Directed screen of Francisella novicida virulence determinants using Drosophila melanogaster

Abstract

Francisella tularensis is a highly virulent, facultative intracellular human pathogen whose virulence mechanisms are not well understood. Occasional outbreaks of tularemia and the potential use of F. tularensis as a bioterrorist agent warrant better knowledge about the pathogenicity of this bacterium. Thus far, genome-wide in vivo screens for virulence factors have been performed in mice, all however restricted by the necessity to apply competition-based, negative-selection assays. We wanted to individually evaluate putative virulence determinants suggested by such assays and performed directed screening of 249 F. novicida transposon insertion mutants by using survival of infected fruit flies as a measure of bacterial virulence. Some 20% of the genes tested were required for normal virulence in flies; most of these had not previously been investigated in detail in vitro or in vivo. We further characterized their involvement in bacterial proliferation and pathogenicity in flies and in mouse macrophages. Hierarchical cluster analysis of mutant phenotypes indicated a functional linkage between clustered genes. One cluster grouped all but four genes of the Francisella pathogenicity island and other loci required for intracellular survival. We also identified genes involved in adaptation to oxidative stress and genes which might induce host energy wasting. Several genes related to type IV pilus formation demonstrated hypervirulent mutant phenotypes. Collectively, the data demonstrate that the bacteria in part use similar virulence mechanisms in mammals as in Drosophila melanogaster but that a considerable proportion of the virulence factors active in mammals are dispensable for pathogenicity in the insect model.

FIG. 1.

Flow chart visualizing the sequence of experiments. The various assays and analysis methods are marked by rectangular boxes, and groups of bacterial mutants are marked by ovals. Assay readouts used for multivariate analysis are indicated by dotted arrows.

FIG. 2.

Phenotypic features of attenuated and hypervirulent (FTN_0119) F. novicida transposon insertion mutants of nonannotated genes, as determined by various assays. If not otherwise noted, representative results from one experiment are shown for the mutants, and the averages are shown for U112. The legend at the bottom applies to panels A, B, and E, and numbers refer to FTN designations (e.g., 0096 represents the FTN_0096 mutant). (A) Survival of infected flies. (B) Bacterial growth in liquid culture at 29°C. The averages and standard errors of the means (SEM) are shown for all mutants (n = 3). (C) Bacterial proliferation in adult flies on day 1 (white), day 3 (gray), and day 5 (black) postinfection. Day 5 values were not determined for the FTN_1412 mutant. (D) Bacterial proliferation in J774 cells 45 to 60 min (white) and 24 h (black) after infection. Note that for the FTN_0119 mutant, bacterial numbers were higher at 16 h (8.05 log₁₀ CFU/well). (E) Bacterial cytotoxicity, as measured by the release of lactate dehydrogenase from J774 cells into the culture medium.

FIG. 3.

Hierarchical cluster analysis of phenotypic features of attenuated and hypervirulent (inset) F. novicida mutants. Readout variables from all five analyses listed in Tables 1 and 2 were standardized. The Manhattan distance was used to calculate the distances between the mutants, and Ward's method was used to calculate the distances between clusters. Circled numbers identify clusters of phenotypically similar attenuated mutants, and numbers used to identify genes refer to FTN designations (e.g., 1240 represents FTN_1240). See the text for further details.