Mutations of Francisella novicida that alter the mechanism of its phagocytosis by murine macrophages

Abstract

Infection with the bacterial pathogen Francisella tularensis tularensis (F. tularensis) causes tularemia, a serious and debilitating disease. Francisella tularensis novicida strain U112 (abbreviated F. novicida), which is closely related to F. tularensis, is pathogenic for mice but not for man, making it an ideal model system for tularemia. Intracellular pathogens like Francisella inhibit the innate immune response, thereby avoiding immune recognition and death of the infected cell. Because activation of inflammatory pathways may lead to cell death, we reasoned that we could identify bacterial genes involved in inhibiting inflammation by isolating mutants that killed infected cells faster than the wild-type parent. We screened a comprehensive transposon library of F. novicida for mutant strains that increased the rate of cell death following infection in J774 macrophage-like cells, as compared to wild-type F. novicida. Mutations in 28 genes were identified as being hypercytotoxic to both J774 and primary macrophages of which 12 were less virulent in a mouse infection model. Surprisingly, we found that F. novicida with mutations in four genes (lpcC, manB, manC and kdtA) were taken up by and killed macrophages at a much higher rate than the parent strain, even upon treatment with cytochalasin D (cytD), a classic inhibitor of macrophage phagocytosis. At least 10-fold more mutant bacteria were internalized by macrophages as compared to the parent strain if the bacteria were first fixed with formaldehyde, suggesting a surface structure is required for the high phagocytosis rate. However, bacteria were required to be viable for macrophage toxicity. The four mutant strains do not make a complete LPS but instead have an exposed lipid A. Interestingly, other mutations that result in an exposed LPS core were not taken up at increased frequency nor did they kill host cells more than the parent. These results suggest an alternative, more efficient macrophage uptake mechanism for Francisella that requires exposure of a specific bacterial surface structure(s) but results in increased cell death following internalization of live bacteria.

Figure 1. Francisella LPS structure of lipid A and core.

Schematic drawing of a portion of the LPS including lipid A, the core and one O-antigen repeat (Modified from [4]). Indicated in the drawing are the regions of the LPS that are affected by the mutant alleles described in this study. Both wzx and htrB contain transposon insertions while the remaining mutations are gene deletions.

Figure 2. The presence of cytochalasin D (2 µM) during infection decreased LDH release in all but three of the J774 macrophage-like cells infected with F. novicida transposon mutant strains.

(A) J774 macrophages were infected with each of the 12 hypercytotoxic transposon mutants or wild-type U112 either in the presence or absence of cytochalasin D (cytD). The levels of LDH in the extracellular medium were determined 12 hours post-infection (p.i.). The levels of LDH release from the mutant- or U112-infected J774 cells were normalized to the level of LDH release from uninfected macrophages lysed with detergent. Parent strain U112 does not promote cell death at 12 hours p.i. (B) The level of LDH release from J774 macrophages infected with U112 at a MOI of 100 was determined 24 hours p.i. LDH release was determined from macrophages infected with U112 both in the presence and absence of cytD. In (A) and (B), each column is an average of three individual infections (±s.d.). The experiment was repeated twice and yielded similar results.

Figure 3. Strains containing deletion mutations in lpcC, manB, and manC induce early cytotoxicity in primary macrophages.

Bone marrow-derived macrophages (BMDM) derived from BALB/c mice were infected with the deletion mutants or parental strain MFN245 at a MOI of 100. The level of LDH release from infected macrophages was determined 10 hours p.i. as described in Figure 2. Each column is an average of three individual infections (±s.d.). Repetition of this experiment yielded similar results.

Figure 4. High numbers of mutant bacteria were visualized intracellularly in infected J774 macrophages even in the absence of actin polymerization.

