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.

IglG and IglI of the Francisella Pathogenicity Island Are Important Virulence Determinants of Francisella tularensis LVS

“…Indeed, a study on Listeria monocytogenes has provided direct evidence in support of this hypothesis (65). Moreover, recent publications on F. tularensis have demonstrated enhanced inflammatory properties of several mutants, many of which appear to have cell wall defects (42,45,75). Thus, a straightforward explanation for the correlation between phagosomal escape and inflammasome activation may be that an increased number of bacteria in the cytoplasm will lead to more lysed bacteria as well.…”

Microinjection of Francisella tularensis and Listeria monocytogenes Reveals the Importance of Bacterial and Host Factors for Successful Replication

“…To study the prerequisites of intracellular growth, we included the murine J774 macrophage-like cell line, since it has been widely used to investigate various aspects of L. monocytogenes and F. tularensis host cell infections in the past and therefore will serve as a comparison with previously published studies (14,(56)(57)(58)(59)(60)(61)(62)(63). The available evidence indicates, however, that J774 cells do not possess as potent an antimicrobial capacity as do various forms of primary macrophages (64), and in support of this view, we have previously observed that the ⌬iglI mutant replicated readily in J774 macrophages but not in BMDM (27).…”

Comparative Phosphoproteomics Reveals Components of Host Cell Invasion and Post-transcriptional Regulation During Francisella Infection

“…This observation suggests that although ⌬lpcC, ⌬manB, and ⌬manC are also defective in membrane components, the mechanism of cell killing may be different than the mutant strains studied by Peng et al (29). Also surprising was that ⌬lpcC, ⌬manB, and ⌬manC strains were phagocytosed at a much higher rate than WT F. novicida via a mechanism that does not require actin polymerization (34). Actin polymerization has thus far been demonstrated to be involved in internalization of Francisella by either phagocytic or endocytic pathways (21,22,35); therefore, the finding that ⌬lpcC, ⌬manB, and ⌬manC mutant strains are able to invade host cells independent of actin filaments remains to be understood.…”

“…To identify genes that are involved in host immune evasion, we recently screened a comprehensive mutant library containing over 3000 F. novicida strains for mutants that were hypercytotoxic (34). Among the identified strains four F. novicida genes were involved in LPS core synthesis and assembly.…”

“…Among the identified strains four F. novicida genes were involved in LPS core synthesis and assembly. These mutants are unable to synthesize the LPS core and thus produce LPS with a very short glycan compared with the long polysaccharide chains of the wild-type (WT) (34). Interestingly, three mutants (⌬lpcC, ⌬manB, and ⌬manC) are hyper-cytotoxic to RAW 264.7 cells, a murine macrophage-like cell line that lacks the production of ASC (apoptosis-associated speck-like protein containing a caspase recruiting domain), a key component of the inflammasome signaling.…”