Importance of PdpC, IglC, IglI, and IglG for Modulation of a Host Cell Death Pathway Induced by Francisella tularensis

Abstract

Modulation of host cell death pathways appears to be a prerequisite for the successful lifestyles of many intracellular pathogens. The facultative intracellular bacterium Francisella tularensis is highly pathogenic, and effective proliferation in the macrophage cytosol leading to host cell death is a requirement for its virulence. To better understand the prerequisites of this cell death, macrophages were infected with the F. tularensis live vaccine strain (LVS), and the effects were compared to those resulting from infections with deletion mutants lacking expression of either of the pdpC, iglC, iglG, or iglI genes, which encode components of the Francisella pathogenicity island (FPI), a type VI secretion system. Within 12 h, a majority of the J774 cells infected with the LVS strain showed production of mitochondrial superoxide and, after 24 h, marked signs of mitochondrial damage, caspase-9 and caspase-3 activation, phosphatidylserine expression, nucleosome formation, and membrane leakage. In contrast, neither of these events occurred after infection with the ⌬iglI or ⌬iglC mutants, although the former strain replicated. The ⌬iglG mutant replicated effectively but induced only marginal cytopathogenic effects after 24 h and intermediate effects after 48 h. In contrast, the ⌬pdpC mutant showed no replication but induced marked mitochondrial superoxide production and mitochondrial damage, caspase-3 activation, nucleosome formation, and phosphatidylserine expression, although the effects were delayed compared to those obtained with LVS. The unique phenotypes of the mutants provide insights regarding the roles of individual FPI components for the modulation of the cytopathogenic effects resulting from the F. tularensis infection.

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

“…S2 in the supplemental material), whereas the ⌬iglA and ⌬iglC mutants showed no growth, which was in agreement with previously published data (27,55). Significant growth of LVS and the ⌬iglG and ⌬pdpE mutants was observed also in BMDM, whereas none of the other three mutants showed any replication (see Fig.…”

“…The ⌬iglG mutant replicates efficiently in the J774 macrophage cell line and in primary macrophages, whereas the ⌬iglI mutant replicates only in the former cell type (27). We previously noted, however, that the latter two mutants induced much less prominent host cell cytopathogenic effects than did the parental strain, suggesting a requirement for the encoded proteins in modulating the host cell death pathway induced by F. tularensis (27,55). In contrast, the ⌬pdpE mutant is one of the few FPI mutants that exhibits wildtype phenotypes with regard to replication and cytopathogenicity in monocytic cells (27).…”

IglE Is an Outer Membrane-Associated Lipoprotein Essential for Intracellular Survival and Murine Virulence of Type A Francisella tularensis

“…For example, whereas both LVS iglI and iglG mutants showed essentially wild-type growth in J774 macrophages, only the iglG mutant was proficient for growth in peritoneal exudates and BMMs (22). Differences in intracellular replication and induction of host cell death pathways were also observed following infection of J774 macrophages with pdpC, iglC, iglG, or iglI mutants of LVS (56). Consistent with this result, Long et al found that while Schu S4 iglI and IglJ were essential for phagosomal escape and alteration of endosomal trafficking, a mutant lacking pdpC was only partially defective in this regard (30).…”

Identification of Mechanisms for Attenuation of the FSC043 Mutant of Francisella tularensis SCHU S4

“…Although no effector functions have been assigned to the proteins of the FPI, many of those studied so far have been found to be essential for phagosomal escape and intracytosolic replication of the pathogen, as well as modulation of the host response (14,(27)(28)(29)(30)(31)(32)(33)(34)(35). Several recent publications have addressed the role of PdpC by investigating the phenotypes of specific pdpC mutants or a spontaneous mutant (14,(35)(36)(37). Mutants derived from strain LVS or SCHU S4 have both demonstrated unique phenotypes, since they showed incomplete phagosomal escape, growth in only a small subset of macrophages, intermediate cytopathogenic effects, and marked attenuation in vivo but modulation of the macrophage inflammatory response similar to that of the parental strains (14,35,36).…”

“…Several recent publications have addressed the role of PdpC by investigating the phenotypes of specific pdpC mutants or a spontaneous mutant (14,(35)(36)(37). Mutants derived from strain LVS or SCHU S4 have both demonstrated unique phenotypes, since they showed incomplete phagosomal escape, growth in only a small subset of macrophages, intermediate cytopathogenic effects, and marked attenuation in vivo but modulation of the macrophage inflammatory response similar to that of the parental strains (14,35,36). Very recently, a spontaneously attenuated SCHU S4 mutant was described (37).…”