J774 macrophages were infected with the three deletion mutants or parental strain MFN245 in four-well microscope chambers for two hours at an MOI of 100 either in the absence (A) or presence (B) of cytD. The cells were fixed in 4% paraformaldehyde, permeabilized, and probed with a rabbit polyclonal antibody against Francisella followed by a secondary goat anti-rabbit antibody conjugated with Alexa 488 (green). J774 nuclei were identified by staining DNA with DAPI (blue). Cells were imaged with an Applied Precision DeltaVision deconvolution microscope system. Experiment was repeated six times with similar results and representative images are shown. Eukaryotic cell boundary can be observed in the phase-contrast images of the same fields. Scale bar 10 µm (lower left corner). X-Z stack images show that bacteria were within cells.

Figure 5. Inhibiting actin polymerization did not reduce the number of intracellular mutant bacteria.

J774 macrophages were infected with ΔlpcC, ΔmanB or ΔmanC deletion mutants or parental strain MFN245 at a MOI of 100. Cells were infected either in the presence or absence of cytD. At two hours p.i., the macrophages were washed and treated with gentamicin to kill extracellular bacteria. Cells were lysed and the lysates plated on CHA plates. Colonies were counted two days after incubation and the numbers of CFU/well were calculated and converted to a log scale. Each column is an average of three individual infections (±s.d.). Repetition of this experiment yielded similar results.

Figure 6. Increasing the number of internalized wild-type bacteria did not increase the cytotoxicity of the strain.

(A) J774 macrophages were infected with ΔlpcC, ΔmanB, ΔmanC, or parental strain MFN245, as well as with complemented mutant strains expressing a wild-type copy of the gene in trans. The cells were infected for 10 hours at three different MOI. The level of LDH release from infected macrophages was determined as described in Figure 2. (B) J774 macrophages were infected with wild-type U112 at a MOI of 10,000 for two hours either in the presence or absence of cytD. Francisella (green) and macrophage nuclei (blue) were visualized as described in Figure 4. Both (A) and (B) were repeated a total of three times and yielded similar results.

Figure 7. Dead bacteria do not promote cell death but are internalized similarly to live strains.

(A) Formaldehyde-fixed ΔlpcC and MFN245 infected J774 macrophages at various MOI. Francisella (green) and macrophage nuclei (blue) were visualized as described in Figure 4. (B) J774 macrophages were infected with live mutant or parental bacteria and with strains that were fixed with 4% formaldehyde at a MOI of 100. LDH release was determined 12 hours p.i. for the mutant strains and 24 hours p.i. for wild-type strain as described in Figure 2. This experiment was repeated once with similar results.

Figure 8. Viable bacteria are required for the cell toxicity observed in the mutant strains.

J774 macrophages were infected with ΔlpcC, ΔmanB, and ΔmanC mutant strains at a MOI of 100. Ciprofloxacin, a bacteriocidal and host cell membrane permeable antibiotic, was added concurrent with infection (0 h) or at one of six time points following initial infection (1 h–6 h). LDH release levels were determined 12 hours p.i. as described in Figure 2 and compared to LDH release from infected macrophages not treated with ciprofloxacin (No). This experiment was repeated once with similar results.

Figure 9. Lipopolysaccharides (LPS) prepared from ΔlpcC, ΔmanB, ΔmanC, and ΔkdtA lack the O-antigen and contain a defect in the core.

Lipopolysaccharides were purified from U112; strains containing deletions in lpcC (FTN1253), manB (FTN1417), manC (FTN1418), kdtA (FTN1469), wbtA (FTN1431) and strains containing transposon mutations in wzx (FTN1420) and htrB (FTN0071) and analyzed on a gradient SDS-PAGE gel. The inverted image is shown in the figure.

Figure 10. Deleting LPS biosynthesis gene kdtA resulted in a cytotoxicity and localization phenotype similar to the ΔlpcC, ΔmanB, and ΔmanC mutants.

J774 macrophages were infected with ΔwbtA (FTN1431), ΔkdtA (FTN1469), transposon mutated FTN1420 (wzx), parental strain MFN245, or ΔkdtA complemented in trans with wild-type kdtA at a MOI of 100 for 10 hours either in the presence and absence of cytD. LDH release levels were determined as described in Figure 2. (B) Francisella (green) and macrophage nuclei (blue) were visualized in macrophages two hours after infection with the mutant strains as described in Figure 5. This experiment was repeated twice with similar results.