotype controls; results shown are from one fly strain transgenic for either UAS-CecA or UAS-CecB; for each UAS-construct similar results were obtained from two additional independent fly strains. Median values of three independent experiments with 20–80 flies per experiment are shown, error bars show standard error of mean.

Fig. 2

Survival of wild-type (OR) and Relish^E20 immune deficient (Rel) D. melanogaster flies infected with F. novicida U112 or U112-derived mutant strains as indicated. a–c 30–45 wild-type or 40–55 Relish^E20 mutant flies per experiment were infected by injection. d–f 25 wild-type or Relish^E20 mutant flies per experiment were infected by pricking. Median values of three independent experiments are shown, error bars show standard error of the mean. For statistical analysis of the data see online supplementary tables S2 and S3.

Fig. 3

In vivo proliferation of F. novicida U112 and U112-derived mutant strains in wild-type (OR) or in Relish^E20 immune deficient (Rel) D. melanogaster flies. a–c Flies were infected by injection. d–f Flies were infected by pricking. Median values of three independent experiments are shown based on homogenates from 5 flies per sample and time point. For statistical analysis of the data see online supplementary tables S2 and S3.

Fig. 4

Blocking phagocytosis improved the survival of lpxF mutant-infected flies but not that of manB mutant-infected flies. a Survival of wild-type flies injected with either F. novicida U112 or U112-derived mutant strains as indicated. Prior to infection flies were injected with latex beads (B) to block phagocytosis of bacteria; control flies were injected with buffer (Co). b Growth of Francisella in wild-type flies. Flies were treated as described in a. Median values of three independent experiments with at least 35 flies per survival experiment and 4 flies per sample for viable count analysis are shown, error bars show standard error of the mean. For statistical analysis of the data see online supplementary table S4.